Introduction

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Chapter 1

Introduction

Introduction

2

The existence of human society depends on the ability of plants to harness

light and produce oxygen and organic matter. Domestication of plants for agriculture

resulted in development of many great civilizations in the past, Asian civilization

based on rice, Middle eastern on wheat and barley, and American on maize

(Giovanni, 2008; Michael and Mary, 1999; Michael and Laurence, 2006). Over the

past few thousand years, more than half of the suitable land on Earth has been

converted for agricultural use. Agriculture today is a global business, and a necessity

for the production of food, drinks and other vital commodities such as building

materials, fibers, drugs and medicines (Jason, 2004). New products from plants are

constantly being sought and developed, and plants are crucial for maintaining the

environment, both globally in maintaining our atmosphere, and locally in the form of

recreational facilities. Today, also plants dominate our lives and economy, just as they

have done in all civilizations.

Tuber crops are important group of staple food in the tropical world (Sheafer

and Moncada, 2008). In West Africa, East Africa, the Caribbean, South America,

India and South East Asia, one or more of the tropical tuber crops feature as major

food items in the diet of the people, and in some of these regions, tropical tubers

constitute the major staple food. Meanwhile, tropical tuber crops have received scant

attention from researchers. The main reason for this is that research in tropical

countries has historically been most vigorous in the cash and export crops like tea,

rubber, cocoa, oil-palm, coffee, etc. These crops featured significantly in International

trade, even though many of them were consumed little in the producing countries. The

tuber crops, on the other hand, enjoyed extensive local consumption and importance

but was neglected by the agricultural authorities mainly because they did not earn

foreign exchange (Bradshaw, 2010).

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This situation is now changing, gradually because those parts of the world

where tropical tuber crops are important in the diet are precisely the parts where

population growth is highest, and the threat of large-scale starvation is ever-present. It

has therefore become of great importance, to improve the quantity and quality of

those crops which the people already consume

Recently there have been increases in the volume of tropical tuber crops that

enter into international trade (Wheeling, 2007). Most of these crops or their processed

forms are being sent to developed countries. This increased trade is a stimulus for

further research both in the producing and importing countries. The formation of

International Society of Tropical Root Crops during the last decade is only one

manifestation of this increased importance. The increased importance of tropical tuber

crops has also been expressed in the curricula of various Agricultural Colleges and

Institutions in the tropics.

Root and tuber crops provide a substantial part of the world's food supply, and

are also an important source of animal feed and industrial products. The consumption

of root and tuber crops as food in developed countries is considerably smaller than it

is in developing countries, but their use as animal feeds is relatively higher. On a

global basis, approximately 55% of root and tuber crop production is consumed as

food, while the rest is used as animal feed or for industrial processing for products

such as starch, distilled spirits, and a range of minor products. The relatively high cost

of transportation, processing, and storage, as well as the considerable time needed in

food preparation, frequently makes unprocessed root and tuber crops less attractive to

urban consumers.

There are five major groups of root and tuber crops cultivated across the world

viz. Cassava (Manihot esculenta), Sweet potato (Ipomoea batatas). Potato (Solanum

Introduction

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tuberosum), Yams (Dioscorea spp.) and edible Aroids (Colocasia esculenta and

Xanthosoma spp.) known variously as Taro (Colocasia) and Tannia (Xanthosoma),

but often referred to as cocoyams. A comparison of the characteristics of major root

and tuber crops is presented in Table 1.1. The annual production of tuber crops

throughout the world over the last decade is summarized in Figure 1.1 and the

average contribution by each of the major root/tuber crops on total world production

in 2009 (latest data available) is presented in Figure 1.2 a and 1.2 b respectively.

Figure 1.1: Tuber crops production across the World during 2000-2009 period

(Data from FAOSTAT; down loaded on 04.04.2011).

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Characteristics

Root/Tube Crop

Cassava

Potatoes

Sweet potatoes

Yam

Taro

Growth period (months)

9-24

3-7

3-8

8-11

6-18

Annual/perennial Perennial Annual Perennial Annual Perennial

Optimal rainfall (cm) 100-150 50-75 75-100 115 250

Optimal temperature (°C) 25-29 15-18 >24 30 21-27

Drought resistance yes no yes yes no

Organic matter requirement low high low high high

Growth in water-logged Soil

Planting material

no

stem

no

tubers cutting

no

vine cutting

no

tubers

yes

corms/cormels

Storage time in ground post

Storage life

long

short

short

long

long

short

Long

long

moderate

variable

Table 1.1: Comparison of the characteristics of major Root/Tuber crops

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Figure 1.2 (a): Contribution of major food crops throughout the world in 2009. Data from FAOSTAT (down loaded on 04.04.2011)

Figure 1.2 (b): Root/tuber crops production throughout the world in 2009. Data from FAOSTAT (down loaded on 04.04.2011)

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Cassava (Manihot esculenta Crantz):

Cassava (Manihot esculenta Crantz) is a dicotyledonous perennial plant

(Figure 1.3) originated in North-East Brazil, with an additional centre of origin in

Central America (Rogers, 1963; Hillock et al., 2002). From its places of origin, the

plant has spread to various parts of the world, and it is today cultivated in all tropical

regions of the world. The major cassava producing countries are presented in Figure

1.4. There are 98 species of Manihot recognized (Rogers and Appan, 1973), including

popular cultivating varieties under M. esculenta (eg: Malayan clone-M4 of India) and

wild varieties like M. anomala, M. caerulescence, etc. . The taxonomic position of the

Manihot esculenta Crantz is presented in Table 1.2. It is assumed that cassava

originated by hybridization between two wild Manihot species followed by vegetative

reproduction of the hybrid (Nassar, 2000).

Figure 1.3: Manihot esculenta Crantz

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Figure 1.4: Major cassava cultivating regions in the World

Taxonomic Rank

Nomenclature

Kingdom

Plantae

Order Malpighiales

Family Euphorbiaceae

Subfamily Crotonoideae

Tribe Manihoteae

Genus Manihot

Species M. esculenta

Table 1.2: Scientific classification of cassava

Growth Cycle of Cassava:

Cassava is a propagated almost exclusively from stem cuttings, but in nature and in

the process of plant breeding, propagation by seed is quite common (Hillock et al.,

2002). If the cuttings are planted in moist soil under favourable conditions, they

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produce sprouts and adventitious roots within a week. Flowering of the plant may

commence as early as sixth week after planting, although the exact time that

flowering starts will depend on the cultivar and on the environment. Once begun,

flowering will continue intermittently for the rest of the plants life. Tuber formation

also commence by the eighth week after the planting, as long as the environment

conditions (e.g. photoperiod) are favorable. The tubering process involves essentially

the onset of secondary thickening in some of the adventitious roots which were

previously fibrous in nature. As the thickening progresses, the roots swell and soon

most of the bulk of the root is occupied by fleshy tuber material. Usually, the

secondary thickening commences at the proximal part of the root and progresses

towards the distal portions. Thus, the tuber is fattest at the proximal part in which the

thickening had occurred for a longer time, and it tapers slowly towards the distal

portions.

Cassava Root and Tuber

In cassava grown from cuttings, adventitious roots usually arise from the base of the

cutting. Those roots later develop into fibrous root systems which are the main feeder

roots of the plant. The roots may penetrate to depths of 50-100 cm (Cours, 1951).

Later on, some of these fibrous roots begin to swell and become tuberous. Apparently,

all the fibrous roots are initially active in nutrient absorption; but once any of them

becomes tuberous, its ability to function in nutrient absorption decreases considerably

(Adrian et al., 1969). A mature cassava tuber may range in length from 15-100 cm

and in weight from 0.5-2.0 kg, depending on variety and growing conditions. Popular

high yielding variety (Sree Rekha) and high starch variety (Sree Harsha) in Kerala are

shown in Figure 1.5.

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Figure 1.5: Cassava tuber- (Left) Sree Rekha- a high yielding top cross hybrid

vaeirty, (Right) Sree Harsha, a high starch triploid variety.

The angle and depth of penetration of the tuber, as well as the colour of the

tuber surface, are also varietal characteristics, although brown is the predominant

tuber surface colour. In comparison to the tubers of Yam, sweet potato, or cocoyam,

the cassava tuber is physiologically inactive. No eyes or buds are present on it, and

none will develop on any part of the tuber even when it is stored for long periods, or

planted. As such, the cassava tuber cannot be used as a means of propagation. The

tuber flesh is composed of about 62% water, 35% carbohydrate, 1-2% protein, 0.3%

fat, 1-2% fiber, and 1% mineral matter. Most of the carbohydrate fraction is starch,

which makes up 20-25% of the tuber flesh (Purseglove, 1968). Among the minerals in

the tuber, phosphorus and iron predominate, with minimal amounts of calcium. The

tuber is relatively rich in Vitamin C (35 mg per 100g fresh weight), and contains

traces of Niacin and Vitamins A, B1, and B2, but the amounts of thiamine and

riboflavin are negligible (Chavez et al, 2005). The protein of the cassava tuber is rich

in arginine. The cassava tuber contains small but significant amounts of cyanogenic

glucosides. The two main cyanogenic glucosides are linamarin and lotaustralin. Both

glucosides are highly soluble in water, and tent to decompose when heated to

Introduction

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temperatures above 150 °C. The enzyme linamarase (linase), which is also present in

the cassava plant hydrolyze both glucosides to produce hydrocyanic acid (Prussic

acid), which is highly poisonous to humans and animals. The rate of this hydrolysis

can be accelerated by soaking the tubers in water, cutting them, or by raising the

temperature up to 75 °C, above 75 °C, the enzyme is destroyed. The concentration of

linamarase is higher in the peel than in the tuber flesh. The concentration of Prussic

acid in cassava tubers ranges from 10 to 490 mg /kg tuber. As the plant gets older, the

prussic acid content of the tuber increases, attains a peak, and then begins to decline

(Sinha and Nair, 1968).

Cassava Stem and Leaves

The cassava plant grows as a shrub with the stem reaching heights of up to 4

m in some varieties, or only attaining heights of 1m or so in some of the dwarf

varieties. The colour of the stem surface is usually greyish or brownish. In older parts

of the stem, prominent node like leaf scars are present, marking the nodal positions

where the leaves are attached. The distance between leaves varies with cultivars and

also with environmental conditions, being shortest when adverse environmental

conditions exists, and longest when growth conditions are favorable (Hillock et al.,

2002). The pattern of branching of the cassava plant varies with different cultivars. In

some, the main stem grows for a while and then produces three branches

simultaneously. Each of these branches then grows and later again produces three

branches simultaneously, and so on (Jones, 1959). In other cultivars, there is less

regularity in the pattern of branching. In any case, the angle of the branches with the

main stem determines whether the plant has an erect or spreading form. Another

important branching characteristic is how close to the ground the initial branches

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arise. The best type of cassava would be one in which the initial branches are a

reasonable distance from the ground, so that, in the field, the crop stand is relatively

open beneath the canopy of leaves. Such branching allows room for cultural

operations between the rows.

The leaves of cassava are spirally arranged on raised nodal portions on the

stem. The leaf of cassava is deciduous, and, even under the best cultural conditions,

each leaf exists for only a few months before it falls off.

Cassava: Reproductive Biology:

The cassava plant is monoecious; bearing separate male and female flowers on the

same plant. The time interval from planting to flowering may vary from 1 to more

than 24 months and depends on the genotype and environmental conditions (Byrne,

1984). Male and female flowers are borne in a single branched panicle, with female

flowers at the base, and male flowers toward the tip. The flowers are small, with the

male flower being about 0.5 cm in diameter, and the female flower slightly larger.

Male and female flowers of cassava plant are presented in Figure 1.6.

Figure 1.6: Male (left) and Female (right) flowers of cassava plant

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Flowers usually begin to open around mid-day, and remain open for about one day.

Cassava exhibits protogyny, where female flowers open first and the male flowers

follow 1 or 2 weeks later on a given branch. Therefore, when male flowers open, the

female flowers on the same branch have been fertilized or have aborted. However,

because flowering on a single plant may last for more than two months, both self- and

sib-fertilization may occur, with the proportion of each dependent on the genotype,

the environment, and the presence of pollinating insects (Kawano, 1980, Jennings and

Iglesias, 2002, Ceballos et al., 2004). Flowering in cassava is strongly influenced by

environmental factors. A particular clone may produce no flowers in one

environment, produce only aborted flowers or fail to produce viable seed in another

environment. The pollen grains of cassava are quite large in size and sticky, and wind

pollination appears to be of little consequence. Several species of wasp and

honeybees (Apis cerana, Apis mellifera, etc.) are the main pollinators in different

continents (Kawano, 1980). Cassava pollen shows size dimorphism, the larger grains

being 130 to 150 microns in diameter, whereas the smaller grains range from 90 to

110 microns (Plazas, 1991). In some varieties, the larger grains are more abundant

and have better germination percentages (60%) under in vitro conditions (2 hours at

40°C) than the smaller ones.

Smaller grains typically germinate less efficiently than large ones, and may

have less than 20% viability. Cassava pollen loses viability rapidly after it is shed.

Leyton has reported nearly 97% seed set with newly-collected pollen, 56% seed set

with pollen stored for 24 hours at 25°C, and 0.9% seed set (one seed from 102

pollinations) after 48 hours of storage (Leyton, 1993). In practice, breeders take care

to perform pollinations within one hour after collection of pollen to avoid loss of

viability of the pollen.

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For some clones, induction of flowering appears to depend on long

photoperiods up to 16-hour day length associated with temperatures of around 24 ºC

(Keating, 1982; Alves, 2002). Flowering is also dependent on plant habit. A flower

bud typically forms when the plant branches, so that highly branched genotypes are

more prolific than those with a sparsely branched habit. Flower bud formation is

preceded by apical branching, which is a prominent visual indication of incipient

flowering, and may be used to identify plants in the pre-flowering stage.

After pollination and subsequent fertilization, the Cassava ovary develops into

young fruit. It requires 3-5 months after pollination for the fruit to mature. The mature

fruit is capsule, globular in shape, with a diameter of 1-1.5 cm. The endocarp is

woody, and there are three locules, each containing a single seed. When the fruit is

mature and dry, the woody endocarp splits explosively to release and disperse the

seeds.

The cassava seed is ellipsoidal and 1-1.5 cm long. It has a brittle testa which is

grey, mottled with dark blotches. There is a large caruncle at the micropylar end of the

seed. Germination of the seed requires a long time, and that duration can be shortened

by filing the micropylar end until the white embryo is just visible.

Fertilized seed is viable two months after pollination, and fruit becomes

mature about one month after that, or about three months after pollination (Ceballos et

al., 2004). The fruit is a trilocular schizocarp, and seeds are ovoid-ellipsoidal,

approximately 100 mm long and 4 to 6 mm thick (Alves, 2002). Dehiscence is

explosive; the seed initially falls close to the mother plant, but then may be further

dispersed by ants, which carry an unknown percentage of the seed to their nests in the

soil. Through these two mechanisms of autochory followed by myrmecochory, a seed

may be dispersed up to several meters from its place of origin (Elias et al., 2001).

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Seed production and viability are variable, depending largely on the quality of the

female parent (Kawano, 1980). Jennings (1976) reports that one viable seed per fruit

is normally achieved in controlled pollinations, from a maximum of three possible in

the trilocular ovary. Ceballos et al. (2004) indicate that one to two viable seeds are

obtained from each hand-pollination. Newly harvested seeds are dormant, requiring 3

to 6 months of storage before they germinate (Jennings and Iglesias, 2002).

Cassava seeds are adapted to ant-dispersal, with large energy reserves that

allow deep burial and a long dormancy period (Pujol et al., 2002). Seeds can remain

viable for up to 1 year, although germination percentages may decline substantially

after 6 months (Rajendran et al., 2000).

Seed germination is favored by dry heat and complete darkness. Ellis (1982),

working with two-dimensional temperature gradient plates, found that germination

occurred most often when temperatures exceeded 30°C for part of the day, with a

mean temperature of at least 24°C. They have suggest that an alternating temperature

regime of 30°C for 8 hours and 38°C for 16 hours for at least 21 days is the most

appropriate for determination of cassava seed viability under laboratory conditions.

Botanical seed is not typically used for commercial propagation. Genetically, any

particular cassava genotype is extremely heterogeneous, and propagation from sexual

seed results in wide and unpredictable diversity of phenotypes, which is of interest to

breeders but presents difficulties in propagation (Ceballos et al., 2004).

Vegetative propagation

Cassava is normally propagated by means of stem cuttings, which are known

horticulturally as ‘stakes’. Stakes are typically at least 20 cm long, and have 4 to 5

nodes with viable buds.

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Breeding Schemes in Cassava:

As in most crop breeding activities, cassava genetic improvement starts with the

production of new recombinant genotypes derived from selected elite clones.

Scientific cassava breeding began only a few decades ago, and the divergence

between landraces and improved germplasm is not as wide as in other crops.

Therefore, accession in for germplasm bank collected from different research

institutions play a more relavant role in cassava than in other crops that have been

scientifically bred for longer periods of time. Parental lines are selected based mainly

on their performance per se and not much progress has been made to use general

combining ability as a criterion for parental selection. The multiplication rate of

cassava planting material is low as five to ten cuttings can be obtained from one plant.

This implies a lengthy selection process, and in fact it takes about six years from the

time the botanical seed is germinated until enough planting material is available for

multilocation replicated trials.

A typical selection cycle in cassava begins with the crossing of elite clones

and finishes when the few clones surviving the selection process reach the stage of

regional trials across several locations. There is some variation among the cassava-

breeding programmes in the world with respect to the number of genotypes and plants

representing them through the different stages.

Strong emphasis on highly heritable traits (plant type, branching habits and

reaction to diseases, harvest index and dry matter content) is applied during the early

phases of selection, (Hahn et al., 1980, 1990, Hershey, 1984; Kawano, 1998; Ceballos

et al., 2004). As the number of plants representing each genotype increases, the

weight of selection criteria shifts towards low heritability traits such as root yield. The

clones that show outstanding performance in the regional trials are released as new

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17

varieties and, eventually, incorporated as parents in the crossing nurseries. With that

the selection cycle is finished and a new one begins.

Cassava: Genetics and Cytogenetics:

Studies on the genetics of cassava have been limited and breeders have concentrated

on obtaining the basic information required for effective genetic improvement of the

crop. Cassava and the wild species of Manihot have 2n = 36 chromosomes (Nassar,

1978). Regular bivalent formation has been reported in the pollen mother cells, with

few meiotic abnormalities. The completely paired pachytene bivalents vary in length

from 19.3 to 40.0 microns. The haploid chromosomal complement has three

functional nucleolar chromosomes and six chromosomal types represented in

duplicate. This information has been used to suggest that the present-day cultivated

types are allopolyploids of crosses between two closely related forms. Their two basic

diploid parental taxa while possessing six chromosomal types in common, differ in

three chromosomes of their complement. Hence the present-day cultivars may be

considered as segmental allopolyploids (Magoon et al., 1969). Similar pachytene

studies have been carried out on M. glaziovii and a comparison with the karyotype of

cassava showed many common features, including the same number and a similar

morphology of chromosomes (Krishnan et al., 1970).

Extensive diversity exists for most traits examined to date. This may be due to

introgression of wild species germplasm and to the many environments and uses for

which cassava has been selected over thousands of years. Precise genetic control has

been characterized for relatively few traits in cassava. Single gene control has been

demonstrated for leaf lobe width, root surface colour, albinism, stem collenchyma

colour, stem growth habit, root flesh pigmentation and male sterility (Hershey and

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18

Ocampo, 1989). A broad range of agronomically important traits have been studied

for their inheritance patterns. Results to date indicate that nearly all these traits are

under multigenic control, with a high proportion of additive genetic effects (Iglesias et

al., 1994). Genetic variability within cassava for some traits, such as resistance to post

harvest deterioration may be limited for breeding objectives. The application of

molecular techniques, such as gene tagging and the identification of gene products,

will complement conventional approaches to genetic studies of agronomically

important traits. A molecular genetic map of cassava was constructed recently by

Fregene et al., (2008).

Diseases and Pests of Cassava:

A number of diseases and pests adversely affect the overall yield and productivity of

cassava in different continents. Cassava is affected by virus, bacteria and fungi.

Cassava is attacked by more than 30 pathogens causing various degrees of losses

(Lozano and Booth, 1974; Calvert and Thresh, 2002; Thottappilly et al., 2003).

Viral Diseases:

Cassava Mosaic Disease (CMD):

Cassava mosaic disease (CMD) is the major viral disease in cassava, caused by

cassava mosaic virus (CMV), which is spread by the vector whitefly (Bemisia tabaci).

The disease is also transmitted when cuttings from infected stem portions are used for

propagation. The disease is prevalent in all cassava producing areas of Africa, India

and Indonesia, but is of relatively rare occurrence in South America. Symptoms of the

disease include a whitish or yellowish chlorosis of the young leaves, accompanied by

leaf distortion and a reduction in size of the leaves. The growth of the entire plant is

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19

stunted, and the number of axillary branches is increased. Normal increase in tuber

weight is disturbed, so that tuber yield at harvest is considerably reduced.

Several control measures have been suggested for CMD. The most promising

control measure is the use of resistant varieties. Controlling the whitefly, careful

selection of planting material and inactivation of CMV by means of a heat treatment

are also suggested.

Cassava Brown Streak Disease (CBSD):

This is the second major viral disease of cassava caused by cassava brown streak virus

(CBSV). This disease was first reported from Mozambique in 1936. It is most

prevalent in the coastal areas of East Africa, where it sometimes results in serious

yield reductions. The disease is characterized by leaf chlorosis, and brown streaks in

the stem cortex.. The white fly, Bemisia, is probably the vector of the virus, but it can

also be transmitted mechanically. The whiteflies Bemisia afer and Bemisia tabaci are

the leading candidates as the vector of CBSV.

Bacterial Diseases:

Cassava Bacterial Blight:

This disease usually results in heavy yield reductions, and, where infection is severe,

the crop may be lost completely. The symptoms appear on the leaves as angular spots,

blight, and wilting. Cassava bacterial blight is caused by the bacterium Xanthomonas

manihotis. The disease has a wide spectrum of symptoms, including angular leaf

spots, blighting, wilting, vascular necrosis of the stem, dieback, and production of

exudates (Maraite, 1993). Crop losses of 12 to 80% have been reported. The

movement of infected, asymptomatic stems is a major means of pathogen dispersal.

The disease was first reported in South America (Lozano, 1986) and has sub

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20

sequently been reported in Africa and Asia. The disease can be controlled through

cultural practices, including the use of uninfected planting material and resistant

cultivars (Boher and Verdier, 1994; Lozano, 1986).

Bacterial Stem Rot:

Bacterial stem rot is a relatively new bacterial disease reported from Colombia (CIAT,

1976). The casual organism is Erwinia spp., which penetrates through wounds.

Infection is restricted to the stem. Infected plants show dark necrosis, followed by

wilting, and then die-back. Erwinia carotovora var. carotovora is the causative agent

for stem rot of Cassava.

Fungal Diseases:

Brown Leaf Spot:

Brown leaf spot disease occurs quite commonly in cassava plantations in South

America, Asia, and Africa. The disease is characterized by large, brown, irregular,

necrotic spots which appear on older leaves and infected leaves tend to drop early.

The disease is caused by the fungus Cercospora henningsii. The disease can be

controlled by treating early with cuprous oxide suspended in mineral oil and applied

at the rate of 20 litres hectare-1 (Galato, 1963).

Phyllosticta leaf spot:

This disease is caused by an organism belonging to the species of Phoma. The fungus

grows best at about 20°C, and the disease is therefore most prevalent in cooler regions

or cooler seasons of the year. The disease is characterized by the presence of

concentric rings on the upper surface of infected leaves. Several resistant varieties

have been identified in Colombia and their use is recommended in areas where the

disease is prevalent.

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21

White thread:

This disease is caused by the fungus Fomes lignosus. It causes rotting of the tubers,

and the surface of the tuber is covered by a white network of mycelia. Control

measures include harvesting the tubers early, and burning of the field before cassava

is planted.

Cassava anthracnose:

This disease is characterized by wilting and death of the young stems and leaves after

sunken spots appear on them.

Super-elongation disease:

It is characterized by extreme elongation of the internodes of young stems, and a

distortion of young stems, petioles, and leaf mid-ribs. The disease is caused by a

species of Sphaceloma (CIAT, 1976).

Insects and Mites:

The Variegated grasshopper, Green spider mite, Red spider mite, Web mite, Cassava

hornworm, Cassava scale, White flies and Termites are the various insects and mites

that affect the cassava plant.

Other diseases and pests of Cassava:

The root-knot nematode, a nematode with a wide range of hosts, also attacks cassava.

Infested plants show the characteristic knots on their roots at about five months after

infestation. Control is by the use of resistant varieties.

Mammals, particularly rodents, wild pigs, and monkeys, may also attain local

importance as pests of cassava. They feed on cassava roots and also help to spread

various tuber rots. Control is by setting traps for them.

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22

Economic Importance of Cassava:

The economic importance of cassava is dependent on many factors like heavy calorie

yield, ease of production, ideal planting material, hardy plant characteristics and ease

of breeding. Cassava is used as a basic food staple for human consumption, starch

source, and also for animal feed. It is mainly consumed as boiled tuber or flour

obtained by processing the root. The roots are very rich in carbohydrates, mainly

starch (about 80% dry weigh). They also contain vitamin C, beta-carotene, calcium,

potassium, and food fibers, but are very low in protein (0.5-1.5% fresh weigh) and fat

(about 0.17%). The leaves are consumed less than tubers and have a large amount of

protein (about 7% fresh weight). The high concentration of potentially toxic

cyanogenic glucosides is of great concern that hinders the use of unprocessed cassava.

The use of cassava leaves and tender shoots for human consumption is widespread in

Africa. Cassava leaves are nutritious food. The leaves are much richer than the tuber

in proteins and vitamins. However, the leaves have to be sufficiently detoxified before

being eaten.

The processed forms of the cassava tuber fall into four general categories like

meal, flour, chips and starch (Sreenivasan and Anaatharaman, 2005). The meal and

flour forms account for the bulk of cassava used for human food in the tropics.

Cassava chips and cassava starch are mainly industrial products that are little used for

direct human consumption. There is a high demand for starch in industry, and cassava

starch has been used to fill part of this demand. The production of cassava starch for

industrial use is a highly specialized and highly mechanized process. Grocery tapioca

(Sago) is essentially cassava starch produced by a special process.

In India nearly 60% of cassava is used industrially in the production of sago,

starch and dry chips (Sreenivasan, 2007). Hi-tech starch factories have been

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23

established in Tamil Nadu for manufacturing several modified starches from cassava.

These include pre-gelatinized starch (for the confectionery and textile industries),

oxidized and cationic starches (for the paper industry), textile grade modified starch

with good tensile and adhesive strength (for the textile industry), and paper grade

starch with ink water resistance, etc. The projected demand of cassava starch in India

is predominantly in the adhesive sector, especially in the corrugation gums, paper and

textile industries. The demand-supply gap in the industrial sector is expected to be 1.5

× 106 t of cassava tubers demanding 750 000 Ha to be brought under cassava

cultivation (Sreenivasan, 2007).

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Cassava Statistics:

Harvested area, Yield and Production; Global, Asian and Indian scenario:

Cassava is mainly cultivated in the sub-Saharan Affrica, Asia and Latin

America. According to the latest report Nigeria is the largest producer of Cassava in

the World (FAOSTAT, 2009). The statistics of cassava harvested area, in the world,

total in Asia and especially in India during last decade is presented in Figure 1.7.

Figure 1.7: (Top) Cassava harvested area during 2000-2009 in the World and Asia

(Bottom) Cassava harvested area during the same period in India.

Data Source FAOSTAT (down loaded on 14.03.2011)

Introduction

25

Figure 1.8 and Figure 1.9 shows statistics of Cassava yield (Hg/Ha, Hg = 0.1 kg) and

Production throughout whole world, total in Asia and in India during last 10 years.

Figure 1.8: Cassava Yield during 2000-2009 Period in the World, Asia and in India.

Data Source FAOSTAT (down loaded on 14.03.2011)

Figure 1.9: Cassava production (Tonns) during 2000-2009 Period in the World, Asia

and in India.

Data Source FAOSTAT (down loaded on 14.03.2011)

Introduction

26

A consolidated data on top (in terms of quantity and value) ten cassava producing

country in the World is shown in Figure 1.10. Nigeria is higher producer followed by

Brazil and India ranks 9th in the list.

Figure 1.10: Top (in terms of quantity and value) ten cassava producing countries

Data from FAOSTAT (down loaded on 04.04.2011)

Cassava Research Net Work Across the World:

Throughout the World, the increasing importance worldwide of cassava for human

consumption, animal feed and industrial applications leads to an increasing need for a

wide range of genetic resources to develop better quality cultivars. Therefore, cassava

biodiversity deserves international support and cooperation for its collection,

conservation, study and use. Two major organization involved in cassava research

programmes are described as follows.

Introduction

27

Global Cassava Development Strategy (GCDS) and related organization:

The Global Cassava Development Strategy and implementation plan was initiated by

Food and Agricultural Organization of United Nations (FAO) and International Fund

for Agriculture Development (IFAD) in 1996. GCDS addresses many of the critical

problems associated with cassava. It provides information on strategic research and

development activities undertaken by its partners in areas related to genetic

improvement and biotechnology, improved production systems, post harvest and

processing, environmental considerations, institutional development and policy

research. GCDS also focuses on supporting the development of action plans on global

cassava improvement, global cassava post harvest and global cassava marketing. A

major suggestion of GCDS was that cassava could become the raw material base for

an array of processed products that will effectively increase demand for cassava and

contribute to agricultural transformation and economic growth in developing

countries. GCDS also suggested that genetic improvement of cassava should be

related to the use of cassava.

Important partner organization of GCDS includes Food and Agriculture

Organization of the United Nations (FAO), International Fund for Agricultural

Development (IFAD), International Development Research Centre (IDRC),

International Centre for Tropical Agriculture (CIAT), International Institute for

Tropical Agriculture (IITA), International Cooperation Centre of Agricultural

Research for Development (CIRAD), Natural Resources Institute , International

Laboratory for Tropical Agricultural Biotechnology (ILTAB) and Brazilian

Agricultural Research Corporation.

CIAT, which has been active in cassava research since the 1970s, is one of the

leaders in innovation that has made a large contribution to the crop's success in many

Introduction

28

parts of Asia and Latin America. CIAT maintains a collection of more than 6000

cassava accessions for the FAO including landraces from Asia and Latin America,

clones selected by the CIAT and the IITA, and several wild Manihot species.

Consultative Group on International Agricultural Research (CGIAR):

The Consultative Group on International Agricultural Research (CGIAR), established

in 1971, is a strategic partnership of diverse donors that support 15 international

Centres, working in collaboration with large number of government and civil society

organizations as well as private businesses around the world. Eleven of the CGIAR

Centres maintain international Genebanks. These preserve and make readily available

a wide array of plant genetic resources, which form the basis of global food security.

The objectives of CGIAR are “food for people”, “environment for people” and

“policies for people”.

One of the important outcomes of the research under CGIAR was biological

control of the cassava mealybug and green mite, both devastating pests of a root crop

that is vital for food security, succeed in sub-Saharan Africa. The economic benefits

of this work alone, estimated at more than $4 billion. Moreover, two CGIAR research

centers IITA and CIAT have developed elite varieties of cassava with improved

qualities. IITA has also discovered spontaneous polyploids in cassava, which are

characterized by enormous vigor and variation in form and structure. Selections from

triploids "super cassava" have doubled the yields of existing improved varieties with

normal chromosome numbers. IITA has also introduced to Africa a wider genetic base

for cassava improvement, focusing on materials with resistance to mites, mealy bugs,

cassava bacterial blight, tolerance to drought, low cyanogen potential, and good

cooking quality. Together, IITA and CIAT launched a famous biological control

Introduction

29

campaign of the mealybug and are cooperating with the International Fund for

Agricultural Development in the formulation of a global development strategy for

cassava.

International Crop Research Institute for the Semi Arid-Tropics (ICRISAT),

Hyderabad, Anthrapradesh in India is one of centres under the consortium of CGIAR.

Cassava Research in India:

The average yield of cassava in India is nearly 2.7 times higher that of the World

average (3, 43,678 Hg/Ha and 1, 26,448 Hg/Ha respectively) as per current

FAOSTAT data. The research and development in the area of root and tuber crops in

India has played a major role in improving the status of cassava in terms of

production, yield, quality, disease management, marketing, etc. Research on tuber

crops in India is undertaken mainly at the Central Tuber Crops Research Institute

(CTCRI) Thiruvananthapuram, one of the primier institutions under Indian Council of

Agricultural Research (ICAR). One of the major activities of CTCRI includes genetic

resource management where root and tuber crops germplasm is conserved at field

Gene banks and the genetic diversity and purity of the collections are being assessed

periodically through morphometric as well as molecular marker assisted methods. A

total of twelve improved high yielding cassava varieties have been released since

1971 from CTCRI of which H-165 and H-226 are most popular varieties in Tamil

Nadu and Andhra Pradesh. A number of varieties/cultivars from CTCRI are grown in

different regions of the country. A number of high value products from cassava have

been developed at CTCRI to increase the market potential and to sustain and promote

cassava cultivation to more areas.

Introduction

30

Cassava cultivation in India:

India with an annual tuber crops production of nearly 45 million MT from an area of

2.2 million Ha (Yield = 202213 Hg/Ha), where cassava contributes nearly 21% (~9.5

million tonnes) (FAOSTAT, 2009 data). Although cassava is cultivated in about 13

states of India, major production is from the southern states of Kerala and Tamil

Nadu. Table 1.3 summarizes the area, production and yield of cassava from major

cultivating States in India.

State

Area

(′000 Ha)

Production

(′000 T)

Yield (T/Ha)

Kerala

109.3

2563.5

23.454

Tamil Nadu 97.2 3425.5 35.242

Anthra Pradesh 18.1 111.5 6.16

Meghalaya

Assam

Karnataka

4

2.8

0.8

21.5

13.4

15.8

4.9

4.78

19.5

Table 1.3: Area, Production and Yield of Cassava in different States of India

The time of cultivation of cassava plant in the different states in India is presented in

Table 1.4. As a result of changing life-styles, the influx of money sent home by

Indians working in the Gulf States, and a shift to cultivation of cash crops like rubber

and plantation crops, the area under cassava in Kerala has gradually decreased over

the past 30 years.

Introduction

31

State

Rain fed

Irrigated

Kerala

April-May September-October

December-January

Tamil Nadu June March

Anthra Pradesh June March

Madhya Pradesh June September (higher yield)

Table 1.4: The time of cultivation of cassava plant in the different states in India Ref: Edison

There are a number of biological constraints to cassava yield improvement.

These include a low multiplication rate, bulky planting material required for

cultivation, rapid drying out of stakes and incidence of cassava mosaic disease (CMD)

and root rot. One of the most fascinating strategies to overcome the low multiplication

rate ant bulkiness of planting material is the true cassava seed program (TCSP). Work

on TCSP was initiated in India almost two decade ago, and has advantages such as a

150 times increase in propagation rate, longer viability of seed, and non-transmission

of mosaic virus through seed.

The shift in focus of cassava from a food to an industrial crop has led to a

change in the breeding strategy for cassava as well. For the industrial zones of

TamilNadu and Andhra Pradesh, importance is being given to develop high starch

varieties with CMD tolerance, early harvestability and better post-harvest storage life.

Management strategies to improve cassava production include agronomic

interventions, such as the development of low input and mycorrhizal technologies,

natural resource utilization and water management. As a management strategy for

Introduction

32

CMD, branching types are also preferred due to better canopy spread with

consequently lower yield reduction from the disease. Testing and popularization of

cassava varieties through outreach programs like the Lab to land Program (LLP),

Institution-Village Linkage Program (IVLP) and on farm trials are another approach

for enhancing cassava production.

Cassava Cultivation in Kerala:

Cassava, which was introduced into India by the Portuguese during the 16th

century as a food crop, is gradually changing its role as an industrial raw material. The

importance of cassava as a food crop was well recognized in Kerala, during the 20th

century, when famine struck India at the time of the Second World War. The crop

integrated well with the traditions and culture of the people of Kerala. Adaptability to

poor soils, an ability to establish in high as well as low rainfall areas, and relative

resistance to pests and diseases are a few of the factors that helped to anchor cassava

in India. Cassava is cultivated in India in about thirteen states with major production

in the south Indian states of Kerala (1, 42,000 Ha) and Tamil Nadu (65,700 Ha). It is

now a major industrial crop in Tamil Nadu and is also gaining importance in Andhra

Pradesh, where it serves mostly as raw material for starch extraction. The phenomenal

growth in the starch and sago trade over the years has also helped in creating rural

employment in Tamil Nadu.

It was reported that cassava is cultivated in all the agro-climatic zones of

Kerala. Farmers utilize all types of cropping systems namely as pure crop, intercrop,

mixed crop and homestead. The study conducted by Sasankan et al., also revealed

high productivity of cassava in low land system (Sasankan et al., 2008). Many cassava

cultivars exist in each locality where the crop is grown. The cultivars have been

Introduction

33

distinguished on the basis of morphology (e.g., leaf shape and size, plant height,

petiole color, etc), tuber shape, earliness of maturity, yield, and the content of

cyanogenic glucoside of the roots. This last-named characteristic has been used to

place cassava cultivars into two major groups; the bitter varieties, in which the

cyanogenic glucoside is distributed throughout the tuber and is at a high level, and the

sweet varieties, in which the glucoside is confined mainly to the peel and is at a low

level.

The flesh of the sweet varieties is therefore relatively free of the glucoside,

although it still contains some (Purseglove, 1968). In general, sweet cassavas tend to

have a short growing season; their tubers mature in 6-9 months, and deteriorate

rapidly if not harvested soon after maturity. The bitter cassava, on the other hand,

require 12-18 months to mature, and will not deteriorate seriously if left unharvested

for several months thereafter. The exact level of glucoside in a particular cultivar will

vary according to the environmental conditions under which the plant is grown. An

introduction from Malaya, called M4 is a popular variety used for culinary purpose in

Southern Kerala.

Need for Cassava Improvement:

The current efforts on cassava improvement mainly focus on varieties resistant to

pests and diseases particularly mosaic and bacterial blight diseases, high yielding,

producing good quality tubers with high starch and protein content, contain low levels

of prussic acid, early maturing varieties, long storage period, minimum of foliage and

adapted to a wide range of environmental conditions, etc.

Introduction

34

The introduction of cultivars from other localities or countries has been one of the

major methods of cassava improvement. Hybridization, followed by selection among

the progeny, is another useful method for improving cassava.

Creation of new genetic diversity:

The first significant large-scale creation of new diversity occurred through controlled

crossing carried out by plant breeders. Hybridization in cassava is relatively easy

(Kawano, 1980) and open pollination schemes are extensively used to increase the

amount of hybrid seed produced. In some regions the principal constraint to crossing

is shy flowering and to date practical methods have not been developed to deal with

non-flowering types. Flowering is controlled by the complex interaction of a range of

genetic and environmental factors. In some areas cassava will flower all year long

while in other locations flowering is seasonal. Cassava is monoecious, with pistillate

flowers opening about two weeks before staminate flowers. Normally, few seeds are

obtained (an average of I to 1.5) and acquiring large numbers of seeds is a labour

intensive and tedious process. Most genotypes appear to suffer drastic inbreeding

depression. Vegetative propagation to preserve superior heterozygotes greatly

simplifies breeding and, whichever breeding method is used, heterozygocity needs

either to be maintained or restored prior to subsequent vegetative propagation. Most

programmes use some form of recurrent selection appropriate for the accumulation of

many genes of minor effect. All existing commercial cultivars of cassava are probably

highly heterozygous.Although difficulties occur at the time of flowering, IITA has

performed extensive intercrossing among wild species and between cassava and wild

species. M. glaziovii has been used as a source of African mosaic-virus resistance in

early East African breeding programmes (Nichols, 1947).

Introduction

35

Mutation has been tried sparingly for cassava. One constraint is the need for

selfing to achieve expression of recessive mutations occurring in the heterozygous

state. This is very difficult for a large number of genotypes due to the asynchronous

opening of staminate and pistillate flowers. Mutation could have more practical

applications when haploid cells, such as microspores, can be regenerated into

plantlets. Haploids and doubled haploids will be a significant research tool for

cassava, with possible applications in genetics, evolution studies, expression of

recessive genes and in a breeding system for true cassava seed.

Polyploid induction, through colchicine-induced tetraploids, has been a subject

of considerable research in India (Graner, 1941; Magoon et al., 1969). The clones

produced generally exhibited the gigas characters associated with polyploidy, such as

increases in leaf breadth and thickness, stomata size, length and girth of petiole and

flower size. Pollen sterility was high, but fertile pollen grains were much larger in size

(180 to 196 microns) compared to diploids (125 to 140 microns). Considerable

genotypic variation was demonstrated in response to polyploidy. Some clones became

weak and could not be maintained, while others were maintained easily for several

generations. Improved yield potential from polyploidy has not been found to be

promising in any of the programmes. In India it was reported that a 42 percent

increase in protein content was achieved by polyploid induction which subsequently

disappeared with continued vegetative propagation.

At IITA work focused on changes in ploidy through inducing production of

unreduced gametes, mainly through interspecific crosses. Four spontaneous

tetraploids and two triploids were isolated from crosses between M. pruinosa or M.

glaziovii and cassava. A majority of the interspecific crosses produced diploid pollen,

Introduction

36

but their frequencies varied with cross-combinations and with genotype within

respective cross combinations (Han et al., 1990). The presence of multivalent in the

polyploids suggests that pairing and crossing over are taking place between cassava

and its related Manihot species. There are no strong incompatibility barriers to

interspecific hybridization, though considerable selection of parent cassava clones is

necessary, which could indicate a highly fluid gene pool within the genus Manihot

and likely widespread introgression among species.

Virtually no somaclonal variation has been detected from extensive studies on

plants propagated from meristem culture or from somatic embryos (Szabados et al.,

1987). Since either of these processes result in considerable variation in many species,

this result suggests that cassava is genetically very stable at these levels of tissue

organization. Regeneration of cassava from individual cells is not possible at present

and it is not known whether genetic stability would be similar for regenerated single

cells.

Genetic markers of cassava:

Genetic markers have become fundamental tools for understanding the inheritance

and diversity of natural selection. They provide Gregor Mendel, the father of modern-

day genetics, the tools for his ground breaking experiment on heredity and more

recently markers have made possible the construction of genetic maps, the cloning of

genes known only by their phenotypes and position on a genetic map, and whole

genome sequencing. The earliest genetic markers in cassava are morphological

(Sumarani et al., 20004), and these markers are governed by alleles with a major

phenotypic effect with little or no environmental effects (Harshey and Ocampo,

1989). The second generations of markers were biochemical, such as Isozymes and

Introduction

37

they provided a useful tool for genetic fingerprinting and the study of genetic diversity

in cassava (Hussain et al., 1987; Ramirez et al., 1987; Ocampo et al., 1992; Lefevre

and Charrier, 1993a, Pillai et al., 2000). Isozymes have been applied to characterizing

relationship among accessions of African cassava germplasm (Lefevre and Charrier,

1993b; Wanyera et al., 1994) and finger printing of the international cassava

collection held at CIAT (Ocampo et al., 1992). The alpha beta esterase system was

found to be most informative, providing 22 alleles, which have complmented

morphological description for the identification of duplicates in the collection at CIAT

(Ocampo et al., 1995).

With the discovery of the molecular basis of natural variation, molecular or

DNA markers have rapidly gained importance in the study of genes, genomes and

genetic diversity. They represented a limitless source of neutral markers for the

quantitative assessment of genetic diversity and “signposts” in gene and genome

mapping. Their abundance in any organism facilitates the resolution of genetic

relationship and genome/gene mapping unknown until the coming of molecular

markers. Cassava genetic improvement could be made more efficient through the use

of easily assayable molecular genetic markers or DNA markers that enable the precise

identification of genotype without the confounding effect of the environment, thereby

increasing heritability. The selection of progenies based on genetic values derived

from molecular marker data substantially increases the rate of genetic gain, especially

if the number of cycles of evaluation or generations can be reduced. Another

application of DNA markers in cassava breeding is reducing the length of time

required for the introgression of traits from wild relatives (Melchinger, 1994),

The prominent molecular marker systems include minisatellites, restriction

fragment length polymorphisms (RFLP) (Botstein et al., 1980) and randomly

Introduction

38

amplified polymorphic DNAs (RAPD) (Williams et al., 1990). Others are

microsatellites, also known as simple sequence repeat markers (SSR) (Litt and Lutty,

1989) amplified fragment length polymorphisms (AFLP) (Vos et al., 1995) and DNA

sequencing of the internal transcribed spacer (ITS) of ribosomal DNA (Baldwin,

1992) and single-copy nuclear genes. More recently DNA chip or oligonucleotide

arrays have been added to an ever growing list of molecular markers (Fodor et al.,

1993). All these marker systems, other than DNA chip have been used in cassava to

assess genetic diversity, genome mapping and gene tagging.

Microsatellites or Simple Sequence Repeat (SSR):

Among the different molecular markers, Microsatellites, or Simple Sequence Repeats

(SSRs) (Tautz and Rentz, 1984: Tautz, 1989), are the most widely applied class in

genetic studies Chavarriaga-Aguirre et al., 1999; Olsen et al. 2001). SSR can be

defined as any one of a series of very short (2-10 bp), middle repetitive, tandemly

arranged, highly variable DNA sequences dispersed throughout fungal, plant, animal

and human genomes. These repeat motifs are flanked by conserved nucleotide

sequences from which forward and reverse primers can be designed to PCR-amplify

the DNA section containing the SSR. The microsatellite alleles, amplified products of

variable length, can be separated through gel electrophoresis and visualized by

Ethidium bromide staining. SSRs are abundant in plants, occurring on average every

6-7 kb (Cardle et al., 2000). They have high level of polymorphism, and are adaptable

to automation (Donini et al., 1998). SSRs are user friendly, highly polymorphic

markers suitable for purity control (Nandakumar et al., 2004), differentiation of plant

cultivars (Bredemeijer et al., 1998: Struss and Plieske 1998), elimination of duplicates

(Lund et al. 2003) and selection of appropriate parents based on genetic distance in

Introduction

39

breeding programmes (Zhou et al., 2006). When first introduced, development of SSR

markers was expensive but now new and efficient methods of repetitive sequence

isolation have been identified which had led to reduced costs and microsatellite-

technique has been increasingly applied in genetic diversity studies.

The present study exclusively uses SSR markers for the DNA analysis and

finger printing for variability and diversity study of Indian cassava cultivars.

A more exhaustive description of molecular marker based genetic studies

particularly in cassava is described in the following chapter-Review of literature.

Introduction

40

Objectives of the present study:

The objectives of the present study are –

1. To study the molecular variability and diversity available in local varieties of

cassava (Manihot esculenta Crantz) and identification of distant varieties for

planning hybridization program.

2. DNA fingerprinting of released varieties of cassava, with a view to protect the

plant breeder’s right. DNA finger printing of popular, local varieties selected

by farmers with a view to protect the Farmer’s Right.

3. DNA analysis of Cassava Mosaic free accessions and also whitefly resistant

accessions to confirm their resistance. Screening apparently resistant of

hybrid populations to confirm actual resistance and escapes.

Introduction

41

Outline of the Thesis:

The present study on “DNA analysis as a tool for Genetic Studies and Molecular

Breeding in Cassava (Manihot esculenta Crantz)” as outlined in this thesis is

presented in seven chapters. The first chapter is a general introduction that give an

over view on Cassava (Manihot esculenta Cranz). The second chapter, Review of

literature gave an elaborate description on genetic markers especially molecular

markers with emphasis on SSR marker studies in Cassava. The third chapter describes

the genetic variability in cassava from a typical cassava growing high land area-

Idukki in Kerala using SSR markers. The fourth chapter presents variability of

cassava among subsamples from old and new collections from CTCRI germplasm

collection using SSR markers. Fifth chapter deals the genetic variability available

among selected cultivars from North Kerala varieties using SSR markers. The sixth

chapter describes genetic variability among CTCRI released varieties and central

Kerala varieties through DNA finger printing by SSR markers. The seventh chapter

deals with application of SSR markers for identifying CMD and whitefly resistant

varieties of cassava. The major observations and a consolidated summary are

provided at the end.

Introduction

42

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Introduction

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