1 Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/6307/6/06...Review of Literature 1.1 Medicinal plants and their importance Medicinal plants are a precious

Review of Literature

1.1 Medicinal plants and their importance

Medicinal plants are a precious natural resource, as they provide raw material

for pharmaceutical industry, modern and traditional forms of medicine and

generate employment and income in addition to conservation of bio-diversity

and traditional knowledge (Balick and Mendelson 1992, Lambert et al. 1997).

The significance of medicinal plants is highlighted by the fact that world wide

more than 50, 000 plant species, covering about 13% of all flowering plants, are

estimated to be medicinally important (Schipmann et al. 2002). In India, over

8,000 plant species are used for traditional and modern systems of medicine

(Anonymous 2000). Their relevance is further accentuated by the fact that

traditional medicine is important for the poor and marginalized sections of the

society, especially in the remote areas where modern medical facilities are not

available (Belt et al. 2003). This is validated by case study of India where per

capita annual consumption of drugs is Rs. 124 only ( US $ 3), which happens to

be the lowest in the world. It has been estimated that in developing nations

approximately 80 percent of the people totally depend on traditional medicines

(Holley and Cherla 1998, Anonymous 2000).

Moreover, interest in herbal medicines and natural products has increased

substantially in developed as well as developing nations. This scenario is well

reflected by the available trade figures. The international medicinal plant trade,

estimated at US $ 60 billion, is presently growing at a rate of 7 percent per year

(Belt et al. 2003). This is besides the fact that a substantial part of the trade goes

unrecorded, partly because much of it is illegal. Further, a large proportion of

medicinal plant material is used domestically (India) and these figures are again

not documented, hence the total trade of medicinal plants remain far greater

than the suggested figures.

The leading suppliers of the world market are China, Singapore, Brazil, India

and Egypt. China and India constitute more than 40 percent of the global

biodiversity. China earns US $ 5 billion per year from herbal trade, while India

manages US $ 1.43 billion only, which is estimated to increase by US $ 2.2

billion by the year 2010 (Anonymous 2000). The international demand is largely

1

Preface

TERI University-Ph.D. Thesis, 2006

from the United States and European Union (EU), with Germany as the leading

trade centre (Belt et al. 2003).

1.1.1 Employment and income generation

Bearing in mind the importance of medicinal plants and their trade estimates

and the ironical fact that most of the raw material is collected from wild,

medicinal plant cultivation and management has become highly remunerative in

economic terms for the small-scale growers. This is illustrated by a case study on

Atis (Aconitum species) cultivation by a local NGO called Society for Himalayan

Environmental Research (SHER) to demonstrate Medicinal and Aromatic Plants

(MAPs) cultivation and conservation to local communities in Uttarkashi district

of Uttaranchal, India (Karki et al. 2003). The results revealed that as compared

to traditional crop of potato, which yields a net profit of $ 200/ha, the net profits

from cultivation of Aconitum atrox were around $1600/ha and $ 6000/ha from

Aconitum heterophyllum cultivation.

In a similar study, conducted in Ghese village of Chamoli district,

Uttaranchal, India, cultivation of a highly prioritised medicinal plant, Picrorhiza

kurrooa (commonly called kutki) was conducted on an experimental basis

(Nautiyal and Nautiyal 2004). According to the calculations, the net benefit to

the farmer that comes from conventional crops such as, potato and rajma

(Phaseolus vulgaris) is a mere US $280 and US $118 per acre, respectively. On

the contrary, kutki cultivation would yield a net profit of US $ 1961. These

studies highlight the fact that sustainable management of the traditionally used

plants not only has implications in conservation of biodiversity but also provide

critical resources to sustain livelihoods.

Besides cultivation, proper value addition and subsistence oriented

application of medicinal and aromatic plant (MAP) can create a large number of

jobs in rural areas. Conversion of socio-cultural traditions and indigenous

knowledge into livelihood means and opportunities would also help in

conserving the traditional knowledge base. One prominent example is the

development of “Jeevani” medicine based on the traditional knowledge of the

Kani tribals of Kerala. The tribals used fruits of a tree that they called

“aarogyapyappacha” which kept them energetic and agile during their arduous

treks across the forests. The detailed investigation of the plant revealed that it

contained certain glycolipids and non-steroidal compounds that had anti-stress,

anti-hepatotoxic and immuno-modulatory / immuno-restorative properties.

Eventually, the drug “Jeevani” was formulated from this plant, which was later,

identified as Trichopus zeylanicus, with three other plants’ ingredients. The

Preface


license to manufacture “Jeevani” was given to a private company for a fee of one

million rupees ( US $ 22000) for a period of seven years. The Kani tribals

received fifty percent of the licence fee as well as fifty percent of the royalty

obtained from sale of the drug.

1.1.2 Traditional knowledge

Seen from a global perspective, India has one of the richest traditions of using

medicinal plants in health care (Lambert et al. 1997, Anonymous 2000).

However, most of the traditional and indigenous knowledge associated with the

rich natural resource of the country remains undocumented and is passed down

either by word of mouth from generation to generation or is described in ancient

classical literature, which remains inaccessible to common man. Though our

traditional health systems such as, Ayurveda, Siddha and Unani are very well

documented, unfortunately, the language in which they are coded (Sanskrit,

Tamil and Persian) are not well understood internationally.

The need for protecting Indian traditional knowledge (ITK) has been

vehemently brought forth by many well-known cases of misappropriation of

traditional knowledge that includes the patents granted for medicinal properties

of brinjal (Solanum melongena) bitter gourd (Momordia charantia), neem

(Azadirachta indica) and turmeric (Curcuma longa), to name a few. Therefore,

documentation of existing knowledge on various traditional systems of medicine

that is already available in the public domain has become imperative in order to

safeguard it from patenting of non-original inventions. Further, protecting and

promoting traditional and indigenous knowledge and its documentation will

also help to accrue benefits to the creators, innovators and holders of traditional

knowledge.

With this perspective, National Institute of Science Communication and

Information Resources (NISCAIR) and the Department of Indian Systems of

Medicine and Homoeopathy (ISM&H) have come up with a novel concept of

traditional knowledge digital library (TKDL), in which all information for a

target species is available in world languages such as, English, French, German,

Japanese and Spanish (www.niscair.res.in). This attempt at digitalisation of ITK

gives legitimacy to the existing traditional knowledge and enables protection of

such information from getting patented illegally.

1.1.3 Conservation of biodiversity

Despite the immense importance of medicinal plants, the raw material is still

not cultivated and about 95% of the medicinal plants are collected from the wild.

http://www.niscair.res.in/

Preface


Over 70% of the plant collection involves destructive harvesting mainly because

of the use of plant parts such as, roots, bark, wood and whole plants. According

to an estimate, the various plant parts used by the Ayurvedic industry in India

are as follows: bark 13.5%, roots 29.6%, rhizome 4%, stem 6%, whole plant

16.3%, wood 2.8% and the rest being contributed by flowers, fruits, leaves and

seeds (Anonymous 2000). Factors such as, urbanisation and habitat destruction

have further endangered many of the medicinal plants.

The IUCN Red list of threatened plants published by the World Conservation

Union includes 33,798 species, of which 380 are extinct in the wild, 371 may be

extinct, 6,522 are endangered and the rest are vulnerable or rare (Pye-smith

1998). As per recent IUCN guidelines southern and northern India has around

200 species of medicinal plants that are rare, endangered and threatened

(Shankar 1998). This could mean a tremendous loss to the world of modern

allopathic medicine as well as traditional system of Ayurveda, Unani and Siddha,

which the human race surely cannot afford.

Considering the threat of extinction that looms large on this important

natural resource, germplasm conservation of medicinal plant species has

become necessary for preventing genetic erosion. According to International

Board for Plant Genetic Resources (IBPGR) medicinal plant species figure in the

priority list for in vitro conservation (Staritsky 1997).

Biotechnology has provided innovative tools for cataloguing,

documentation and conservation of medicinal plants. The first and most logical

step towards conservation and sustainable management of this important

natural resource is systematic documentation. This entails assessment of genetic

diversity available among the medicinal plant species and molecular markers

have been effectively employed to realise the above objective. Section 1.3

discusses the most commonly used molecular markers and their relevance in

genetic diversity analysis. Besides aiding in proper documentation, bio-diversity

studies also help in devising pragmatic conservation strategies. Further, the

knowledge of the available genetic diversity of a particular species is essential for

genetic improvement of the crop as it can help in selection and introduction of

desirable traits in the crop of interest.

Plant tissue culture plays relevant role in ex situ conservation and

multiplication of endangered and commercially exploited medicinal plants. The

novel technique provides a viable alternative for managing these valuable

resources in a sustainable manner. It also serves as an alternative means of

secondary metabolite production through cell and organ culture. Most

importantly, micropropagation provides an efficient method for ex situ

Preface


conservation of plant biodiversity and multiplication of the endangered species

from minimum plant material available as detailed in section 1.4.

With these deliberations, Swertia chirayita, a medicinally important herb

from temperate Himalayas was chosen for the present research work. Section 1.2

gives detailed description of the herb along with its medicinal and economic

importance. The concerns raised due to its ruthless collection from the wild have

also been discussed.

1.2 Description of Swertia chirayita

Swertia chirayita belongs to the genus Swertia Linnaeus (1753), a large group

of annual and perennial herbs, representing approximately 135 species, which

are distributed in the mountainous tropical regions of Africa (30 spp), America

(1 sp.), Asia (100 sp.), Europe (3 spp.), and Madagascar (1 sp). In India, 40

species of Swertia are recorded of which Swertia chirayita is considered to be

the most important.

1.2.1 Geographical distribution, habitat, ecology and classification

The plant is a native of temperate Himalayas, found at an altitude of 1,200-

3,000 m (4000 to 10,000 ft.), from Kashmir to Bhutan, and in the Khasi hills at

1,200-1,500 m (4,000 to 5,000 ft., Clarke 1885, Kirtikar and Basu 1984). Figure

1.1 highlights the natural habitat of the species across the trans-Himalayan belt

on the map of India. S. chirayita can also be grown in sub-temperate regions

between 1,500 to 2,100 m altitudes. Unfortunately, there is no consistency in the

literature citing the habit of S. chirayita. Some authors have described chiretta

as an annual (Anonymous 1982, Kirtikar and Basu 1984) while others have

described it as biennial or pluri-annual (Edwards 1993). The plant can be grown

in variety of soils with sandy loam rich in carbon and humus. It is also found in

open ground and recently slash-and-burnt forests (Edwards 1993). S. chirayita

is also reported to grow under the canopy of Acer and Quercus mixed forests,

mostly on a southeast aspect (Bhatt et al. 2006).

The taxonomic position of the plant is as follows

Order: Gentianales

Family: Gentianaceae

Tribe: Gentianeae

Subtribe: Swertiinae

Genus: Swertia

Species: chirayita

Preface


Swertia chirayita (Roxb. ex Fleming) H. Karst., (trade name chiretta) is also

mentioned in the literature as Swertia chirata, Buch. - Ham.; Ophelia chirata

Grisebach.; Agathotes chirayita Don.; Gentiana chirayita Roxburgh. and G.

floribunda Don. The genus Swertia is named in honour of Emanuel Sweert

(rarely spelled Swert, 1552–1612), a Dutch gardener and illustrator who

published an important florilegium in the early 1600s. On the other hand, the

plant got its specific name from the “kirat” tribals and is variously called as

“chirata” or “chirayita”. The plant has an erect, about 2-3 feet long stem, the

middle portion is round, the upper 4-angled, with a prominent decurrent line at

each angle (Figure 1.2). The stems are orange brown (Anonymous 1982) or

purplish in colour (Clarke 1885), and contain large continuous yellowish pith.

The root is simple, tapering and stout, short, almost 7 cm long and usually half

an inch thick (Bentley and Trimen 1880, Clarke 1885, Figure 1.2). The

cytological work done on the species is poor. Khoshoo and Tandon (1963) used

pollen-mother cells for cytological studies in some Himalayan species of

Swertia. The authors counted thirteen bivalents at metaphase I for S. chirayita,

and observed that one of them was bigger than the rest.

The plant is harvested for the drug industry when it sets into flowering in

July-Sept (Bentley and Trimen 1880, Anonymous 1982). Seed setting commence

around October-November and the seeds germinate immediately after shedding.

1.2.2 Medicinal uses of Swertia chirayita

S. chirayita belongs to family Gentianaceae, which records the occurrence of

taxonomically informative molecules, namely the iridoids, xanthones,

mangiferin and C-glucoflavones. The widespread use of this herb in the

traditional medicines have resulted in considerable chemical analysis of the

plant (Table 1.1) and the active principles that confer the plant its medicinal

properties have been identified and isolated (Table 1.2). Detailed reviews

describing the chemical constituents of the Swertia genus have been reported by

Pant et al. (2000) and Jensen and Schripsema (2002). The entire plant is used

medicinally; however the root is reported to be the most powerful (Kirtikar and

Basu 1984). The plant is gathered during the late stages of flowering, commonly

tied up in flattish bundles about 3 feet long and 1.5 to 2 lbs. in weight (Bentley

and Trimen 1880) and is sold in the markets as dried brownish stems with root

and leaves intact.

S. chirayita is used in British and American pharmacopoeias as tinctures and

infusions. According to Ayurveda’s pharmacology (Joshi 2000) chiretta is

described as bitter in taste (rasa). The thermal action (virya) of chiretta is

Figure 1.1 Natural distribution of Swertia chirayita (based on Clarke 1885, Kirtikar and Basu 1984). Plant material for the present study was collected from Chakrata 30o42’N, 77o51’E; Mandal 30o.4’N, 790.1’E; Jageshwar 290.38’N, 79o 40’E; Joshimath 30o34’N 79o34’E; Sikkim 27o 20’N, 880.3’E, also marked on the map. The shaded area represents the habitat of chiretta in the Indian Himalayan Region.

Figure 1.2 Pictorial representation of Swertia chirayita plant representing A) shoot with root B) A flowering twig a) a flowering bud b) flower cut open showing the two nectaries per corolla lobe c) ovary d) longitudinal section of the ovary e) a fertilized ovary at seed set f) the seeds (Bentley and Trimen 1880).

A

B

a

b c d

e

f

CHIRATA, HAM

defined as cooling (shita). It is light (laghu), i.e. easily digestible, and ruksha

(dry). These characteristics drain heat from the blood and liver. Its use has also

been mentioned in Unani medicine (Mukherji 1953). Concoction of chiretta with

cardamom (Elletaria cardamom), turmeric (Curcuma longa) and kutki

(Picrorhiza kurrooa) is given for gastrointestinal infections and with ginger

(Zingiber officinale) it is considered good for fever (Kirtikar and Basu 1984).

When given along with neem (Azadirachta), manjishta (Rubia cordifolia) and

gotu kola (Centella asiatica) it serves as a cure for various skin problems. It is

used in combination with other drugs in cases of scorpion bite (Nandkarni

1976).

Table 1.1 Secondary metabolites of Swertia chirayita

Compounds Chemical

nature

Reference

1,3,5,8-Tetrahydroxyxanthone Xanthone Ghosal et al. 1973

1,3,7,8-tetrahydroxyxanthone Xanthone Ghosal et al. 1973

1,3,8-trihydroxy-5-methoxyxanthone Xanthone Ghosal et al. 1973

1,5,8-trihydroxy-3-methoxyxanthone Xanthone Ghosal et al. 1973

1,8-Dihydroxy-3, 5-

dimethoxyxanthone/Swerchirin

Xanthone Dalal and Shah 1956, Ghosal et

al. 1973, Asthana et al. 1991

1,8-dihydroxy-3,7-dimethoxyxanthone /

7-O-methylswertanin

Xanthone Ghosal et al. 1973, Asthana et al.

1991

1-Hydroxy-3, 5,8-trimethoxyxanthone Xanthone Ghosal et al. 1973

1-Hydroxy-3, 7,8-trimethoxyxanthone Xanthone Ghosal et al. 1973

2,5-Dihydroxyterephthalic acid Aromatic

carboxylic acid

21- -H-hop-22(29)-en-3- –ol Triterpenoid Chakravarty et al. 1991

Amarogentin Seco-iridoid

glycoside

Korte 1955, Chakravarty et al.

1994

Amaroswerin Seco-iridoid

glycoside

Chakravarty et al. 1994

Chiratanin Dimeric

xanthone

Mandal and Chaterjee 1987

Chiratenol Hopane

triterpenoid

Chakravarty et al. 1992a,

Chakravarty et al. 1992b

Chiratol/ 1,5 dihydroxy 3,8-

dimethoxyxanthone

Xanthone Purushothaman et al. 1973a,

Purushothaman et al.1973b,

Asthana et al. 1991

Decussatin Xanthone Purushothaman et al. 1973a,

Purushothaman et al.1973b,

Asthana et al. 1991

Enicoflavine Triterpenoid

alkaloid

Purushothaman et al. 1973a,

Sharma 1982, Asthana et al.


Compounds Chemical

nature

Reference

1991

Episwertenol Triterpenoid Chakravarty et al. 1991

Erythrodiol Hexane extract Chakravarty et al. 1992a, Islam

et al. 1995

Gammacer-16-en- –ol Triterpenoid Chaudhuri and Daniewshi 1996

Gentianine Triterpenoid

alkaloid

Sharma 1982

Gentiocrucine Triterpenoid

alkaloid

Sharma 1982

Kairatenol Hexane extract Purushothaman et al. 1973a

Lupeol Triterepene

alcohol

Mandal et al. 1992

Mangiferin Xanthone Dalal and Shah 1956

Mangostin Xanthone Sharma 1983

Oleanolic acid Triterpenoid Mandal and Chaterjee 1987,

Duke et al. 2002

Pichierenol Swertane

terpenoid

Mandal et al. 1992

Sweroside Seco-iridoid

glycoside


Sweroside-2‟-O-3‟‟, 5‟‟-trihydroxy

biphenyl-2‟‟carboxylic acid ester

Seco-iridoid

glycoside


Swerta-7,9(11)-dien-3- -ol Swertane

terpenoid

Mandal et al. 1992

Swertanone Triterpenoid Korte 1955

Swertenol Triterpenoid Korte 1955

Swertianin/ 1,7,8-trihydroxy-3-

methoxyxanthone

Xanthone Sharma 1982, Islam et al. 1995

Taraxerol Triterpene

alcohol

Korte 1955

Ursoilic acid Triterpenoid Korte 1955

-Amyrin Triterpenoid

alcohol

Duke et al. 2002

-Sitosterol-3- -D-glucoside Sterol Mandal and Chaterjee 1987

-Taraxasterol or Heterolupeol Hexane extract Islam et al. 1995

Compiled in Joshi and Dhawan (2005)


Table 1. 2 The biological activities attributed to Swertia chirayita Activities Reference

Alternative Ray et al. 1996, Duke 2002

Antihelmintic Medda et al. 1999

Antileishmaniac Rafatullah et al. 1993

Anticholinergic Bhattacharya et al. 1976

Anticonvulsant Blaschek et al. 1998

Antiedemic Kumar et al. 2002

Anti-inflammatory Goyal et al. 1981, Chakravarty et al.

1992a, Banerjee et al. 2000

Antimalarial Goyal et al. 1981

Antipyretic Blaschek et al. 1998

Antitubercular Mukherjee et al. 1997

Astringent Nandkarni 1976

Bitter Asthana et al. 1991

Cardio stimulant Blaschek et al. 1998

Cholagogue Kirtikar and Basu 1984

Choleretic Blaschek et al. 1998

CNS Depressant Dalal and Shah 1956

Emollient Sharma 1983, Duke 2002,

Hepatoprotective Mukherjee et al. 1997

Hypnotic Ray et al. 1996, Duke 2002,

Hypoglycemic/antidiabetic Mukherjee and Mukherjee 1978,

Chadrashekar et al. 1990, Bajpai et

al. 1991, Saxena and Mukherjee 1992,

Saxena et al. 1993, Grover et al. 2002

Laxative Ray et al. 1996, Duke 2002,

Secretagogue Kirtikar and Basu 1984

Stomachic Nandkarni 1976

Tonic Nandkarni 1976

Undersedative Kirtikar and Basu 1984

Vermifuge Kirtikar and Basu 1984

Compiled in Joshi and Dhawan (2005)


1.2.3 Trade and economics of Swertia chirayita

Swertia chirayita enjoys a good domestic and international market. The

medicinal plant sector in India is unorganised and it is difficult to get a regular

update of statistics vis-à-vis the demand and supply, collection and economics of

chiretta. The only available data regarding collection and trade of the plant is

for the year 1990-1991 (HMG, Nepal 1992) with respect to Nepal and for the

year 2001-2002 for India (Shah 1999, Brinckmann 2003). The plant has a huge

demand in the international medicinal market and makes important

contribution to the economy of Nepal. About 45% of chiretta in the Himalayan

region is collected from Nepal (Shah 1999).

The trade and economics of chiretta is also influenced by the adulterants of

the herb. The true chiretta can be distinguished from other substitutes and

adulterants by its intense bitterness, brownish purple stem (dark colour),

continuous yellowish pith and petals with double nectaries.

1.2.4 The concerns

The widespread use of S. chirayita in the traditional systems of medicine reflects

the herb‟s pharmacological importance. However, the existing populations are

diminishing by each passing day and therefore, according to the International

Union for Conservation of Nature and Natural resources (IUCN) criteria,

Swertia chirayita has been categorised as critically endangered (Anonymous

1997). This leads to a need for conservation of the plant. National Medicinal

Plant Board, (Government of India) prioritised it for conservation and

cultivation in Uttaranchal state (http://www.nmbp.nic.in) emphasising the need

to develop agro-technology packages for the herb. The novel technique of in

vitro conservation and micropropagation can help in conservation and

production of a large number of disease-free, true to type plants. Despite these

concerns very few in vitro studies (compiled as Table 1.3) have been conducted

on the species of Swertia genus.

Table 1.3 In vitro culture studies in family Gentianaceae: tissue culture and secondary metabolite production

Species Name Tissue culture Reference

Swertia japonica Callus cultures from seedling Miura et al. 1978a

Swertia japonica Callus cultures Miura et al. 1978b

Swertia japonica Cell suspension culture Miura et al. 1986

Swertia pseudochinensis Excised root culture from

seedling

Kitamura et al. 1987

http://www.nmbp.nic.in/


Species Name Tissue culture Reference

Swertia pseudochinensis Callus and excised shoot

cultures, whole plant via

embryogenesis from callus;

production of swertiamarin

from cultured tissues

Kitamura et al. 1988

Swertia pseudochinensis Shoot and root from callus Miura and Kitamura in Miura

(1991)

Swertia chirata Regeneration from root

explants

Wawrosch et al. 1999

Swertia chirata Micropropagation from

mature nodal explants

Ahuja et al. 2003

Swertia chirata Hairy root cultures for

amarogentin production

Keil et al. 2000

Swertia japonica Hairy root cultures for

amarogentin, amoroswerin

and four xanthones

Ishimaru et al. 1990

1.3 Bio-diversity studies in medicinal plants

Biological diversity is a measure of relative „diversity‟ among organisms present

in different ecosystems. Thus the word diversity includes diversity within species

(genetic diversity), among species (species diversity) and comparative diversity

among ecosystems (ecological diversity). A measure of genetic diversity is of

direct consequence in defining the taxonomic grouping of the species, its origin

and evolution; for breeding of superior varieties and most importantly in

conservation of the endangered ones. Genetic diversity is the diversity of genes

within species and results from genetic differences between individuals and is

manifested as a change in DNA sequences, biochemical characteristics and

physiological properties as well as morphological characteristics.

The variations that form the base line of genetic diversity arise from

mutations and recombination events. Various factors such as selection (both

natural and artificial), genetic drift and gene flow act on alleles in different

populations to cause variations in them, thus resulting in diversity. Also, eco-

geographical factors namely the climatic, edaphic and biotic factors, species-

specific factors such as, ploidy level, breeding systems, population size, all have a

definite role to play in determining the diversity available within a species.

Based on biochemical, physiological and morphological characteristics,

conventional tools such as, protein based assay and morphological markers have

been employed for studying genetic variability. However, these characteristics

are merely the phenotypic expression of the genes and are under the control of

environmental effects and developmental stage of the plant. DNA based


molecular markers on the other hand, rely on direct appraisal of the genome and

are therefore neutral to environmental effects and developmental stages. Also,

DNA based markers provide a wide array of molecular assays for evaluation of

genetic diversity and hence are preferred over the classical markers.

1.3.1 Molecular analysis of genetic diversity

The plant genome consists of DNA, the sequence of which is unique for each

genotype. Based on the sequence variation that exists between different species

of the same genus, powerful molecular markers have been developed. In this

section an attempt has been made to describe the most frequently used

molecular assays for the purpose of genetic diversity studies. These include

Restriction Fragment length Polymorphism (RFLP), Amplified Fragment Length

Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD), Simple

Sequence Repeats (SSR) and Inter Simple Sequence Repeats (ISSR) molecular

markers.

1.3.1.1 Restriction Fragment Length Polymorphism (RFLP)

RFLP (Botstein et al. 1980) is a hybridization based molecular marker and

makes use of RFLP probes, which are hybridized to the specific restricted and

electrophoresed DNA samples, resulting in unique hybridization patterns

characteristic to each genotype. The molecular basis of the polymorphism so

detected is attributed to variation in restriction enzyme target site arising from

point mutation or insertion and deletions as summarized below:

Point mutation resulting in loss or gain of restriction enzyme cut site

An insertion or deletion of the DNA between two restrictions sites

A DNA rearrangement where one end of the rearranged segment resides

between two restrictions sites

In RFLP assay, the probes, which are either obtained from cDNA or genomic

library, are species or locus specific and co-dominant in nature. Restriction

fragment length polymorphism helps to screen for multiple alleles for a specific

locus and therefore reveals greater level of heterozygosity and higher

information content. It is highly reproducible and reliable. However, certain

drawbacks of the technique limit its use as a popular tool to assess genetic

diversity of a species. The major drawback is that it analyses very few loci per

assay. Moreover, the technique is expensive, labour intensive and requires the

use of radioactive probes making the procedure technically cumbersome. Also,

RFLP requires DNA in large amounts and purity of the sample is an important

consideration.


With the discovery and introduction of Polymerase Chain Reaction (PCR,

Mullis et al. 1986), a variety of PCR based molecular markers became the

preferred tool over the hybridisation-based markers. This is because the PCR

based molecular markers are technically simple, cost effective and reasonably

sensitive. Further, it can be optimised for automation and multiplexing thus

making large scale fingerprinting a feasible proposition. The most commonly

employed PCR-based molecular markers for fingerprinting studies are described

below.

1.3.1.2 Random Amplified Polymorphic DNA (RAPD)

Williams et al. (1990) invented Random Amplified Polymorphic DNA or RAPD.

This employs the use of standard PCR method. However, only one primer with

an arbitrary nucleotide sequence is used. The primer is usually 10-nucleotide

long and the GC content is almost 50%. To obtain good amplification with a

single arbitrary primer there must be two identical target sequences close to

each other. The distance between the two sites should be within amplifiable

units of 4-5 Kb. The two sites should be present on the two opposite strands in

inverse orientation. The polymorphism obtained by the RAPD primers can

result due to the following reasons:

Insertion of DNA fragment between the two annealing sites, because of

which the original fragment, becomes too large to be amplified

Deletion of DNA fragments carrying the primer annealing sites

Nucleotide substitution, which may affect the annealing of the primers at a

given site due to change in homology, which in turn results in presence or

absence of the fragment or a change in size of the amplified product

Insertion or deletion of small piece of DNA, which may result in change in

size of the amplified product

However, the reproducibility of the RAPD profile is subject to discussion.

There is debate over the genetic basis, reliability and hence usefulness of the

technique (Devos and Gale 1992). It has been reported that even minor changes

in any aspect of PCR amplification reaction can affect the output of the assay in

terms of fingerprint patterns desired (Xu et al. 1995). The DNA quality and

quantity (Williams et al. 1993), choice of DNA polymerase (Schierwater and

Ender 1993), magnesium concentration (Williams et al. 1993, Park and Kohel

1994), choice of thermocycler (Penner et al. 1993), primer concentration

(Williams et al. 1993), use of ethidium bromide vs. silver nitrate for detection of

amplification products (Caetano-Anolles et al. 1998) and presence of RNA (Yoon


and Glawe 1993), all these factors affect the results of the amplification reaction

in the RAPD assay.

Despite the mentioned shortcomings, RAPD has been successfully used as a

molecular marker to gain rapid and precise information about the genetic

diversity in different plant taxa. This is because the technique is quite simple

and fast and generates upto 5 to 10 bands per assay with as little as 10-400 ng of

DNA. Further there is no need for prior sequence information. Additionally, the

use of radioactivity for the detection of polymorphism is not required in RAPDs.

1.3.1.3 Amplified Fragment Length Polymorphism (AFLP)

The AFLP assay (Zabeau and Vos 1993, Vos et al. 1995) involves detection of a

subset of genome‟s DNA restriction fragments by PCR amplification. The

genomic DNA is restricted with a rare cutter like Pst I or Eco RI and by a

frequent cutter such as Mse I or Taq I. The restriction fragments, thus generated

are ligated to specific adapter sequences. These adapters serve as the binding

site for annealing of the primers in the AFLP reaction. The next step involves

selective preamplification of the restricted fragments with primers specific to the

restriction enzymes (Eco RI and Mse I or Pst I and Taq I) with extra nucleotide

at the 3‟ end. This ensures selective amplification of only a subset of adapter

ligated restriction fragments, which carry nucleotide complementary to the

selective nucleotide present in both the primers. Thus the template DNA that is

finally used for AFLP reaction is generated. This subset of preamplified library is

further amplified under stringent PCR conditions with primers carrying +3

selective nucleotides and one of the selective primers is radiolabelled with either

P32 or P33. The reaction products are electrophoresed on polyacrylamide gel and

subsequently autoradiographed. Thus, even a single nucleotide change either on

the restriction site or in the nucleotide adjacent to the restriction sites is

detected by AFLP. Other factors such as deletion, insertion and rearrangement

also affect the presence of the restriction site and its size and lead to

polymorphism that is detected by AFLP.

A single AFLP reaction generates 50-80 fragments in size range of 50 bp to

300 bp. The multiplex-banding pattern obtained, makes AFLP a powerful

marker. Further, the stringent PCR conditions attribute the assay its high

reproducibility. Thus, the AFLP assay combines the efficiency of PCR based

markers such a RAPD with the specificity and reliability of the hybridisation

based markers such as RFLP. Hence, it is the preferred tool for most of the

marker-based studies. The AFLP technique can be applied to DNA of any origin

and complexity.


1.3.1.4 Microsatellite based molecular markers

Microsatellites

Microsatellite or the simple sequence repeats consist of a basic motif of 1-6 base

pair (bp) repeated tandemly in the plant genome (Tautz and Renz 1984). They

occur frequently and randomly in all eukaryotic genome (Tautz and Renz 1984,

Gupta et al. 1994). These simple sequence-repeats (SSRs) are prone to high rates

of mutation, which mainly affects the number of core motifs. The polymorphism

thus presented is called Variable Number of Tandem Repeats (VNTR).

In general, microsatellite regions do not carry any coding functions. Hence,

these are under neutral selection pressure and freely mutate in the genomes.

The mutation rate is correlated to the total size of the array. The repeat remains

invariant for a long time if it contains a few repeated motifs only. When the

tandem copy number exceeds a certain limit, the chances of mutation increase

proportionately (Caetano-Anolles and Gresshoff 1998). Microsatellites are

construed as hyper-variable regions of the genome and hence are an ideal tool to

analyse genetic relatedness between highly similar genotypes at intra species

level. At the molecular level, the mutations in the repeat DNA sequence arise

due to replication slippage, transposition, recombination events, unequal

exchange between sister chromatids during mitosis, meiosis and between

homologous chromosome during meiosis and gene conversion. As these markers

can detect large number of alleles with high reproducibility (Litt and Luty 1989,

Tautz 1989 and Weber and May 1989), they find widespread use in DNA

fingerprinting.

Various microsatellite primed PCR assays have been developed to exploit

high levels of polymorphism inherent in microsatellites for genome analysis.

Selective Amplification of Microsatellite Polymorphic Loci (SAMPL, Morgante

and Vogel, 1994) involves amplification of restricted genomic DNA using

radiolabelled SSR primer and an unlabelled adapter primer. In order to detect

high levels of polymorphism, SSR primers corresponding to compound repeats

are generally preferred for SAMPL analysis. Since compound repeat carry

natural repeat-based anchor system, they show perfect self-anchoring and low

levels of stuttering.

Sequence Tagged Microsatellite Sites (STMS) assay involves use of primers

designed from sequence information of regions flanking the microsatellites.

However, STMS assay requires isolation of microsatellites de novo for most

plant species being examined for the first time and this limits its widespread use

for genetic diversity studies.


Inter Simple Sequence Repeat (ISSR)

On the other hand Inter Simple Sequence Repeat (ISSR) marker assay skips

over the requirement of any prior sequence information, cloning or sequencing

and has been extensively employed for genetic diversity studies.

This microsatellite primed-PCR employ 3‟ and 5‟ anchored 15 to 20-mer

primer and is referred as Inter SSR amplification, ISA or ISSR (Wolff et al.

1995). The primers have an extra nucleotide at the either end. These extra

nucleotides ensure that the primer binds to the opposite end of the

microsatellite, which has the first nucleotide of the flanking sequence

complementary to the anchor (i.e. the extra nucleotide at the 3‟ or the 5‟ end) of

the primer. The amplification occurs only if two suitable microsattelites with

appropriate flanking nucleotides are present within the amplifiable distance.

With this technique, one can target the variation between the SSR sequences or

the sequences that flank the SSRs.

The technique has many inherent advantages because of which it has been

regularly employed for a variety of purposes. The ISSR technique has the

reliability and advantages associated with any SSR marker system along with the

broad taxonomic applications of RAPDs. Abundance of SSRs is exploited to

generate a complex banding pattern. The ISSR technique is simple and

reproducible.

1.3.2 Importance of genetic diversity data

A consistent erosion of genetic diversity of medicinal and aromatic plants have

been reported due to various factors namely, ruthless destructive collections of

the herbs from the wild, destruction of natural ecosystems by human

interventions and monoculture in case of cultivated crops. Hence, it is essential

to catalogue the medicinal plant resources and effectively manage the available

germplasm through in situ and ex situ conservation programs.

Evaluation of genetic diversity using molecular markers aid in devising

realistic conservation strategies and in formulating core collections for

endangered germplasm as one is able to identify regions and populations

representing maximum diversity. These regions and populations can then be

successfully conserved with least population size on a priority basis (Shanker

and Ganeshaiah 1997, Rao and Hodgkin 2002). Further, molecular markers help

to evaluate the extent to which, a collection of germplasm contains significant

gaps in terms of the range of variations covered and in identifying duplicates or

near duplicates in a collection. Elimination of these duplicate samples helps to


confine the collection size to manageable limits in a scientific and rational

manner.

Pragmatically devised conservation strategies help in preserving maximum

genetic diversity found in target species, thereby, conserving the potentially

useful genes that can be utilised for the future breeding programmes. The

available genetic diversity data also helps in forming core collection for a

particular species. These core collections are of help to the plant breeders who in

addition to passport and characterization data, emphasize on the need of

improved evaluation of accessions. The result from diversity studies helps in

understanding the patterns of variations in crop species and identifies groups of

accessions with high diversity or with shared characteristics, which can be used

during breeding programs. For instance, genetic analysis of elite Indian cultivars

of Cymbopogon species revealed that cultivars of citronella and palmorosa

differed very little among themselves (Sangwan et al. 2001). This confirmed that

restricted genetic base was utilized while conducting breeding program of the

species. Hence, in order to broaden the genetic base of the crop, there is a

definite need for exploitation of wild counterparts. The study, thus illustrates,

the relevance of molecular data along with agronomic, morphological and

chemotypic characteristics in defining core collections and in sustainable

management of economically important medicinal and aromatic plant species.

In a nutshell, genetic diversity studies are of great relevance in conservation

of genetic resources and allow scientists in deciding which germplasm to

conserve on a priority basis, thereby, ensuring that the resources are managed in

a sustainable manner and conserved for future use.

Genetic diversity studies have been conducted using different molecular

markers with the above objectives. High levels of genetic diversity were obtained

among different populations of Cymbopogon species (Sangwan et al. 2001),

Changium smyrniodes (Fu et al. 2003) Eryngium alpinum (Gaudel et al. 2000)

and Podophyllum hexandrum (Singh et al. 2004a), rather than within

populations. In these cases loss of populations would mean loss of significant

amounts of genetic variability. Therefore, in such cases it is recommended that

maximum number of population should be included while collecting samples for

conservation purposes in the target species.

Further, the high genetic diversity revealed in endangered and rare plant

species such as Changium smyrnioides (Fu et al. 2003), helped to hypothesize

that it could be possible for the plant species to easily adapt to different

environmental regimes. Given the high levels of genetic variations, relocation of

the species to considerably less vulnerable habitats could be attempted.


Considering the fact that one of the main reasons for the species‟ threatened

status is its habitat destruction, relocation of the species to less vulnerable

locations appears to be a pragmatic approach towards conservation of some of

the endangered species. Further, based on cluster analysis one can decipher the

distribution of the genetic variation in populations collected from different

geographical locations and on the basis of these results it would be more logical

to protect the plant‟s habitat especially those with highest genetic variations (Fu

et al. 2003). Table 1.4 summarizes some of the genetic diversity studies

undertaken for endangered and economically important medicinal and aromatic

plant species.

Table 1.4 Genetic diversity studies conducted on Medicinal and Aromatic Plants (MAPS) using different molecular markers

Plant Marker assay Reference

Aloe species RAPD Darokar et al. 2003

American and Oriental

ginseng

AP-PCR Cheung et al. 1994

Andrographis paniculata RAPD Padmesh et al. 1998

Artemisia annua RAPD Sangwan et al. 1999

Asimia triloba ISSR Pomper et al. 2003

Asparagus species RAPD Shasany et al. 2003

Azadirachta indica RAPD Deshwal et al. 2005

Azadirachta indica AFLP Singh et al. 1999

Bacopa monnieri RAPD Darokar et al. 2001

Cardoon AFLP Portis et al. 2005

Changium smyrniodes RAPD Fu et al. 2003

Chicorium species RAPD and RFLP Besnard et al. 2001

Coptis species RAPD Cheng et al. 1997

Cymbopogon martini RAPD, enzyme and SDS-

protein polymorphism

Sangwan et al. 2003

Cymbopogon species RAPD Sangwan et al. 2001

Cymbopogon species RAPD Khanuja et al. 2005

Cymbopogon winterianus

and C. nardus

RAPD Shasany et al. 2000a

Digitalis obscura L. RAPD Nebauer et al. 1999

Echinacea angustifolia AFLP Still et al. 2005

Elaeagnus angustifolia RP-HPLC Qiang et al. 2006

Epimedium species RAPD and RFLP Makai et al. 1996

Eryngium alpinum AFLP Gaudel et al. 2000



Eucalyptus species RFLP Keil and Griffin 1994

Eurycoma longifolia Single Nucleotide

polymorphism (SNP)

Osman et al. 2003

Garlic RAPD Shasany et al. 2000b

Glehnia littoralis RFLP Mizukami et al. 1993

Glycyrrhiza species RAPD and RFLP Yamazaki et al. 1994

Humulus lupulus ISSR Danilova et al. 2003

Humulus lupulus AFLP Seefelder et al. 2000

Medicago citrina AFLP Juan et al. 2004

Mentha species RAPD Shasany et al. 2002a

Mentha species RAPD and AFLP Shasany et al. 2005

Moringa oleifera AFLP Muluvi et al. 1999

Ocimum sanctum Morphological and

protein markers

Ahmad and Kahliq 2002

Ocimum species RAPD Singh et al. 2004b

Ocimum species Morphological,

karyotypic, chemical and

molecular markers

Sabine et al. 2002

Olea europa RAPD Belaj et al. 2002

Olea europa RAPD, AFLP, RFLP and

SSR

Rosa et al. 2003

Olea species AFLP and Microsatellite Bandelj et al. 2004

Panax notoginseng AFLP Kiers et al. 2000

Panax quinquefolius RAPD Bai et al. 1997

Panax species RAPD Shaw and But 1995

Pelargonium graveolens RAPD Shasany et al. 2002b

Phyllanthus amarus RAPD Jain et al. 2003

Pimenta longa RAPD Wadt et al. 2004

Piper longum RAPD Philip et al. 2000

Piper nigrum RAPD Pradeepkumar 2003

Piper nigrum RAPD George et al. 2005

Plantago ovata Morphological markers Lal et al. 1999

Podophyllum hexandrum RAPD Sharma et al. 2000

Podophyllum peltatum Isozyme and RAPD Lata et al. 2002

Swertia species Isozyme Verma and Kumar 2001

Taxus wallichiana RAPD Saikia et al. 2000

Terminalia arjuna AFLP Sarawat et al. 2005

Valeriana wallrothii AFLP and chloroplast

SSR

Grassi et al. 2004



Valerians Chloroplast markers Bell and Donoghue 2005

Vetiveria zizaniodes RAPD and AFLP Lal et al. 2003

Withania somnifera AFLP Negi et al. 2000

Even as diversity studies help in selecting what to conserve and in devising

appropriate conservation strategies, tissue culture provides the options of in

vitro conservation of plant species and its multiplication in large numbers for

sustainable use. In the following section, different techniques of

micropropagation are discussed with reference to medicinal plants.

1.4 Micropropagation of medicinal plants

Micropropagation can be used as a routine method of multiplication in species

where conventional modes of propagation are inadequate to meet the demands

of the planting stock, either due to poor seed set or low germination. Also,

micropropagation is a tool for cloning of elite plants. Even in species where

vegetative multiplication is possible, tissue culture has been important for

multiplication of disease certified material and multiplication of elite clones. The

latter requires culture initiation from mature explants. There are several reports

of shoot regeneration from explants having pre-existing meristems such as

nodal segments (Selvakumar et al. 2001, Arya et al. 2002, Benniamin et al.

2004), shoot tips (Bhattacharya and Bhattacharya 2001, Thiem 2003) and

vegetative buds (Borthakur et al. 1998, Narula et al. 2003).

1.4.1 Explant selection and establishment of aseptic cultures

The technique of micropropagation starts with selection of suitable explant from

a desirable donor plant and its successful establishment in axenic culture

conditions. The choice of the donor plant is important in case of medicinal

plants, as with any other species, because growth of the plant varies with the

genotype and also the content of secondary metabolites may vary among

different genotypes of the same species.

The process of surface sterilization is critical for raising aseptic cultures.

Heavy infections have been reported during the initiation stages of many

medicinal plants, such as, Curcuma species (Balachandran et al. 1990), Alpinia

galanga (Borthakur et al. 1998), Bacopa monnieri (Tiwari et al. 1998) and

Saussurea lappa (Joshi and Dhar 2003). In most cases, infections (bacterial and

fungal) at the initiation stage can be correlated to the sanitary state of the place

where the donor plant is growing. For example, in explants derived from plants


growing in marshy areas and from underground plant parts such as rhizomes

and tubers, the contamination frequencies are usually high.

Another cause of persistent fungal and bacterial contamination in the

cultures is systemic or endemic infections present in the donor explant. In fact,

high rates of endemic contaminations in black pepper have proven to be a major

constraint for successful utilisation of tissue culture technique for the species

(Mathews and Rao 1984). In Trichopus zeylanicus, 70% of the explants were

lost due to bacterial infection during culture initiation stage (Krishnan et al.

1995) and hence aseptic seedlings were used for culture initiation instead of

mature explants.

The rate of contamination is also dependent on the season during which the

material is collected. This was evident in the study conducted by Chaturvedi et

al. (2004), where the contamination in nodal cultures of Azadirachta indica was

directly related to the season during which the material was collected. The study

concluded that the cultures initiated in March-May showed higher bud break

and less contamination than those raised in June-October or November-

February. Therefore, the selection of young and tender shoot tips in

comparatively drier seasons can reduce the problems of contamination to a

considerable extent.

Overnight fungicide treatment is also effective in cases of severe infections

(Chetia and Handique 2000). The severity of the treatment is, however, limited

by the nature of the explant. Some times explant shows high sensitivity to a

specific surface sterilant. The explants of Trichopus zeylanicus and Bacopa

monniera turned brown when treated with 0.01-0.1% mercuric chloride

(Krishnan et al. 1995, Tiwari et al. 1998). In Rauvolfia tetraphylla, the increase

in the treatment time and concentration of mercuric chloride to control the

growth of microflora resulted in extensive leaching of the phenolics (Sharma et

al. 1999). The sensitivity of the explant to the sterilisation process thus limits the

establishment of axenic cultures.

In order to control the incidences of contamination, antimicrobial agents

such as antibiotics and fungicides are generally added in plant tissue culture

media. Alternatively, pre-treatment is given in the form of pulse treatment,

wherein heavy dosage of the antimicrobial agent is given for a short period of

time. However, it is desirable to obtain axenic culture on antibiotic free media

because antibiotics may hinder growth and even survival of the explants. The

application of antibiotics also raises environmental concern relating to the

selection of antibiotic resistant strains of bacteria. Additionally, this process is


time consuming and labour intensive (Teng and Nicholson 1997, Teng and Teng

2000).

Teng et al. (2002), in a very interesting study investigated the influence of

explant preparation on culture initiation and subsequent regeneration response

of the root explants of Panax ginseng and P. quinquefolius. The freshly collected

roots were found to be extremely difficult to disinfect due to abundant soil borne

organisms associated to the root surface as well as inside the root. The authors,

however, could successfully establish the aseptic cultures from the non-surface

disinfected tissues. The process involved aseptic removal of the internal tissue of

the root including the pith and vascular bundles and inoculating the explant

material on the culture medium. In comparison to non-disinfected internal

tissues, the surface-sterilised explants revealed evidence of latent

contamination. The process of sterilisation also imposed a carry over effect on

explants and adversely affected subsequent regeneration steps.

Hence, apart from the choice of donor plants, the explant used, the season

during which initiation is conducted as well as the process of sterilisation also

influences the success of culture establishment to a considerable extent.

1.4.2 Modes of multiplication

Once the axenic cultures are established, next step is to develop methods for

continuous multiplication. Depending upon the presence and absence of pre-

existing meristem in the explant, the shoot bud induction may be divided into

two major heads; induction from explant having pre-existing meristem

(enhanced axillary proliferation) and from explants devoid of pre-existing

meristem (shoot regeneration or somatic embryogenesis).

1.4.2.1 Axillary multiplication

Axillary buds present in the axis of each leaf have the potential to develop into

shoots (Bhojwani and Razdan 1996). However, due to apical dominance, these

buds remain dormant. Application of cytokinins or removal of the apical

meristem eliminates apical dominance, thus stimulating the growth of lateral

buds. The lateral buds, as a result of continuous presence of cytokinins in the

culture media, also develop axillary buds. This process finally yields enhanced

axillary multiplication. Hence, the initial multiplication rate is usually low in

axillary multiplication. However, once the initial establishment phase is

accomplished, a logarithmic increase is attained in shoot multiplication.

Further, the multiplication rate achieved during axillary multiplication is

dependent on various factors of which, the first and most obvious is the choice of


the explant used. The superior in vitro response of the nodal explants compared

to apical shoot buds has been reported in Woodfordia fruticosa (Krishnan and

Seeni 1994), Clerodendrum colebrookianum (Mao et al. 1995), Gmelina

arborea (Kannan and Jasrai 1996), Rauwolfia micrantha (Sudha and Seeni

1996), Melia azedarach (Thakur et al. 1998) and Vitex nugendo (Sahoo and

Chand 1998).

Micropropagation of endangered plants from mature explants is, however,

limited because of the difficulty in procuring sufficient authentic material for

culture initiation (Wawrosch et al. 1999). Even when the explants from mature

mother plant are obtained, it is usually extremely difficult to obtain axenic

cultures on account of persistent contamination and associated problem of

browning (Wawrosch et al. 1999, Ahuja et al. 2002a). In such cases, alternative

sources such as seeds, aseptic seedlings and nodal explants from seedling-

derived in vitro cultures serve as the explant material. For instance, intact

seedling and explants derived from it have been used for axillary multiplication

in case of Murraya koenigii (Bhuyan et al. 1997), Swertia chirata (Wawrosch et

al. 1999), Allium wallichi (Wawrosch et al. 2001a), Lilium nepalense (Wawrosch

et al. 2001b), Pueraria lobata (Thiem 2003) and Saussurea obvallata (Joshi

and Dhar 2003).

Plant growth regulators are, perhaps, one of the most prominent factors that

influence rate of multiplication by axillary branching. For instance, the type and

concentration of cytokinins used, is known to have marked effect on shoot

regeneration. Various cytokinins such as, 2iP, BAP, Kn, TDZ and Zeatin, have

been regularly employed to achieve the same. For instance, though shoot bud

induction was obtained on basal medium in Aconitum carmichaeli, the optimal

number of shoots obtained increased to seven per explant only on incorporation

of BAP at a concentration of 5.0 mgl-1 (Hatano et al. 1988).

Thidiazuron (TDZ) is a substituted phenylurea (N-phenyl-1, 2,3 thidiazol-5-

urea) and a potent bio-regulator that influences in vitro morphogenesis and has

been demonstrated to be more effective over a wide range of species than most

of the other conventional cytokinins. Its mode of action has been attributed to

its ability to induce cytokinin accumulation and enhance the accumulation and

translocation of auxins within the TDZ exposed tissue (Murch and Saxena

2001). Faisal et al. (2005) also employed thidiazuron for shoot multiplication in

Rauvolfia tetraphylla L.

In the literature, BAP is reported to be most potent and thus used for large

number of species. For instance, in Picrorhiza kurrooa (Upadhyay et al. 1989),

Clerodendrum colebrookianum (Mao et al. 1995), Crateva magna (Benniamin


et al. 2004), Vitex negundo (Sahoo and Chand 1998) and Orthosiphon

stamineus (Waileng and Laikeng 2004) BAP was more effective than TDZ and

2-iP or Kn. The optimal cytokinin required for specific morphogenic response

varies with the plant system under investigation. In case of Cephaelis

ipecacuanha (Jha and Jha 1989) and Anemopaegma arvense (Pereira et al.

2003), Kn proved to be superior to 2-iP and BAP, while in Bixa orellana 2-iP

was found superior to BAP and Kn (Sharon and D‟ Souza 2000).

In many cases the synergistic effect of two or more cytokinins is known to

give the most optimal multiplication rate. In Rauvolfia tetraphylla, a

combination of cytokinins (BAP and Kn) was found essential for healthy shoot

development (Sharma et al. 1999). Similarly, synergistic effect of cytokinins and

auxins is reported to have a profound impact on the growth of the cultures as

well as multiplication rate. For instance, in Picrorhiza kurrooa, shoot tip

explants cultured on MS medium containing 5.0 mgl-1 Kn, on an average,

produced 50 shoots at the end of each passage of 6 wk duration. The addition of

IAA (1.0 mgl-1) revealed marked improvement in shoot growth, leaf size and

stem thickness without any evidence of adventitious shoot formation (Lal et al.

1988). Similarly, Sudha and Seeni (1994) reported that shoot multiplication was

directly related to cytokinin activity (both BAP and 2-iP) along with the

synergistic effect of IAA, in Adhatoda beddomei. In case of Aristolochia indica

(Manjula et al. 1997), multiple shoots induced on medium supplemented with

BAP alone, were weak and fragile. Therefore, NAA was incorporated with BAP

and this proved to be stimulatory, producing 45 shoots in 16 weeks. This

stimulatory effect of auxins on shoot proliferation has been observed in many

other medicinal plants, such as, Glehnia littoralis (Hiraoka and Oyanagi 1988),

Cephaelis ipecacunha (Jha and Jha 1989), Rauwolfia micrantha (Sudha and

Seeni 1996), Syzygium alternifolium (Khan et al. 1997), Yucca aloifoila (Atta-

Alla and van Staden 1997), Dioscorea bulbifera (Narula et al. 2003), Puereria

lobata (Thiem 2003) and Saussurea lappa (Joshi and Dhar 2003).

Thus, it may be inferred that the endogenous level of growth regulators

largely influences the requirement of the exogenous hormones in the plant

system. The levels of endogenous growth regulators on the other hand, vary with

the plant tissue, plant type and physiological state of the plant and also with the

genotype of the species under consideration.

Beside growth regulators, the ability to form multiple shoots is also

dependent on different factors such as size of the explant, its orientation,

temperature, intensity of illumination and the type of cultures used in the study.

In Digitalis lanata, meristem without leaf primordia (length of shoot tip up to


0.3 mm) did not develop into shoots; instead they formed a green, nodular

tissue (Diettrich et al. 1990). Similarly, the shoot tip of Stevia rebaudiana

without leaf primordium failed to grow and turned brown. On the other hand,

90% of explants with 6-8 leaf primordia (1. 5-2.0 mm long) formed single

shoots, but those in between 0.3-1.5 mm length (with 2-4 leaf primordia) formed

multiple shoots (Tamura et al. 1984). In case of nodal explants, shoot induction

was affected by position of the node on mature explants in Gymnema elegans

(Komalavalli and Rao 1997). Nodes at 3rd and 4th positions were more responsive

than those procured from distal and proximal portions of the shoot.

On the other hand, in Bixa orellana, size of the nodal explant was an important

factor in producing optimum number of shoots (Sharon and D‟souza 2000).

Smaller explants, approximately 0.5 cm long initiated more multiple shoots than

the longer (1.5 cm long) nodal explants.

Explant orientation was found to play an important role during shoot

proliferation in Rauwolfia micrantha (Sudha and Seeni 1996). In Adhatoda

beddomei, split halves of shoots were used for subculture; this resulted in

regeneration of 23-27 shoots from each of the two halves, which was higher than

the numbers (15-17 shoots) obtained from culture of the intact nodes (Sudha and

Seeni 1994). Similarly Wawrosch et al. (2001a, 2001b) reported a higher

response of longitudinally split shoot halves as compared to whole shoot

explants in Allium wallichi Kunth. and Lilium nepalense D. Don, irrespective of

the culture media.

In some species, such as, Ocimum americanum and O. sanctum (Pattnaik

and Chand 1996) Vitex negundo (Sahoo and Chand 1998), Gymnema elegans

(Komalavalli and Rao 1997) and Azadirachta indica (Chaturvedi et al. 2004),

frequency of shoot proliferation and growth was greatly influenced by the season

during which the explants were collected. Since, it is during the spring and

monsoon seasons, that the plants show vigorous growth, the explants collected

during these seasons are more responsive as was also seen in the above-

mentioned examples.

In contrast to the above studies, shoot proliferation from axillary buds of

Lavandula latifolia was not affected by seasonal fluctuations in the stock plants

but was dependant on the macronutrient composition and the type and

concentration of cytokinin tested (Sanchez-Gras and Calvo 1996).

The plantlets maintained in vitro are photosynthetically not active and their

energy demands are met by the carbohydrate source in the medium. Despite

this, both intensity and duration of light influences the morphogenetic response

of the explants. In Digitalis lanata for instance, the number of shoots formed


was dependent on light quantity and quality. At a qualitative level, light sources

producing high intensity in the blue and red regions of the spectrum were found

to be more favourable for shoot bud induction (Diettrich et al. 1990).

Temperature conditions maintained in the plant growth room constitute an

important culture condition, which needs to be optimised for may plant taxa.

For instance, the effect of temperature on multiple shoot formation was

investigated in Cnidium officinale and it was found that at 15 2 0C

temperature, multiple shoot regeneration was retained in BAP containing

medium for 12 generations. However, at 25 2 0C, the sub-cultured shoots kept

proliferating without any decline in the ability to form shoots even after 2 years

of subculture (Pant et al. 1996).

Gelling agent is yet another factor which determines the success of a

micropropagation protocol. Conventionally, semisolid medium with 0.6 to 0.8%

of agar is used in most micropropagation protocols. Agar being an inert material

offers support to the explant and the growing tissues and permits free flow of

nutrients. Liquid media however, have been favourably used to obtain increased

multiplication fold and better cluster vigour in many plant species. However,

one of the major problems in liquid cultures is to provide aeration to the

explants growing in cultures. An efficient method using floating membrane rafts

was developed for micropropagation of Aconitum napellus (Watad et al. 1995),

which resulted in shoot proliferation that was 45% higher than that obtained on

semi-solid media. Similar response has been reported in Indian rhubarb

(Rheum emodi) where liquid shake culture medium required half the culture

medium and lesser time to induce shoot proliferation comparable to that

obtained in semi-solid cultures (Lal and Ahuja 1989). In their study, a stable rate

of regeneration was maintained for more than 2 years in shake cultures, whereas

the explants kept on static liquid cultures submerged and failed to induce

multiple shoots and eventually turned brown. In case of Isoplexis canariensis

also, proliferation of shoots was found to be greater in liquid medium than on

semi-solid medium (Arrebola et al. 1997). Mitra et al. (1998) described the

simultaneous use of the rotatory and static culture media for rapid

multiplication of Catharanthus roseus from nodal explants. Arrebola and

Verpoorte (2002) also obtained optimal shoot multiplication for Isoplexis

isabelliana on liquid media.

The lower shoot multiplication rate obtained on agar solidified medium in

comparison to liquid medium of the same composition could be due to lower

water potential and other physical factors of agar affecting the diffusion of ions

and compounds. Further, it has been proposed that growth regulators and other


nutrients are also released slowly from agar-solidified medium, which might

have some influence on plant growth and survival.

Role of additives in multiplication of medicinal plants has been well

explored. Additives such as ascorbic acid (50-200 mgl-1), coconut milk and GA3

were reported to significantly increase the number of shoots in Gymnema

elegans (Komalavalli and Rao 1997). Addition of adenine hemi-sulphate in

combination with low levels of NAA to the culture medium was found to

improve shoot multiplication in Lavandula stoechas (Nobre 1996). In

Gymnema sylvestre (Anu et al. 1994) and Uraria picta (Anand and Kutty 1995),

adenine sulphate was used for shoot regeneration, while adenine was used in

Cephaelis ipecacuanha (Jha and Jha 1989) Azadirachta indica (Arya et al.

1995) and Siatia grosvenorii (Liang et al. 1995).

Exogenous GA3 is known to stimulate flower, root and shoot morphogenesis.

Zhang and Leung (2002) reported that the presence of GA3 in the medium

enhanced in vitro shoot development in Gentiana species. GA3 has also been

reported to have a rejuvenating effect on mature tissues (Shakirova et al. 2001).

Similar effect was also observed in Scopolia parviflora in which addition of GA3

to culture media promoted shoot proliferation from rhizomes (mature tissues).

In cultures of Tylophora indica, addition of ascorbic acid to the plant growth

regulator-supplemented medium was found to be essential for initial bud-break

and further shoot multiplication (Sharma and Chandel 1992). Initial wash for

ten minutes in antioxidant solution (citric acid and ascorbic acid 250 mgl-1 each)

helped in reducing browning of buds, nodes and hypocotyl and leaf segments,

which were employed for culture initiation in Minthostachys andina (Castillo

and Jordan 1997). Further, the strong browning effect observed in in vitro

cultures, was partially overcome and morphogenic response was enhanced by

using one of the following antioxidants: glutathione at 15 and 25 mgl-1; L-

cysteine at 15 mgl-1; PVP at 50 mgl-1 or amino-oxy-acetic acid at 10 mgl-1. The

effect of additives such as maize extract, phloroglucinol and adenine sulphate

has also been described by Butiuc-Keul and Deliu (2001) in Arnica montana, a

medicinal plant of Europe and North America.

1.4.2.2 Shoot regeneration

There are several reports discussing adventitious shoot regeneration and factors

affecting it in medicinal plant species from explants devoid of meristems.

Adventitious shoot regeneration is known to affect the genetic uniformity by

causing large-scale genome rearrangements thereby resulting in somaclonal

regenerants. The occurrence of somaclonal variants is, however, of great


significance for genetic improvement. Further, efficient plant regeneration

protocol is pre-requisite for the application of in vitro genetic transformation

technique.

Shoot organogenesis takes place either, directly or, through callus formation

from explants such as leaf, petiole, cotyledon leaves, internode, root and

hypocotyl. For instance, young leaves were the best source of explants, both for

caulogenesis and regeneration in Artemisia absinthium (Nin et al. 1996).

Similarly, shoot bud induction was observed from all over the surface of leaf

explants in Withania somnifera. However, the frequency of explants responding

to regeneration varied with the size and the type of culture vessel used (Kulkarni

et al. 1996). It has been suggested that amount of ethylene and CO2 present in

culture vessel influences organogenesis. The culture vessels with cotton plugs

allow free exchange of gas and hence are more conducive for growth of tissues in

vitro.

In Bacopa monnieri, in vitro-derived leaves were found to be superior

explant material for shoot regeneration in comparison to field-grown plants

(Shrivastava and Rajani 1999). The source of the leaf explant as well as the

choice of gelling agents was found to influence shoot-induction and eventual

shoot growth in Bacopa monnieri (Shrivastava and Rajani 1999). The tissue

culture reports in Bacopa monnieri confirmed a high-frequency regeneration

system for repeated harvesting of the shoots from the original explant, which

could be exploited further in commercial breeding and genetic improvement

through genetic engineering (Tiwari et al. 1998, Shrivastava and Rajani 1999,

Tejavathi and Shailaja 1999).

The choice of growth regulators employed also influence the response of the

explant towards shoot bud regeneration. A combination of GA3 and BAP induced

multiple shoot formation in leaf explants of Saussurea lappa (Arora and

Bhojwani 1989) and Naregamia alata (John et al. 1997), whereas BAP alone

was found optimal for shoot regeneration in Mentha arvensis (Phatak and

Heble 2002). Most of the other studies have reported combined use of auxin and

cytokinin for improved efficiency of shoot regeneration, such as, in leaf explants

of Valeriana wallichi (Mathur and Ahuja 1991), Artemisia absinthium (Nin et

al. 1996), Withania somnifera (Kulkarni et al. 1996) and Thapsia garnica

(Makunga et al. 2003). Auxins alone were found to induce shoot regeneration

from leaf explants of Drosera rotundifolia (Bobak et al. 1995) and Eucomis

poleevansii (McCartan and Van 1995). Addition of adenine sulphate (5.0 mgl-1)

to the culture medium increased the growth of shoot buds in case of leaf and

hypocotyl explants of Psoralea corylifolia (Saxena et al. 1997). In Celastrus


paniculatus, Nair and Seeni (2001) reported regeneration in medium

supplemented with BA along with IAA or NAA from leaf base and internode

explants. However, in Bacopa monniera growth regulators were not found

indispensable for shoot regeneration, wherein Tiwari et al. (2006) reported

direct shoot regeneration by culturing internodes and leaf explants on MS

medium supplemented with trimethoprim (an antibiotic) or bavistin (a

fungicide).

Internodes have been frequently employed for inducing organogenesis.

Internode explants of Cephaelis ipecacuanha were reported to show direct shoot

regeneration from epidermis when cultured on GR-free MS medium; the

presence of 0.01-0.1 mgl-1 BAP or 0.1 mgl-1 kinetin in the medium increased the

number of shoots produced per internode segment (Yoshimatsu and Shimomura

1991). Misra and Chaturvedi (1993) reported that the regeneration potential of

first internode of Rosmarinus officinalis was more than that of second

internode. Within an internode it was found greater in the upper end than in the

lower end. Also, as expected, the concentrations of auxin and inorganic salts

present in the medium played an important role in determining regeneration

frequencies. Similarly, Kanjilal and Datta (2000) reported shoot organogenesis

on Knudson media supplemented with BAP and NAA from stem section of

Geodorum densiflorum.

Despite the fact that establishing aseptic cultures from root explants is a

cumbersome exercise, excised roots have been successfully used for inducing

adventitious shoots in many plant species. Adventitious shoots were directly

formed without callus formation in Cephaelis ipecacuanha from the cut ends of

root segments cultured in dark on growth regulator-free B5 semi-solid medium

(Yoshimatsu and Shimomura 1994). On the other hand, presence of BAP (0.2-

0.4 mgl-1) was found most conducive for shoot bud induction in root explants of

Piper longum (Bhat et al. 1995). The use of cytokinins resulted in shoot bud

induction from root explants in Swertia chirata, however, hyperhydricity was

evident in the regenerated shoots (Wawrosch et al. 1999). Hence, a two-step

system (an initial supplement of BAP followed by hormone-free medium) was

employed for shoot regeneration from root explant of S. chirata. Chaudhuri et

al. (2004) studied the effect of different cytokinins, explant age and explant type

on Tylophora indica regeneration. Apical and sub-apical root segments

promoted direct shoot organogenesis on BA supplemented media at a

concentration of 10.72 M.

Indirect shoot regeneration has also been reported in many medicinal plants.

Martin (2002) studied regeneration in Holostemma ada-kodien from basal end


of the nodal explants on MS media supplemented with 1.5 mgl-1 of BAP. It was

found that the addition of AgNO3 prevented abscission of leaves and shoot tips,

which otherwise hindered proper growth of the plants. Shoot regeneration was

induced in disk tissues of young leaves of Hydrastis canadensis when callus

induced on NAA + TDZ supplemented media was transferred to media

supplemented with either BAP and Kn combined or TDZ alone (Bedir et al.

2003). Sharada et al. (2003) reported indirect organogenesis in Celastrus

paniculatus from cotyledonary leaf derived callus.

1.4.2.3 Somatic embryogenesis

In plant tissue culture, somatic embryogenesis is the process of embryo

initiation and development from vegetative and non-gametic cells (Bhojwani

and Razdan, 1996). Somatic embryos (SEs) are bipolar structures produced

from the tissues of somatic origin. Their induction is the result of acquisition of

totipotency by mature plant cells when they are exposed to a suitable

concentration of plant growth regulators. Plants generated via somatic

embryogenesis generally give rise to truly clonal populations as compared to

shoots produced from regeneration processes with intervening callus phase.

These plants are derived from meristematic cells, which are by nature

genetically stable and a strong selection occurs in favour of genetically normal

cells during somatic embryo development (Vasil 1994). Somatic embryogenesis

may also occur indirectly via an intervening callus phase (Murhty and Saxena

1998, Krishnamurthy 1999 and Hariharan et al. 2002).

Somatic embryogenesis, ever since it was first reported in carrot (Reinert

1958, 1959 and Steward et al. 1958), has been regarded as a suitable system for

the investigation of various morphological and physiological phenomena

(Nomura and Komamine 1995).

The technique has been successfully employed for micropropagation of elite

genotypes of forest-trees (Gupta et al. 1993), ornamentals (Yantcheva et al.

1998) and endangered medicinal plants (Wakhlu and Sharma 1998, Das et al.

1999a and Choffe et al. 2000, Park and Facchini 2001 and Martin 2004). It is

also considered an alternative method for mass multiplication through

encapsulation in the form of synthetic seeds and ex situ conservation

(Redenbaugh et al. 1991 and Jung et al. 2004). Somatic embryogenesis through

plant cell suspension cultures offers a promising tool for unlimited production of

plants with functional root and shoot meristems. Further, there lies immense

scope for process optimisation and control (Vasil 1994), which can overcome the


reported problems of uneven and problematic conversion of somatic embryos to

robust seedlings (Janick 1993).

Most protocols for somatic embryogenesis employ 2,4-D, a strong auxin or

an auxin-cytokinin combination in the primary culture medium (proliferation

media) to support both cell proliferation and induction of embryogenesis. The

pro-embryogenic mass present in cultures maintained on a medium containing

auxins generally expresses all the genes necessary to complete the globular stage

of embryogenesis (Zimmerman 1993). Despite the predominant role of 2,4-D in

SE induction, successful use of other auxins such as NAA along with TDZ in

Pimpinella tirupatiensis (Prakash et al. 2001), IAA along with BA and TDZ in

Echinacea purpurea (Choffe et al. 2000), NAA and BAP in Catharanthus roseus

(Junaid et al. 2006) have also been reported.

Interestingly, in case of Piper nigrum (Joseph et al. 1996) and Panax

ginseng (Choi et al. 1997), exogenous growth regulators were neither essential

for callus formation nor for proliferation, and were also not required for the

development of somatic embryos. These observations suggest that endogenous

hormones may regulate the entire process of somatic embryogenesis.

It has been observed that the process of somatic embryogenesis continues on

a basal medium after the initial somatic embryo development on hormone-

supplemented media. It is usually considered that exogenous growth regulators

must be omitted for normal somatic embryo development in tissue cultures of

many plant species (Ammirato 1983). These observations have been

corroborated by studies in Acanthopanax senticosus (Gui et al. 1991),

Catharanthus roseus (Kim et al. 1994), Glehnia littoralis (Hirai et al. 1997),

Ceropegia candelabrum (Beena and Martin 2003), Andrographis paniculata

(Martin 2004) and Rotula aquatica (Chithra et al. 2005).

In some species, such as, Panax ginseng (Shoyama et al. 1988), Bunium

persicum (Wakhlu et al. 1990), Bacopa monniera (Tiwari et al. 1998), and

Sapindus trifoiatus (Borad et al. 2001), somatic embryos were induced by an

auxin, while the presence of a cytokinin favoured their maturation and

germination.

In Begonia gracilis, red light (45 µmol m-2s-1) enhanced somatic

embryogenesis in comparison to continuous dark (Castillo and Smith 1997),

while in Oldenlandia umbellata, diffused light conditions were effective for

somatic embryogenesis (Rao and Bahadur 1990). Similarly in Salvia species, low

light intensity (50 µmol m-2s-1) favoured callus formation and induction of

somatic embryos (Kintzios et al. 1999). However, in some cases such as


Corydalis yanhusuo (Sagare et al. 2000) and Aloe barbadense (Wu 2000) light

was found to inhibit formation of embryogenic callus.

Carbon source and concentration has a marked influence on maturation and

subsequent germination capacity of somatic embryos. Choi et al. (2002) and

Choi and Jeong (2002) reported that sucrose levels play a significant role in

subsequent maturation and germination of Panax ginseng somatic embryos. At

a low concentration of sucrose (1.5%), maturation and germination of the

somatic embryos occurred immediately. However, at 6% sucrose concentration,

somatic embryos did not germinate although this could be overcome by GA3

treatment. On the other hand, Datta et al. (2006) reported improvement in

embryo quality on pre-treatment of explants with 1.0 M sucrose at 4 0C in Taxus

wallichiana. A reduction in sucrose level from 3.0 to 1.5% raised somatic

embryo formation in Piper nigrum (Joseph et al. 1996), while a higher sucrose

level of 5.8% was found optimal in R. vomitoria (Tremouillaux-Guiller and

Chenieux 1991), 10% in P. ginseng (Asaka et al. 1994), 5% in Murraya

paniculata (Jumin and Nito 1995) and 7% was found optimal in Taxus

wallichiana (Datta et al. 2006).

Reduced nitrogen in proper combination with nitrate enhanced somatic

embryogenesis in Digitalis lanata (Reinbothe et al. 1990) and Glehinia littoralis

(Hirai et al. 1997). Certain additives have been used for induction and

germination of somatic embryos. For instance, coconut water at 2-10% was

added in the medium for somatic embryo induction in Begonia gracilis (Castillo

and Smith 1997) and absicisic acid (10.5 mgl-1) was used in Digitalis lanata

(Reinbothe et al. 1990) and Taxus brevifolia (Chee 1996). ABA in liquid MS

media was also employed for somatic embryogenesis in Aralia cordata

(Kangseop et al. 2002). Ascorbic acid at 0.1% concentration in Oldenlandia

umbellata (Rao and Bahadur 1990) and charcoal at 1% in Taxus brevifolia

(Chee 1996) were found optimal for induction of somatic embryogenesis. In case

of Heracleum candicans, mature embryo formation was significantly affected by

pH of the medium and by addition of 2.0 mgl-1 AgNO3 and 1.0 mg l-1 ABA in MS

medium (Wakhlu and Sharma 1998). In Andrographis paniculata, the embryo

maturation was influenced by addition of silver nitrate to half strength MS liquid

media (Martin 2004).

In many species such as Panax ginseng (Arya et al. 1991), Aconitum

heterophyllum (Giri et al. 1993), Cymbopogon flexuosus (Nayak et al. 1996),

and Tylophora indica (Chaudhuri et al. 2004), growth regulators such as GA3,

BAP and IBA have been used for somatic embryo germination and plantlet

conversion. A chilling treatment (-2 oC for over 8 weeks) was found to be an


essential pre-requisite for germination of P. ginseng somatic embryos (Choi et

al. 1999).

Various studies have demonstrated that embryogenic cell suspension

cultures derived from a broad spectrum of plants, including medicinal plants

can be grown in bioreactors. The cultures of embryogenic cell suspensions of

Euphorbia pulcherrima (Preil et al. 1988) were used to study the parameters

influencing somatic embryogenesis. Park and Facchini (2001), reported somatic

embryogenesis from embryogenic cell suspension cultures of Eschscholzia

californica and described the effects of media composition, gyratory shaker

speed, various carbon sources, different cytokinins and AgNO3 on efficiency of

somatic embryogenesis. In fact, suspension cultures were found to be superior

to semi-solid media in terms of embryogenic callus production in Rotula

aquatica (Chithra et al. 2005). Recently, Nair and Dutta (2006) reported a

standardised single flask system for proliferation, maturation and conversion of

secondary somatic embryos in black pepper (Piper nigrum) L.

Mass propagation via organogenesis of Stevia rebaudiana, an important

source of non-caloric sweetener named stevioside, was achieved in a 500 l

bioreactor (Akita et al. 1994). Choi et al. (2002) standardised mass production

of Siberian ginseng plantlets through large-scale tank culture of somatic

embryos and reported approximately 12,000 embryos per 500-ml flask after 4-

weeks of culture. The production was scaled up to 10 litres in a bubble-lifting

tank culture system. Besides the culture conditions and growth regulators

balance, the aeration and agitation of the cultures is considered equally

important. Albeit the considerable progress that has been made in the past,

improvements in bioreactor design and culture conditions are still required.

It can be concluded that presently the technology of embryogenic cell

suspensions in bioreactors is still in the nascent stages and cannot be

successfully taken up for commercial ventures.

1.4.3 Elongation and rooting

Once the objective of mass multiplication is achieved, the regenerated shoots are

rooted on suitable culture media or treated as micro-cuttings for ex vitro

rooting. Since shoot multiplication is achieved on a high cytokinin

supplemented medium, it is imperative to bring the endogenous levels in favour

of auxin. Also, the shoots should be elongated to a specific length before being

used for root induction. The inoculation of shoots on a basal medium or on

media supplemented with auxin, helps in shoot elongation.


In some medicinal plants, such as Acmella oppostifolia (Salgado-Garciglia et

al. 1996), Alpinia galanga (Borthakur et al. 1998), Ochreinauclea missionis

(Dalal and Rai, 2001), Scopolia parviflora (Kang et al. 2004) and Tylophora

indica (Chaudhuri et al. 2004), elongation of regenerated shoots was found

adequate on basal medium.

However, in many other cases shoot elongation is favoured by the addition of

additives. For instance, GA3 promoted shoot elongation in Ocimum

americanum (Pattnaik and Chand 1996) and Vitex negundo (Rani and Nair,

2006), adenine sulphate in O. sanctum (Pattnaik and Chand 1996), ascorbic

acid in Psoralea corylifolia (Saxena et al. 1997), coconut milk in Gymnema

elegans (Komalavalli and Rao 1997) and calcium pantothenate along with biotin

in Azadirchata indica (Joshi and Thengane 1996).

Interestingly, Shibata et al. (1996) reported shoot elongation when the

shoots were transferred to MS medium supplemented with a much lower

concentration of sucrose (1%) and solidified with 0.2% gelrite in Croton

sublyratus. Consequently, the use of basal medium for shoot elongation renders

the micropropagation protocol economical and simpler.

Rooting of in vitro developed shoots also show variable response depending

on the plant species. In some plant species in vitro rooting is largely genotype

dependent with shoots rooting spontaneously during multiplication stage and

thereby obviating the inclusion of separate rooting stage. In other species, basal

medium encourages root formation. Also, in some species root formation is

often inhibited by cytokinins, which are important for shoot multiplication

(Debergh and Maene 1981, George and Sherrington 1984). Rooting is

significantly affected by the ionic strength of the medium and hence low ionic

strength medium such as White's (1963) are also frequently employed for in

vitro rooting due to the need for only a small amount of total nitrogen (George

and Sherrington 1984). Kang et al. (2004) employed basal B5 medium

(Gamborg et al. 1968) for root induction in Scopolia parviflora. However, in

most of the medicinal plants, reduced strength is mostly confined to MS

medium (Koroch et al. 1997, Ajithkumar and Seeni 1998, Prakash et al. 1999,

Nair and Seeni 2001, Joshi and Dhar 2003). In Croton sublyratus, in vitro root

induction was best optimised when shoots were inoculated in 1/10 MS medium

supported by vermiculite (Shibata et al. 1996). Similarly, 90% of the excised

shoots of Coleus forskohlii formed roots on MS basal medium within 20 days

(Sen and Sharma 1991). On the other hand, the common practice to transfer

rootable shoots from high strength medium to lower strength solutions did not

work in Cephaelis ipecacuanha (Jha and Jha 1989).


Sahoo and Chand (1998) reported that excised shoots of Vitex negundo,

which is a woody aromatic medicinal shrub, failed to produce roots on full or

half strength media (with or without growth regulator) even after 40 days of

culture initiation. On the other hand, 94% of shoots induced rooting when 1/2

strength MS medium was augmented with IAA and IBA each at 1.0 mgl-1, thus

emphasising the role of auxins during root induction. Similarly, in Bixa

orellana, hormone-free B5, 1/2 MS or MS medium failed to develop good roots

and for inducing healthy root system, 12 week-long sub-culturing was required

on MS medium supplemented with 0.01 and 0.5 mgl-1 NAA (Sharon and D‟souza

2000).

In Coleus forskohlii and Hemidesmus indicus, basal medium failed to induce

rooting, but various auxins like IAA and IBA readily induced rooting (Sharma et

al. 1991, Patnaik and Debata 1996). Thus, the role of auxins in root induction has

been reaffirmed as also illustrated in Dioscorea bulbifera (Nair and Seeni 2001),

Ochreinauclea missionis (Dalal and Rai 2001), Crateva magna (Benniamin et

al. 2004) and Mandevilla illustris (Biondo et al. 2004). However, in several

studies, root suppression has been noticed on addition of these growth

regulators. For instance, in Hybanthus enneaspermus, 9.6 M IBA suppressed

rooting and caused extensive callusing at the basal portion of differentiated

shoots (Prakash et al. 1999), whereas 4.8 M IBA was found optimal for root

initiation.

Pulse treatments or dipping of rootable shoots in solution of root inducing

growth regulators such as NAA and IBA has been widely used for root initiation

in many medicinal plants, such as, Rauvolfia tetraphylla (Sharma et al. 1999)

and Swertia chirata (Wawrosch et al. 1999), Lagerstromia parviflora (Tiwari et

al. 2002) and Thapsia garganica (Makunga et al. 2006). In Gmelina arborea

2.0 mgl-1 IBA resulted in senescence and eventual abscission of leaves, however

100% rooting occurred when cut ends of microshoots were pulse treated with 50

mgl-1 IBA and transferred to vermiculite directly (Kannan and Jasrai 1996).

Luna et al. (2003) reported a two-step rooting in Ilex dumasa. An initial three-

week inoculation on quarter strength semisolid MS media supplemented with

0.15% sucrose and 7.3 M IBA was required, followed by transfer to media

supplemented with 20 mM codavarine without any growth hormone.

The concept of in vivo (ex vitro) rooting was conceived in the early eighties,

when Debergh and Maene (1981) described the advantages of ex vitro rooting as

compared to in vitro rooting. Since in vivo rooting combines both the rooting as

well as acclimatisation stages, it effectively reduces aseptic handling and labour

cost incurred otherwise. Ex vitro rooting has been reported for many medicinal


plants such as, Maytenus aquifolium (Pereira et al. 1994), Isoplexis canariensis

(Arrebola et al. 1997), Bacopa monniera (Tiwari et al. 1998) and Vitex negundo

(Kannan and Jasrai 1998). Arya et al. (2002) treated the clonally produced

shoots of Celastrus paniculatus with a solution containing 100 mg l-1 each of

indole-3-butyric acid (IBA) and naphthoxy-acetic-acid (NOA). These auxin-

pulsed shoots were subsequently treated with 10 mg l-1 solution of chlorogenic

acid for 3 minutes. Of these, 83% shoots rooted ex vitro.

In all these mentioned cases ex vitro induced roots were structurally and

functionally much better as compared to those induced in vitro leading to higher

transplantation success in the former case. Thus, ex vitro rooting leads to better

results and also saves an in vitro step and therefore curtails the cost of in vitro

production of plants.

1.4.4 Hardening and field establishment

In vitro regenerated plantlets are finally acclimatised in a green house prior to

their transfer to natural habitat or fields. The process of hardening and

acclimatisation is a critical step because it finally determines the success of the

micropropagation protocol and thus influences its economics as well. In spite of

this, research in this area is restricted to few commercially exploited species, and

in many instances results are not published but retained confidential by the

industry.

In vitro developed plantlets although green in colour, have heterotrophic

mode of nutrition since they have been maintained in the presence of carbon

source and supplemented with all the necessary macro and micronutrients in

the culture medium. The plants are kept under controlled conditions of

temperature and close to 100% humidity. Once the plants are transferred to pots

with potting mixes of inorganic supplements in a green house, the regenerated

plants have to switch their growth pattern to autotrophic mode. However,

irrespective of the nature of plant species, it is generally accepted that during

this transition phase, cuticle formation, its thickness and stomatal development

are poor. These are coupled with inefficient chlorophyll ratios and lower surface

area, which affects the plant‟s photosynthetic rate. Therefore, as a routine, the in

vitro propagated plants are required to be kept under high relative humidity for

the first few days after being taken out of culture vessels. For instance, in

Hedeoma multiflorum glass covers were used to ensure high humidity around

the plants in the initial stages of acclimatisation and were gradually opened for

longer duration day by day during the hardening process (Koroch et al. 1997).

Similarly, high humidity conditions were required for four weeks in Excoecaria


agallocha (Rao et al. 1998) in order to obtain 95-100% survival. In Trichopus

zeylanicus, 80% of the plants transferred directly to the nursery were lost;

whereas those hardened for 4 weeks in humidity chamber showed 85% survival

(Krishnan et al. 1995). In Thapsia garganica inclusion of 2% polyethylene glycol

(w/v) in the growth medium or ventilation of cultures prior to acclimatisation

resulted in better hardening success (Makunga et al. 2006). These instances

suggest the importance of acclimatisation step, absence of which, results in

drastic mortality during final transplantations.

However, as an exception, some plant species do not require acclimatisation

before transplantation. For instance, plantlets of Glehnia littoralis did not

require any covering on transfer to field as they had well-developed cuticle on

the surface of leaves and small ratio of surface area to fresh weight of leaf blade

(Hiraoka and Oyanagi 1988). Krishnan and Seeni (1994) also reported transition

of Woodfordia fruticosa (a rare medicinal plant) plantlets to field without

hardening. Thus, in certain exceptional cases as mentioned here, acclimatisation

step can be foregone, without much loss.

Usually acclimatisation temperatures are akin to the ideal growing

conditions of the species under consideration. Low temperature conditions were

found essential during acclimatisation of medicinal plants such as Aconitum

napellus (Watad et al. 1995), Swertia chirata (Wawrosch et al. 1999) and

Valeriana wallichi (Mathur et al. 1988) that inhabit 1,500 to 3,000 m altitudes

in temperate Himalayas. The plant survival was optimal at 17 to 20 0C

temperature and most of the plantlets died at 25 0C temperature.

Different potting mixes also has significant role in determining survival

percentage in some plant species. Sahoo and Chand (1998) used four different

substrates (vermiculite, vermicompost, soilrite mix and garden soil) separately

for hardening of Vitex negundo plantlets inside the environmentally controlled

growth chamber. The percentage survival was highest in vermicompost (93%)

followed by soilrite mix (80%), garden soil (63.3%) and vermiculite (60%).

It is thus evident that each stage of micropropagation has its own

significance while developing a viable micropropagation protocol. Every step of

micropropagation is influenced by different factors, and in absence of

understanding the significance of these factors the seemingly innocuous step can

itself become the limiting step during optimisation of a viable tissue culture

protocol.


1.5 Somaclonal variations and their detection by molecular markers

Micropropagation techniques offer the possibility of cloning millions of

plants under disease free conditions. However, the technique must be carefully

applied due to the reported incidences of somaclonal variation (D‟Amato 1978,

Skirvin 1978, Larkin and Scowcroft 1981, Larkin et al. 1984, Swartz 1990 and

Rani et al. 1995).

Somaclonal variation can be associated with different types of genetic

changes such as the numerical and structural chromosomal changes, expansion

or contraction of the genetic regions, activation of transposable elements,

changes in DNA methylation patterns including, point mutations (Scowcroft

1984 and Phillips et al. 1990). Among these, the chromosomal rearrangements

resulting due to late or delayed replication of heterochromatin during cell

division phase of clonal multiplication steps are considered the most prevalent

(Rani and Raina 2000).

Chromosomal breakage in tissue culture is affected by breakdown of cell

cycle controls and is also induced by alteration in the methylation pattern

(Phillips et al. 1990, 1994). Single nucleotide changes arise due to deamination

of the methylated cytosine resulting in a C to T or G to A transition or due to

errors in the DNA replication or repair machinery (Bretell et al. 1986 and Dennis

et al. 1987). All these changes are interrelated events, which ultimately result in

the loss of normal cell controls, and hence lead to in vitro produced somaclonal

variations.

Since somaclonal variations are manifested in the form of morphological,

cytological or chemotypic changes, therefore, different marker systems such as

the morphological, cytological and biochemical markers have been used to

screen out these variants. However, of late, molecular markers (described in

section 1.3.1) are routinely used for the analysis of any somaclonal variations

that might have resulted during the micropropagation period. Table 1.5 provides

a list of micropropagated medicinal plants whose genetic uniformity was

assessed by using different molecular markers.

Shenoy and Vasil (1992), Kidwell and Osborn (1993), and Nelke et al. (1993)

have reported the successful use of RFLP to ascertain clonal fidelity of tissue-

cultured plantlets of Napier grass, alfalafa and red clover respectively. Molinari

et al. (2003) used RFLP technique for analysis of regenerated plants of

Paspalum simplex. An analysis of the regenerated plant‟s genome revealed an

absence of variation for apomixis controlling regions, whereas various degree of

variations were detected for DNA methylation sites of the same loci. Jaligot et al.


(2002) employed methylation sensitive RFLP for characterisation of oil palm

showing somaclonal variation associated polymorphism.

RAPD technique employs random primers for screening the genome and has

been used to detect the off types from micropropagated plants such as Panax

notoginseng (Shoyama et al. 1997), turmeric (Salvi et al. 2001) Lilium (Varshney

et al. 2001) and Musa species (Newbury et al. 2004).

In AFLP, DNA fragments are amplified using primers specific to adapters

that are ligated to these fragments. Vendrame et al. (1999) made comparisons

between and within embryogenic cell lines/ cultures (cultures derived from

single cells or small groups of similar cells) of pecan (Carya illinoinensis) using

AFLP technique. Variations were detected between different culture lines,

however, embryos derived from individual culture lines showed similar banding

profiles. Singh et al. (2002) availed AFLP markers for establishing clonal

fidelity of tissue culture-raised neem plants. Seven primer combinations

revealed a monomorphic pattern across the mother tree and its progeny.

Microsatellite, a class of repetitive DNA sequences, are known to be

abundant and hypervariable sequences and therefore an interesting tool to

investigate variation in sequences during tissue culture processes. Chowdari et

al. (1998) made an attempt to assess the effect of tissue culture and regeneration

on DNA variation at microsatellite loci in R2 progeny of callus derived rice

plants. The technique employed enabled successful monitoring of DNA changes

in successive generations of somaclonal variants. Palombi and Damiano (2002)

detected genetic variations induced in micropropagated plants of Actinidia

deliciosa by SSR markers. Primers designed on microsatellite sequences are

used for amplification in ISSR marker assay. Leroy et al. (2000, 2001),

employed ISSR markers for detecting in vitro generated genetic instability in

cauliflower.

Table 1.5 Molecular marker assisted studies for assessing the genetic fidelity of some of the tissue-cultured medicinal plants

Plant Molecular

assay

Comments Reference

Aegle marmelos Isozymes

assay

No somaclonal variation were

observed in the isozyme patterns on

the polyacrylamide gel

Ajithkumar and

Seeni 1998

Arnica montana Isozyme assay Isoesterase profile of the regenerants

and the mother plant was compared

Butiuc-Keul and

De 2001

Azadirachta indica AFLP No somaclonal variation detected Singh et al. 2002

Chlorophytum

arundinaceum

RAPD Five arbitrary decamers were

employed for assessing genetic

stability

Lattoo et al. 2006


Plant Molecular

assay

Comments Reference

Codonopsis

lanceolata

ISSR and

RAPD

15.7 and 24.6% genomic variation was

detected with ISSR and RAPD

markers, respectively in the 63

regenerants analysed

Guo et al. 2006a

Curcuma anada RAPD 10 random primers generated 103

scorable bands, including nine which

were absent in the control

Prakash et al.

2004

Curcuma longa

(Turmeric)

RAPD 16 decamer primers were used and no

polymorphism was detected

Salvi et al. 2001

Cymbopogon species RAPD Molecular diversity in relation to the

oil-chemotypic variations

Sangwan et al.

2001

Cymbopogon species RAPD The somalcones identified by RAPD

analysis were employed for selecting

elite somaclones

Nayak et al. 2003

Digitalis obscura RAPD Variations observed in banding profile

of mother plants and regenerants

derived from cryo-preserved tissue

Sales et al. 2001

Dioscorea floribunda RAPD Ten RAPD primers produced a total of

5120 bands of which only one was

polymoprhic, revealing genetic

stability

Ahuja et al. 2002b

Drosera anglica and

D. binata

RAPD 20 primers were used to screen 15

randomly selected plants of each

species. No variation was seen in D.

binata, however, 0.08%

polymorphism revealed in D. anglica

Iojkowska and

Kowiak 2004

Hybanthus

enneaspermus

RAPD No variation was observed among the

micropropagated plants vis-à-vis the

mother plants

Prakash et al.

2001

Hypericum

perforatum

Chromosome

count and

VNTR

No changes in chromosome numbers

detected but variations were detected

in the non-coding sequences

Urbanova et al.

2006

Hypericum

perforatum

RAPD Natural genetic variation, ascribed to

alloploid origin and apomictic mode of

reproduction in the plant, was detected

Haluskova and

Kosuth 2003

Mandevilla velutina Biochemical

analysis

Heterogeneity concerning

morphology, differentiation, carbon

assimilation was observed on the cell

cultures

Maraschin et al.

2002

Mentha arvensis RAPD 99.9% homogeneity in the regenerated

plants with respect to the mother

plants

Shasany et al.

1998

Myrtus communis DNA

methylation

patterns

analysed by

HPLC assay

No DNA methylation variations

observed among the micropropagated

and field transferred plants

Parra et al. 2001

Panax notoginseng RAPD Somaclonal variation among somatic Shoyama et al.


Plant Molecular

assay

Comments Reference

embryos 1997

Plumbago rosea and

Plumbago zeylanica

RAPD No polymorphism detected among the

regenerated plantlets

Rout 2002

Plumbago zeylanica RAPD 20 arbitrary primers were analysed

and revealed no polymorphisms

among the regenerated plants.

Rout and Das

2002

Rheum rhaponticum Leaf Leaf trichomes, stomatal characters

and epidermal subcellular features

were analysed for detecting variants

Zhao et al. 2006

Robinia ambigua ISSR 32 selected ISSR primers revealed

10.62% polymorphism in the 41

morphologically normal plants

analysed

Guo et al. 2006b

Tylophora indica RAPD 20 random primers were used in the

study and all scored bands were

monomorphic

Jayanthi and

Mandal 2001

Zinziber officinales RAPD 15 primers were used to screen the

progenies; no variation was detected

Rout et al. 1998

It is thus evident that different molecular markers provide good options of

analysing genetic uniformity of micropropagated plantlets, which can screen

different parts of the genome.

1.6 Biochemical analysis of medicinal plants

Biochemical analysis of medicinal plants is an essential prerequisite for

determining the phyto-chemical biodiversity of the target species. Large

variations can be found in the active principles of medicinal plants growing in

different geographical locations. Intra- and inter-population chemotypic

profiling of wild medicinal plants is very essential as it helps to define elites or

the better performers among the different samples collected. However, most of

the medicinal plants, excluding Papaver somniferum, P. bracteatum, Cinchona

spp. Digitalis lanata, Chamomilla recutita and Mentha piperita are still

genetically “wild” types (Tyler 1988, Chomchalow 1993). Also, biochemical

analysis of the micropropagated plants is recommended in establishing the

biochemical profile of the regenerants vis-à-vis the donor plant.

Several research groups have attempted to achieve the above objectives.

Eighteen populations of Hypericum perforatum were tested for morphological

as well as biochemical phenotypes for two consecutive years. In the study, a

hyperosid-rich chemo-variety with low rutin content was distinguished

involving two accessions of different origins. The study also determined three


kinds of hypericin production and accumulation tendencies during the three-

year cycle of the plant. Further, it was also established that harvesting with

flowering top is advantageous for obtaining maximum biochemical yield of this

herb. Kirakosyan et al. (2004) also analysed 15 genetically distinct populations

and 10 cultivars of Hypericum perforatum (also called St. John‟s wort) from

Armenia and North America to identify superior plant germplasm vis-à-vis

secondary metabolite content. The study concluded that the population from

Armenia accumulated higher levels of hyperforin where as the North American

samples were rich in hypercins. Chemical characterisation of basil (Javanmardi

et al. 2002) established drastic variations between different accessions analysed.

Unusual basil accessions were identified during the study and these can serve as

genetic resource for crop improvement.

Once the candidate elites have been established, their cultivation becomes a

commercially lucrative option as it renders greater control over quality and

supply of the raw material. Variation in phyto-chemical profile due to

environmental effects is a critical factor while considering the cultivation of

medicinal plants. The use of controlled environment can overcome cultivation

difficulties and can help to manipulate the phenotypic variations in bioactive

compounds. For instance, shade grown Mentha piperita was found to have

lower essential oil content (1.09 and 1.43%) and lower menthol content (57.5

and 61.8%) compared with Mentha piperita grown in direct sunlight

(McChesney 1999). Similarly, cool grown Papaver somniferum (poppy) contains

more morphine but has lower alkaloid content than the warm grown poppy

(McChesney 1999). Thus, an understanding of how environmental factors affect

phytochemical production will be of great importance towards optimising field

growth conditions for maximal recovery of the phyto-medicinal compounds.

In vitro studies conducted on medicinal plants such as Eclipta alba (Franca

et al. 1995) and Coleus forskohlli (Sharma et al. 1991) have established the utility

of tissue culture for larger scale production of medicinal plants with metabolite

profile similar to or at times comparably higher than that of the donor plants.

Micropropagated plants of Coleus forskohlii were considered to be potential

source of forskolin, because their metabolite content was found equal to wild

plants (Sharma et al. 1991). In Ocimum basilicum, Dwivedi et al. (2000)

observed that original profile of the in vitro lines was maintained after sub-

culturing for eight passages. In Corydalis ambigua different genotypes showed

variable metabolite profile, however they were not significantly different from

the corresponding alkaloid content in wild plants (Hiraoka et al. 2001). Tissue

culture raised plants of Mentha arvensis showed similar essential oil profile as


that of the parent plants (Phatak and Heble 2002). Leveille and Wilson (2002)

on analysis of tuber tissue of micropropagated Harpagophytum procumbens

detected iridoids at concentrations comparable with those found in wild plant

material. Castillo et al. (2002) reported similar valepotriate content in samples

of in vitro cultures in comparison to the roots and rhizomes of wild plants of

Valeriana edulis.

In Hyoscyamus niger, hyoscyamine and scopolamine was found in the roots

of the whole plant, however in callus cultures only hyoscyamine was detected

(Eemay et al. 2003). Sivakumar and Mukundan (2003) detected diterpene

glycosides by high-pressure thin layer chromatography (HPTLC) technique in

Stevia rebaudiana. Highest sweetener content was detected in callus cultures.

The above-mentioned studies indicate that the active metabolite content of

the micropropagated plantlets remains comparable to that of the plants growing

in the wild and hence corroborate the efficacy of the micropropagation

techniques for commercial production of medicinal plants. Murch et al. (2004)

identified 26 chemically distinct germplasm lines of Scutellaria baicalensis

(Huang-quin) in population derived from in vitro regeneration. Such studies

document the role of in vitro manipulation of medicinal plants combined with

metabolic screening for production of new germplasm.

Thus, medicinal plant biotechnology needs to design and implement a

holistic approach involving different technologies, as described above to achieve

the dual objective of conservation as well as sustainable utilisation and

management of the valuable resource.

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1 Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/6307/6/06...Review of Literature 1.1 Medicinal plants and their importance Medicinal plants are a precious