REVIEW OF LITERATURE - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/18626/5... · REVIEW OF LITERATURE In Punjab (an Indian State), rough lemon (Citrus jambhiri Lush.) is being

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REVIEW OF LITERATURE

In Punjab (an Indian State), rough lemon (Citrus jambhiri Lush.) is being used

as major root stock for a number of Citrus spp. like mosumbies, kinnows, oranges,

grape fruits etc. for its high vigour and well adaptation to climate of Punjab as well as

resistance to Citrus tristeza virus (cause of an epidemic). However, the main drawback

is its sensitivity to Phytophthora, which leads to major losses in an orchard if proper

phytosanitary conditions are not followed. Phytophthora species have been shown to

cause some serious soil borne diseases of citrus including damping off of seedlings in

the seedbed, root and crown rot in nurseries, foot rot and brown rot of fruits.

2.1. CITRUS DISEASES CAUSED BY PHYTOPHTHORA SPP.

Phytophthora is a cosmopolitan Oomycete of warm temperate, subtropical, and

tropical environments. It has a broad host range including both herbaceous (e.g.

tobacco, tomato, carnation) and woody (e.g. Citrus and Eucalyptus) hosts.

Phytophthora spp. cause the most serious and economically important soilborne

diseases of Citrus crops. Tree and crop production losses occur from damping-off of

seedlings in the seedbed, root and crown rot in nurseries, foot rot and fibrous root rot

and brown rot of fruits in groves. Foot rot results from infection of the bark near the

ground level producing lesions on the trunk or crown roots that can girdle and kill the

tree. Phytophthora spp. also attack and cause the decay of fibrous roots, especially on

susceptible rootstocks in nurseries. In bearing groves, fibrous root rot damage causes

tree decline and yield losses. The most important species include Phytophthora

parasitica Dastur (P. nicotianae), P. citrophthora and P. palmivora. P. parasitica has

been reported to cause citrus diseases in India (Kamat, 1927; Uppal and Kamat, 1936;

Kumbhare and Moghe, 1976; Lele and Kapoor, 1982; Naqvi, 1988; Naqvi, 2000b) and

Florida (Zitko and Timmer, 1994; Widmer et al., 1998; Graham and Menge, 2000).

Other Phytophthora spp. viz. P. boehmeriae, P. cactorum, P. cinnamon, P. citricola, P.

citrophthora, P. dreschleri, P. hibernalis, P. megasperma, P. palmivora and P. syngiae

have been reported pathogenic on citrus from different Citrus growing areas of World

................................................................................................................................Review of literature

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(Boccas and Leville, 1978; Erwin and Rebiero, 1996; Naqvi, 2002b). P. citropthora and

P. parasitica are wide spread in tropical and subtropical areas of citrus causing foot rot,

root rot, gummosis and brown rot of citrus.

2.1.1. History and distribution

Phytophthora is most destructive pathogen of Citrus trees. It had been causing

severe crop losses to citrus orchards much before its discovery in real scientific terms.

In 1836, large scale destruction of citrus plants because of Phytophthora infection was

recorded in Azore Island, much before the potato famine of Ireland in 1845, and after

31 years of famine Anton de Bary discovered the fungus as Phytophthora means “plant

destroyer”. The first appearance of Phytophthora epidemic of citrus was reported in

Azore Island during 1832-1836 (Bonavia, 1988). Later Phytophthora epidemics of

citrus were reported in 1841 from France, in 1845 from Purtagal, in 1855-1889 from

Italy, in 1860-1879 from Australia, in 1871 from Spain, in 1875 from California, in

1876 from Florida, in 1869-1880 from Greece, in 1906 from Cuba, in 1911 from

Paraguay, in 1917 from Brazil, in 1920 from Mexico and in 1935 from Trinidad

(Fawcett, 1936).

An extensive survey of citrus orchards and citrus nurseries in India has been

conducted under National Network Project on Phytophthora disease of Horticulture

Crops (Citrus) funded by Indian Council of Agricultural Research during 1997-2002.

Citrus cultivation belt of Vidarbha and Marathwada region of Maharashtra, Punjab,

Madhya Pradesh, Andhra Pradesh and North Eastern States of India were surveyed to

assess the impact of Phytophthora diseases. Two mating type (A1 and A2) of

Phytophthora parasitica (nicotianae) have been recorded from the orchards of Nagpur

mandarin in Nagpur district (Naqvi, 2000b, 2002b). P. parasitica Dastur has been a

common species associated with citrus disease in Assam (Chowdhury, 1951) and in

North-West India (Bajwa, 1941; Paracer and Chahal, 1962). Kapoor and Bakshi (1967)

reported that 14-18% decline in sweet orange was due to foot rot in Abohar (Punjab).


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In Madhya Pradesh adjoining to Vidharbha region of Maharashtra, India 20-

50% Nagpur mandarin plants were found affected and causing severe decline due to P.

parasitica, P. citrophthora along with P. palmivora (Naqvi, 2000b). In Andhra

Pradesh, 20-100% acid lime plantation was severly affected with P. parasitica along

with P. citropthora and P. palmivora. Kinnow growing areas of Punjab state, 10-80%

plants of C. sinensis Osbeck and 10-100% plants of kinnow mandarin ranging from 12-

25 years old, were showing symptoms of diseases caused by P. parasitica, P.

citrophthora and P. palmivora due to accessive flood irrigation (Naqvi, 2002a, 2002b).

Northeastern states of India are considered the natural home of citrus and place of origin

of several Citrus species. In Tripura State, the citrus cultivation is mostly confined to

Jampui Hills and sankan area about 140 Km from Agartala. In Tripura and Mezoram

State, almost all the citrus orchards are seriously infested/infected with P. parasitica, P.

citrophthora and P. palmivora causing gummosis, root rot and leaf fall (Naqvi, 2000b,

2002b).

2.1.2. Symptomatology

A survey of Central India for Phytophthora diseases of Nagpur Mandarin and

other Citrus spp. has revealed that P. parasitica, P. citrophthora along with P.

palmivora are causing root rot and crown rot of C. jambhiri (rough lemon)- a major root

stock for the area and foot rot and leaf fall of nagpur mandarin (Naqvi, 1988, 2000a,

2002a, 2002b). A 1987 survey of nurseries in Florida indicated that 8 of 15 field

nurseries were infested with P. parasitica, whereas only 1 of 13 of the greenhouse

operations was infested (Zitko et al., 1987). Several species of Phytophthora have been

associated to this syndrome but P. citrophthora and P. parasitica (sin. P. nicotianae)

have been identified as the most destructive species causing foot rot (gummosis) and

root rot, often considered as two different diseases affecting citrus production

worldwide (Klotz, 1973; Timmer and Menge, 1993; Zitko et al., 1991; Martins et al.,

2001; Siviero et al., 2002; Timmer et al., 2003; Vernière et al., 2004).

In Chile, P. citrophthora and P. parasitica were reported on lemon and sweet

orange associated with foot rot and P. citrophthora to citrus brown rot on lemon fruits


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(Besoain et al., 1998; Mujica and Vergara, 1980). However, a complete characterization

of the Phytophthora species involved in these syndromes and their relative importance

and distribution remain unknown in Chile. In Spain P. citrophthora was shown to affect

clementine (Citrus clementina Hort. ex Tan.) cultivars and their hybrids (Alvarez et al.,

2008a,b, 2009) resulting in considerable losses. Diseased trees showed cankers and gum

exudations on the above-ground parts on the scions, especially on the major limbs,

whereas rootstocks generally remained healthy. The trunk and secondary branches were

affected by the expansion of these lesions and eventually, the entire tree collapsed and

died (Alvarez et al., 2008a). Phytophthora branch canker has also been described in the

winter rainfall citrus-growing areas of South Africa affecting mainly clementine

cultivars (Schutte, 2007). Phytophthora causes the following common diseases in Citrus

cultivars.

2.1.2.1. Damping-off

Damping-off of seedlings in nursery bed is a widespread problem of citrus

industry and frequently occurs in citrus orchards where phytosanitory conditions are

difficult to maintain. More than 20% seedling mortality has been observed in central

India due to Phytophthora infection (Naqvi, 1996, 2000a). Damping-off of Citrus

seedlings is caused by Phytophthora parasitica, P. citrophthora and P. palmivora.

Typical symptoms of damping-off result when the soil or seed-borne fungus penetrates

the stem just above the soil line and causes the seedling to topple. Phytophthora spp.

also cause seed rot or pre-emergence rot. Infected seedlings are killed rapidly when

moisture is abundant and temperatures are favourable for fungal growth (Klotz et al.,

1969). Plants usually become resistant to damping-off once true leaves have emerged

and the stem tissue at the soil line has matured.

2.1.2.2. Foot rot and gummosis

The most serious diseases caused by Phytophthora spp. are foot rot and

gummosis (Fawcett, 1913; 1936). Foot rot results from an infection of the scion near the

ground level, producing lesions which extend down to the budunion on resistant


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rootstocks. Scaffold root rot or crown rot below ground may occur when susceptible

rootstocks are used. Infected bark remains firm with small cracks through which

abundant gum exudation occurs. Citrus gum, which is water-soluble, disappears after

heavy rains but is persistent on the trunk under dry conditions. Lesions spread around

the circumference of the trunk, slowly girdling the tree. Badly affected trees have pale

green leaves with yellow veins, a typical girdling effect. If the lesions cease to expand

or the fungus dies, the affected area is surrounded by a callus tissue. Nursery trees and

young orchard trees of small trunk circumference can be rapidly girdled and killed.

Large trees may be killed likewise, but typically the trunks are partially girdled and the

tree canopy undergoes defoliation, twig dieback, and short growth flushes. On

susceptible rootstocks, lesions may occur on the crown roots below the soil line and

symptoms in the canopy develop without obvious damage to the trunk above ground

(Timmer and Menge, 1993; Timmer et al., 2003; Alvarez et al., 2008a).

2.1.2.3. Fibrous root rot

Phytophthora spp. infect the root cortex and cause a decay of fibrous roots. The

cortex turns soft, becomes somewhat discoloured and appears watersoaked. The fibrous

roots slough their cortex leaving only the white thread-like stele, which gives the root

system a stringy appearance. Root rot can be especially severe on susceptible rootstocks

in infested nursery soil. Root rot also occurs on susceptible rootstocks in bearing

orchards where damage causes tree decline and yield losses. In advanced stages of

decline, the production of new fibrous roots cannot keep pace with root death. The tree

is unable to maintain adequate water and mineral uptake and nutrient reserves in the

root are depleted by the repeated fungal attacks. This results in the reduction of fruit

size and production, loss of leaves and twig dieback of the canopy (Timmer and Menge,

1993; Timmer et al., 2003).

2.1.2.4. Brown rot of fruit

Phytophthora infection of fruit produces a decay in which the affected area is

light brown, leathery and not sunken compared to the adjacent rind. White mycelium


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forms on the rind surface under humid conditions. In the orchard, fruits near the ground

become infected when splashed with soil containing the fungus. If favourable

conditions of optimum temperature (75-82°F) and long periods of wetting (18 plus

hours) continue, the disease spreads to fruit throughout the canopy. Most of the infected

fruit soon abscise, but those that are harvested may not show symptoms until after they

have been held in storage for a few days. If infected fruit is packed, brown rot may

spread to adjacent fruit in the container. In storage, infected fruit have a characteristic

pungent, rancid odour. Brown rot epidemics are usually restricted to areas where

rainfall coincides with the early stages of fruit maturity (Timmer and Menge, 1993;

Timmer et al., 2003).

2.1.3. Disease cycle

The disease cycle of P. parasitica begins with the production of sporangia

which release large numbers of zoospores, chlamydospores and oospores. With time

and appropriate conditions zoospores encyst and germinate to form mycelia. The

optimum temperature for mycelial growth is 86°-90°F. Sporangial production is favored

by small deficits in matric water potential (5 to -70 KPa), but not by saturated

conditions unless sporangia are produced on citrus root pieces. The optimum conditions

for sporangium formation probably represent a compromise between requirements for

free water and aeration. Nutrition depletion and light also stimulate sporangial

production from mycelium. Indirect germination of sporangia to produce zoospores

requires free water and is stimulated by a drop in temperature. Under moist conditions

sporangia may also germinate directly by growth of germ tubes, but the correlation

between soil saturation and severity of Phytophthora root rot suggests that indirect

germination is more important in the root disease cycle.

Chlamydospore production by P. parasitica occurs under unfavourable

conditions for fungal growth, i.e. nutrient depletion, and low oxygen levels and

temperature (59-64°F). Water requirements for germination of chlamydospores are

similar to those for sporangia. Chlamydospores of P. parasitica appear to become

dormant below 59°F, so exposure to temperatures of 82-90°F is used to stimulate


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germination. Nutrients that are acquired from soil extracts and excised citrus roots are

known to stimulate chlamydospore germination. The requirements for oospore

germination are thought to be nearly identical to those of chlamydospores. Oospore

maturation appears to be an important factor in germinability of Phytophthora spp.

Periods of alternating high and low temperatures may also be a prerequisite for uniform

germination (Timmer and Menge, 1993; Timmer et al., 2003).

2.1.4. Epidemiology

Phytophthora inhabits the soil and is a water loving fungus. It survives in soil

through small thick walled spores (chlamydospores) and oospores which can tolerate

dry summer conditions. The chlamydospores are developed in cool and dry conditions

with limited soil moisture, poorly aerated soil with high CO2 concentration and in

paucity of actively growing roots since Phytophthora species are primarily parasites but

are poor saprophytes in soil. Under favourable conditions of high moisture and

temperature, infected roots produce sporangia which in turn release motile zoospores.

The zoospores swim in water for short distance by flagellar movement or may be

carried away by rain or irrigation water. Zoospores are attracted to the zone of

elongation of new roots by nutrients in exudations. Upon contact with the root,

zoospores encyst, germinate and then infect in the area of the zone of root elongation

(Duniway, 1983; Khew and Zentmeyer, 1973, 1974). Once the fungus has entered the

root tip, the infection may advance in the cortex resulting in rot of the entire rootlet. The

cycle can repeat itself as long as conditions are favourable and susceptible tissue is

available.

In case of P. parasitica, the fungus most likely survives unfavourable periods in

root debris. The rotted cortex is sloughed and the fungus produces chlamydospores

which may persist in the soil for long periods (Tsao and Ocana, 1969). When favourable

conditions return, chlamydospores germinate indirectly to produce sporangia and

zoospores or directly to produce mycelium. When both mating types are present

oospores may also be produced which aid survival of the fungus. Foot rot or gummosis

of the trunk occurs when zoospores or other propagules are splashed onto the trunk


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above the bud union. A wound and moisture on or around the base of the trunk are

necessary for infection. Wounds are susceptible to infection for up to 14 days. Foot rot

lesions do not usually produce inoculum for subsequent infections and thus are of no

epidemiologic significance. Even though Phytophthora spp. in Citrus are usually

present, epidemics can develop in certain cases. When Phytophthora spp. are first

introduced into a grove or nursery, spread to other plants may be epidemic. However,

epidemics of foliage blight and brown rot are much more common, especially on highly

susceptible hosts such as lemons. When conditions are favourable, fruit and young

foliage may become infected by propagules splashed from the soil. Secondary

infections may then be caused by inoculum from the above-ground parts of the plant

which is dispersed by rain splash or wind-blown rain. This seldom occurs with P.

parasitica which does not readily produce aerial sporangia, but is more common in P.

citrophthora and other species which produce abundant sporangia on fruit and leaf

surfaces. Brown rot is most common in areas where these species are present and when

prolonged winter rains occur (Graham and Timmer, 1992; Naqvi, 1999a, 1999e, 2000a,

2000c).

2.1.5. Means of spread

The primary means by which Phytophthora spp. are spread through citrus

orchards is by use of infested nursery stock (Graham and Timmer, 1992; Naqvi, 1999a,

1999e, 2000a, 2000c). Ristaino and Gimpertz (2000) reported the mechanisms of

dispersal of Phytophthora species viz. (i) dispersal from root to root in soil involves

either root growth to inoculums, inoculum movement to roots or root to root contacts.

(ii) Inoculum dispersal in surface water. (iii) Splash dispersal from soil to aerial parts of

the plant. (iv) Aerial dispersal from sporulating lesions on leaves, stem, fruits or other

aerial parts of the plant and (v) dispersal by human or invertebrate activities including

movement of soil, plants or propagules. The pathogen may be present in soil or infected

roots even though disease symptoms are not readily apparent. The fungus is also carried

in soil on equipment when vehicles move from infested to non-infested groves or

nurseries. Propagule densities decline sharply when soil is air-dried, reducing the

probability of spread. Irrigation water may also move the pathogen from area to area.


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Within groves, dispersal by irrigation water occurs especially where furrow or flood

irrigation is used. Surface water following heavy rains may carry the fungus as it drains

from the grove. More serious problems can arise in irrigated Citrus areas where run-off

water carries the pathogen into canals or ponds. Use of water from these sources may

then contaminate previously noninfested areas. Wind is not a major factor in dispersal

of Phytophthora spp. However, wind-borne soil carries Phytophthora spp. and may

recontaminate fumigated soils. Windblown rain can disseminate sporangia produced on

the surface of aboveground plant parts. Environmental factors also play an important

role in disease development, severity, dispersal and surviaval of Phytophthora in

causing epidemics (Govindarao, 1954; Cheema et al., 1954). Phytophthora parasitica

was prevalent in warmer and drier climate of Vidarbha, Ahmednagar and Kodur at 30◦-

32◦C temperature and rainfall below 1150-1300 mm (Lele and Kapoor, 1982). In Citrus

nurseries of Vidarbha region, population of P. parasitica and P. palmivora increases

with increase in temperature while population of P. citrophthora decreases with

increase in temperature. The soil temperature in central India remains between 20-29◦C

through out the year. Thus, Phytophthora remains active throughout the year in citrus

nurseries and irrigated orchards of high region (Naqvi, 1990, 2002b). The critical

threshold temperature for Phytophthora parasitica is 33◦C or above in Arizona. A

fivefold increase in zoospore motility was observed for P. citrophthora at 24◦C than at

30◦C, temperature that respectively prevents and favours colonization while an 11-fold

increase was detected for zoospores of P. parasitica at favourable temperature

compared to inhibitory soil temperatures of 30 and 36◦C respectively (Matheron and

Porchas, 1996).

2.1.6. Isolation and detection

For disease management purposes, it is frequently desirable to know whether

Phytophthora spp. are present and if so, at what level. Fruit and leaf baiting methods

were developed many years ago for detection of Phytophthora spp. (Grimm and

Alexender, 1973; Klotz et al., 1958). These are usually considered qualitative tests, but

propagule densities can be quantitated by use of more complex techniques. They are

relatively simple and require minimal equipment and supplies. Subsequently, media


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have been developed for the selective isolation of Phytophthora spp. from soil to

determine propagule densities (Tsao, 1960; 1970; 1983). Methods for sample collection

and handling have been developed. Propagule densities are highest where fibrous root

densities are greatest. Thus, populations diminish with depth and with distance from the

tree. It is also important to recognize that Phytophthora populations are not uniform

across the orchard. Spatial distribution studies in Florida indicate a more random

distribution of the fungus in a grove. For routine propagule density determinations,

samples should be collected at random in the orchard. Propagule densities vary

considerably between sample times and populations may show seasonal trends of

abundance. Genus specific polyclonal and monoclonal antibodies have been produced

against Phytophthora. For detection and quantification of Phytophthora parasatica, P.

citrophthora and P. palmivora many mediums have been used viz. Modified Kerr’s

medium (Hendrix and Kuhlman, 1965), MacCain medium (McCain et al., 1967),

Masago medium (Masago et al., 1977), PVPH medium (Tsao and Guy, 1977), PARPH

medium (Mitchell et al., 1986) and BHMPVR medium (Bist and Nene, 1988). The

number of selective agar media containing antibacterial antibiotics and selective fungal

agents have been proved successful in Phytophthora isolations from soil and plant

tissues (Eckert and Tsao, 1960; Kuhlman and Hendrix, 1965; Flowers and Hendrix,

1969; Tsao and Ocana, 1969; Sneh, 1972; Fugisawa and Masago, 1975; Masago et al.,

1977; Tsao and Guy, 1977; Kueh and Khew, 1982). Leaflets of Albizzia falcatarea were

used as baits to isolate Phytophthora palmivora (Butl). Butt. MF4 from soil (Anandaraj

and Sharma, 1990).

Diagnostic kits using enzyme-linked immunosorbent assay (ELISA) have been

developed for detection of Phytophthora spp. in roots and in soil debris. The ELISA

method is highly sensitive and can detect the presence of Phytophthora at lower

population densities than dilution plating onto selective media (Miller and Martin, 1988;

Miller et al., 1990; Skaria and Miller, 1989). Theoretically, the tissue baiting and the

ELISA soil assay should be the most sensitive since they utilize larger volumes of soil.

However, in one study the leaf baiting and selective media were about equally sensitive

(Timmer et al., 1993). Although it is possible to quantify Phytophthora spp.


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populations, the significance of those populations remains yet to be determined.

Populations may vary considerably during a season suggesting a need to sample

repeatedly through the year as well as from year to year. Propagule densities in most

Florida groves range from 1-20 propagules/cm3, but occasionally range as high as 100-

200 propagules/cm3. Threshold densities have not been established, but populations of

less than 5 propagules/cm3 have been considered insignificant. Increases in fibrous root

densities and in some cases, yield have been observed following fungicide treatment of

orchards with 10 propagules/cm3 or higher.

Recently, for quick and reliable identification of Phytophthora species, internal

transcribed spacers (ITS) based identification have been developed to identify known

species of Phytophthora and unknown isolates quickly (Cooke et al., 1996; Cooke et

al., 2000; Crawford et al., 1996; Cooke and Duncan, 1997; Lee et al., 1993; Levesque

et al., 1998; Ristaino et al., 1994). An identification website has also been developed to

facilitate Phytophthora workers where user generated ITS digest profiles of any

unidentified taxa may be entered to the search page for comparison with the database.

The website (www.phytid.com) includes a database of 48 Phytophthora species.

However, some species remain as polyphyletic assemblages awaiting more rigorous

analysis (Braiser and Hanson, 1992). There is need to optimize PCR assays for

detection of different propagule types in soil, if these assays are to be used for precision

agriculture application (Ristaino and Gumpertz, 2000). Molecular detection methods of

Phytophthora and their impacts on plant disease management have been reviewd

(Martin et al., 2000).

2.1.7. Pathogenicity testing

Zoospore suspensions have been successfully used by several workers to

establish the pathogenicity of Phytophthora spp. on their hosts (Mehrotra, 1972;

McDonald and Duniway, 1978; Sastry, 1982; Dutta, 1984; Subramanian, 1993).

Sowic et al. (2001) reported the effects of culture filtrates (CFs) and

homogenates of Verticillium dahliae and Phytophthora cactorum on in vitro growth and

development of strawberry shoots of cvs. Elsanta, Real and Senga Sengana. The


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filtrates of fungal cultures as well as filtrates of homogenated mycelia did not affect

strawberry shoot growth and development, whereas inoculation with mycelia of V.

dahliae and P. cactorum resulted in leaf yellowing, growth inhibition, inhibition of root

formation and after several days, the death of infected plants. However, fungal growth

on the culture media was irregular and the infection rate of individual plants was

variable.

Siviero et al. (2002) reported inoculation methods of Phytophthora parasitica

on seedlings and young plants of Citrus spp. to determine the resistance of citrus

rootstocks to Phytophthora gummosis. The following inoculation methods were tested:

non-wounded host-pathogen contact, bark removal, insertion of culture disc under bark,

insertion of inoculated needle or toothpick in the collar region and the culture disc

method. The assessment of resistance to gummosis in citrus seedlings produced in vitro

was achieved by inserting an inoculation in collar region. The disc method and insertion

of disc under the bark were the best inoculation methods for young plants under both

orchard and greenhouse conditions. The best variable for disease assessment had been

the measurement of the total lesion area. Lesion length can also be used when

pathogenesis tests were carried out in nurseries and in the field.

Vial et al. (2006) reported pathogenicity testing of different isolates of

Phytophthora spp. A total of 27 pathogenic isolates of Phytophthora were obtained in

2003 and 2004. Morphologically, 20 isolates were identified as P. citrophthora;

however, they were distinctively divided into sterile and fertile isolates. The latter were

morphologically similar to P. citrophthora sensu lato but, they induced oospore

production when pairing against P. cinnamomi A2. These fertile isolates exhibited a

98% match with several P. citrophthora strains and were considerably less aggressive

than sterile isolates. Phytophthora inundata, a relatively new described species, was

identified on the basis of analysis of the ITS regions of rDNA. Isolates of P. inundata

were weakly pathogenic on citrus and possibly were found as secondary pathogens

without importance on foot and root rot development on Citrus trees. P. citrophthora

was the cause of foot and root rot found on Chilean citrus groves, planted in temperate

and relatively dry weather conditions. The isolation and pathogenicity of P.


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citrophthora on Citrus macrophylla and carrizo citrange, considered resistant

rootstocks, could be suggesting that these isolates have overcome resistant genes

associated with Phytophthora resistant citrus rootstocks.

Savita et al. (2011a) studied the effect of culture filtrate (CF) of P. parasitica

on rooting response by culturing shoots regenerated from calli on MS medium

containing 0.5 mg/l NAA and different concentrations of CF (5–100%). Two types of

controls were set: MSA- MS rooting medium containing autoclaved CF and MS0-MS

rooting medium without CF. When regenerated shoots were cultured on rooting

medium without CF they showed 91.66% rooting response whereas culture of shoots on

MSA medium showed rooting in 86.11% cultures. A dramatic fall in rooting response

(47.22%) was observed when shoots were cultured on rooting medium containing 5%

CF. At 10% CF only 19.45% shoots showed rooting. Whereas no rooting response was

observed with 15% and higher concentrations of CF. 100% necrosis of shoots was

observed with 25% and higher concentrations of CF.

2.1.8. Economic importance

Phytophthora species are responsible for significant economic losses to citrus

industry. Besides foot rot and brown rot, root rot caused by Phytophthora alone reduces

46% yield of citrus plants in California and Citrus industry losses around 12.9 million

dollar annually due to this pathogen (Anonymous, 1989; Menge, 1993). To combat this

problem in India, Indian Council of Agricultural Research (ICAR) launched a National

Network Project on Phytophthora Diseases of Horticultural Crops (PHYTONET) in

1977. Phytophthora causes serious disease to Citrus and infect almost every part of

citrus plants (Naqvi, 2003).

2.1.9. Disease management

The disease is transmitted from soil, hence the use of Phytophthora resistant/

tolerant budwood or saplings is the recommended principle to check the spread of the

disease in the virgin areas. The “Budwood Certification Programme” including

surveillance, indexing and identification of mother trees resistant to Phytophthora


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should be strengthened for raising disease free planting materials. The marked

Phytophthora resistant trees should be multiplied and distributed to nurserymen for

producing Phytophthora resistant seedlings. Strict plant quarantine measures should be

enforced to check the movement of contaminated budwood and planting material in the

new planting sites. Some of the effective fungicides for Phytophthora root rot diseases

include Subdue MAXX, Chipco Aliette, Banrot and Truban which can be used for

effective disease management.

2.2. TISSUE CULTURE STUDIES IN CITRUS SPECIES

Studies on citrus tissue and organ culture in vitro were initiated in early 1950s

with the aim of genetic improvement of the species as well as to obtain virus free plants.

In vitro techniques have the potentiality to multiply for large scale propagation.

Plantlets can be obtained through direct plant regeneration, indirect plant regeneration

through callus and through somatic embryogenesis.

2.2.1. Direct plant regeneration

Regeneration through callus has one major limitation in the sense that

regenerants produced via callus mediated regeneration exhibit somaclonal variations

which is not desirable in an attempt to produce uniform planting material. On the other

hand, tissue culture methods inducing direct shoot regeneration possesses less

probability of somaclonal variations among regenerants in comparison to callus

mediated regeneration pathways. Furthermore, the availability of an efficient

regeneration system is also a necessary pre-requisite for genetic improvement and

genetic resource conservation. Several researchers have reported regeneration in

different species of Citrus using different explants.

Edriss and Burger (1984) reported direct shoot formation from root segments of

troyer citrange (Citrus sinensis [L.] Osbeck x Poncirus trifoliata L. Raf.) on Murashige

and Skoog’s (MS) medium supplemented with 10 mg/l BA + 1 mg/l NAA. Sim et al.

(1989) reported shoot induction from different vegetative parts of Citrus mitis seedlings


20

(epicotyl, leaf, shoot tip and nodal segments) and mature plants. A high frequency (66–

100%) of shoot regeneration was obtained from shoot tip and nodal segments of mature

plants cultured on medium with or without benzylaminopurine (BA). Bud induction

from roots was examined in 3 systems: (a) intact roots on whole seedlings; (b) intact

roots on decapitated (shootless) seedlings; (c) excised roots. The highest yield of buds

was obtained from intact roots on whole seedlings cultured in medium with 0.5 mg/1

BA, from which 275 shoots were obtained from 13 cultures in 9 weeks. Regenerated

shoots were rooted in basal medium and the plantlets were successfully transplanted to

soil.

Tisserat et al. (1989) reported that juice vesicles isolated from 120-180 day old

fruits of different Citrus spp. were capable of proliferation via adventitious vesicle

branching in vitro. Gibberellic acid (GA3) levels of 1-100 mg/l greatly enhanced

adventitious vesicle branching while delaying vesicle senescence as evidenced by the

vesicle’s ability to retain its green colour for up to 4 to 6 months in culture. Vesicles

grown on media without GA3 readily matured, turning opaque within 2 months in

culture and rarely produced adventitious branches. Additions of 0.1 mg/l

α-naphthaleneacetic acid (NAA) or 1.0 mg/l BA to media containing 10 mg/l GA3

further enhanced vesicle branching in some species and cultivars. Adventitious juice

vesicles have been induced from a variety of Citrus species, including Citrus grandis

(L.) Osb., C. hystrix DC., C. limon (L.) Burm. f., C. medica L., C. paradisi Macf., C.

reticulata Blanco and C. sinensis (L.) Osb. Adventitious vesicle branches originate from

primordia initiated on the surface of the cultured vesicle. These primordia commonly

occurred on the terminal meristem region of the vesicle. However, some species (e.g. C.

grandis and C. paradisi) produced adventitious vesicle branches from their bodies and

stalks as well. Distinct vesicle branches begin to appear on preformed vesicles after 30-

60 days in culture.

Bhat et al. (1992) established root culture of lime, C. aurantifolia, in MS

medium containing 3% sucrose. De novo shoot bud initiation was recorded in basal

medium at a low frequency during three years of continuous culture. The effect of

indole-3-acetic acid (IAA) and BA investigated on root growth and shoot


21

morphogenesis. Inclusion of BA in the culture medium had an important effect on root

growth and shoot elongation. The frequency of shoot regeneration was only slightly

improved by the growth regulator treatment. Shoot bud differentiation occurred at the

proximal region of the root at or just distal to the cut end. Histology of shoot

regeneration revealed the endogenous origin of the shoot buds from the pericycle tissue

of the root. The regenerated plants contained normal diploid chromosome number (2n =

18) and were successfully established in soil.

Goh et al. (1995) reported shoot regeneration from root segments of Pummelo

(C. grandis) with 0.89 µM BA and shoot regeneration from epicotyl segments with 2.2

µM BA. Maggon and Singh (1995) reported promotion of adventitious bud regeneration

by absicic acid (ABA) in combination with BA in epicotyl and hypocotyl explants of

sweet orange (Citrus sinensis L. Osbeck var. mousambi). Epicotyl and hypocotyl

segments (1–1.5 cm) from 2 weeks old sweet orange seedlings germinated in vitro were

placed horizontally on MS basal medium. Adventitious shoot regeneration from the cut

ends occurred only in epicotyl explants. Addition to the MS medium of BA up to 2 mg/l

had a promotive effect, both in terms of the frequency of responding explants and the

number of shoots per explant, while higher concentrations of BA had a depressive

effect. GA3 at 0.04 and 0.4 mg/l with or without 2 mg/l BA suppressed shoot bud

regeneration in both types of explants. ABA alone suppressed shoot regeneration when

added to basal MS medium, but in combination with 2 mg/l BA it had a strong

promotive effect, particularly at 0.2 mg/l. A remarkable feature of this ABA effect was

that the bulk of the buds regenerated away from the cut ends directly from the epidermis

without any apparent callus formation. These shoot buds did not develop further even

8–10 weeks after their transfer to the basal MS medium, but showed normal growth on

medium containing 0.04 mg/l GA3.

El-Morsy and Millet (1996) reported shoot induction from nodal segments of C.

aurantium on MS medium supplemented with 0.5 mg/l Kinetin (KN) + 0.5 mg/l GA3 +

0.2 mg/l indole-3-butyric acid (IBA). Roots were induced from shoots on MS medium

supplemented with NAA 2 mg/l. Gutierrez et al. (1997) used Murashige and Tucker’s

medium supplemented with BA 5 mg/l to regenerate shoots from internodal stem


22

segments of sour orange (C. aurantium). Normah et al. (1997) reported multiple shoot

induction from shoot tips, nodal segments and hypocotyls of C. halimii on MS medium

supplemented with 4.4-11.1 µM BA, 2.2 µM BA and 2.2-11.1 µM BA respectively.

These shoots were separated and individual shoots were transferred on rooting medium.

Best rooting response was observed on MS medium supplemented with 2.7 µM NAA.

Garcia-Luis et al. (1999) reported that morphogenic pathway of adventitious

bud and shoot regeneration at the ends of troyer citrange epicotyl cuttings is determined

by polarity and explant orientation. In explants planted vertically with the basal end

inserted in the medium, bud formation at the apical end occurs by direct organogenesis.

Bud growth and subsequent shoot formation is markedly increased by the addition of

BA to the medium. This growth regulator also increases the number of buds formed.

When the epicoyl cuttings come in contact with the culture medium, both the apical end

and the basal end of the cutting form a vigorous callus with many xyllary elements. In

these calli, buds differentiate by a process of indirect organogenesis. This indirect

regeneration pathway requires the addition of BA to the medium and the number of

buds formed is higher at the apical end than at the basal end of the cuttings. This

pathway of regeneration is reduced as the position of the cuttings during incubation

deviates from the normal upright vertical position. Thus, for the basal end of the

cuttings, the number of buds and shoots formed is higher when the explants are placed

vertically than when they lie on the surface of the medium. For the apical end, this

number is higher in explants placed horizontally than when inserted vertically in the

medium in an inverted position.

Le et al. (1999) designed a method for direct in vitro bud regeneration of

trifoliate orange (Poncirus trifoliata L. Raf). Transverse thin cell layer (tTCL) explants

excised from the stem internodes of 1 year old young plants of P. trifoliata regenerated

bud in vitro on a medium containing BA 1-50 µM and N-phenyl-N’-1,2,3-thidiazol-5-yl

urea (thidiazuron, TDZ) (0.1–10 µM). The optimal concentrations for bud induction

were 25 µM BA and 1 µM TDZ leading to 87 and 72% of responsive tTCLs and 24 and

15 buds per tTCL, respectively. A higher percentage of responsive tTCLs and a higher

frequency of bud regeneration were obtained with BA and TDZ combined. With a


23

combination of 10 µM BA and 1 µM TDZ, 90% of responsive tTCLs forming 37 buds

per tTCL were obtained. Shoot elongation occurred after a transfer onto a medium

containing 1 µM GA3. Rooting of individual shoots was induced using 5 µM NAA. One

hundred per cent of rooted shoots developed normally after transfer to the greenhouse;

no phenotype variation was observed. High numbers of regenerated viable plants could

be produced directly without callus formation from tTCL after 9 weeks of culture.

Moreira-Dias et al. (2000) reported bud differentiation by direct organogenesis

at the apical end of troyer citrange (Citrus sinensis L. Osbeck X Poncirus trifoliata L.

Raf.) epicotyl cuttings inserted vertically in a semi-solid culture medium without any

hormone addition. The number of buds regenerated was slightly, but significantly,

increased when the incubation was performed in the light as compared to dark and by

the addition of BA (2.2 to 22 µM) to the medium. Bud sprouting and subsequent shoot

formation required the addition of BA and was increased by light to a higher extent than

bud formation. The best response was obtained with the highest BA concentration

tested (22 µM). Regeneration through the indirect organogenic pathway at the two

edges of the epicotyl cuttings when in contact with the culture medium did not occur in

the absence of BA, which was an absolute requirement for callus development. The best

regeneration response was obtained when the explants were incubated in the light in the

presence of 4.4 µM BA and an auxin. IAA 5.8 µM was more effective in increasing

shoot formation than NAA 0.54 µm. Higher NAA concentrations inhibited shoot

formation. Incubation in the dark or increasing the BA concentration (22 µM) increased

markedly callus growth, but inhibited both bud differentiation and sprouting, almost

completely suppressing shoot formation. The conditions during regeneration affected

the rooting of the regenerated shoots. Rooting of 86% of the shoots was achieved in a

medium with 2.7 µM NAA and 2.6 µM IBA. All the rooted explants acclimated and

survived transplanting. Under the optimal conditions tested, the proliferation rate

obtained through the indirect regeneration pathway ranged from 60 to 86 plants per

seedling.

Bordon et al. (2000) reported the influence of light, hormones and explant

orientation on in vitro regeneration in epicotyl cuttings and compared the influence in


24

four Citrus species (C. aurantium, C. macrophylla, C. reshni and C. sinensis) and

hybrid troyer citrange (C. sinensis × Poncirus trifoliata). Explants were planted in

various orientations on MS medium supplemented with 100 mg/l myo-inositol, 10 mg/l

thiamine-HCl, 10 mg/l pyridoxine, 1 mg/l Nicotinic acid; 30 g/l sucrose; 4.4 µM BA

and 0.54 µM NAA. Root formation occurred both in the presence of NAA or low

concentrations (2.2 to 4.4 µM) of the cytokinin BA, but no buds were formed. Paudyal

and Haq (2000) reported in vitro propagation of Pummelo (C. grandis (L.) Osb.) using

shoot tip explants from seedlings. Maximum shoots per explant were obtained on

medium supplemented with 1.8 µM BA. Addition of 5.8 µM GA3 to shoot

multiplication medium during second subculture, enhanced the shoot elongation

response significantly. Over 75% rooting response was observed when shoots were

transferred to half strength MS medium containing 1.3, 2.7, or 5.4 µM NAA.

Al-Khayari and Al-Bharany (2001) established a micropropagation protocol for

lime, Citrus aurantifolia Christm Swing., using nodal explants of mature trees. Nodes

were cultured on MS medium containing IBA at 0, 0.5 and 1 mg/l combined with BA at

0, 0.25, 0.5, 1 and 2 mg/l and KN at 0, 0.5 and 1 mg/l. Best results for multiple shoot

formation (8 shoots per node) were obtained with 1 mg/l BA and 0.5 mg/l KN. The

concentration of IBA had little effect on shoot multiplication. Shoot elongation

appeared to favour 0.25 mg/l BA combined with 1 mg/l KN. Shoot elongation and leaf

size were inhibited in response to high levels of BA. Transfer of shoots to a rooting

medium induced the highest percentage of rooting, 56%, on 1 mg/l IAA. Plantlets

survived in soil and exhibited normal growth in a greenhouse. Begum et al. (2001)

reported in vitro clonal propagation of pummelo (Citrus grandis L.).

Kotsias and Roussos (2001) reported in vitro shoot proliferation and rooting on a

modified Driver–Kuniyuki (DKW) basal medium in lemon (Citrus limon (L.) Burm, f.

cv. Interdonato) seedlings. BA alone (1, 2 and 4 mg/l) and in combination with either

orange juice (10%, v/v), silver nitrate (3 mg/l), GA3 (0.1 mg/l at the establishment stage

and 0.5 mg/l at all combinations during the proliferation stage) or ABA (0.2 mg/l only

at the establishment stage) were used to stimulate shoot formation during the

establishment and the proliferation stage. The combination of BA with ABA gave a


25

high rate of shoot formation, while GA3 and silver nitrate enhanced shoot elongation.

When these shoots were transferred to the rooting stage, the effect of application of two

different auxins IBA and NAA was examined, as well as different methods of

application (auxin added to the basal medium and auxin application by dipping the base

of the explant in auxin solution). Dipping the base of the explants in a 50% ethanol

solution of IBA at 1000 mg/l for 5s resulted in 80% rooting with subsequent 90%

survival of these explants, during acclimatization under mist.

Moriera-Dias et al. (2001) reported adventitious shoot regeneration in epicotyl

cuttings of troyer citrange (Citrus sinensis X Poncirus trifoliata) varied with the

conditions of illumination. The response to illumination and to growth regulators

differed for the direct and the indirect (through callusing) pathways of regeneration.

Shoot formation through the direct organogenic pathway decreased as the concentration

of BA in the medium was increased in the range 2.2-22 µM, when the explants were

incubated in the dark or under an 8 h daylength. For explants incubated under a 16 h

daylength, the number of shoots formed increased with BA concentration. Optimal

conditions of incubation for shoot formation through the direct pathway were either an

8 h daylength with a photon flux density of 74 µM m-2 s-1 in the presence of 2.2 µM

BA, or a 16 h daylength with a photon flux density of 37 µM m-2 s-1 in the presence of

22 µM BA. Irrespective of the conditions of incubation, shoot formation through the

indirect organogenic pathway was suppressed by the addition of 22 µM BA to the

medium. Optimal conditions for shoot formation through this pathway were incubation

under an 8 h daylength at a photon flux density of 74 µM m-2 s-1 in the presence of 2.2

µM BA. At the optimal conditions indicated, the addition of the synthetic auxin NAA

(0.54 µM) reduced shoot formation. Irrespective of the pathway of regeneration, the

number of shoots formed decreased markedly with the distance of the cutting from the

cotyledonary node.

Al-Bahrany (2002) reported production of multiple shoots from nodal segments

of lime (Citrus aurantifolia (Christm.) Swing) on MS medium supplemented with BA,

KN and NAA. The highest number of shoots (nine shoots per node) were produced on a

medium containing 2 mg/l BA, 1 mg/l KN and 1 mg/l NAA. Depending on the


26

concentration of BA and KN, NAA either inhibited, stimulated or did not affect shoot

multiplication, which also depended on the cytokinin level. Maximum shoot length was

obtained from treatments containing 0.5 mg/l BA combined with 1 mg/l KN (4.6 µM)

and 0.5 mg/l NAA. The largest leaves of resultant shoots were produced on a medium

containing 0.5 mg/l each of KN and NAA. Transferring in vitro shoots to rooting media

containing IBA and NAA produced complete plantlets. The highest rooting percentage

was obtained on a medium containing either 1 mg/l NAA alone or 0.5 mg/l NAA

combined with 2 mg/l IBA, whereas the highest number of roots was produced on a

treatment containing both 2 mg/l NAA and 2 mg/l IBA. Roots elongated most on

treatments containing 0.5 mg/l of either NAA or IBA. Shoot growth associated with the

rooting phase was the highest in response to 2 mg/l IBA or 0.5 mg/l NAA. Plantlets that

survived acclimatization (82%) exhibited normal growth in soil under greenhouse

conditions.

Almeida et al. (2002) defined organogenesis and plant regeneration protocols for

sweet orange varieties Natal, Valencia and Hamlin (Citrus sinensis L. Osbeck) and

Rangpur lime (Citrus limonia L. Osbeck). Teguments of seeds were removed and were

germinated in vitro and maintained in the dark for three weeks, followed by one week at

16-h photoperiod (40 µmol m-2s-1) and 27±2°C. Organogenesis was achieved by

introducing epicotyl segments on MT medium with 25 g/l sucrose and different BA

concentrations. After adventitious bud growth, the shoots were transferred to MT

medium with either NAA or IBA (1 mg/l), or absence of auxin, for rooting. The best

results were obtained with 1 mg/l BA for bud induction and 1 mg/l IBA for rooting for

all three sweet orange cultivars. The use of 0.5- 2.5 mg/l BA, followed by 1 mg/l IBA

were the best growth regulator combinations for bud induction and rooting,

respectively, for Rangpur lime.

Kobayashi et al. (2003) developed an efficient system for in vitro plant

regeneration from thin transversal stem sections of explants (1–2 mm) using mature

tissues of sweet orange cv. Pera. Explants were cultured on different media to evaluate

the frequency of regeneration and size of buds. A high percentage of explants (54%

with 3.1 buds/explant) producing large buds (1–4 mm) was observed when the explants


27

were cultivated for 2 weeks on MS medium and then transferred to Woody Plant

medium (WPM). Both media were supplemented with 1.8 µM BA and 0.7 µM GA3.

Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting.

Regenerated plants started to flower after 12 months in the greenhouse, confirming their

mature nature.

Begum et al. (2004) reported shoot proliferartion from nodal segments of in

vitro germinated seedlings of three pummelo varieties [Var-1 (pink pulp), Var.-2 (white

pulp colour) and Var.-3 (red pulp)] were cultured on half strength medium for axillary

shoot proliferation. A large number of shoot buds were produced when such four weeks

old culture were subcultured on half stength MS medium containing 1.0 mg/l BA. Roots

were induced when the isolated individual shoots were cultured on half strength MS

medium supplemented with 0.1 mg/l each of NAA, IBA or IAA. 100% root induction

was observed with 0.1 mg/l NAA.

Costa et al. (2004) studied in vitro morphogenesis responses of epicotyl explants

from ‘Cravo’ rangpur lime (Citrus limonia Osb.), ‘Foster’ grapefruit (C. paradisi

Macf.), and ‘Pera’ sweet orange (C. sinensis (L.) Osb.). Further analysis was performed

in ‘Cravo’ rangpur lime and ‘Foster’ grapefruit aiming to verify the in vitro

morphogenesis of five distinct regions of the epicotyl under different treatments. The

same general pattern of morphogenic gradient along the epicotyl axis was observed in

both citrus cultivars, with greater organogenic response as the distance of the explants

from the cotyledonary node increased. This morphogenic gradient was influenced by

factors related to plant material, composition of the culture medium and conditions of

incubation. The regions of the epicotyls farthest from cotyledons could be used as a

source of explants in experiments of genetic transformation of the genotypes evaluated

aiming to improve the efficiency of production of transgenic Citrus plants.

Rani et al. (2004) reported micropropagation of kinnow (Citrus nobilis x Citrus

deliciosa) through nodal segments on MS medium supplemented with different

concentrations and combinations of KN, adenyl sulphate (ADS), BA, 2-isopentyle

adenine (2-iP), NAA and malt extract (ME). Maximum shoot induction (91.67%) was


28

noted with 2-iP at 1 mg/l. Shoot multiplication was done on the same medium.

Multiplied shoots rooted best on MS medium supplemented with NAA (3 mg/l). Rooted

plantlets were established in soil after hardening and showed normal morphology.

Khawale and Singh (2005) reported a protocol for storage and regeneration of

plantlets of Citrus reticulata Blanco and Citrus jambhiri Lush. from the cotyledonary

segments. MS basal medium fortified with 2.0 mg/l BA + 0.5 mg/l KN + 1.0 mg/l

NAA, gave the highest frequency of adventitive embryony. The regenerated plantlets

were hardened and tested for clonal fidelity or variation using randomly amplified

polymorphic DNA (RAPD) markers. The results showed that plantlets regenerated

through this pathway were found to be true to the mother type.

Mukhtar et al. (2005) initiated to explore the role of explant type, growth

substances and organic additives in regeneration process of kinnow mandarin [Citrus

reticulata (Blanco)]. In vitro shoot tips and nodal segment explants were cultured on

MS media supplemented with different concentrations of KN and BA to produce

multiple shoots. The results revealed that the shoot tip explants cultured on MS media

supplemented with 1 mg/l of BA and 1.5 mg/l of KN gave highest shoot percentage.

Usman et al. (2005) reported in vitro multiple shoot induction from nodal

explants of Citrus cultivars, kinnow, sweet lime and succari. Six week old seedlings

were used as source of explant for nodal and internodal segments (3-5 mm in length)

between cotyledonary leaves and first two leaves. These segments were taken as

explants and cultured on MS medium supplemented with NAA (0.1 mg/l) and ME (500

mg/l) with varying concentrations of BA (0.1-10 mg/l) for shoot induction. Sweet lime

explants showed higher shoot regeneration (64%) when cultured at the lowest

concentration of BA (0.1 mg/l) followed by succari, while kinnow explants depicted the

lowest shoot regeneration capacity (47.50%). Significant increase in shoot regeneration

was observed with increase in the concentration of BA. Among cultivars, kinnow

explants gave the highest shoot regeneration (78.09%) followed by sweet lime and then

succari. Highest number of shoots (4.28) per explants was observed in kinnow followed

by sweet lime and succari (3.78 and 3.23 shoots respectively). The explants derived


29

shoots were excised and sub-cultured on MS medium supplemented with ME 500 mg/l

used as basal medium, further modified with different levels of NAA in the medium

significantly increased the rooting percentage. Kinnow showed the best rooting (91%)

followed by sweet lime (79%) at 10 mg/l NAA.

Ali and Mirza (2006) reported direct micropropagation of rough lemon (Citrus

jambhiri Lush.) from leaves, roots, stem segments and cotyledons from 5 weeks old in

vitro grown seedlings. These explants were tested on shoot regeneration medium having

several different concentrations of hormones individually or in combinations. Among

these explants only stem explants showed optimal shoot regeneration (83%) on MS

medium supplemented with BA 3 mg/l and 76% with BA 0.5 mg/l. Very little (13%)

shoot regeneration response was observed with cotyledons on MS medium

supplemented with BA 3 mg/l, while roots and leaf explants did not produce any shoots

on any media tested. For rooting of these regenerated shoots, they cut-off shoot

segments were cultured on MS medium supplemented with 0.5 mg/l NAA or 1 mg/l 2,

4-D. Best rooting response (70%) was observed on 0.5 mg/l NAA without callus

induction.

Almeida et al. (2006) induced adventitious bud development in epicotyl

segments of valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in

vitro for three weeks in the dark, followed by one week at 16-h photoperiod. Epicotyl

segments were cultured on Murashige and Tucker (1969, MT) culture medium

supplemented with 1.0 mg/l BA for organogenesis. Samples were observed by light and

scanning electron microscopy from day 0 to day 25. The adventitious buds originated

directly from the cambial region on the cut ends of the explants.

Dejam et al. (2006) reported in vitro propagation of Bakrai (C. reticulata Blanco

× C. limetta Swing.) using epicotyl expalnts. Expalnts were excised from in vitro grown

seedlings, cultured on basal MS medium supplemented with various concentrations of

BA and NAA. Maximum number of cultures with adventitious shoots (83%) was

obtained on MS medium supplemented with BA 2 mg/l. However, maximum number of

shoot buds (20.33) was obtained with BA 4 mg/l but, most of these buds remained


30

quiescent. Addition of 0.5 mg/l NAA increased shoot number and shoot length but

higher NAA concentrations has an inhibitory effect on shoot regeneration, particularly

with 1 mg/l BA. The highest number of shoots (5.5) and longest shoots (9.68 mm) were

obtained with concentrations of 2 and 1 mg/l BA + 0.5 mg/l NAA, respectively. The

shoots were rooted on half strength MS medium supplemented with IBA (0.5-10 mg/l).

Highest percentage of rooting (91.67%) and the maximum number (1.71) and length

(20.33 mm) of roots were obtained with IBA 10 mg/l.

Garcia-Luis et al. (2006) studied the effect of orientation as regards to gravity,

and that of contact with the medium of culture, on shoot regeneration at the cut edges of

epicotyl explants of troyer citrange (Citrus sinensis (L.) Osbeck X Poncirus trifoliata

(L.) Raf.). The shoot regeneration pathway was not affected by the orientation of the

explants as regards to gravity and was determined by explant polarity and the contact

with the culture medium. At the apical edge of the explants, the contact with the

medium shifted the pathway of shoot regeneration from a direct one to an indirect one,

with formation of callus. This callus formation was cytokinin-dependent, but the change

in the pathway of organogenesis was not caused by the increase in cytokinin availability

resulting from the contact with the medium. In contact with the medium regeneration at

the basal edge of the explants occurred through an indirect pathway after callus

formation. No regeneration occurred, at the basal edge, if contact with the medium was

prevented. The orientation of the explants as regards to gravity affected shoot formation

through the direct pathway of organogenesis. The number of buds differentiated and

growing shoots increased when the orientation of the explants departed from the vertical

upright position.

Duan et al. (2007) aimed to optimize the organogenesis of epicotyl segments

and to efficiently obtain transgenic plants of ‘Bingtang’ sweet orange (Citrus sinensis L.

Osb.), an elite citrus cultivar in China. Organogenesis was induced from epicotyl

segments of 2 weeks old seedlings of this cultivar. Two important factors influencing

organogenesis in vitro were hormone combination (IAA and BA) and cut modes. IAA

had a positive effect on bud formation only when BA was used at the concentration of

2.0 mg/l and an inhibitive effect was observed with higher or lower concentration of


31

BA. The number of regenerated buds reached up to 8.9 per explant with the

combination of IAA 0.2 mg/l and BA 2.0 mg/l. Among cut modes, oblique cut

performed the best for its effect on the number and quality of regenerated shoots and its

convenience to manipulate. With the optimized hormone combination and oblique cut

mode, citrus transformation with green fluorescent protein (GFP) gene was performed

and twelve independently transformed plant lines were achieved. Southern blot

hybridization confirmed the stable integration of GFP gene into the citrus genome. The

successful transformation of this cultivar revealed that it is possible to introduce other

genes with agronomic traits into it. Furthermore, these GFP expressing transgenic plants

could serve as a visual marker material for citrus somatic fusion and sexual

hybridization.

Altaf et al. (2008) reported direct shoot regeneration from nodal segments of

Citrus jambhiri on MS medium supplemented with BA 3 mg/l. They observed 70%

rooting response from regenerated shoots on MS medium supplemented with NAA 0.5

mg/l. Miah et al. (2008) established a protocol for direct multiple shoot regeneration

from both in vitro grown seedlings and mature plants of Citrus macroptera. Both nodal

and shoot tip explants taken from in vitro grown seedlings were cultured on MS

medium supplemented with different concentrations of BA and KN either singly or in

combinations. Both these explants were capable to regenerate and produce in vitro

multiple shoots. Maximum number of shoots was obtained from nodal explants on MS

medium supplemented with 1.0 mg/l BA. BA alone was found superior to KN. Ex vitro

rooting in pot soil (mixed with biogas slurry derived from cow-dung) was most

successful compared to in vitro rooting on half strength of MS medium supplemented

with different concentrations of NAA and IBA.

Sharma et al. (2008) achieved direct shoot formation from the nodal segments of

kinnow (Citrus nobilis Lour x Citrus deliciosa Tenora) on MS medium supplemented

with 2-iP (1 mg/l) and ME (800 mg/l). Shoot tips from these nodal sprouts were further

excised and micrografted on to rough lemon in vitro. Khan et al. (2009) made attempts

to investigate culture of leaf explants derived from in vitro seedlings of two sweet

orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of


32

several factors, including culture medium, lighting condition, explant age and genotype

on regeneration response were examined based on three parameters, percentage of

explants producing shoots, mean number of shoots per explant and shoot forming

capacity. Culture of explants on shoot inducing media (SIM) composed of MT salts

supplemented with different growth regulators gave rise to disparate shoot regeneration,

in which SIM1 (MT + 0.5 mg/l BA + 0.5 mg/l KN + 0.1 mg/l NAA + 3% sucrose +

0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of

shoots from leaf explants. Highly significant difference in the response of shoot bud

regeneration was noted between the two cultivars, with Bingtangcheng being more

responsive than Valencia. Culture of explants from fully developed leaves led to better

shoot regeneration capacity in comparison to undeveloped ones. However, the two

lighting conditions used herein did not cause significant difference in shoot

regeneration. Phenotypic observation and RAPD analysis confirmed that all the

regenerated plants from both genotypes were genetically identical to their donor plants,

suggesting absence of detectable genetic variation in the regenerated plants.

Tavano et al. (2009) studied in vitro organogenesis in Citrus volkameriana and

C. aurantium considering three explant types: epicotyl segments, internodal segments,

and hypocotyl segments with attached cotyledon fragment. The explants were cultured

on medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5,

and 2.0 mg dm-3 BA, incubated firstly in darkness for 4 weeks and then transferred to

16-h photoperiod for 2 weeks. A higher number of shoots per explant were obtained

with epicotyl segments, regardless of the BA concentration. For C. volkameriana the

highest percentage of responsive epicotyl segments (42%) was obtained in EME with

1.0 mg/dm3 BA, while for C. aurantium (59%) in EME with 0.5 mg dm-3 BA. The

organogenesis efficiency was best with the use of the hypocotyl segment with attached

cotyledon fragment (77% for C. volkameriana and 75% for C. aurantium). With this

explant the morphogenesis occurred only in the hypocotyl region. The in vitro

organogenesis was characterized by histological analyses showing that the morphogenic

process started in the cambium region near the explant cut end.


33

Jajoo (2010) developed an efficient and highly reproducible plant regeneration

protocol from nucellar embryos of Citrus limonia. MS medium was used for plant

regeneration from nucellar embryos. It was noted that BA at a concentration of 2.22 µM

induced highest number of multiple shoots (18.26 shoots per explants). Transfer of

individual shoots to root inducing MS medium supplemented with IBA (2.46 µM) and

BA (1.11 µM) resulted in rooting in 78.80% cultures with an average of 5.53

roots/shoot. Plantlets thus obtained were successfully transferred to green house for

further observations.

Pe'rez-Tornero et al. (2010) investigated the influence of the basal medium and

different plant growth regulators on micropropagation of nodal explants from mature

trees of lemon cultivars. Although the basal medium did not affect any of the variables,

explants on DKW medium were greener. Several combinations of BA and GA3 were

used to optimise the proliferation phase. The number of shoots was dependent on the

BA and GA3 concentrations and the best results were obtained with 2 mg/l BA and 1 or

2 mg/l GA3. The best results for productivity with respect to number of shoots and the

average shoot length were obtained with 2 mg/l BA and 2 mg/l GA3. The transfer of in

vitro shoots to rooting media, containing different concentrations of IBA and IAA

produced complete plantlets. Lemon shoots rooted well in all rooting combinations. The

highest rooting percentages were obtained on medium containing 3 mg/l IBA alone or

IBA in combination with 1 mg/l IAA. Root length was greater when only 3 mg/l IBA

was used. The success during the acclimatisation was close to 100% and the plantlets

exhibited normal growth in soil under greenhouse conditions.

Niedz and Evens (2010) studied the effect of BA and meta-topolin (mT) on the

three responses i.e. shoot quality, numbers of epicotyl explants producing shoots and

the number of shoots greater than 2 mm from citrus rootstock US-812 (Citrus reticulata

x Poncirus trifoliata) epicotyl explants. The experiment was designed as a mixture-

amount. The two mixture components, BA and mT were varied proportionally from

0 BA: 1 mT to 1 BA: 0 mT and the amount of total cytokinin varied from 1 to 50 µM.

Models developed for each of the three measured responses were highly significant.

Results were as follows: first, the three responses were similar over the design space;


34

second, no synergistic or antagonistic interactions were detected; and third, BA and mT

resulted in similar responses, but at substantially different amounts – BA at 1 µM was

essentially equivalent to mT at 13.25 – 37.75 µM.

Saini et al. (2010) studied the effect of explants and cobinations of plant growth

regulators on the efficiency of plant regeneration in C. jambhiri Lush. The epicotyl

segments placed horizontally on culture media proved to be best for shoot bud

formation. The bud induction frequency of 83.975 was obtained on MS medium

supplemented with BA at 0.5 mg/l with an average of 8.6 buds per explant. Maximum

proliferation of shoots was observed on MS medium supplemented with BA 0.5 mg/l

and GA3 1.0 mg/l. The elongated shoots were rooted on MS medium containing NAA 1

mg/l + IBA 1.0 mg/l with 77% rooting response.

Samarina et al. (2010) investigated the effect of sterilization agents and the

composition of the nutrient medium on lemon explants regeneration, micropropagation

and rooting. Nodal segments of eight year old lemon trees cvs. Novoaphonsky,

Beskolyuchy, Udarnik, Meyer lemon were taken as explants during spring. Veltolen

0.2%, 0.2% HgOCl and hypochlorite Ca 7% solutions were used as disinfectants at

different exposures. The best result for surface sterilization was obtained when shoots

were dipped in Veltolen solution by about 40 min. In this case 58.7% sterile explants

were received. Several combinations of BA with NAA were used for optimization of

cell proliferation and micropropagation. Maximum effect at the stage of proliferation

was obtained by adding to the MS basal medium 0.1 mg/l BA and 0.5 mg/l NAA.

Cultural media consisted of MS minerals and vitamins with 1 mg/l BA and 0.1 mg/l

NAA resulted in multiplication of regenerated plants. Rooting of regenerants was

obtained on ½ MS medium supplemented with 0.5 mg/l NAA alone.

Singh and Rajam (2010) explored sweet orange (Citrus sinensis L Osbeck, var.

Nagpur) for efficient multiple shoot regeneration and rooting in different media. The

influence of phytohormones and carbon source on in vitro morphogenesis of sweet

orange epicotyl explants was investigated. Among various concentrations and

combinations of auxins (IAA and NAA) and cytokinins (BA, KN, Zeatin and TDZ)


35

tried, MT (Murashige and Tucker, 1969) medium fortified with 6-benzylaminopurine

(BA) at 1 mg/l without auxin had a strong promotive effect on shoot regeneration and

elucidated best morphogenic response from one-month-old etiolated epicotyl explants.

A 100% regeneration frequency was obtained and multiple shoots with an average of

8.24 shoots per explant were produced on all the explants. Root formation was seen in

response to all the three auxins viz. IBA, NAA and IAA, but best response with rapid

induction was observed under the influence of IBA at 1 mg/l. Sucrose was observed to

be at par with maltose as carbon source to support shoot regeneration.

Germana et al. (2011) examined the encapsulation of carrizo citrange uninodal

microcuttings (3–4 mm long) and evaluated the influence of the calcium alginate

coating, short time storage at cold temperature and different sowing substrates on the

viability and regrowth of the explants. A secondary aim was to develop an efficient

protocol to induce root formation in the microcuttings. The results showed that

encapsulation did not negatively affect the viability, providing a satisfactory regrowth,

and storage potential for 30 days at low temperature. No differences in viability and

regrowth were detected between the two different sowing substrates tested (agar-

solidified medium and paper filter).

Marques et al. (2011) studied the effects of BA and NAA treatments on

adventitious bud and shoot regeneration from internodal branch segments of 12 months

old plants of Citrus aurantium L. cv. Brazilian. High rates of bud initiation and shoot

development were obtained both with BA supplemented medium, in the range from 1

mg/l to 3 mg/l and with 0.1 mg/l NAA supplemented medium. NAA concentrations

above 1 mg/l significantly reduced bud initiation and shoot elongation. The results

obtained using different in vitro culture vessels such as Petri dishes, tubes and glass

culture jars were compared. The highest adventitious bud induction was observed in

Petri dishes for internodes cultured on 2 mg/l BA supplemented medium, with 95%

responsive explants forming 9.0±2.4 adventitious buds. The adventitious buds observed

in Petri dishes reached a maximum height of 1mm with no further development, while

some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm


36

in height. A shoot regeneration gradient of the internodes collected along the branch

axis was noticed, with basal ones exhibiting higher regeneration frequency.

Singh et al. (2011) reported that sterculia gum can be used as an alternative to

agar as gelling agent to decrease the cost of tissue culture protocol. The study was

conducted on C. jambhiri Lush. Savita et al. (2012) reported an effiecient

micropropagation protocol for C. jambhiri Lush. using nodal segments as explant.

Maximum multiple shoot regeneration response (75%) was observed on MS medium

supplemented with BA at 3 mg/l. Shoots were multiplied for 30 days on fresh medium

with similar composition. A total of 67% cultures showed multiplication with optimum

number (4.02) and height of shoots (1.81 cm) with BA (3 mg/l) alone. Maximum

rooting response (87%) was observed with NAA at 0.5 mg/l. Transverse sections of

shoots obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal

segments) showed similar anatomies. RAPD analysis confirmed that all the regenerated

plants were genetically identical to their donor plant, suggesting absence of detectable

genetic variation in the regenerated plantlets. Summary of literature on direct

regeneration studies of different Citrus spp. is given in Table 2.1.

2.2.2. Indirect plant regeneration through callus

Plant tissue culture has emerged as a powerful tool for propagation and

improvement of many woody plant species including Citrus. In vitro culture has the

potential to eliminate diseases and provides scope for development of new cultivars

through somaclonal variations (Hammschlag et al., 1995). Production of callus and its

subsequent regeneration are the prime steps in crop plants to be manipulated by

biotechnological means and to exploit somaclonal variations (Islam et al., 2005).

Despite its rich genetic resources, scientists encounter difficulties in citrus hybridization

breeding due to high heterozygosity, incompatibility, sterility and nucellar embryos

(Shen et al., 1998). These obstruct scientific efforts to develop new varieties with

enhanced production and quality characteristics and rootstocks with improved

resistance to abiotic and biotic stresses. With the development of biotechnology, genetic

transformation and protoplast hybridization have been established to circumvent those


37

breeding obstacles in many fruit trees (Deng and Liu, 1996). For citrus, embryogenic

callus is widely used in genetic transformation and protoplast hybridization since it can

easily regenerate plants (Deng and Liu, 1996; Hao, 2000; Hao and Deng, 2002). Citrus

embryogenic calluses can be maintained in culture at one month intervals for a long

period (Yi and Deng, 1998; Hao and Deng, 2002). However, frequent subculture of

many cultures is labour intensive and costly (Ashmore, 1997; Engelmann, 1997). To

solve this problem, short and medium term storage methods have been developed to

reduce growth and increase subculture intervals. Tissue culture protocols have been

described for a number of Citrus spp. through callus (Ali and Mirza, 2006; Altaf et al.,

2008; Altaf et al., 2009a,b; Khan et al., 2009; Laskar et al., 2009; Singh and Rajam,

2009 and Savita et al., 2010, 2011a, 2011b).

Kobayashi et al. (1984) isolated protoplasts from nucellar callus of ‘Troviata’

orange (Citrus sinensis Osbeck) and cultured on MT medium containing 0.15 µM

sucrose, 0.45 µM glucose and 0.6% agar, but lacking phytohormones. Colonies with

26.3% plating efficiency were observed after 6 weeks culture. When the protoplasts

were cultured in the presence of exogenous phytohormones, reduction in colony

formation was observed. Some colonies were developed into green embryoids, which

eventually formed plants. Edriss and Burger (1984) Isolated epicotyl, root meristem and

root segment tissues of ‘Troyer’ citrange [Poncirus trifoliata (L.) Rat. × Citrus sinensis

(L.) Osbeck] and established in continuous culture to compare their regeneration

potential. Callus was obtained from these explants on MS medium containing NAA (10

mg/l) and BA (0.1–10 mg/l). Formation of shoots from root segments was direct

without callus formation on MS medium containing BA (10 mg/l) and NAA (1 mg/l).

Shoot formation from epicotyl callus occurred on MS medium containing 0.25 mg/l BA

and 0.1 mg/l NAA. Formation of shoots from epicotyl segments occurred on MS

medium containing BA (0.5 mg/l) and NAA (0.1–1.0 mg/l), while rooting of

regenerated shoots occurred in treatments containing 2.0 mg/l NAA alone.

Beloualy (1991) reported callus induction from embryos excised from seeds of

Poncirus trifoliata, Citrus aurantium and carrizo citrange (Citrus sinensis × Poncirus

trifoliata) on MT medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D 2


38

mg/l), BA (5 mg/l) and casein hydrolysate (1000 mg/l). Sucrose 50 g/l and 0.8% agar

were added. Adventitious shoots and emryoids were induced from callus on MT

medium supplemented with NAA (1 mg/l), BA (5 mg/l), casein hydrolysate (100 mg/l)

and sucrose 50 g/l in case of Poncirus trifoliata and carrizo citrange, while in case of C.

aurantium NAA (1 mg/l), BA (10 mg/l), casein hydrolysate (100 mg/l) and sucrose 50

g/l were used to induce embryoids and adventitious shoots. Rooting was promoted by

supplement of 1 mg/l GA3 or NAA (1 mg/l). Saito et al. (1991) maintained suspension

culture from nucellar tissue of C. sudachi on MT medium containing BA (5 mg/l).

Francisco et al. (1992) reported callus induction from leaf and stem segments of

30 different genera including 28 citrus relatives on non-embryogenic callus induction

medium i.e. MTC [MT medium + 2,4-D (0.663µM) + KN (0.22 µM)] with and without

activated charcoal; MTNC [MT medium + KN (0.22 µM) + NAA (5.60 µM)] with and

without activated charcoal. A soft and friable callus was obtained from several genera

including Aegle, Afraegle, Atlantia, Basamocitrus, Citropcis, Citrus, Microcitrus and

Zanthoxylem.

Chakravarty and Goswami (1999) reported best callus induction on MS medium

containing BA and 2,4-D from explants incubated in the dark. High levels of MS

vitamins enhanced callus initiation and growth. These calli could be maintained in

culture for upto two years and plantlets regenerated from them on transfer to light.

Greening of callus and regeneration of shoot-buds from callus occurred on transfer to

MS medium with BA. These shoots grew further in media with both BA and GA3.

Rooting of shoots occurred on media with NAA or IBA and the rooted plantlets could

be transferred to soil. Regeneration of shoots and roots decreased with increase in age

of callus. Cytological study of the regenerants revealed no change in chromosome

number. Thus, genetically uniform plantlets could be regenerated continuously from

established callus upto two years old, thereby helping in multiplication and conservation

of the species.

Tadeo et al. (1995) studied the effect of I-aminocyclopropane- l-carboxylic acid

(ACC) treatment on callus formation from internodal explants of troyer citrange (C.


39

sinensis (L.) Osb.× Poncirus trifoliata). The in vitro culture of explants in media

supplemented with 0.05, 0.5 and 2 µM ACC resulted in rapid growth of whitish soft

friable calli, whereas no calli were observed in the absence of ACC. The calli were

evident 3-5 days after culture and callus growth reached a stationary phase 11 days after

culture. A rapid and transitory increase in the rate of ethylene production occurred at the

highest ACC concentrations used. Callus formation correlated with amounts of ethylene

released by the explants. The lowest ACC concentration (0.05 µM) stimulated callus

growth in only l0-20% of the explants, with callus weighing 100 mg and the explants

produced low ethylene levels at day 3 of culture (33 nl g-1 FW h-l). The highest ACC

concentrations (0.5 and 2 µM) stimulated callus growth in HI-75% of the explants, with

callus weights of 155 and 210 mg, respectively. The explants produced high levels of

ethylene at day 3 of culture (330-430 nl g-1 FW h-l). The inhibition of ethylene

biosynthesis or action with cobalt chloride and silver thiosulfate, respectively, reduced

callus formation in the ACC-treated explants in a concentration-dependent manner.

Taken together, these observations indicate that ethylene synthesized from ACC is

responsible for callus formation.

Gloria et al. (1999) reported embryogenic callus induction from nucellar tissue

of seven citrus cultivars i.e. ‘Cravo’ mandarin and ‘Ponkan’ mandarin (Citrus reticulata

Blanco); ‘Natal’ sweet orange, ‘Serra d’agua, sweet orange, ‘Perra’ sweet orange,

‘Valencia’ sweet orange (Citrus sinensis L. Osbeck); Murcott tangor (Citrus reticulata

Blanco × Citrus sinensis L. Osbeck). Three different media tested were; EME [MT

modified with the addition of ME (500 mg/l)]; ½ EME [Half conc. of MT

macronutrients + Half conc. of BH3 macronutrients + ME (500 mg/l) + glutamine (1.55

g/l)] and EBA [MT modified with the addition of ME (500 mg/l) + BA (0.44 µM) +

2,4-D (0.04 µM)]. Soft friable calli were obtained from ‘Carvo’ and ‘Ponkan mandarin’

(Citrus reticulata Blanco), Citrus reticulata Blanco × Citrus sinensis L. Osbeck

(Murcott tangor), ‘Serra d’agua and ‘Valencia’ sweet orange (Citrus sinensis L.

Osbeck) 120 days after callus induction.

Fleming et al. (2000) reported an alternative method for transforming sweet

orange (C. sinensis L. Osbeck) using plasmid DNA encoding the non-destructive


40

selectable marker enhanced green fluorescent protein (GFP) gene introduced using

polyethylene glycol (PEG) in protoplasts isolated from nucellar derived suspension

culture. Callus was induced from nucellar tissue on MT medium containing glutamine

1.55 g/l + 0.5 g/l ME. Regeneration of transformed protoplasts was observed with

13.3µM BA + 0.045 µM 2,4-D and rooting was obtained on ½ MT medium containing

0.11 µM NAA + 0.5 g/l activated charcoal (AC).

Das et al. (2000) standardized a protocol for micropropagation of elite plants of

sweet orange (Citrus sinensis) through nucellar embryo culture. MS medium

supplemented with NAA (1 mg/l) or 2,4-D (1 mg/l) promoted callus development in

both nucellar and zygotic embryos. GA3 (1 mg/l) enriched medium induced plantlets

initiation but their growth was very poor. No significant differences were observed

between initial growth patterns of nucellar and zygotic seedlings developing from the

same ovule. Five to six shoots were obtained from collar region of both categories of

embryos in MS medium supplemented with BA (1 mg/l) within 60 days of inoculation.

The number of plantlets was almost doubled after their transfer to the same medium and

culture for another 30 days. Higher doses of BA resulted in initiation of callus directly

from the embryos. The regenerated shoots (2-3 cm) could be rooted on MS medium

supplemented with either only NAA (0.75 mg/l) or NAA (0.50 mg/l) and IBA (2.0

mg/l). A number of plantlets could be obtained from a nucellar embryo grown shoot

within a limited time period.

Tao et al. (2002) studied the effect of 2,4-D, NAA, 2,4,5-trichlorophenoxy

acetic acid (2,4,5-T), 4-chlorophenoxyacetic acid (4-CPA), 2-methyl-4-chlorophenoxy-

acetic acid (MCPA) on callus induction from leaf explants taken from in vitro raised

seedlings in C. grandis (L.) Osbeck (pummelo). Begum et al. (2003) used cotyledon

explants from in vitro raised seedlings of three varities of pummelo for callus induction

on half strength MS medium supplemented with 1 mg/l BA + 5 mg/l NAA. These calli

were transferred on half strength MS medium supplemented with cytokinin only.

Maximum shoot regeneration was observed with BA 1 mg/l in three varieties of

pummel. For rooting, shoot cuttings were cultured on half strength MS medium


41

supplemented with NAA, IBA or IAA. Maximum rooting response was observed with

1.0 mg/l NAA.

Ali and Mirza (2006) studied the effect of various concentrations and

combinations of 2,4-D, BA and NAA on regeneration of rough lemon (Citrus jambhiri

Lush.) explants. Optimal callus induction response was observed on MS medium,

supplemented with 2,4-D 1.5 mg/l from all types of explants, with stem explants

showing the highest response (92%). Maximum shoot regeneration response (70 %)

from callus was observed on MS medium supplemented with BA 3 mg/l. Direct shoot

regeneration was highest in stem segment explants on MS medium with BA 3 mg/l. MS

medium supplemented with NAA 0.5 mg/l provided 70 % rooting response.

Molina et al. (2007) reported that ilumination did not affect the pathway of

shoot regeneration at the cut edges of epicotyl explants of troyer citrange, but

signigficantly affected the number of developed shoots and the response to exogenous

cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic

pathway without callus formation. For explants incubated in the light, this regeneration

did not require cytokinin addendum, but the number of shoots formed was significantly

increased by BA, but not by zeatin or KN. Incubation in the dark almost suppressed

shoot formation at the apical end. The addition of BA or KN, but not zeatin, restored

shoot formation in the dark to the value obtained in the light. At the basal end of the

explants, shoot regeneration occurred through an indirect organogenic pathway after the

formation of primary callus. In explants incubated in the light, callus formation and

shoot growth was supported by a low (0.5–1 mg/l) BA concentration and by zeatin. KN

did not support callus growth. Shoot formation was higher in the presence of BA (0.5–1

mg/l) than of zeatin, but was inhibited by a high BA (5 mg/l) concentration. Incubation

in the dark increased callus growth and shoot formation at the basal cut as compared to

explants incubated in the light.

Laskar et al. (2009) reported shoot regeneration from leaf derived callus in

Citrus indica Tanaka. Regenerative calli were induced on MS medium supplemented

with 0.01 mg/l TDZ + 0.1 mg/l NAA. Shoots were regenerated on WPM medium


42

supplemented with 0.5 mg/l BA + 0.25 mg/l TDZ + 0.25 mg/l NAA. These shoots were

rooted on MS medium supplemted with 1.0 mg/l NAA. Sharma et al. (2009)

standardized a protocol for in vitro propagation of citrus rootstocks viz. rough lemon,

cleopatra mandarin, pectinifera and troyer citrange. The shoot tip explants of these

rootstocks were found better for callus induction than the nodal segments.

Savita et al. (2010) established a protocol for micropropagation of C. jambhiri

via callus induction and regeneration. Leaf segments, nodal segments and root segments

excised from in vitro raised seedlings were used as explants. Maximum callus induction

(98.66%) was observed from leaf segments on MS medium supplemented with 2, 4-D

(4 mg/l). For nodal segments, maximum callus induction (96%) was observed with 2, 4-

D (1 mg/l) and from root segments, it was 48.66% on MS medium supplemented with

2, 4-D (2 mg/l). Callus raised from leaf segments showed maximum regeneration

(57%) with NAA (0.5 mg/l) + BA (1 mg/l) whereas nodal segments showed better

regeneration (71.89 %) with NAA (0.5 mg/l) + BA (3 mg/l). However, callus from root

segments did not regenerate. Regenerated shoots were rooted on MS medium

supplemented with different PGRs and best response (71%) was observed with NAA

(0.5 mg/l).

Savita et al. (2011b) developed an efficient miropropagation protocol for Citrus

jambhiri Lush. using cotyledons as explants. Maximum callus induction (91.66%) was

observed on MS medium supplemented with 2,4-D (2 mg/l) in combination with ME

(500 mg/l). Green healthy calli were cut into small pieces and cultured on MS medium

for regeneration. Maximum shoot regeneration (87.50%) was observed with BA (3

mg/l). Effect of increasing age of callus was also studied which showed that callus

retained regeneration capacity (58.33%) even after 420 days of culture. Regenerated

shoots were separated out and cultured on rooting medium. Maximum rooting response

(91.67%) was observed on half strength MS medium supplemented with NAA (0.5

mg/l). After hardening and acclimatization the plantlets were transferred to the field and

showed 67% survival. Summary of literature on micropropagation of Citrus

(regeneration through callus) is given in Table 2.2.


43

2.2.3. Somatic embryogenesis

Somatic embryogenesis is the process of a single cell or group of cells initiating

the developmental pathway that leads to reproducible regeneration of non-zygotic

embryos capable of germinating to form complete plants. A remarkable homology is

known to exist between somatic and zygotic embryos (Redenbaugh et al., 1988). In

Citrus species, this phenomenon has been studied in detail by Rangaswamy, 1961;

Sabharwal, 1963; Rangan et al., 1968; Vardi et al., 1975; Kochba and Spiegel-Roy,

1977; Tisserat and Murashige, 1977; Kunitake et al., 1991; Gill et al., 1995; Carimi et

al., 1995, 1998,1999; Perez et al., 1998; Chaturvedi et al., 2001; Ricci et al., 2002;

Fiore et al., 2002; Guo and Grosser, 2005; Shimada et al., 2005.

The embryogenic potential of citrus varied with genotype and type of explant,

but better results were usually obtained from regeneration protocols involving the use of

explants of ovular origin. Embryogenic cultures derived from the shamouti orange have

been described as proembryogenic masses (PEMs) (Button et al., 1974) rather than non-

differentiated cell masses (Gavish et al., 1991). Embryos, pseudobulbils and

embryogenic callus were obtained by in vitro culture of undeveloped ovules excised

from ripe fruits of the navel orange group and lemon (Starrantino and Russo, 1980).

Large number of seedlings were produced by subculturing the embryos, pseudobulbils

and embryogenic callus on fresh MS medium with added 6-methyl aminopurine. The

technique of growing isolated citrus embryos in artificial media was described by

Hurosvilli (1957). Rangan et al. (1968) reported culture of nucellar tissues in C.

grandis. Nucellar tissue isolated from monoembryonic varieties gave rise to 10-15

embryos per culture, which developed into complete plantlets. The plants arising from

embryogenesis of nucellus in vitro were found to be free from most of the pathogenic

viruses (Bitters et al., 1970).

Chaturvedi and Mitra (1972) produced embryoids and complete plants from

unpollinated ovaries and from ovules of in vivo grown emasculated flower buds of

Citrus spp. Explants of unpollinated ovaries of Citrus aurantifolia and C. sinensis, but

not of C. maxima, a monoembryonic species, produced embryoids and complete plants


44

under certain cultural conditions. Subsequent experiments showed that ovary wall tissue

was more prone to produce these responses. In long-term culture, the responses

gradually decreased resulting in complete cessation of embryoid formation. This

cessation occurring earlier in C. aurantifolia than in C. sinensis. Explants of nucellus

contained in longitudinal halves of ovules taken from in vivo grown emasculated flower

buds of C. auranti-folia produced three types of responses, namely, (a) an increasing

mass of proliferating embryoids, (b) friable, spongy and pale-white tissue along with

callusing integumentary tissue and (e) mixed responses of (a) and (b). The explants with

continuously increasing mass of proliferating embryoids contained most of the stages of

zygotic embryogenesis. Using different culture media, a good number of embryoids

could be induced in explants of friable and spongy tissue of nucellar origin, but they did

not develop into seedlings. No embryoids could be induced from callusing

integumentary tissue regardless of medium employed.

Kobayashi et al. (1983,1985) examined the effects of protoplast density and

mannitol concentration on cell division and embryoid formation using protoplasts

isolated from 6-year old nucellar callus of 'Trovita' orange (Citrus sinensis Osb.).

Somatic embryogenesis in nearly direct manner was observed only at a combination of

low cell densities (4 × 104/ml) and low mannitol concentrations (0.4 M). Orange

protoplasts (cells) showed embryogenic potential and repression of embryogenesis

occurred when protoplasts were cultured at a high density and/or under high osmotic

pressure.

Grosser et al. (1988) used MT medium containing 500 mg/l ME to maintain

suspension culture of ‘Hamlin’ sweet orange (C. sinensis). MT medium containing 1

mg/l GA3 and 15 mg/l coumarin was used for somatic embryo germination and 5 mg/l

BA for shoot multiplication.

Sim et al. (1988) isolated protoplasts from embryogenic suspension cultures of

Citrus mitis and cultured in a medium without any plant growth substances. Somatic

embryos developed directly from protoplasts without an obvious intervening callus

phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast


45

suspension (density 2 x 106/ml) cultured for 35 days. Upon transferring the embryoids

to medium with 1.0 mg/l GA3, they developed into plantlets. Rooted plantlets were

obtained in 3 months after protoplast isolation.

Vardi and Gulan (1988) reported callus growth on MT medium with 4% sucrose

and embryo induction with 2% glycerol. Somatic embryo maturation was observed on

MT medium supplemented with ME (1500 mg/l) and 4% sucrose. Addition of NAA to

MT medium promotes root formation and axis elongation of normal shaped embryos

induced from protoplasts of C. limon, C. paradisi, C. reticulata and C. sinensis.

Ohgawara et al. (1989) and Kobayashi et al. (1991) used MT liquid medium containing

10 mg/l BA to maintain suspension culture of cells from ovules of navel orange and

somatic embryos were initiated on MT medium supplemented with GA3 (5 mg/l).

Gmitter and Hu (1990) used various formulations of MT medium including

doubled MT inorganic component concentration (2 MT) supplemented with 2,4-D (1-2

mg/l), KN (5 mg/l) and GA3 (2-10 mg/l) to test their ability to induce callus from

endosperm of C. sinensis, to induce globular embryogenic callus from primary callus

and to allow embryo development and plantlet production from proliferating calli

respectively.

Nito and Iwamasa (1990) tried various concentrations of NAA, KN and GA3 in

MS medium to induce callus from juice vesicles of Satsuma (Citrus unshiu Marc.).

Belouly (1991) regenerated complete plantlets from callus derived embryo culture of

Citrus aurantium, Poncirus trifoliata and carrizo citrange (C. sinensis × Poncirus

trifoliata) via somatic embryogenesis and organogenesis. Isolated embryoids were

differentiated on medium containing 1000 to 1500 mg/l ME. Both embryoids and

shoots were obtained by the use of plant growth regulators, NAA and BA. The highest

frequency was obtained with 1 mg/l NAA + 5 mg/l BA in Poncirus trifoliata and

citrange. Rooting was promoted by a supplement of 1 mg/l GA3 or 1 mg/l NAA. GA3

enhanced stem elongation and rooting in embryoids and NAA stimulated adventitious

root formation.


46

Augustin and Millo (1995) cultured unfertilized ovules of troyer citrange treated

with a chemical mutagen (EMS) on MS medium supplemented with GA3 (1 mg/l) and

ADS (27 mg/l) for somatic embryo induction. Carimi et al. (1995) reported somatic

embryogenesis from styles (including the stigma) of Citrus aurantium L. (cvs. ‘AA 12’,

‘AA 30’ and ‘AA31’), C. deliciosa Tenore (cvs. ‘Avana’ and ‘Tardivo di Ciaculli’), C.

paradisi Macf. (cvs. ‘Marsh seedless’ and ‘Star Ruby’) and C. sinensis (L.) Osb. (cvs.

‘Bonanza’, ‘Brasiliano 92’, ‘Sanguinello’ and ‘Valencia’). Explants were excised from

flower buds which were collected during full bloom and cultured on MS basal medium

supplemented with 146 µM sucrose, 500 mg/l ME and 13.3 µM BA as well as the same

medium without BA. Callus development was observed from the style base 4 weeks

after treatment initiation and embryogenesis occurred 2-3 months later. Embryogenesis

has been induced from the style derived callus of all the cultivars tested except for the

cultivars ‘Avana’ and ‘Star Ruby’. The best results for callus growth and embryo

regeneration were obtained from explants of ‘Brasiliano 92’ cultured on medium

containing BA. Somatic embryos were isolated from callus and placed on MS medium

supplemented with 146 µM sucrose, 500 mg/l ME and 0.27 µM NAA where they

formed entire plants. Two months later plants were successfully established in soil.

Gill et al. (1995) initiated callus cultures from leaf, epicotyl, cotyledon and root

segments of in vitro grown nucellar seedlings of Citrus reticulata Blanco ‘Local

Sangtra’. Callus initiation was seen on cut ends of all the explants within 10 days of

culture. The epicotyl segments were the best explants for callus induction. Best callus

induction occurred on MS medium containing NAA (10 mg/l) and KN (0.5 mg/l). MS

medium supplemented with NAA (10 mg/l), KN (1 mg/l) and vitamins (10 X normal

MS levels) were best for induction of somatic embryogenesis. The epicotyl-derived

callus exhibited significantly higher (83.0%) mean somatic embryogenesis than calli

derived from other explants. Most of the somatic embryos germinated like normal

embryos, but some only produced shoots. The time taken for root initiation from

separated shoots was 7-10 days. The number of roots per shoot and root length also

varied with culture media.


47

Carimi et al. (1998) reported somatic embryogenesis and plant regeneration

from undeveloped ovules and stigma/style explants of sweet orange navel group [Ctrus

sinensis (L.) Osb.]. Explants were cultured on three different modifications of MS

medium with 500 mg/l ME or 500 mg/l ME + 4.6 µM KN or 500 mg/l ME + 13.3 µM

BA. Sucrose (146 µM) was used as carbon source. High frequency of somatic

embryogenesis was observed with ME (500 mg/l) + 13.3 µM BA.

Carimi et al. (1999) reported callus induction, somatic embryogenesis and plant

regeneration in six different Citrus species [Citrus deliciosa Ten. (cv ‘Avana’), C. limon

(L.) Burm. (cv ‘Berna’), C. madurensis Lour. (cv ‘CNR P9’), C. medica L. (cv ‘Cedro

di Trabia;), C. tardiva Hort. ex Tan. (cv ‘CNR P6’), C. sinensis (L.) Osb. (cv

‘Ugdulena7’)] from cultures of pistil transverse thin cell layer explants. Explants were

cultured on three different media: MS basal medium; MS medium + 500 mg/l ME; MS

medium + 500 mg/l ME + 13.3 µM BA.

Perez et al. (1999) reported somatic embryogenesis in different Citrus spp.

(Citrus deliciosa Ten. (C. reticulata Blanco × C. sinensis (L.) Osb.), (C. paradisi Macf

×C. tangerine Hort. ex Tan.), (C. clementina Hort. ex Tan. × C. paradise Macf. × C.

tangerina Hort. ex Tan.), (C. nobilis Lour. × C. deliciosa Ten.), (C. clementia Hort. ex

Tan. × (C. paradise Macf ×C. tangerina Hort. ex Tan.) × (C. clementina Hort. ex Tan.)

× (C. paradisi Macf. × C. tangerine Hort. ex Tan.) using ovules as explants. Somatic

embryos were obtained on MS medium supplemented with 500 mg/l ME + 50 g/l

sucrose.

Carimi et al. (2000) reported a new method for rearing the citrus flower moth

(Prays citri Mill.) (Lepidoptera, Yponomeutidae) on lemon [Citrus limon (L.) Burm.]

callus induced from lemon stigma and style explants cultured on MS medium

supplemented with 500 mg/l ME, 13.3 µM BA and 146 µM sucrose. Somatic

embryogenesis and plant regeneration was also obtained from the cultures of styles and

stigmas of lemon. Obukosia and Waithaka (2000) reported somatic embryogenesis in

Citrus sinensis and Citrus limon from nucellar embryos on MS medium supplemented

with 10% coconut water or 0.4 g/l casein hydrolysate. Yang et al. (2000) reported callus


48

induction and embryoid regeneration from the endosperm culture of C. grandis (L.)

Osb. on MS medium consisting of 1% sucrose, 1µM BA. Embryogenesis and rooting

were induced with addition of 30 µM GA3 to basal MS medium.

D’Onghia et al. (2001) cultured stigmas and styles of C. reticulata, C. sinensis

and C. reticulata × C. sinensis on MS medium supplemented with 6 mg/l BA to induce

somatic embryogenesis. Bespalhok et al. (2001) described a protocol for shoot

regeneration of sweet orange cvs. Pera, Valencia and Folha Murcha from thin sections

excised from juvenile explants.

Tomaz et al. (2001) reported somatic embryogenesis from nucellus-derived

callus cultures of five Citrus cultivars, including three (Caipira, Seleta Vermelha, and

Valencia) of sweet oranges (C. sinensis L. Osbeck), one each of rangpur lime (C.

limonia L. Osbeck), and cleopatra mandarin (C. reticulata Blanco) (lines I and II).

Callus lines maintained on MT medium consisting of 50 g/l sucrose were transferred to

MT medium supplemented with different carbohydrate sources like galactose, glucose,

lactose, maltose or sucrose at 18, 37, 75, 110, or 150 µM, or glycerol at 6, 12, 24, 36, or

50 µM. Globular embryos were observed after approximately 4 weeks in several

treatments. Cultures of valencia, caipira sweet oranges and cleopatra mandarin (line I)

showed higher numbers of embryos on medium containing galactose, lactose and

maltose. Histological studies showed somatic embryos in all developmental stages with

a normal histodifferentiation pattern. The other two cultivars (rangpur lime and

cleopatra mandarin, line II) formed very few embryos, which did not develop further

following the globular stage. Some of the abnormalities observed were lack or

dedifferentiation of protoderm and absence of apical meristems and procambial strands.

Embryos that followed the normal sequence of development were easily converted into

plants. Non-embryogenic cultures continued as proliferating callus cultures, eventually

forming a few embryos which did not convert into plants.

Fiore et al. (2002) reported callus induction and somatic embryogenesis from

stigmas and styles of C. sinensis and C. limon. Best response was observed with stigma

of C. limon on MT medium supplemented with 4 µM 4-CPPU [N-(2-chloro-4-pyridyl)-


49

N’-phenylurea. Madhav et al. (2002) reported somatic embryogenesis from nucellus

tissues in C. reticulata on MT medium supplemented with 500 mg/l ME and 0.5 mg/l

BA. Miah et al. (2002) reported callus induction and plant regeneration through somatic

embryogenesis from immature ovules on MS medium supplemented with 500 mg/l ME

in Citrus macroptera Mont. Var. anammensis (‘Sat Kara’). Han et al. (2002) obtained

somatic embryos from aborted seeds of C. unshiu on MS medium supplemented with 5

µM KN + 0.2% gellan gum.

Neidz et al. (2002) reported that somatic embryos initiated from embryogenic

callus of sweet orange [C. sinensis L. Osbeck cv. ‘Hamlin’] generally results in

abnormal morphogenesis of somatic embryos. To normalize development, glycerol-

induced globular stage somatic embryos were cultured on 6000–8000 MW cutoff

cellulose acetate, 400000 MW cutoff cellulose acetate, nitrocellulose, polyvinylidene

fluoride (PVDF), cellulose filter paper or positive or neutral charged nylon membranes.

Only the two cellulose acetate membranes resulted in the development of normal, two-

cotyledon, bipolar, heart-shaped embryos and no aberrant teratoma-like structures.

Heart-shaped embryos developed and germinated normally on Murashige and Tucker

basal medium with 0.5% sucrose and 1 µM GA3. Culture of embryogenic callus directly

onto cellulose membranes also resulted in the development of normal heart-shaped

embryos, indicating that glycerol induction of globular-stage embryos is not necessary.

Heart-shaped embryos were not observed when the osmotic potential of the medium

was increased by the addition of 2.5–15% PEG; neither were they observed when the

matric potential of the medium was increased by increasing the gelling agent

concentrations of agar and gelrite from 0.8% to 3% and 0.15% to 0.9%, respectively.

Ricci et al. (2002) reported somatic embryogenesis in C. reticulata, C. sinensis

and C. nobilis × C. deliciosa on MT medium supplemented with ME and different

carbohydrates (galactose, maltose, lactose, sucrose and glucose). Wu and Mooney

(2002) reported callus induction and somatic embryogenesis from ovules excised from

immature fruits of citrus cultivars on MT medium containing 50 mg/l sucrose, 500 mg/l

ME and 7.5 g/l agar. This callus was treated with colchicine to induce chromosome

doubling in order to produce autotetraploid plants.


50

Germana et al. (2005) reported the influence of light quality on anther culture of

Citrus clementina Hort. exTan., cultivar Nules. After one month of cultivation in

darkness, four light qualities were tested: White, Red, Far-Red and Blue. Continuous

darkness and white light under photoperiod of 16 hrs were used as control conditions.

Gametic embryoids and embryogenic callus were obtained only under photoperiodic

conditions of White light, suggesting that the alternation of light and dark can be used

for the process of gametic embryogenesis in Citrus.

Mukhtar et al. (2005) used nodal segments and leaf discs of sweet orange

(Citrus sinensis L.) cv. Musambi and lime (Citrus aurantifolia ) cv. Kaghzi nimbu to

obtain aseptically raised plantlets on MS medium supplemented with 2,4-D (2 mg/l) and

coconut milk (20%). Callus induction was highest when shoot segments of lemon were

cultured on MS media supplemented with 2, 4-D and coconut milk. Embryo

proliferation was highest when they were cultured on MS medium supplemented with

1.5 mg/l of KN. Shoot induction was highest when embryos were cultured on MS

medium supplemented with 2.0 mg/l of BA in both species. Highest rooting percentage

in musambi was obtained at concentration of 1.5 mg/l NAA. Singh et al. (2005) used

MS medium supplemented with 9.29 µM KN to induce somatic embryogenesis from

ovules in kinnow (C. nobilis × C. deliciosa).

Carra et al. (2006) investigated the possibility that three diphenylurea (DPU)

derivatives, N-phenyl-N¢-benzothiazol-6-ylurea (PBU), N,N¢-bis-(2,3-methilendioxy-

phenyl) urea (2,3-MDPU) and N,N¢-bis-(3,4-methilendioxyphenyl) urea (3,4-MDPU),

stimulate the induction of somatic embryogenesis in three Citrus species. The

hypothetical embryogenic activity was assessed using stigma and styles of Citrus

myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three

compounds influenced the production of somatic embryos differently as regards the

concentrations tested and the Citrus species. PBU was able to induce somatic

embryogenesis at all the concentrations tested and in all the three species with

percentages that ranged from 44 (C. limon) to 85% (C. myrtifolia). 2,3-MDPU and 3,4-

MDPU were completely unable to induce the production of somatic embryos in C.

myrtifolia while both the compounds at the higher concentration (12 µM) acted


51

positively in both C. madurensis and C. limon (68% of embryogenic explants). The

phenylurea derivatives, used for the first time in this study to induce somatic

embryogenesis in plants, showed a higher embryogenic performance when compared

with BA, a classical adenine-cytokinin, and with N-(2-chloro-4-pyridyl)-N¢-phenylurea

(CPPU), a classical DPU derivative.

Chiancone et al. (2006) investigated the influence of polyamines on anther

culture of Citrus clementina, cv. Nules, with particular attention to the free, soluble and

insoluble-conjugated polyamine levels. Putrescine, spermidine and putrescine plus

spermidine, were added to the standard induction medium. Before culture, spermidine

was the most abundant among the free polyamines detected in anthers. The exogenous

supply of either putrescine or spermidine, either independently or combined, effected

greater uptake and accumulation of polyamines. The addition of 2 µM spermidine to the

medium stimulated gametic embryogenesis in clementine Nules, whereas putrescine did

not influence embryo production. Regenerants were mostly tri-haploids; a few doubled-

haploids and no haploid plants were obtained.

El-Sawy et al. (2006) initiated embryogenic cultures from undeveloped ovules

(8-weeks old after anthesis) of several Citrus species on three different media: basal MS

supplemented with ME alone (M1 medium) or in combinations with BA (M2 medium)

or KN (M3 medium). Differences were observed in callusing percentage, embryogenic

callus and the number of embryos among species, cultivars and culture media under

investigation. Also, embryo germination was determined. Somatic embryogenesis via

ovules depended on the genotype and the culture medium. The most effective treatment

for embryo production varied among genotypes as the highest percentage of

embryogenesis was obtained with local mandarin on M1 medium (100%) and for

shamouti orange on M2 medium (44.4%) but M3 medium was the best for Washington

navel orange (100%). In general, M1 medium gave the best response for most

genotypes.

Kayim and Kemal (2006) studied the effects of several carbohydrates i.e.

glycerol, sorbitol, mannitol, lactose, glucose, and galactose at different concentration


52

ranges (1–5%) on embryogenesis of ovule-derived calli from three species and four

varieties of Citrus; clementine mandarin (Citrus clementina), Washington navel orange

(C. sinensis), kutdiken and Zagara bianca lemon (C. limon). The best results of embryo

formation from calli of different citrus varieties were obtained in 4% and 5% glycerol

concentrations. Sixty seven Washington navel orange embryos and 137 Zagara bianca

lemon embryos were obtained at a concentration of 4% glycerol. Clementine mandarin

and kutdiken lemon produced significant numbers of embryos 808 and 453,

respectively, with 5% glycerol. Embryogenesis was also stimulated at fewer rates with

other carbohydrates, such as lactose and sorbitol, at various concentrations in all citrus

calli utilized in this study.

Moiseeva et al. (2006) reported organization of initial stages of somatic

embryogenesis in tissue culture of Citrus sinensis cv. Toracco at the organismal level.

The callus (pre-embryogenic mass) was induced from nucellus tissue on MT medium

containing 1% sucrose, 2% glycerol and 500 mg/l ME.

Niedz et al. (2006) isolated protoplasts from nucellar-derived embryogenic

callus of sweet orange (C. sinensis L. Osbeck) cultured in alginate beads for 5–30 days,

and the resulting protoplast derived calli (p-calli) released by liquefaction and cultured

on semi-permeable membranes overlaid on MT culture medium. Somatic embryos did

not develop from 5- to 10-day-old p-calli but did develop from 15-, 20-, 25-, and 30-

day-old p-calli. There were no significant differences in the numbers of embryos

produced among the 15- to 30-day-old p-calli and no abnormal embryo morphologies

were observed. The minimum size of p-calli to form embryos was 77.84 mm in

diameter. Embryos were smaller from p-calli than those produced from embryogenic

callus; p-calli-derived embryos ranged in size between 0.5 and 0.8 mm, while embryos

derived from embryogenic callus ranged between 1 and 2 mm.

Singh et al. (2006) reported callus induction from nucellus tissues of kinnow

(C. nobilis × C. deliciosa) on MS medium supplemented with 9.02 µM 2,4-D, and 400

mg/l ME in combination with 7.56 µM ABA or 500 mg/l ME was used for somatic

embryogenesis. Siragusa et al. (2007) reported regeneration of somatic embryos from


53

calamondin (Citrus madurensis Lour.) style stigma explants cultured in the presence of

BA and three synthetic phenylurea derivatives, N-(2-chloro-4-pyridyl)-N-phenylurea

(4-CPPU), N-phenyl-N¢-benzothiazol-6-ylurea (PBU) and N,N¢-bis-(2,3-

methilendioxyphenyl)urea (2,3-MDPU). The phenylurea derivative compounds tested at

micromolar level (12 µM) were able to induce a significant percentage of responsive

explants as compare that obtained with BA and hormone-free conditions.

Singh et al. (2007) investigated the effect of storage conditions on per cent

germination of encapsulated and non-encapsulated somatic embryos of kinnow

mandarin (Citrus nobilis Lour × C. deliciosa Tenora). Different batches of encapsulated

and non-encapsulated embryos were preserved at room temperature, 4◦C, in liquid

nitrogen as such and by embedding in liquid paraffin. In the encapsulated somatic

embryos stored at room temperature in sealed Petri plates, percentage of germination

was 24.99%, but 5.55% in non-encapsulated embryos after 3 days of storage.

Encapsulated embryos stored in vials containing liquid paraffin at room temperature

were germinated at 18.05% after 60 days of storage, while it was 13.88% in non-

encapsulated embryos after 45 days of storage. Encapsulated somatic embryos stored at

4◦C in sealed Petri plates remained viable for up to 75 days with 6.94% germination,

whereas non-encapsulated embryos remained viable for up to 45 days with 24.99%

germination. Encapsulated embryos stored at 4◦C in vials filled with paraffin germinated

at 11.11% after 120 days of storage, but 5.55% in non-encapsulated embryos after 90

days of storage. Encapsulated and nonencapsulated embryos stored in liquid nitrogen

showed 58.33 and 51.38% survival, respectively, after 7 months of storage. The

plantlets developed from these embryos were transplanted after acclimatization and

showed normal growth.

Altaf et al. (2008) reported somatic embryogenesis from immature ovules. The

callus was formed on explants cultured on MS medium supplemented with KN 2 mg/l +

2,4-D 2 mg/l. Embryos were obtained in MS + ME 0.5 mg/l + ADS 25 mg/l. Wu et al.

(2009) reported that embryogenic callus culture of Citrus sinensis gave rise to a large

number of embryos on the embryo inducing medium (EIM) containing glycerol,

whereas very few embryos were observed on the callus growth medium (CGM).


54

Exogenous application of 1 µM putrescine to EIM together with 5 µM a difluoro methyl

ornithine led to dramatic enhancement of endogenous polyamines, which successfully

restored somatic embryogenesis. All of these, collectively, demonstrated that free

polyamines, at least spermidine and spermine, were involved in glycerol-mediated

promotion of somatic embryogenesis, which will open a new avenue for establishing a

sophisticated system for somatic embryogenesis based on the modulation of

endogenous polyamines. Summary of literature on somatic embryogenesis in Citrus

spp. is given in Table 2.3.

2.3. INDUCED MUTAGENESIS

The discoveries during the late 1920s that the genetic material is amenable to

changes excited geneticists who saw new opportunities for both basic and practical

applications. During the 1950s, induced mutagenesis was widely pursued in the US,

Europe, Japan and China. In India, Swaminathan and his team at the Indian Agricultural

Research Institute, New Delhi initiated a major programme on mutagenesis in crop

plants. These studies were broadly aimed at understanding the process of mutations,

testing the efficacy of various mutagens, identifying optimum dose and the best method

of treatment for different crop species; isolation of mutants of basic and applied value;

elucidating the biological effects of radiation-treated medium, seeds and vegetative

propagules on the organisms consuming them. Chemical mutagens mostly used for

mutation induction in plants belong to class of alkylating agents (ethyl methansulfonate

(EMS); diethylsulfate (DES); ethyleneimine (EI); ethyl nitroso urethane (ENU); ethyl

nitrosourea (ENH), methyl nitroso urea (MNH) and azides (Heslot, 1977).

2.3.1. EMS as chemical mutagen for crop improvement

EMS is one of the most widely used chemical mutagen in mutation studies.

Carlson (1970) selected auxotrophic mutants in haploid cells using EMS as the

mutagen. Nabors et al. (1975) selected NaCl-resistant cell lines from Nicotiana

tabacum L. cell suspension culture treated by the mutagen EMS and grown in a medium

containing 0.03 M NaCl. Cells derived from these lines even resisted concentrations as


55

high as 0.09 M NaCl in the medium. The mutant was resistant to seed germination

inhibition by spermine.

Epp (1987) suggested incubation of shoot-tips for 1 hour in 12.41-37.23 mM

EMS. Omar et al. (1989) used EMS as mutagen to induce variations in banana by

treating shoot tips of two banana clones: SH-3362 (AA); and GN-60A, a mutant of

Grande Naine (AAA) and then regenerated adventitious buds. The best response was

achieved with 24.69 mM and 3 h of incubation.

Lu and Jia (1994) treated embryogenic calli of millet with 0.5% EMS for 2.5 h,

the regenerated plants exhibited strong resistance to NaCl stress. Pius et al. (1994) used

EMS (Ethyl methane sulfonate) to induce variation in fingermillet. Embryogenic tissues

were treated with 0.5 and 1% EMS. The calli were immersed into filter sterilized

solution of EMS for ½, 1 and 2 h, washed four times with sterile distilled water and

transferred to MS basal medium.

Venkatachalam and Jayabalan (1995a) established callus cultures from

immature leaf explants of two cultivars of Arachis hypogaea (VRI-2 and TMV-7) on

MS medium supplemented with 2.0 mg/l of NAA and 0.5 mg/l of BA. Three week old

calli were subjected to mutagenic treatments (gamma rays: 50-250 Gy and EMS: 5-25

raM). Mutagen-treated calli were subcultured to fresh medium containing various

concentrations (25-100% v/v) of pathotoxic culture filtrate (CF). Calli were challenged

in vitro with pathotoxic CF of the fungal pathogen and were assessed by visible growth

ratings expressed as the percent response to the doses/concentrations of mutagen.

Selected mutagen-treated calli showed resistance in vitro on media containing

Cercosporidium personatum pathotoxic CF. Resistant calli were then transferred to MS

regeneration medium supplemented with BA (2.0 mg/l) and NAA (0.5 mg/l) for shoot

bud regeneration. The progeny of the plants produced 13 disease-resistant plants (R2) in

both the cultivars. Among the eight R 2 populations studied, 70.2-82.5% of the plants

exhibited enhanced resistance. This study suggested that groundnut plants with

resistance to C. personatum can be selected from mutagen-treated callus of tikka leaf


56

spot-susceptible cultivars using host-specific pathotoxic CFs of C. Personatum through

in vitro technology.

Alcantra et al. (1996) determined optimal conditions for mutagenesis of

Capsicum annuum L. Seeds of the cultivar Keystone Resistant Giant No. 3 were treated

with 0.5%, 1.0%, and 1.5% EMS and exposed for 3, 6, and 9h at 5°C, 10°C, 15°C and

20°C. Several unique and interesting mutants were generated in this study. In the M

generation, seeds treated with 1.5% EMS at 20°C for 9 h had the lowest germination

percentage among 36 treatments, but the observed differences in germination

percentage were not significant. This study suggests that concentration and duration of

seed exposure to EMS could be increased to induce even greater number of mutants.

Mirza and Saeed (1997) isolated a spermine-resistant mutant of Arabidopsis

thaliana L. Henth. from M2 population of EMS mutagenized seeds. Subhash et al.

(1997) reported EMS to induce lincomycin resistance in Capsicum annuum.

Mutagenized cotyledons were cultured on shoot regenerating medium containing

lincomycin (100 mg/l). Approximately 14% of regenerated shoots were chlorophyll

deficient and about 4% of regenerated shoots were green from mutaganized cotyledons.

The regenerated green plants were resistant to lincomycin but sensitive to

chloramphenicol, kanamycin, spectinomycin and streptomycin. Reciprocal crosses were

made between resistant and sensitive plants. Inheritance of lincomycin resistance was

transmitted as a non-Mendelian trait. Lincomycin resistance is a first selectable and

maternally inherited organelle encoded genetic marker described in chilli pepper.

Bhagwat and Duncan (1998) reported mutagen treatment with EMS in banana

(Musa spp. AAA group) to produce variants for tolerance to Fusarium wilt. The

mutagens tested were EMS (100-300 mM), DES (10-25 mM) and sodium azide (1.1-4.6

mM). Shoot apices were immersed in a buffered solution for 30 or 60 minutes. Optimal

doses (2.3 mM NaN3 for 30 min, 25 mM DES for 60 min and 200 mM EMS for 30

min) were determined on the basis of number of parameters such as the survival rate of

treated apices, the regeneration frequency and the phenotypic variation among the

regenerated shoots.


57

Rai and Kumar (2000) reported a recessive EMS-induced mutation inherited in

Mendelian fashion which caused monocotyledonous embryo formation and seed

germination on high salt medium in Catharanthus roseus which is a dicotyledonous

plant. Availability during embryo development of exogenously supplied cytokinin KN

suppressed the mutant phenotype. These observations suggest that, in C. roseus, (i)

insufficiency in endogenous KN may lead to monocotyledonous embryo patterning and

(ii) dicotyledonous embryo formation requires a critical amount of KN in certain cells

of early embryos.

Jabeen and Mirza (2002) used seeds of Capsicum annuum for mutagenesis with

EMS. They used 0.01, 0.1 and 0.5% concentration of EMS for different time duration

i.e. 3 and 6 h. 0.5% concentration with 6 h duration exposure was highly toxic and

produced adverse effect on seed germination. Latado et al. (2004) reported induction of

mutations in chrysanthemum using EMS in immature floral pedicels, followed by the

induction and production of adventitious buds in vitro. Preliminary studies on the

sensitivity of pedicels to EMS revealed that the LD50 was close to 0.82% (v/v).

Immature pedicels of chrysanthemum cv. Ingrid (dark pink color) were treated with

0.77% (0.075 M) EMS solution for 1 h and 45 min, which was followed by rinsing in

water for 15 min and surface disinfection. Afterwards, they were cultivated on MS

medium (salts and vitamins) amended with 1 g/l of hydrolyzed casein, 1 mg/l BA and 2

mg/l IAA. A total of 910 plants were obtained from the pedicels treated with EMS and

were evaluated at the flowering stage. Forty eight mutants (5.2%) were obtained,

deviating in petal colour (pink-salmon, light-pink, bronze, white, yellow and salmon

color). Most of them (89.6% of the total) were phenotypically uniform. The results

showed the efficiency of EMS to induce in vitro mutation of chrysanthemum.

Oliveira et al. (2004) used 0.010 mol dm-3, 0.015 mol dm-3 and 0.020 mol dm-3

of EMS to induce mutations in seeds of sunflower. Seeds were pre-treated with

potassium phosphate buffer of pH 8.0 for 8h. They were then treated with EMS solution

for 16 h, rinsed and were sown in the field at the Embrapa Soybean experimental

station, in Londrina, PR, Brazil and M1 plants were harvested in bulk. M2, M3 and M4

plants were screened for disease resistance under natural infection in the field. Plants


58

were evaluated for Alternaria leaf spot symptoms, using a diagrammatic scale from 0

(no symptoms) to 5 (maximum infection). Before flowering, plants showing no

symptoms of Alternaria leaf spot (grade 0) or less than 5% diseased leaf area (grade 1)

were bagged for self-pollination. Self-pollinated plants and open-pollinated plants from

150 Gy and 165 Gy populations with no or mild disease symptoms were selected. In

the second experiment, sunflower seeds of the genotypes HA 300 and HA BR 104 were

treated with ethyl methanesulfonate (EMS) at 0.015mol dm-3. Selected M2 and M3

were screened for disease resistance in the field. From the EMS treatment, 300 M3

plants with no disease were recovered.

Svetleva and Crino (2005) investigated the influence of EMS and N-nitroso-N´-

ethyl urea (ENU) mutagenic treatments on three time sub-cultured calli obtained from

leaf petiole explants of 7-day old sterile plants. Calibrated sterile seeds of the common

bean Bulgarian variety Plovdiv 11M were pre-cultivated on MS basal medium

supplemented with 1 µmM BA. Then, both mutagens EMS and ENU were applied for

different times such as 15, 30, 60 and 90 min on the explants at the concentrations of:

2.5×10-2 M and 6.2×10-3 M, respectively. Times of the mutagenic treatments influenced

callus growth, calli from 30 min treatment with both mutagens showed the highest

weights. In both cases, the 90-min mutagen application caused a too relevant effect

either on callus browning or growth inhibition. In general, ENU showed a stronger

effect than EMS. The effect of subcultures on callus growth was higher than mutagenic

treatments. Interactions between these factors checked by correlation ratio (η%) were

quite low.

Swamy et al. (2005) induced streptomycin-resistant plantlets showing

chloroplast encoded mutants in Solanum surattense from mutagenised (EMS and

gamma-rays) cotyledon explants. Chloroplast encoded streptomycin resistant – shoots

were developed from green (unbleached) sectors of the cotyledons. The streptomycin-

resistant plants were similar to parental plants in morphology and ploidy level.

Reciprocal crosses between streptomycin-resistant and the original streptomycin

sensitive plants have shown the non-Mendelian transmission under the control of


59

chloroplast DNA. These antibiotic resistant plants are useful in designing biochemical

selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.

Venkataiah et al. (2005) studied the effects of atrazine on cotyledon cultures of

Capsicum annuum (L.) cv. G4 with a view of establishing a system for in vitro selection

of resistant mutants. At low levels, herbicide produced little growth inhibition, some

chlorophyll loss occurred associated with the production of albino shoots. At 20 mg/l

atrazine bleaching was more pronounced and was accompanied by the development of

necrotic spots; however, efficient bleaching was associated with severe suppression of

growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant

plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg/1).

Differential morphogenetic responses were observed when the levels of sucrose (0.5–

5%) were altered. Shoot regeneration was maximum in 2% sucrose and the regenerating

ability decreased with a further increase in sucrose concentration (3–5%). However,

lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of

explants and permitted the selection of herbicide-resistant plants. Complete atrazine-

resistant plantlets were obtained after rooting of regenerated green shoots on rooting

medium containing 10 mg/l atrazine, 1.0 mg/l IAA and 0.5% sucrose. Leaf segment

assay of differentiated plants revealed that all regenerants were resistant to the atrazine.

Reciprocal crosses between atrazine-resistant and sensitive plants showed a non-

Mendelian transmission of resistance trait.

Wani and Khan (2006) treated seeds of mungbean (Vigna radiata (L.) Wilczek)

var. PDM-11, presoaked in distilled water for 9 h with 0.1%, 0.2% EMS or 0.01%,

0.02% hydrazine hydrate (HZ) for 6 h to induce mutations. A significant increase in

mean values of the fertile branches per plant, pods per plant and seed yield per plant

was noticed in mutants of mungbean. Luan et al. (2007) reported in vitro screening for

salt tolerance and plant regeneration of sweet potato (Ipomoea batatas L.) by using

EMS. Calli initiated from leaf explants were treated with 0.5% EMS for 0, 1, 1.5, 2, 2.5

and 3 h, followed by rinsing with sterile distilled water for four times. Preliminary

experiments showed that 200 mM NaCl could be used as selection pressure. Salt

tolerant calli were sub-cultured on medium supplemented with 200 mM NaCl for


60

selection of mutant cell lines and this process repeated 5 times (20 days each). The

selected calli were transferred onto somatic embryo formation medium (MS medium

supplemented with 4 mg/l abscisic acid (ABA), 10 mg/l GA3). After 15 days, somatic

embryos were transferred onto MS medium supplemented with 0.05 mg/l ABA, 0.2

mg/l zeatin for regeneration. Plants designated as ML1, ML2 and ML3 were regenerated

from the somatic embryos formed by calli treated with 0.5% EMS for 2 and 2.5 h. After

propagation, salt tolerance of these mutants was investigated. Data suggested that the

mutants were more salt tolerant than control plants.

Basu et al. (2008) developed early maturing fenugreek mutants with a

determinate growth habit to ensure uniform maturity within the 100 frost free days

available on the Canadian prairies. Seeds from Tristar Fenugreek, a forage cultivar

developed for production in western Canada were treated with 10–300 µM EMS for 2–

24 h and plants were selected for determinate growth habit, early maturity and high seed

yield. This mutation breeding approach has detected new breeding material exhibiting

early seed maturity coupled with high seed yield, seed quality and determinate growth

habit.

Van et al. (2008) reported regeneration of Plants from EMS-treated. Immature

embryo cultures in Soybean [Glycine max (L.) Merr.]. 2 to 4 mm long immature

cotyledons were placed in induction medium after EMS treatment and the numbers of

somatic embryos formed per explant were counted four weeks after culture initiation. In

this study, genotypic differences in the efficiency of somatic embryogenesis from

immature embryos among four cultivars treated with different concentrations of EMS

for six hours were observed. Cultivars, Sinpaldalkong 2 and Jack, displayed highly

efficient somatic embryogenesis regardless of EMS concentration, whereas very low

efficiency or no survival was observed in Jinju 1 and Iksannamulkong cultivars.

Preculture duration did not influence the efficiency of somatic embryogenesis. Because

Sinpaldalkong 2 exhibited the best somatic embryogenesis, much higher concentrations

of EMS were used to test somatic embryo formation under different periods of time in

this cultivar. Three and six hour treatments with both 1 and 2 mM EMS yielded higher

embryo formation than longer periods of time. Increasing the time with embryos in 2


61

mM EMS caused a reduction in somatic embryogenesis in Sinpaldalkong 2, but many

chlorophyll-deficient soybean variants were identified in the M1R0 and M2R1

generations. In addition to Jack, Sinpaldalkong 2 is a good genotype for plant

regeneration from EMS-treated immature embryo cultures.

Khan and Goyal (2009) induced mutations in two mungbean varieties, K-851

and PS-16 using EMS and gamma rays as mutagens. Selection studies were conducted

to improve the yield and to generate genetic variability in different quantitative traits

viz., fertile branches per plant, pods per plant and seed yield per plant. Mean values in

traits increased significantly over the controls and genetic parameters were recorded

higher for the mutants isolated in M5 generation.

Okubara et al. (2009) reported genetic resistance/tolerance to Rhizoctonia solani

AG-8 and R. oryzae in wheat (Triticum aestivum L. em Thell) germplasm ‘Scarlet-Rz1’.

Scarlet-Rz1 was derived from the allohexaploid spring wheat cultivar Scarlet using

EMS mutagenesis. Tolerant seedlings displayed substantial root and shoot growth after

14 days in the presence of 100–400 propagules per gram soil of R. solani AG-8 and R.

Oryzae in greenhouse assays. BC2F4 individuals of Scarlet-Rz1 showed a high and

consistent degree of tolerance.

Dai et al. (2011) reported that EMS treatment inhibited callus proliferation in

Viburnum dentatum L., particularly in the early stage of callus recovery. However, no

significant difference in shoot regeneration among different treatments was observed,

indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their

capacity to grow and regenerate in the regular medium. Kulkarni (2011) exposed seeds

of horse gram [Mocrotyloma uniflorum (Lam) Verdcourt] to various doses of EMS,

Sodium azide (SA) and NMU. The results indicated the reduction in seed germination

67.67% to 47.65% with EMS, 69.66% to 50.33% with SA and 72.33% to 57.59% with

NMU, whereas in control it showed 86% seed germination. Dry/Fresh Wt. ratio (of 8

days old seedling) shows great reduction from 0.102 to 0.079 with EMS, 0.110 to 0.081

with SA and 0.091 to 0.071 with NMU as against 0.108 in control. Pollen fertility was

also reduced with the increase in the concentration of the mutagen. It was noticed


62

86.4%, 74.3% and 54.6% with EMS; 87.8%, 78.9% and 61.3% with SA; 89.3%, 73.6%

and 64.7% with NMU. In general, greater shift in mean and variance was observed in

treatment with higher doses of mutagen.

2.4. IN VITRO SELECTION FOR DISEASE RESISTANCE IN PLANTS

USING PHYTOTOXINS/CULTURE FILTRATES OF PATHOGENS AS

SELECTIVE AGENTS

Toxins are considered to be the special weapons of the plant pathogens to evade

or overcome the inherent resistance strategies of host plants (Kimura et al., 2001).

Culture filtrates (CF), as well as other biotic elicitors (polysaccharides, proteins,

glycoproteins, unsaturated fatty acids, cytoplasm of parasitic and non-parasitic bacteria

and fungi), are able to induce the production of phytoalexins in vitro. The elicitor-

induced accumulation of antimicrobial phytoalexins deserves special attention for

explaining plant fungal parasite interactions (Barz et al., 1988). Substances that trigger

most plant defence reactions (e.g. exogenous elicitors) may originate from mycelial

walls or CF (Knogge, 1996a). The phytotoxicity of numerous secondary metabolites

contained in CF of many pathogenic fungi has been demonstrated in bioassays on

plants. Most of these compounds are non-host-selective phytotoxins that interact with

different targets of the physiology of plants (Abbas et al., 1995). Progress in the field of

in vitro selections is often complicated by: (1) the substances contained in filtrates may

not yet have been completely characterized; (2) the expression of resistance to the toxin

in vitro may vary from that shown to the pathogen in plant; and (3) the level of desired

resistance is not obtainable via toxin influence (Lebeda et al., 1988). Part of the

published studies in this field were focused on basic research; they defined the effects of

particular substances contained in the selective agent in the cultures and tried to find

correlations between their in vitro and in vivo effects (Behnke, 1980; Fereol, 1984;

Buiatti et al., 1985; Connel et al., 1990; Hunold et al., 1992; Huang and Hartman, 1998;

Jayasankar et al., 1999; Hamid and Strange, 2000; Jayasankar et al., 2000; Hollmann et

al., 2002). Valuable and significant results were achieved concerning the induction of

substances involved in plant pathogenesis, e.g. b-glucanases, peroxidases (Broekaert et

al., 2000; Huang, 2001; Singh et al., 2003), glucanases and chitinases (Jayasankar and


63

Litz, 1998), lignin deposition and accumulation of polyphenolic compounds (Cvikrova´

et al., 1992; Rodeva et al., 2000). Other studies dealt with the effects of these agents on

cells or tissues in vitro and compared them with the in vivo response of plants after

inoculation with fungi or bacteria (Bonnet et al., 1985; Buiatti et al., 1987; Kleijer et

al., 1997; Koike and Nanbu, 1997; Jayasankar and Litz, 1998; Fernandez et al., 2000).

The results were often different, positive and/or negative, depending on the particularity

of the plant pathogen interactions involved.

Many studies on the use of specific toxin for in vitro selection of somaclonal

variants resistant to toxin have been carried out on maize plants (Bretell and Thomas,

1980; Umbeck and Gengenbach, 1983; Gengenbach et al., 1997) and oats (Rines and

Luke, 1985). In vitro selection with CF or non-specific toxins has often given

contrasting results. During 1980 to 1990 many studies reported that the selection of in

vitro calli for insensitivity to CF or toxins gives an increase in the percentage of

resistant regenerants (Sacristan, 1982; Ling et al., 1985). However, in some studies the

frequency of in vitro selection for resistance to toxic medium was not improved (Shahin

and Spivey, 1986).

Behnke (1979) selected dihaploid calli from Solanum tuberosum which were

resistant to the CF of Phytophthora infestans. Each of the selected calli was resistant to

all four pathotypes of Phytophthora used in these experiments. The resistance was not

lost through regeneration and the induction of new callus. Behnke (1980) selected calli

from Solanum tuberosum, which were resistant to the CF of Fusarium oxysporum.

Lower fungus growth correlated with resistance to the CF on the level of calli tissues.

Taylor and Secor (1990) selected callus cultures of S. tuberosum by inoculation and

bilayer culture with Erwinia carotovora subsp. caraotovora. Calli resistant to E.

carotovora were identified, number of regenerants were too low to determine

correlation between in vitro response and tuber resistance.

Thanutong et al. (1983) reported in vitro selection of calli of tobacco in the

presence of toxin isolated from Pseudomonas syringae pv. Tabaci and Alternaria

alternata. Assay of R1 generation derived from toxin resistant calli indicated the


64

inheritance of resistance. Bonnet et al. (1985) studied phytotoxicity of individual filtrate

fractions isolated from CF of Phytophthora cryptogea on the leaves of N. tabacum.

Tuzun and Kuc (1987) proved transfer of induced resistance via tissue culture by

inoculating callus cultures of N. tabacum with fungus Peronospora tabacina. Ishida and

Kumashiro (1988) reported that AT toxin of A. alternata induced typical symptoms on

leaves of susceptible cultivars of N. tabacum and cells of resistant cvs. survived toxin

treatment better than those of susceptible cultivar. Field evaluation for tolerance among

cvs. was expressed at the cultured cell level.

Fereol (1984); Peros and Chagvardieff (1987) reported production of disease

resistant plants of Saccharum officinarum (Sugar cane) by treating calli and

micropropagated plants with CF of Ustilago scitaminea. The study showed correlation

between in vitro response and resistance of plants. Mohanraj et al. (2003) reported in

vitro selection of resistant calli of sugercane on the media containing phytotoxin of

Colletotrichum falcatum.

Buiatti et al. (1985) studied the correlation between in vivo resistance to

Fusarium oxysporum f. sp. dianthi and in vitro response to fungal elicitors and toxic

substances, phenylalanine ammonialyase and phytoalexin accumulation in "in vitro"

cultures of three susceptible and four resistant Dianthus caryophyllus cultivars.

Cultivars showing varying degrees of resistance in vivo either tolerated higher CF

concentrations or showed high phenylalanine ammonia-lyase (PAL) enzyme activity

and phytoalexin production when treated with Fusarium elicitor, or responded

positively to both treatments. No such responses were shown in tissue cultures of

susceptible cultivars.

Rines and Luke (1985) studied the effect of Toxin-victorin isolated from

Helminthosporium victoriae on the growth of calli of Avena sativa. This study revealed

that insensitivity to toxin was heritable; specific resistance can be selected in tissue

cultures of oats. Gupta et al. (1986) reported the effect of CF of Alternaria pori on seed

germination in Alium cepa. Seed germination and seedling vigour reduced after filtrate

treatment. Gaurd et al. (1988) exposed the calli and shoot cultures of Allium cepa to the


65

CF of Pyrenochaeta terestris. Calli exposed to the filtrate reflected the degree of whole

plant susceptibility.

Scala et al. (1985) used filtrate and mycelial cell wall components-elicitors of F.

oxysporum f. sp. Lycopersici for in vitro selection of callus cultures of L. esculentum.

Correlation between in vivo resistance and in vitro hypersensitive response and

phytoalexin induction were observed, but tolerance to toxic filtrate in vitro was not

indicator of in vivo resistance. Shahin and Spivey (1986) reported in vitro selection of

calli of L. esculentum for resistance to Fusarium oxysporum in the presence of fusaric

acid (FA). Single dominant gene type of resistance to Fusarium wilt was obtained after

in vitro selection on FA.

Barlass et al. (1986) produced downy mildew resistant plants of Vitis vinifera by

co-cultivation and dual culture of in vitro raised shoots with Plasmopara viticola.

Selected plants were resistant to the pathogen and technique can be used for screening

and selection for host resistance. Fanizza et al. (1995) cultured shoots of several table

and wine grape cultivars in vitro on a medium supplemented with polysaccharides

obtained from a CF of Botryotinia fuckeliana through differential ethanolic

precipitations. The general effects of polysaccharides resulted in leaf yellowness and in

a reduction of fresh and dry weight. Differential response of assayed cultivars to

polysaccharides seemed to be not related to their bunch susceptibility to grey mould

under field conditions.

Beech and Gessler (1986) reported that ultrastructural interactions

between Venturia inaequalis and callus cultures from scab susceptible and resistant

apple varieties, were similar. Host cell wall changes, appositions and invagination of

host plasmamembrane at sites of close contact with fungal hyphae were regularly

observed. The host cell alterations as well as many fungal structures corresponded to

those known in young leaves of susceptible apple varieties. Joung et al. (1987) reported

selection of resistant shoots of M. domestica by inoculation with Gymnosporangium

juniper-virginianae. Shoots from resistant variety were not affected by the fungus;

differences in resistance between cotyledonary or embryo axis shoots were observed.


66

Cucuzza and Kao (1986) developed an in vitro assay using excised cotyledons

of alfalfa that accurately detects resistance to Colletotrichum trifolii. The cotyledons

were sprayed with a spore suspension and resistant and susceptible reactions were

determined after incubation for 14 days at 24◦C with a 16-hr light period. Susceptibility

is based on the presence of sporulating acervuli. Five isolates of C. trifolii reacted

similarly in the assay, and the range of effective inoculum was 2,500–10,000 spores per

spray. Excised cotyledons and the seedlings from which the cotyledons were obtained

were simultaneously tested in vitro and in a greenhouse. Saranac AR and Saranac had

86.5 and 96.2% agreement of observations, respectively, between the two screening

techniques. Results indicated the usefulness of this technique as an alternative to

greenhouse screening methods.

Vardi et al. (1986) used nucellar calli from four Citrus cultivars with known

resistance to the Phytophthora citrophthora pathogen to test the pathogen's response to

CF. Sensitivity of the four calli to CF of the fungus was in reverse order to what is

known on the susceptibility of the cultivars in vivo. Sensitivity of protoplasts derived

from the same four calli to 2,4-dichlorophenoxyacetic acid (2,4-D) was in the same

order as that of calli to CF. Protoplasts derived from calli selected for tolerance to CF

showed a higher plating efficiency with increasing concentration of CF in the medium.

TLC and GLC analysis showed the presence of IAA in the CF. Results indicate that CF

of P. citrophthora cannot be used as a selection tool in vitro. Gentile et al. (1992) used

CF and partially purified toxin (PPT)- malseccin, non-specific phytotoxin of Phoma

tracheiphila for selection of disease resistant calli of Citrus limon (lemon) and C.

sinensis (orange). CF increased the weight of calli, PPT reduced the growth of calli;

HPLC analysis proved low presence of KN and GA3, and high amount of IAA. In citrus

selective toxic effect exists and PPT can be used for screening and also for in vitro

selection.

Buiatti et al. (1987) used CF of Fusarium and cell wall components of Fusarium

and Phytophthora for selection of resistant cell lines of Lycopersicon esculentum

(tomato). Cells selected with Fusarium elicitor showed the same behavior as those with

Phytophthora elicitor; low and high phytoalexin producing clones were selected.


67

Kramer et al. (1988) developed toxin tolerant cell lines of L. esculentum by in vitro

selection of cell lines in the presence of toxin isolated from Clavibacter michiganensis.

Bulk et al. (1991) selected resistant somaclonal variation derived from leaves,

cotyledons and hypocotyles of L. esculentum in the presence of suspension culture of

Clavibacter michiganensis subsp. michiganensis. Limited potential of somaclonal

variation as a source of resistance to bacterial cancer was proved. Kodama et al. (1991)

reported in vitro selection of calli of L. esculentum for resistance to AL-toxin of

Alternaria alternata. Storti et al. (1992) reported in vitro selection of calli of L.

esculentum resistant to F. oxysporum in the presence of dual culture and cell wall

components of pathogen. Resistant cultivars had higher content of polysaccharides (that

lead to phytoalexin synthesis and increased peroxidase activity and callose) and were

tolerant to FA.

Chawla and Wenzel (1987a) reported calli selection of two genotypes of barley,

‘Dissa’ and W 193 for resistance against fusaric acid, a pathotoxin of Fusarium. Callus

was induced from 7 to 10 days old immature embryos. 80% of the calli were killed

during first selection cycle on medium containing 0.8 mM fusaric acid. After 4 selection

cycles 8 to 11% resistant calli were selected and regenerated. Chawla and Wenzel

(1987b) selected calli of Hordeum hordeum (barley) and Triticum aestivum (wheat) for

resistance to toxic filtrate of Helminthosporium sativum by continuous cycles of

recurrent cultivation on media with and without filtrate. 6–17% surviving calli of toxin-

tolerant lines showed one more isozyme than unselected sensitive calli; majority of

regenerated selected plants were less sensitive to H. sativum. Wenzel and Foroughi-

Wehr (1990) reported that in vitro selection did not possess significant improvement in

disease resistance when suspension cultures of H. vulgare (barley), T. aestivum (wheat)

and S. tuberosum (potato) were exposed to extracts of H. sativum, Fusarium coeruleum,

F. sulphureum, Phytophthora infestans and fusaric acid. Hunold et al. (1992) reported

selection of callus cultures of H. vulgare for resistance in the presence of toxin of

Drechslera teres. Nine progenies of S2 showed a correlation between toxin tolerance

and resistance against the pathogen.


68

Ireland and Leath (1987) treated intact plants of M. sativa with CF of

Verticillium albo-atrum and reported that symptoms on stems and leaves of alfalfa after

filtrate treatment were typical for field-infected plants; the filtrate has a potential for use

in screening for resistance. Cvikrova´ et al. (1992) reported selection of resistant calli of

M. sativa by giving challenge of CF of F. oxysporum. Results revealed that cell cultures

derived from genotype susceptible to F. oxysporum accumulated double the amount of

phenolic acids than the resistant ones. Alfalfa protoplast and cell cultures were selected

with fungal cell-wall components and heat-released elicitors for resistance to V. albo-

atrum (Koike et al., 1993b; Koike and Nanbu, 1997). Nedenı´k and Rˇ epkova´ (1998)

reported in vitro selection of calli resistant to fusaric acid, which showed enhanced level

of resistance than the control.

McComb et al. (1987) cocultured callus of Eucalyptus marginata with agar

blocks of fungal culture of Phytophthora cinnamomi. The study revealed that hyphal

growth on callus is correlated with susceptibility of the plant. Cahill et al. (1992) used

suspension culture of Phytophthora cinnamomi to challenge micropropagated clones of

Eucalyptus marginata. Primary roots of resistant micropropagated lines were able to

restrict and confine colonisation by P. cinnamomi. Stukey et al. (2007) reported

selection of Phytophthora cinnamomi resistant plants of jarrah ortest (Eucalyptus

marginata). Plantlets were planted in a former bauxite mine-site in the jarrah forest and

inoculated with P. cinnamomi. Mortality after 13 years in resistant clones was 0–30%,

while that of susceptible clones was 40–100%. Mean heights of resistant clones after 13

years were 7.8–13.6 m, while heights of surviving susceptible clones were 0.9–6.7 m.

The resistance character of the seedling ortets was transmitted consistently to the

clones. The field mortality of clones of some rare, apparently resistant seedlings

selected from susceptible half-sib families was low after 1 year, but approached that of

the susceptible clones after 2 years. The results showed that Phytophthora-resistant

jarrah ortets could be selected using stem inoculation of glasshouse-grown seedlings;

the resistance of the resulting clones was validated in the field in an inoculation trial.

Heath-pagliuso et al. (1988) produced resistant lines of Apium graveolens by

inoculating roots of somaclones in the suspention culture of Fusarium oxysporum.


69

Heath-pagliuso et al. (1989) reported that somaclonal lines (S1 progeny) of Apium

graveolens showed significantly more resistance to F. oxysporum by inoculation of

soaking roots in suspension culture of F. oxysporum. Heath-pagliuso and Rappaport

(1990) tested S1 and S2 somaclonal lines Apium graveolens for the inheritance of

resistance to Fusarium by inoculating the roots of somaclones in the suspention culture

of Fusarium oxysporum. Evenor et al. (1994) studied the effect of CF of Septoria

apiicola on callus of Apium graveolens (celery). Calli showed enhanced degree of

tolerance to CF in selection.

Sjodin et al.(1988); Sjodin and Glimelius (1989) reported selection of disease

resistant plants of Brassica juncea, B. napus, B. carinata and B. nigra (rape), B.

oleracea (rape), Nicotiana tabacum (tobacco) and Solanum tuberosum (potato) from

protoplast, calli and intact plants selection on medium containg toxin-sirodesmine PL

extracted from Phoma lingum. Clear correlation between resistance to P. lingam and

insensitivity to sirodesmin PL was present; toxin could be used to distinguish resistant

and susceptible material both in vitro and in vivo. Hodgkin (1990) selected disease

resistant plants of B. napus (rape) by pollination with pollens treated with toxic extract

of Alternaria alternata. Progeny of 25% selected plants showed pollen resistance

(possibility of utilisation for resistance breeding). Pedras and Biesenthal (2000) used

toxins- phomalide and destruxin isolated from Phoma lingam and Alternaria brassicae

respectively, for selection of disease resistant plants of B. juncea, B. napus (rape) and

Sinapis alba (mustard) using callus cultures. The study revealed differential

phytotoxicity of phomalide and direct correlation with plant disease resistance;

destruxin showed less clear relationship to disease resistance. Rresistant plants of

Brassica oleracea var. botrytis through selection of callus cultures against the CF of

Xanthomonas campestris pv. Campestris were peoduced by Mangal and Sharma (2002).

Mutagenised calli were selected on 30% CF, high level of correlation between

resistance of calli to the filtrate and resistance of regenerated plants to pathogen was

observed.

Connel et al. (1990) studied the cytotoxic effects of CF of Verticillium albo-

atrum on the growth of the protoplasts and cell suspension cultures of Humulus lupulus


70

(hop). Cytotoxicity of the CF correlated both with the virulence of the isolates and the

resistance of the cultivars.

Hammerschlag (1990) reported selection of regenerants of peach (Prunus

persica L. Batsch.) cvs. Sunhigh (susceptible to leaf spot) and Redhaven (moderately

resistant to leaf spot) for resistance to Xanthomonas campestris pv. pruni, the causal

agent of bacterial leaf spot using a detached-leaf bioassay. Regenerants obtained from

calli produced on two ‘Sunhigh’ embryos, #61 and #156, and on three ‘Redhaven’

embryos were evaluated. Sixty-four percent of the regenerants derived from ‘Sunhigh’

embryo #156 and 13% of the regenerants derived from ‘Sunhigh’ embryo #61

demonstrated significantly greater spot resistance than ‘Sunhigh’. Regenerants with

resistance greater than ‘Redhaven’ were also obtained from both ‘Sunhigh’ embryos.

The frequency of variation in the ‘Sunhigh’ seedling population, with respect to the

response to bacterial leaf spot, was not so great as that exhibited by the regenerants

derived from ‘Sunhigh’ embryo #156. None of the ‘Redhaven’ seedlings or any of the

regenerants derived from ‘Redhaven’ embryos were more resistant than ‘Redhaven’.

These studies suggest that the frequency of somaclonal variation is genetically

determined and that screening for somaclonal variation may be a feasible approach to

obtaining leaf spot-resistant peach plants.

Pijut et al. (1990) exposed callus culture and stem cuttings of Ulmus americana

(elm) to CF of Ceratocystis ulmi. Reduction in callus growth of susceptible variety on

media with filtrate and correlation between callus reaction and cut stem assay was

observed.

Saindrenan et al. (1990) reported that Phytophthora cryptogea produces elicitor

active component(s) which caused necrosis, accumulation of the phytoalexins kievitone

and phaseollidin and electrolyte leakage from cowpea leaf tissues. Elicitor activity of

CF was enhanced upon treatment of the fungus with 2.44 µM phosphonate, a dose

which slightly effected fungal growth in vitro. The necrosis-inducing activity of CF was

diminished after heating, treatment with pronase and protease, whereas active

substance(s) was/were periodate-sensitive. The fact that phosphonate improved fungal


71

cells in vitro to overproduce compound(s) which elicited a resistance response,

supported the hypothesis that the toxophore may act indirectly in vivo interfering with

the processes of pathogenicity.

Wolf and Earle (1990) treated leaf mesophyll protoplasts of susceptible and

resistant corn (Zea mays L.) and non-host oats (Avena sativa L.) with HC toxin. This

toxin, a cyclic tetrapeptide produced by the fungus Helminthosporium carbonum race 1,

is a pathogenicity factor for the disease Helminthosporium leaf spot. The effects of HC

toxin on susceptible protoplasts differed from those in other toxin/protoplast systems in

which toxin rapidly decreased viability. In contrast, HC toxin promoted long-term

viability (i.e. staining with fluorescein diacetate) of protoplasts cultured in a minimal

medium. The first effect of HC toxin, observed after 36 h or more depending on the

culture medium used, involved changes in the distribution of chloroplasts within the

protoplasts. Increases in budding of protoplasts occurred after 48 h of exposure to toxin.

Effects of HC toxin on chloroplast distribution and protoplast shape might be due to

enhanced nutrient uptake. As in other types of studies with HC toxin, protoplasts from a

susceptible genotype were affected by low toxin concentrations (1 µg/ml), whereas

protoplasts from resistant genotypes (host and non-host) showed similar effects at

higher toxin concentrations (10 µg/ml). Chlamydocin, a cyclic tetrapeptide similar to

HC toxin produced by the fungus Diheterospora chlamydosporia, also altered

chloroplast distribution in corn protoplasts, with no differential effect on susceptible and

resistant protoplasts.

Ahmed et al. (1991) reported in vitro selection of calli of spring and winter

wheats (Triticum aestivum L.) for Fusarium resistance, using the double-layer culture

technique. Potato-dextrose agar medium in vials was inoculated with mycelia

of Fusarium graminearum and F. culmorum. After one week, fungal cells were killed

by autoclaving and the agar medium containing the thermostable toxic metabolites was

overlayered with MS callus-growing medium. Later, wheat calluses were placed on the

upper medium for 4–5 weeks, and from the surviving calluses plants were regenerated.

R2 seedling populations from self-fertilized R1 plants of 4 varieties were tested


72

for Fusarium resistance by artificial infections in the greenhouse, and 3% of the

regenerated R2 plants have been found to be more resistant than the original cultivars.

Mezzetti et al. (1992) tested two methods for early screening of disease

resistance in apple rootstocks and cultivars. The capacity of Phytophthora cactorum CF

to act as selective agent was tested both on in vitro proliferating shoots and on cell

membrane polarization using an optical probe (Merocyanine 540). With both assays,

four rootstocks (M.26, MM.106, MM.111, Mark), five cultivars ('Gala', 'Liberty',

'McIntosh', 'Empire', 'Jonathan') and some M.26 and MM.106 regenerants were tested.

Both methods were able to characterize different tolerance to CF among the rootstocks,

which correlated with their known field resistance. The second method could be

considered more efficient for genotypes discrimination and also for host-pathogen

interaction studies.

Takahashi et al. (1992) obtained resistant clones of Fragaria vesca by

inoculation of fungus (A. alternata) to wounded plantlets of F. vesca. Orlando et al.

(1997) developed a novel strategy in selecting strawberry (Fragaria x ananassa L.)

plants with resistance to Rhizoctonia fragariae and Botrytis cinerea. Purified pectic

enzymes produced by R. fragariae were used in vitro to select morphogenetic calluses.

Both regenerated shoots and plants were tested in vitro and in vivo with R. fragariae

and B. cinerea. The in vitro resistance of shoots regenerated under selection pressure

was confirmed by in vivo tests with runner plants either by root immersion in a

suspension of R. fragariae mycelium before potting the plants in sterile soil or by

spraying the leaves with several strains of B. cinerea spores. The increase of resistance

against pathogens was correlated with the increase of phenolic compounds, particularly

orthodihydroxyphenols.

Bruins et al. (1993) tested four types of wheat plant material i.e., seedlings,

coleoptile segments, anther-derived callus and anther-derived embryos at different

concentrations of deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-ADON). DON

inhibited growth of all types of plant material. Seedling growth response to 4 × 10−5M

DON of a large set of genotypes did not differentiate between tolerant and sensitive


73

genotypes according to observed Fusarium Head Blight (FHB) resistance level in the

field. In general, coleoptile segments showed a growth reduction at 10−5M DON. A

concentration of 10−4M DON appeared to be the optimum concentration to differentiate

between haploid wheat calli for DON tolerance. However, growth analysis data of 40

callus clones did not show any correlation with the known FHB resistance levels of the

original donor genotypes and populations. Regeneration of the anther-derived embryos

in the embryo selection experiment was decreased 100-fold on DON-containing

medium. Averaged across the callus and embryo selection experiments, green plant

regeneration showed a decrease of approximately 20-fold on medium containing toxin.

Cerato et al. (1993) reported the selection of callus of seven potato cultivars

with CF of Phytophthora infestans (Mont.) de Bary. Regenerated plants were tested for

late blight resistance. Cells selected in one selection cycle were resistant to CF. In vivo

screening of clones regenerated from selected cells was done in two steps: on whole

plants assessing foliage late blight, and on detached leaves, assessing single factors of

horizontal resistance. Koike et al. (1993a, 1996) reported in vitro selection of calli of S.

tuberosum and S. melongena (eggplant) for resistance against the CF of V. albo-atrum.

Trillas-Gay and Araus (1993) reported in vitro selection of calli of D.

caryophyllus for resistance against CF of Fusarium oxysporum. Cells of resistant calli

showed proliferation of endoplasmic reticulum cisternae, nuclei and concentration of

free Ca2+ increased in resistant carnation callus after treatment with filtrate. Trillas- Gay

and Azcon-Bieto (1995) observed different respiratory pattern of two cvs. differing in

degree of susceptibility to F. oxysporum when callus cultures of D. caryophyllus were

exposed to CF and cell wall components of F. oxysporum.

Hammerschlag et al. (1994) reported phenotypic stability of bacterial leaf spot

resistance in peach (Prunus persica (L.) Batsch) regenerants, either selected at the

cellular level for insensitivity to a toxic CF of Xanthomonas campestris pv. pruni or

screened at the whole plant level for resistance to X. campestris pv. pruni. A detached-

leaf bioassay was used to evaluate the original regenerants again after three years in the

greenhouse and also after a two to three year cycle of tissue culture propagation. Peach


74

trees derived through micropropagation from the original regenerants were also

evaluated after one to three years growth in the field. Although leaf spot resistance was

retained in some regenerants over time in the greenhouse, following in vitro

propagation, and under field conditions, resistance was either lost or not expressed in

others. Regenerants # 19-1 and #156-6, derived from embryo callus of bacterial spot

susceptible `Sunhigh', were significantly more resistant than `Sunhigh'. High levels of

resistance were exhibited in greenhouse plants and field-grown trees of regenerant #

122-1, derived from embryo callus of moderately resistant 'Redhaven'.

Keller et al. (1994) reported in vitro selection of embryogenic calli of wheat on

media containg toxin of Septoria nodorum. Good correlation between field resistance

and embryo resistance in vitro was observed. Ahmed et al. (1996) selected calli of

resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties using CFs

of Fusarium graminearum and F. culmorum and evaluated the regenerants for

resistance up to R3. Resistant calli were transferred again to fresh MST for further two

selection cycles. The surviving calli produced less fertile regenerated lines (Ro) than the

non-selected ones. Among 18 R, lines tested for Fusarium-resistance in the seedling

stage by artificial inoculation in the green house, two (11.1%) were significantly more

resistant, one (5.6%) was more susceptible than the original cultivar and the rest

(83.3%) behaved similarly to the donor plants. Kleijer et al. (1996) inoculated

embryogenic calli of triticale with F. culmorum and F. graminearum. No influence of

toxins produced by both Fusarium strains on regeneration was observed.

Mackay et al. (1994) developed an in vitro selection system using roridin E as a

selection agent in Cucumis melo. Vacuum infiltration of callus with the toxin solution

was the only successful selection method at the concentrations tested. Primary callus

(callus originating directly from the explant) was not sensitive to roridin A or E at the

concentrations used. Secondary callus (callus produced from primary callus) exhibited a

differential response to roridins A and E similar to that of detached-leaf assays.

Electrolyte leakage studies of callus were not conclusive in establishing the membrane

as the site of toxin action or useful for screening tolerance in vitro. A small percentage


75

of callus from tolerant and susceptible cultivars survived repeated exposure to roridin E

at 50 µg"mÅl-1.

Brosch et al. (1995) reported that HC toxin, the host-selective toxin of the maize

pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 µM.

Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did

not affect histone acetyltransferases. After partial purification of histone deacetylases

HD1-A, HD1-B and HD2 from germinating maize embryos, it was demonstrated that

the different enzymes were similarly inhibited by the toxins. Inhibitory activities were

reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by

modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,

10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the

uncompetitive type and reversible. HC toxin, in which the epoxide group had been

hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had

been reduced to the corresponding alcohol, the modified toxin was less active than

native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of

highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in

susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and

the myxomycete Physarum polycephalum were also inhibited, indicating that the host

selectivity of HC toxin is not determined by its inhibitory effect on HD.

Cassels and Walsh (1995) reported selection of somaclones derived from shoots

and leaves of Helianthus tuberosus (sunflower) on medium containg metabolites of

Sclerotinia sclerotorum. The results revealed that selection on media was successful,

strong correlation between growth in vitro on calcium free media and field resistance

was observed. Kintzios et al. (1996) studied the effect of toxin isolated from A.

alternata on the growth of callus cultures of H. tuberosus. The calli selected in the

presence of toxin could not regenerate further.

Dan and Stephens (1995) reported selection of resistant plants of Asparagus

officinalis (asparagus) using CF of Fusarium oxysporun and F. proliferatum.

Somaclones originated from protoplast culture were selected for resistances in the


76

presence of CF. Somaclonal lines were more resistant to CF of F. oxysporum than

vegetatively propagated plants.

Matsumoto et al. (1995) selected tolerant variant of banana with fusaric acid

(FA) after chemical mutagenesis. Multiple shoots were selected for tolerance on the

medium containg different concentrations of FA. Trujillo and De-Garcia (1996)

inoculated callus and intact plants of banana with Cercospora musae and

Mycosphaerella musicola. Both calli and plants showed similar levels of survival after

inoculation. Okole and Schulz (1997) obtained the fungus resistant plantlets in a double

selection system. This involved in a first step the use of a fungal crude filtrate and in

the second step the purified host-specific toxin 2,4,8-trihydroxytetralone extracted

from the fungus Mycosphaerella fijiensis, the causal agent of Black Sigatoka

disease. Resistant plantlets obtained from the double selection system were

inoculated with conidia of M. fijiensis in a growth chamber to reproduce Black

Sigatoka symptoms. Compared to non-treated control plantlets, which were highly

susceptible to the fungus, 10.7-19.3% toxin-resistant plantlets which arose from

tissues that went through the double selection system were resistant against M.

fijiensis. This technique of using micro-cross sections for selection on fungal toxins

seems to be amenable to different Musa genotypes for the production of fungus-

resistant plants.

Venkatachalam and Jayabalan (1995b) established callus cultures from

immature leaf explants of Arachis hypogaea on MS medium supplemented with 2.0

mg/l of NAA and 0.5 mg/l of BA of the susceptible cultivars namely VRI-2 and TMV-

7. Three-week-old calli were subjected to mutagenic treatments (gamma rays: 50-250

Gy and EMS: 5-25 raM). Mutagen-treated calli were subcultured to fresh medium

containing various concentrations (25-100% v/v) of pathotoxic CFs. Calli were

challenged in vitro with pathotoxic CF of the fungal pathogen and were assessed by

visible growth ratings expressed as the percent response to doses/concentrations of

mutagen. Selected mutagen-treated calli showed resistance in vitro on media containing

Cercosporidium personatum pathotoxic CF. Resistant calli were regenerated with BA

(2.0 mg/l) and NAA (0.5 mg/l). The progeny of the plants produced 13 disease-resistant


77

plants (R2) in both the cultivars. Among the eight R2 populations studied, 70.2-82.5%

of the plants exhibited enhanced resistance. This study suggested that groundnut plants

with resistance to C. personatum can be selected from mutagen-treated callus of tikka

leaf spot-susceptible cultivars using host-specific pathotoxic CF of C. personatum

through in vitro technology.

Jin et al. (1996) selected embryogenic calli of Glycine max on the medium

containing different concentrations of crude CF of Fusarium solani. Regenerants

showed lower disease ratings than susceptible cultivars. Li et al. (1999) tested the

phytotoxicity of CF of Fusarium solani f. sp. glycines, the fungus causing sudden death

syndrome (SDS) of soybean (Glycine max), with a viability stain of soybean

suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-

cultured cells from a SDS-susceptible soybean cultivar were exposed to cell-free CF of

F. solani f. sp. glycines or other F. solani isolates for 2, 4, 6, and 8 days and then

stained with 0.1% phenosafranin. The percentage of dead soybean suspension-cultured

cells was greater (P<0.001) with filtrates prepared from F. solani f. sp. glycines than

from other F. solani isolates, and dead cells increased over time and with higher

concentrations of CF. Cuttings of soybean seedlings with their stems immersed in CF of

F. solani f. sp. glycines isolates developed SDS-like foliar symptoms, but not when

immersed in filtrates of other isolates. There was a positive correlation (r=0.94,

P<0.001) between soybean foliar symptom severity and percentage of stained soybean

suspension cultured cells. Both methods were used to determine the phytotoxicity of

fungal CFs. Rodeva et al. (2000) inoculated intact plants of Glycine max with Fusarium

solani f. sp. glycines and its filtrate. Calli were obtained from selected resistant plants of

G. max and were exposed to CF of pathogen. Field performance and callus reaction to

fungal CF showed enhanced resistance against the pathogen.

Remotti and Loffler (1996) selected shoots, callus cultures and intact plants of

Gladiolus species on medium containing different concentrations of fusaric acid. Shoot

assay and ion-release with intact cormels gave significantly coinciding results. These

selected plants showed enhanced resistance to Fusarium solani f. sp. gladioli. Part of

the fusarium resistance was based on insensitivity to fusaric acid.


78

Nyange et al. (1997) used hypocotyl-derived calli of genotypes and segregating

populations of Coffea arabica, differing in susceptibility to Colletotrichum kahawae to

produce cell suspensions and protoplasts which were exposed to partially purified

culture filtrate (PPCFs) prepared from the pathogen. The growth and viability of PPCF-

treated cells and protoplasts were measured using packed cell volume, fluorescein

diacetate staining and a colorimetric assay involving the tetrazolium salt MTT.

Differential responses of cells and protoplasts were influenced by genotype, time of

exposure and PPCF concentration. Protoplasts of resistant genotypes responded

differentially from more susceptible genotypes as early as 4h after challenge with the

phytotoxin, suggesting that they were more sensitive than cell suspensions to the

treatments. Protoplasts exposed to PPCFs from C. kahawae may therefore be used to

screen and select genotypes resistant to, or tolerant of coffee berry disease.

Wedge and Tainter (1997) reported that Dogwood anthracnose, caused by the

fungus Discula destructiva Redlin, is a severe disease of flowering dogwood (Comus

florida L.) and Pacific dogwood (C. nutta Uii Aud.). Selection of callus for insensitivity

to D. destructiva metabotites was done by placement of individual cultures on media

amended with progressively higher concentrations of a partially purified culture filtrate

(PPCF) containing low molecular weight compounds. Following this selection process,

cultures were challenged in a dose-response format with PPCF to determine whether the

sensitivity of the callus to the CF had changed. During the selection period, the fresh

weight of callus grown on medium containing 2,4-D and amended with PPCF was

always less than that of callus grown on medium amended with the same concentration

of potato-dextrose broth (PDB, negative control). Fresh weight of callus was greater on

medium containing NAA amended with PPCF than on medium with the same

concentration of PDB. Callus selected in the presence of NAA showed decreased

sensitivity to toxic metabolites at higher concentrations of CF.

Hidalgo et al. (1998) examined two pineapple varieties differing in resistance to

phytotoxic effect of CF of Fusarium subglutinans. The cultivars were Perolera (more

resistant to pathovars of Fusarium subglutinans) and Smooth Cayenne (more

susceptible). The phytotoxic effect of CF was assessed in tissue cultured pine apple


79

plantlets (by electrolyte leakage and placing the CF on wounded leaf segments) and

callus (inhibition of growth). Smooth Cayenne proved to be the more sensitive cultivar

in each test, whereas Perolera showed resistance to the CF and its callus grew in the

presence of high concentrations of CF that were completely toxic to Smooth Cayenne.

These results show that plants can display cellular resistance to the CF. Borras et al.

(2001) reported in vitro selection of resistant plants of Ananas comosus (pine apple)

using CF of Fusarium subglutinans. CF allowed the selection of resistant plants to

fungus; susceptible plants were sensitive to CF.

Jayasankar and Litz (1998) selected embryogenic nucellar cultures of two

polyembryonic mango cultivars, “Hindi” and “Carabao”, for resistance to the CF and

phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated

from mango leaves. The cultures were recurrently selected either with progressively

increasing concentrations of CF or by continuous challenge with the same concentration

of either CF phytotoxin. Mycelium growth was inhibited when the pathogen was

cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth

medium containing macerated resistant embryogenic cultures did not inhibit mycelium

growth, confirming that extracellular antifungal compounds were involved in the

defense response. Enhanced secretion of chitinase and glucanase was observed in the

plant growth medium in which resistant embryogenic cultures and regenerated somatic

embryos were grown in comparison with the controls.

Jayasankar et al. (1998) did RAPD analysis of embryogenic cultures of two

mango cultivars, 'Hindi' and 'Carabao', that had been selected for resistance to the CF of

Colletotrichum gloeosporioides. In vitro selection caused changes in RAPD markers in

the selected embryogenic cultures with respect to the unchallenged control cultures and

the stock plants. The differences involved both the absence and the presence of

additional RAPD markers in the resistant lines, although the former was most

commonly observed. The absence of differences between the unchallenged control of

either cultivar and DNA from the leaves of parent trees confirmed that the changes were

not due to prolonged maintenance in liquid cultures.


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Yang and Huang (1998) developed an efficient in vitro selection system for scab

resistance by using in vitro screening for tolerance to DON. Immature embryos of two

wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11,

and their reciprocal F1 hybrids were cultured on MS medium supplemented with 2,4-D

2 mg/l and 0.6×10-4 M DON for callus induction. The responses of callus induction and

plant regeneration to 0.6×10-4 M DON differed significantly between resistant and

susceptible varieties, according to observed scab resistance levels at the plant level in

the field. The percentage of callus formation of resistant variety Sumai 3 on induction

medium containing DON was higher than that of susceptible variety Mianyang 11.

Regeneration of DON-tolerant calli on DON-containing differentiation medium differed

significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant

calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was

decreased 3-fold on DON-containing medium. The somaclonal lines had lower disease

scoring (reaction index, infected spikelets and disease incidence), shorter plants and

better yield components than Sumai 3, a famous Chinese resistant variety.

Cristinzio and Testa (1999) analyzed in vitro resistance of ten potato cultivars

(Agria, Ajax, Desire, Liseta. Kennebec, Majestic, Monalisa, Prima, Spunta and Tonda

di Berlino) to Phytophthora infestans using 8 fungal strains. An assay based on

electrolyte leakage was used for screening leaves and tuber tissues with fungal CFs.

With almost all cultivars the resistance of leaves did not correlate with the resistance of

tubers. Cultivar Ajax appeared, the least susceptible in both leaf and tuber tests, while

the cv. Prima was the most susceptible in tuber tests.

Jayasankar et al. (1999) reported that partially purified phytotoxin produced by

Colletotrichum gloeosporioides Penz., presumably colletotrichin, caused anthracnose-

like symptoms on young mango leaves, was toxic to embryogenic suspension cultures

of two mango cultivars, 'Hindi' and 'Carabao'; and inhibited in vitro seed germination of

two nonhosts, lettuce and tobacco. There were linear relationships between

concentration of the partially purified phytotoxin and mortality of mango embryogenic

cultures. Embryogenic cultures grown in the presence of the partially purified

phytotoxin showed significantly lower growth rates than the controls. Similarly,


81

embryogenic cultures grown in the presence of 40% (v/v) fungal CF showed

significantly lower growth rates than unchallenged controls. Medium containing 40%

(v/v) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures,

indicating the production of phytotoxin in vitro. The results suggest that either fungal

CF or purified phytotoxin can be used as in vitro selection agents to screen for

resistance to this fungus.

Danson et al. (2000) reported that infection of rice plants with Rhizoctonia

solani, the sheath blight fungus, at the flowering stage resulted in an increase in acid

invertase activity. Activity of the invertase(s) with optimum pH at 3.5 and 4.5 was

higher in the susceptible plants compared to resistant plants. Healthy control plants had

no change in invertase activity. These results suggested that the fungus produced an

invertase for the hydrolysis of sucrose that resulted in alterations of source-sink

relationships in the colonized cells. Thus, the increased invertase activity in the

susceptible plants regulated the ratio of hexose to sucrose. This observation is further

supported by the lack of a significant invertase increase in the resistant plants.

Fernandez et al. (2000) used a bioassay in which CF of Colletotrichum

lindemuthianum caused dark brown lesions on the lower surface veins of bean leaves, to

study the production of toxic fungal metabolites. Calli from anthracnose-susceptible

bean cultivars ‘Collacia’, ‘Andecha’ and ‘Seronda’ were sensitive to a 12.5% solution

of race 38 filtrate or to a 25% solution of race 7 filtrate. In contrast, calli from

anthracnose-resistant bean genotypes A 247, TU, PI 207262, ‘Collacia’ ‘Tu’, ‘Collacia’

AB 136 and ‘Collacia’ PI 207262 did not develop browning. CF was passed through an

ionic-exchange resin and a gel filtration resin. Toxic activity of fractions from two races

of the fungal pathogen was different, although in both races slight necrosis was

produced by the same nine fractions. Pathogenicity could be related with common

substances and toxicity could be identified with differential compounds.

Hammerschlag (2000) reported a detached-leaf bioassay to evaluate peach

[Prunus persica (L.) Batsch] somaclone 122-1 (derived from callus produced on an

immature embryo of peach cultivar Redhaven) for resistance to several virulent strains


82

of Xanthomonas campestris pv. pruni [E.F. Sm.) Dows] and to a virulent isolate of

Pseudomonas syringae van Hall pv. syringae, causal agent of bacterial canker. The

detached-leaf bioassay was also used to evaluate progeny of 122-1 for resistance to X.

campestris pv. pruni virulent strain XP1. Somaclone 122-1 was significantly more

resistant to most strains of X. campestris pv. pruni than was ‘Redhaven’, and all of its

progeny exhibited high levels of resistance to X. campestris pv. pruni strain XP1.

Somaclone 122-1 exhibited significantly higher levels of resistance to Pseudomonas

syringae pv. syringae than did ‘Redhaven’ and this resistance was retained over time in

the greenhouse and following a 2-year cycle of tissue culture propagation.

Hoss et al. (2000) investigated the host–pathogen interactions of Musa spp.

and Mycosphaerella fijiensis in order to determine the function of secondary

metabolites within the pathosystem of the Black Sigatoka disease. The pentaketide

metabolites flaviolin, 2-hydroxyjuglone, juglone and 2,4,8-trihydroxytetralone (2,4,8-

THT) of the pathogen were identified. The concentration of 2,4,8-THT was

significantly increased by application of the synthetic compound tricyclazole and by

natural activators extracted from the intercellular space of leaf tissue of

resistant Musa cultivars. When inoculated host plants were treated with tricyclazole,

extensive necrosis of both susceptible and resistant Musa cultivar leaves was observed.

Plant defence mechanisms of resistant Musa cultivars were first detected as an

activation of phenylalanine–ammonia lyase and the subsequent accumulation of post-

infectional substances which blocked fungal growth. These results indicated the

bivalent importance of 2,4,8-THT for host-specific reactions, depending on its

concentration at different stages of pathogenesis.

Rao and Padmaja (2000) regenerated Fusarium resistant plants of Cicer

arietinum (chickpea) from callus cultures selected on medium containg CF of F.

oxysporum f. sp. ciceri. Plantlets obtained from filtrate-selected calli showed resistance

to Fusarium CF. Hamid and Strange (2000) used CF and toxins i.e. solanapyrone A, B,

C of Ascochyta rabiei and F. oxysporum for selection of disease resistant shoots of

Cicer arietinum. Sensitivity to the toxins was correlated with susceptibility to the

disease; selection of plants with well expressed glutathion/glutathion-S-transferase may


83

be one means. Singh et al. (2003) protected chickpea (Cicer arietinum) plants from

Sclerotium rolfsii infection when applied singly or in combination as seed treatment

with two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens

strain Pf4 and P. aeruginosa strain Pag. The two PGPR strains induced the synthesis of

specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth

stages of chickpea seedlings with varied amount. The maximum amount of total

phenolics was recorded in all the aerial parts of 4 week old plants. Gallic, ferulic,

chlorogenic and cinnamic acids were the major phenolic acids detected in high-

performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids

in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid

(SA) was induced frequently during the first 3 weeks of growth only. Between the two

strains, Pag was more effective in inducing phenolic acid synthesis applied either singly

or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In

the presence of a CF of S. rolfsii, the two Pseudomonas strains induced more phenolic

acids in treated than in non-treated and control plants.

Reustle and Matt (2000) reported in vitro selection of protoplasts isolated from

calli of V. vinifera on medium containg CF of Botrytis cinerea. With increasing filtrate

concentration the plating efficacy and embryogenic competence of regenerated calli

decreased. Jayasankar et al. (2000) exposed proembryogenic masses of grapevine (Vitis

vinifera L.) `Chardonnay' (clone 02Ch) to the CF of Elsinoe ampelina (deBary) Shear,

the causal agent of anthracnose disease. After four or five cycles of recurrent in-vitro

selection with medium containing 40% fungal CF, putative resistant lines RC1 and RC2

respectively, were established. The selected lines inhibited the growth of E. ampelina

and Fusarium oxysporium (Schlecht.) (isolated from watermelon) in a dual-culture

assay and reduced the growth of mycelium on a conditioned-medium test, thus

suggesting the involvement of extracellular compounds in resistance. Sodium dodecyl

sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis of extracellular proteins from

spent suspension-culture medium showed enhanced secretion of new proteins by

selected lines. A 36-kDa protein was immunodetected by a chitinase antiserum. This

chitinase continued to express constitutively in differentiated somatic embryos and also


84

in the intercellular fluids of plants regenerated from the selected lines. Somatic embryos

from selected lines grew uninhibitedly in a medium containing 40% fungal CF, whereas

non-selected (control) somatic embryos became necrotic and died within a few days.

Plants regenerated from selected lines exhibited resistance to infection by E. ampelina

in both greenhouse tests and detached leaf bioassays.

Chen and Swart (2002) examined five Amaranthus hybridus varieties in vitro for

sensitivity to a CF of Fusarium oxysporum. The phytotoxicity of the CF was assessed

for its inhibitory effect on callus and seedling root growth, as well as on the viability of

callus cells. The five varieties exhibited a significant amount of variation in response to

the CF of the pathogen. Variety 17 was the most sensitive variety in each bioassay,

whereas variety 20 displayed least sensitivity to the CF. Callus of variety 20 grew well

in the presence of concentrations of CF that were toxic to another four varieties and the

percentage mortality of callus cells after exposure to the filtrate was also lowest of the

five varieties. Root growth of variety 20 was also least affected by exposure to the CF

for up to 6 days.

Fujii et al. (2002) isolated two polyketides, decumbenones A and B and versiol

from the CF of the fungus, Penicillium decumbens. Their respective structures were 1-

(2,8-dihydroxy-1,2,6-trimethyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl)-3-hydroxy-1-

propanone and 1-(2,8-dihydroxy-1,2,6-trimethyl-1,2,4a,5,6,7,8,8a-octahydronaphthalen-

1-yl)-3-hydroxy-1-propanone based on NMR spectroscopic data, chemical conversion,

and X-ray analysis. Decumbenone A inhibited melanization in Magnaporthe grisea, the

rice blast pathogen, whereas decumbenone B like versiol did not.

Prachi et al. (2002) used salicylic acid (SA) to induce insensitivity in the callus

cultures of Zingiber officinale against CF of Fusarium oxysporum f.sp. zingiberi. The

treatment of callus cultures with SA (104 µM) prior to selection with CF of the

pathogen-increased survival of callus. Exogenous application of SA resulted in

increased activity of peroxidase and β-1,3-glucanase enzymes in the callus cultures. No

increase in the activity of phenylalanine ammonia lyase was obtained. Two new protein

bands of 97 and 38 kDa molecular weights were obtained by SDS-PAGE analysis of


85

soluble proteins extracted from SA-treated calli. The PR-1 monoclonal antibody used

for immunodetection of induced proteins cross-reacted with the 38 kDa protein band. In

vitro antifungal activity of protein extract of calli treated with SA tested against the

spores of F. oxysporum f.sp.zingiberi showed significant reduction in spore germination

and germ tube elongation. It is concluded that in ginger, SA may result in the induction

of resistance to F. oxysporum f.sp. zingiberi by inducing increased activity of

peroxidase, β-1,3-glucanase and antifungal PR-proteins.

Thakur et al. (2002) used callus culture derived from internodal segments of two

cultivars of carnation susceptible to Fusarium oxysporum f.sp. dianthi for in vitro

selection for resistance to this pathogenic fungus. Resistant lines were selected by

culturing calli on growth medium containing various concentrations of the CF of F.

oxysporum f.sp. dianthi. Resistant calli obtained after two cycles (25 days/cycle) of

selection were used for plant regeneration. About 32% of the plants regenerated from

the resistant calli had acquired considerable resistance against the pathogen in the field.

No phenotypic variation was observed in the selected regenerants.

Zemanek et al. (2002) studied the response of three different peach (Prunus

persica L. Batsch) genotypes to bacterial and fungal CF, wounding and sterile nutrient

broth (NB) treatments by evaluating β-1,3-glucanase mRNA levels. Northern blot

analysis was conducted using the 3′ end of a peach β-1,3-glucanase gene, PpGns1, as a

probe. Autoradiographs were analyzed using a Stratagene Eagle Eye II gel

documentation system. Analysis of the accumulation of mRNAs encoded by β-1,3-

glucanase demonstrated that activation trends were different among the three peach

genotypes. All genotypes, ‹Evergreen›, ‹Stark’s Earliglo›, and ‹White Lady›, showed an

increase in β-1,3-glucanase mRNA following treatment with CF of the bacterial

pathogen Xanthomonas campestris pv. pruni. Two genotypes, ‹Evergreen› and ‹White

Lady›, showed an increase in mRNA levels following treatment with CF of the bacterial

pathogen Pseudomonas syringae pv. syringae, and two genotypes, ‹Evergreen› and

‹Stark’s Earliglo›, showed an increase in mRNA levels following treatment with CF of

the fungal pathogen Monilinia fructicola. Differences in induction patterns were

observed between bacterial and fungal CF treatments. Wounding induced high levels of


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β-1,3-glucanase mRNA in one genotype, ‹White Lady› while treatment with a sterile

nutrient broth showed an increase in mRNA in another genotype, ‹Evergreen›. The use

of gene-specific primers in RT-PCR indicated that PpGns1 and a second closely-related

gene family member, PpGns2, were transcriptionally active and were differentially

regulated.

Collonnier et al. (2003) produced interspecific somatic hybrids between

Solanum melongena L. (2n = 2x = 24) and two accessions of Solanum torvum Sw. (2n =

2x = 24) in view of transferring resistance to two soil-born pathogens, Ralstonia

solanacearum and Verticillium dahliae, from the wild species into the cultivated

eggplant. Tests for resistance performed in vitro by using suspensions of two strains of

R. solanacearum (race 1 and 3) and filtrate of culture medium of one strain of V.

dahliae, revealed that S. melongena was susceptible, whereas both accessions of S.

torvum had high levels of resistance. Except for two hybrid clones, which were found

susceptible to race 3, as was S. melongena, all somatic hybrids tested showed good

levels of bacterial and fungal resistance, either intermediate or as high as that of the

wild parent.

Companioni et al. (2003) developed a rapid and non-destructive procedure to

differentiate field-grown banana resistant from susceptible clones. This procedure

implicated application of CF of Fusarium oxysporum f. sp. cubense race 1 onto banana

leaves. The relationship between duration of the fungal in vitro incubation, and the

fungal culture fresh mass, the CF absorbency, and the Gross Michel (susceptible

cultivar) leaf lesion area (after application of the CF) were similar and at 24 day-

incubation the highest values of the recorded indicators were observed. A comparison

between Gross Michel and FHIA-01 (resistant) was also performed. The most relevant

differences between cultivars were observed at 48 hours after application of the CF, and

in the middle-aged leaves.

El-Kazzaz and Ashour (2004) selected resistant calli in vitro from cucumber

(Cucumis sativus) explants under challenging stress of cucumber wilt pathogen

Fusarium oxysporum f.sp. cucumerinum culture filtrate. The selection protocol has two


87

directions: First one is step-by-step selection from lower to higher selective CF

concentrations; meanwhile the second one is exchangeable continuous cycles with

and/or without CF using the same selective CF concentration until the end of selection

regime. The progenies of in vitro regenerated plants, obtained under CF stress, showed

resistance when exposed to the pathogen infection. The results indicated that resistance

in cucumber to wilt pathogen is controlled by one pair of genes and segregated as 3

resistant: 1 susceptible.

Olieviera et al. (2004) reported that genetic variability for resistance to

Alternaria leaf spot disease (Alternaria helianthi) can be induced by radiation or

chemical mutagens. Genetic variability was induced in cultivated sunflower to select

lines resistant to Alternaria leaf spot. Sunflower seeds of the genotype HA BR 104 were

irradiated with 150 and 165 Gy of gamma rays. Seeds were sown in the field at the

Embrapa Soybean experimental station in Londrina, PR, Brazil and M1 plants were

harvested in bulk. M2, M3 and M4 plants were screened for disease resistance under

natural infection in the field. Plants were evaluated for Alternaria leaf spot symptoms,

using a diagrammatic scale from 0 (no symptoms) to 5 (maximum infection). Before

flowering, plants showing no symptoms of Alternaria leaf spot (grade 0) or less than 5%

diseased leaf area (grade 1) were bagged for self-pollination. Self-pollinated plants and

open-pollinated plants from 150 Gy and 165 Gy populations with no or mild disease

symptoms were selected. In the second experiment, sunflower seeds of the genotypes

HA 300 and HA BR 104 were treated with EMS at 0.015 mol dm-3. Selected M2 and

M3 were screened for disease resistance in the field. From the EMS treatment, 300 M3

plants with no disease were recovered.

Gayatri et al. (2005) reported selection of resistant calli of Curcuma longa L. cv.

suguna against the CF of Pythium graminicolum which causes root rot disease. Callus

was obtained from in vivo vegetative buds when cultured on LSBM (Linsmaier and

Skoog’s Based Medium) supplememted with 2,4-D (3 mg/l) after 45 days of culture.

Callus was challenged with pure CF to select resistant calli within 30 days of culture

which was further subjected to pure CF treatment. Four tolerant cell lines were selected

after 3 cycles of treatment through continuous in vitro selection and subcultured on


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LSBM supplemented with BA (4 mg/l) along with control non-selected callus. Plants

regenerated from tolerant and non-selected calli were screened for disease tolerance by

adopting in vitro sick plot technique. The data obtained from this experiment revealed a

ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus.

Khan et al. (2005) tested the nematicidal potential of CFs of the blue-green alga,

Microcoleus vaginatus (Cyanobacterium) against Meloidogyne incognita on L.

esculentum in pots under greenhouse conditions. Prior to the transplantation of tomato

seedling, roots were dipped in different concentrations (0.2%, 0.5%, 1%, 2%, 10%, 50%

and 100%) of CF of M. vaginatus for 30 min. Root-dip treatment reduced the root

galling and final population of M. incognita and increased vegetative growth of plants

and root-mass production compared with the control. The beneficial effect of root-dip

treatment increased with the increase in the concentration of CF. Root galling and final

nematode populations were reduced by 65.9% and 97.5%, respectively when treated at

the highest concentration.

Palmer et al. (2005) isolated a phytotoxic protein that evoked the typical

symptoms of Verticillium wilt disease in seedlings of Gossypium hirsutum L. (upland

cotton) from CF of Verticillium dahliae. The protein was purified by ammonium sulfate

precipitation, Sephadex- G100 fractionation, and native PAGE. The 18.5 kD a protein,

designated VD18.5, appears to be a single subunit protein with an isoelectric point

between 3 and 5. VD18.5 induced symptoms of leaf dehydration, chlorosis, necrosis

and stem discoloration in seedlings of the disease susceptible cotton cultivar Siokra 1–4.

The LD50 of VD18.5 on protoplasts of Siokra 1–4 was 18 mg/l. VD18.5 had no

noticeable effect on Pima S-7, which is a disease resistant cultivar. Phytotoxic activity

was partially destroyed at high temperature and was abolished by digestion with

proteinase K. Mass spectrometry fingerprinting and protein sequence data from VD18.5

yielded no significant matches when submitted to the Mascot search engine and NCBI

non-redundant protein databases, respectively. These results suggest that VD18.5 is a

novel protein that may be involved in the development of some of the symptoms

associated with Verticillium wilt disease in the cotton plant.


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Boso et al. (2006) studied resistance to downy mildew in different Vitis vinifera

L. cv. albariño clones belonging to the collection of the Mision Biológica de Galicia,

CSIC (Spain). V. riparia, V. vinifera cv. Solaris and V. vinifera cv. Müller Thurgau

were used as controls. Plants were inoculated with Plasmospora viticola in the

laboratory using the leaf disc, whole leaf and whole plant techniques. The results were

compared with those obtained in the field for the same Albariño clones. The most

susceptible group of clones included MBG-2, MBG-14, MBG-12 and MBG-9, while

MBG-13, MBG-3 and MBG-6 formed the most resistant group. The remaining clones

showed intermediate resistance. These results coincide with observations made in the

field. The resistance observed in MBG-12 could have been generated from in vitro

culture because this induces changes in the downy mildew resistance.

Ganeshan and Jayabalan (2006) reported an in vitro selection method that led to

isolation of Fusarium wilt and Alternaria leaf spot disease-tolerant plantlets in cotton

(Gossypium hirsutum L. cv. SVPR2). Embryogenic callus was isolated from hypocotyl

explants of cotton cultured on 5–50% Fusarium oxysporum CF-fortified callus

induction medium. Somatic embryos tolerant to fungal culture filtrate (FCF) were

isolated from this embryogenic callus on somatic embryo regeneration medium fortified

with 40% FCF. Sixteen plantlets were selected as FCF tolerant from 34 somatic

embryos tested, which corresponded to about 47% success rate. The FCF-tolerant plants

were analyzed for disease tolerance by challenging them with spores of F. oxysporum

and Alternaria macrospora. Four plants were selected as F. oxysporum tolerant from a

total of 24 plants tested. The selected plants showed an enhanced survival rate

compared with the control when they were grown in earthen pots inoculated with 1-105

spores/ml of F. oxysporum. From the FCF-tolerant plants, another nine randomly

selected plantlets were challenged with spores of A. macrospora in order to test their

tolerance to Alternaria leaf spot disease. The number of lesions per leaf significantly

decreased from 8.2 to 0.9 and the lesion lengths were also reduced from 2.8 to 1.2 mm

per leaf spot in these plants. Electrophoresis analysis of extracellular proteins from the

FCF-tolerant plants showed enhanced secretion of proteins in the range of 24–36 kDa.

Isozyme analysis of FCF-tolerant plants by using native gels showed the presence of


90

chitinase. Quantitative analysis showed that there was 13-fold increase in chitinase

activity in the selected FCF-tolerant plants compared to the control plants. The results

showed that overexpression of chitinase enzyme leads to enhanced disease resistance

against F. oxysporum and A. macrospora.

Gonzales et al. (2006) tested in vitro culture responses from different explants of

a race-specific resistant cultivar (Red Mexican) and a race susceptible cultivar

(Palmena) to halo-blight pathogen (Pseudomonas syringae pv. phaseolicola). Two kinds

of filtrate obtained from a phaseolotoxin producer wild type and a non-producer mutant

of P. syringae pv. phaseolicola race-7 were used. Callus formation of Red Mexican was

significantly reduced in the presence of phaseolotoxin. Bud-shoot growth was more

sensitive than callus formation to other metabolites present in the pathogen filtrate, but

the presence of phaseolotoxin in the media showed a positive correlation between

resistance to halo blight race-7 pathogen and bud-shoot growth. The results indicated

that differential in vitro responses are influenced by the plant genotype and metabolite

composition and concentration of the filtrate.

Martin-pinto et al. (2006) investigated the interactions between the mycorrhizal

fungi Boletus edulis, Rhizopogon roseolus, Laccaria laccata and Lactarius deliciosus

and damping off pathogens (Fusarium oxysporum and F. moniliforme) in vitro and

mechanisms involved in the protection of damping off in Pinus nigra seedlings. The

effect of filtrates from mycorrhizal species on spore germination of F. oxysporum

varied throughout the tests. At the end of the assay, the inhibitory effect only could be

observed in the L. deliciosus treatment. None of the filtrates reduced spore germination

in F. moniliforme. Finally, three of the four mycorrhizal species grown for 2 months in

the substrate, L. laccata, L. deliciosus and B. edulis, increased the survival of Fusarium

inoculated P. nigra seedlings. Plantlets of Pisum sativum were selected for resistance

against the Fusarium spp. by using CF and autoclaving inactivating filtrate. The effect

of the filtrates was compared; degree of resistance/susceptibility and of regeneration

capacity was evaluated. Regenerants were tested in field conditions and artificially

inoculated substrates; improved resistance evaluated with infection degree was


91

observed (Lebeda and Svabova, 1997; Svabova and Griga, 1997; Svabova et al., 1998;

Svabova and Odstrcilova, 2001).

Rani and Ravikumar (2006) compared sporophytic and gametophytic recurrent

selection with the aim to enhance the level of resistance to Alternaria leaf blight.

Alternaria leaf blight, caused by Alternaria helianthi Hanf., is one of the most important

diseases of sunflower causing significant yield losses in several tropical countries. The

base population was synthesized by random mating three populations, two interspecific

derivatives involving different species of Helianthus and one germplasm accession

based on their partial resistance to disease incidence. The base population was subjected

to 1-2 cycles of both sporophytic and gametophytic selection. The gametophytic

selection was practiced by applying pathogen CF to the stigma and style one hour

before pollination. The selection response was measured by scoring the percent disease

index at flowering, 15 days after flowering, and at physiological maturity and by

quantifying economic yield gain. A significant reduction in mean per cent disease index

values and a gain in seed yield were observed for both the types of selection cycles, but

more so for gametophytic selection. The populations improved through gametophytic

selection appeared to be more promising as the pollen selection allowed the selection of

rare favorable allelic combinations that would hardly be detected at the sporophytic

level. A combination of gametophytic selection and conventional sporophytic selection

should be considered as an effective tool in population improvement programs to

achieve higher levels of resistance in relatively short time.

Duan et al. (2007) tested the metabolites of Helminthosporium gramineum

Rabenth. for their antagonistic effects towards Rhizoctonia solani (AG-1-IA), the rice

sheath blight fungus. The CF and EtOAc extract of the CF and mycelia (termed as crude

toxin) of H. gramineum significantly reduced the in vitro growth of R. solani. The

biologically active metabolite isolated from crude toxin of H. gramineum was identified

as ophiobolin A by spectroscopic analysis. Significant inhibition of mycelial growth of

R. solani by ophiobolin A was detected for all concentrations tested. In the field

experiments, the treatments of crude toxin effectively reduced the development of rice

sheath blight, whereas there were no significant adverse effects on the growth and yield


92

attributes of rice plants. HPLC tests indicated no detectable ophiobolin A remaining in

rice grain for all treatments. These results showed that the crude toxin and metabolite

produced by H. gramineum may be developed as a biocontrol agent for rice sheath

blight control.

El-Kazzaz and El-Mougy (2007) selected resistant calli under in vitro conditions

from explants of watermelon under challenging stress of (0–100%) CF which was

obtained from the wilt pathogen Fusarium oxysporum f.sp. niveum. The selection

protocol had two directions: the first was a step-by-step selection from lower to higher

selective CF concentrations and the second was exchangeable continuous cycles with

and without CF using the same selective CF concentration until the end of the selection

regime. The plantlet regeneration occurred under CF stress. The progenies of in vitro

regenerated plants showed resistance when exposed to the pathogen infections. The

results were clear in that the resistance in cucumber to wilt pathogen was segregated as

1 resistant: 2 moderately resistant: 1 susceptible meaning that it is controlled by one pair

of genes.

Quaglia and Zazzerini (2007) reported in vitro selection of sunflower calli

against CFs of two pathogen (Diaporthe helianthi) isolates (7/96 and 101/96). This

technique may be an effective and rapid tool to discriminate the most virulent D.

helianthi isolate and to screen for host resistance in the early stage of a breeding

programme. Further investigation on the mechanisms involved in defence pathways

showed no induction of salicylic acid and pathogenesis-related proteins in calli,

indicating that the host resistance is not associated with Systemic Acquired Resistance

(SAR) but probably other biochemical mechanisms.

Rodriguez et al. (2007) used reaction to the CF of Alternaria solani (sorauer) as

an indicator in an in vitro screening test to select lines with decreased field

susceptibility to early blight from a population of 1000 putative mutants. Plantlets of cv.

Desire derived from irradiated callus of potato were inoculated in vitro with a CF of A.

solani (sorauer). Of the 45 lines selected and subsequently evaluated under conditions

of natural infection in the greenhouse, six showed lesser degrees of early blight


93

infection than the cv. Desire control. The six lines selected in the greenhouse retained

lower degrees of infection during 2 years of field trials.

Kumar et al. (2008) described an efficient plant regeneration protocol via

organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv.

NARI-6 in fungal culture filtrate (FCF) treated cultures. Cotyledon explants cultured on

callus induction medium with different levels of FCF (10–50%) produced embryogenic

callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus

tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus

cultured on embryo induction medium containing 40% FCF induced 50.2% somatic

embryogenesis. Embryo germination percentage was decreased from 64.5% to 28% in

embryo maturation medium containing 40% FCF. However, nine plantlets from

organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-

tolerant. Alternaria carthami fungal spores (5 × 105 spores/ml) sprayed on the leaves of

FCF-tolerant plants showed enhanced survival rate over control plants which were more

susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased

from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in

organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-

tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size

was also reduced to 2.7–0.5 mm when compared to the control. This study also

examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity

was slightly decreased whereas peroxidase (POD) activity was increased to a maximum

of 42% (0.19 µmol min-1 mg-1 protein) from organogenesis and 47% (0.23 µmol min-1

mg-1 protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD)

activity was also increased to 17% (149 U mg-1 protein) and 19.5% (145 U mg-1

protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis

when compared with control plants.

Saxena et al. (2008) established a protocol for induction of disease resistance in

callus cultures of rose-scented geranium, Pelargonium graveolens cv. Hemanti against

leaf blight disease caused by the fungal pathogen Alternaria alternata. The callus

cultures were raised and maintained on medium supplemented with various


94

concentrations of CF (0%, 4%, 8%, 12%, 16% and 20%) obtained from A. alternata.

Resistant calli were selected and placed on regeneration medium for regeneration. The

regenerants were confirmed for A. alternata resistance by exposing their leaves to the

same concentrations of CF as used previously. While the parental wild type

demonstrated typical susceptibility, the leaves of putative resistant clones remained

green and viable in the presence of toxin and regenerated shoots directly on the toxin-

free regeneration medium (MS supplemented with 5 mg/l KN and l mg/l NAA). The

above experiment demonstrated the induction of disease resistance in rose-scented

geranium plants at the cellular level.

Mala et al. (2011) examined defence responses in embryogenic cell suspension

cultures of Norway spruce (Picea abies [L.] Karst) elicited by intracellular protein and

cell wall fractions (PF and WF, respectively) prepared from mycelia of the fungus

Sirococcus strobilinus Preuss focusing on changes in (soluble and cell wall-bound)

phenolic and stilbene concentrations. Treatment with both preparations induced an

increase in the total contents of phenolic acids in Norway spruce cells and variations in

the levels of stilbene glycosides. More rapid and intense induction of defence response

was observed in cells after WF application. Significantly decreased concentrations of

stilbene glycosides, isorhapontin, astringin and piceid, were determined in PF and WF

treated Norway spruce cell cultures. The total content of stilbene glycosides decreased

within 8 h after WF application to 68% of the amount determined in the control and

within 12 h to 73% of the control in PF-treated cells. These results demonstrate that

both PF and WF prepared from the Sirococcus strobilinus mycelium elicit changes in

the metabolism of phenylpropanoids, which are involved in the defence responses of

plants to pathogens.

Savita et al. (2011a) reported selection of Phytophthora tolerant lines of Citrus

jambhiri and their regeneration. Cotyledon derived calli were cultured on selective MS

medium supplemented with 5–100 % of CF, to estimate the critical concentration of the

selective agent. The survived calli under stress were subcultured for 20 days on callus

multiplication medium (2,4-D 2 mg/l + BA 0.75 mg/l) without CF. After multiplication,

these calli were further exposed to other cycles of selection, which contained the same


95

and the 3 steps higher concentrations of the selective agent (CF) and this procedure was

repeated several times until the selection regime completed. The selected tolerant calli

were transferred to regeneration medium (MS medium supplemented with 3 mg/l of BA

and same concentration of CF on which the calli were selected). Regenerated shoots

were transferred to rooting medium (½ strength MS medium supplemented with 0.5

mg/l of NAA). Under in vivo conditions about 81% of the selected regenerants

exhibited resistance to Phytophthora parasitica, whereas none of the control plants

showed resistance. Summary on literature on in vitro selection for disease resistance in

plants using phytotoxins/culture filtrates of pathogens as selective agents is given in

Table 2.4.

2.5. CHARACTERIZATION OF VARIATIONS (SOMACLONAL/INDUCED)

IN PLANTS RAISED IN VITRO USING MOLECULAR MARKERS

Molecular markers generally refer to biochemical constituents including primary

and secondary metabolites and other macromolecules such as proteins and DNA (Joshi

et al., 1999). Secondary metabolites as markers have extensively been used in quality

control and standardization of botanical drugs. The analysis of genetic diversity within a

species may involve storage proteins such as glutenins, gliadins, hordeins, etc. (Vapa

and Radovic, 1998; Metakovsky, 1991; Shariflou et al., 2001; 1995; Cerny and Sasek,

1996a,b). A piece of DNA or a protein can be used as a marker (Ovesna et al. 2002).

During late 1950s, development of protein electrophoresis techniques provided

selectively neutral markers spanning several genes, thus contributing significantly to the

current understanding of plant genetic diversity (Hunter and Markert, 1957; Smithies,

1955).

Quality checks are essential to assure reproduction of high quality

micropropagated plants and to have end-users confidence. Quality assurance becomes

more important when the plant material is for export. The choice of explants source,

freedom of the donor from viruses, disease causing fungi, bacteria, viroids,

phytoplasmas, vigour and conformity of the variety and eliminination of somaclonal

variations are critical for maintaining plant quality. Variety identification by proper


96

labelling at all stages of micropropagation is essential of suitable tests to maintain

quality control and the assurance of micropropagated material irrespective of the system

used. No industry can prosper without a quality policy based on carefully identified,

relevant and recognized quality specifications and standard methods to test and verify

them. Hence, the plant coming out of any facility should be subjected to quality testing

without discrimination or prejudice. It has been reported that in vitro cultured plantlets

might exhibit somaclonal variations (D’Amato, 1978; Skirvin, 1978; Larkin and

Scowcroft, 1981; Earle and Demarly, 1982; Rani et al., 1995; Hashimi et al., 1997).

These variations are often heritable (Larkin et al., 1984; Breiman et al., 1987) and are

therefore, unwanted in clonal propagation. Thus, it would be very important to be able

to detect these variations quite early in the life of a plant. Many workers have tried to

assess these variations in various plant species through morphological, biochemical and

molecular analysis (Patel and Berlyn, 1982; Renfroe and Berlyn, 1984; Mo et al., 1989;

Shenoy and Vasil, 1992).

Marker-assisted selection is becoming the method of choice in facilitating

tagging of the desirable traits in many crops (Abdel-Tawab et al., 2003). It was evident

that salinity tolerance is a complex trait which is greatly affected by the environmental

factors. However, Marker assisted selection could enhance the identification of wheat

genotypes tolerant to salt stress. This approach would enable the molecular plant

breeders to grasp the promising genotypes with more confidence in their merits as

selection will be based on genetic rather than phenotypic basis with the elimination of

the confounding effects of environmental factors. Moreover, this process is fast, reliable

and cost effective which can reduce the required time for breeding program.

Quality testing of tissue culture raised plants includes testing of clonal fidelity or

establishing true to mother type whichever is applicable and could be accomplished by

testing genetic uniformity at random loci. The utility of DNA markers for genetic

mapping has enabled the location of several genes of agronomic interest within the

context of an existing Restriction Fragment Length Polymorphism (RFLP) map

(Rafelsky and Tingey, 1993). DNA markers have also been effectively used for cultivar

identification (Moore and Durham, 1992). Polymerase chain reaction (PCR) based


97

methods [i.e., Randomly Amplified Polymorphic DNA (RAPD) analysis], are being

used for identifying molecular markers due to their speed and simplicity in comparison

with RFLPs (Tulseiram et al., 1992). Therefore, variety identification based on DNA

fingerprinting, such as RAPD, microsatellites, STR’s (Sequence Tagged Repeats),

AFLP (Amplified Fragment Length Polymorphism) and multi-locus probes, is

potentially useful. Molecular markers have been shown to enhance breeding efforts in

annual and perennial crops, since they are not altered by major environmental effects

and are more reliable than morphological characters for identification of varieties.

2.5.1. RAPD in determining somaclonal or induced variations

The RAPD marker system is based on the PCR amplification of random DNA

segments in the genome using primers (usually 10-mers) of arbitrary nucleotide

sequence (Williams et al., 1990). Allelic variation among individuals is detected as the

presence or absence of the amplification product, visualized as a band after PCR and

electrophoresis (Welsh and McClelland, 1990). Analysis of variation at the nuclear

genome level using RAPDs has advantages over RFLPs, as a single primer produces

several loci, covering a larger portion of the genome (Tulseiram et al., 1992). Among

other perennial fruit species, RAPDs have been used to determine genetic relationships

in Anona spp. (Ronning et al., 1995a), cacao (Theobroma cacao L.) (Ronning et al.,

1995b) and papaya (Carica papaya L.) (Stiles et al., 1993). In Mangifera indica,

RAPDs have been used to determine phylogenetic relationships and for cultivar

identification (Schnell et al., 1995). RAPD markers have also been useful for

confirming somatic hybrids following protoplast fusion between Citrus spp. (Deng et

al., 1995). Somaclonal variations in regenerants of Vitis spp. from protoplasts and of

Prunus persica (L.) Batsch from embryogenic cultures derived from zygotic embryos

were detected by RAPDs by Schneider et al. (1996) and Hashimi et al. (1997),

respectively. Jayasankar et al. (1998) reported variations at genetic level in the resistant

cultures selected against the CF of Colletotrichum gloeosporioides which were analysed

by RAPD markers. RAPD analysis has been used for Brassica (Demeke et al., 1992),

Triticum (Chandrashekhar and Nguyen, 1993; Abdel-Tawab et al., 2003) and cotton

(El-Kady et al., 2006). Moreover, RAPD analysis has been used earlier for genetic


98

diversity analysis of Egyptian barley cultivars (Abdelsalam et al., 1998 and El-Halfawy

et al., 2006).

2.5.2. RAPD analysis in Citrus species

RAPD markers have also been used for the assessment of variations, variety

identification and assessment of genetic diversity in genus Citrus. Iannelli et al. (1998)

identified genotypes of lemon by flow cytometry and RAPD markers. Guo et al. (2000)

identified somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C.

paradisi) for seedless triploid breeding using RAPD markers. Guo and Deng (2001)

produced wide somatic hybrids of Citrus with its related genera and somatic hybrids

were identified by morphology, cytology, isozyme, RAPD and RFLP analyses. Abkenar

and Isshiki (2003) characterized genetic diversity among Japanese acid Citrus (Citrus

spp.) based on RAPD markers. Hao et al. (2004) used RAPD markers for genetic and

epigenetic evaluations of citrus calluses recovered from slow-growth culture.

Anantakrishnan et al. (2006) identified somatic hybris of Sour orange (Citrus aurantium

L.) using RAPD markers. High levels of citrus genetic diversity in Vietnam were

observed for varieties presenting in pummelo, orange and mandarin groups, particularly

in mandarin originated from the south of China, and Vietnam (Webber, 1943; Scora,

1975) showing morphological variability and also identified by RAPD analysis.

Orbovic et al. (2008) carried out analysis of genetic variability in various tissue culture-

derived lemon plant populations using RAPD and flow cytometry. Khan et al. (2009)

reported regeneration and characterization of plants derived from leaf in vitro culture of

two sweet orange (Citrus sinensis (L.) Osbeck) cultivars using RAPD markers. Biswas

and Deng (2010) used RAPD, ISSR, IRAP and REMAP markers for the genetic

analysis of Citrus spp.

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REVIEW OF LITERATURE - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/18626/5... · REVIEW OF LITERATURE In Punjab (an Indian State), rough lemon (Citrus jambhiri Lush.) is being