11

REVIEW OF LITERATURE

Medicinal plants as a group comprise approximately 8000 species and account for about

50% of all the higher flowering plant species of India. Millions of rural mass use medicinal

plants. In recent years the growing demand for herbal products has led to a quantum jump in

volume of plant material traded within and outside the country. Though India has rich

biodiversity and one among the twelve mega diversity centers, the growing demand is putting a

heavy strain on the existing resources causing a number of species to be either threatened or

endangered category (Sharma et al., 2010). About 90% of medicinal plants used by industries are

collected from the wild. While over 800 species are used in production by industry, less than 20

species of plants are under commercial cultivation. Over 70% of the plant collections involve

destructive harvesting because of the use of parts like roots, bark, wood, stem and the whole

plant in case of herbs. This poses a definite threat to the genetic stocks and to the diversity of

medicinal plants. Recently some rapid assessment of the threat status of medicinal plants using

International Union for Conservation of Nature (IUCN) designed ‘Conservation Assessment and

Management Plan’ (CAMP) methodology revealed that about 112 species in southern India, 74

species in Northern and Central India and 42 species in the high altitude of Himalayas are

threatened in the wild (Sharma et al., 2010).

In terms of the number of species individually targeted, the use of plants as medicines

represents by far the biggest human use of the natural world. Plants provide the predominant

ingredients of medicines in most medical traditions. There is no reliable figure for the total

number of medicinal plants on Earth, and numbers and percentages for countries and regions

vary greatly (Schippmann et al., 2002). Estimates for the numbers of species used medicinally

include: 35,000-70,000 or 53,000 worldwide (Schippmann et al., 2002); 10,000- 11,250 in China

(Xiao and Yong, 1998); 7500 in India (Shiva, 1996); 2237 in Mexico (Toledo, 1995); and 2572

traditionally by North American Indians (Moerman, 1998).

The United Nations Conference on Environment and Development (UNCED), held

recently at Rio de Janeiro, Brazil helped to place the loss of biodiversity and its conservation on

12

the global agenda. Resulting in biodiversity becoming a household wood. Biodiversity is a new

term for species-richness (plants, animals, microorganisms) occurring as an interacting biotic

component of an ecosystem in a given area (Sharma et al., 2010).

Micropropagation/Clonal propagation techniques using shoot tip and nodal segments are

must for mass-scale multiplication and conservation of an endangered or threatened and

medicinally important species within short period and limited space. The plants produced from

this method are true to type. Propagation through tissue culture provides solution for mass

propagation of plants in general and threatened plants in particular. There is a need to conserve

plants with medicinal values. Due to ever growing demand, the availability of medicinal plants to

the pharmaceutical companies is not enough to manufacture herbal medicines. The powerful

techniques of plant cell and tissue culture, recombinant DNA and bioprocessing technologies

have offered mankind a great opportunity to exploit the medicinal plants under in vitro

conditions (Sharma et al., 2010).

In clonal propagation, plants are multiplied using nodal segments and shoot meristems as

explants. For rapid in vitro clonal propagation of plants, normally dormant axillary buds are

induced to grow into multiple shoots by judicious use of growth regulators cytokinins and or

auxin and cytokinin combinations. Shoot number increases logarithmically with each subculture

to give greatly enhanced multiplication rates. As this method involves only organized meristems,

hence it allows recovery of genetically stable and true to type progenies (Murashige, 1974; Hu

and Wang, 1983).

For the regeneration of a whole plant from a cell or from a callus mass cytodiffrentiation

is not enough and there should be differentiation leading to organogenesis. This may occur

through shoot bud differentiation (organogenesis) or through somatic embryogenesis. In the

former, shoot buds (monopolar structures) are formed while in the later, somatic embryos

(bipolar structures) are formed both leading to regeneration of whole plant. Callus mediated

organogenesis depends on various factors. The type of callus, growth regulators used for

induction of callus and also callus developed from the type of explant. The cells, although

undifferentiated, contain all the genetic information present in parent plant. By suitable

13

manipulation of growth regulators and contents of the medium, it is possible to initiate the

development of roots, shoots and complete plant from callus cultures (Sharma et al., 2010).

Somatic embryogenesis is the process of formation of embryo like structure from somatic

tissue. The somatic embryo may be produced either directly on the explant or indirectly from

callus or cell suspension culture. For the first time, Haccius (1978) defined somatic

embryogenesis as a non-sexual developmental process, which produces a bipolar embryo from

somatic tissue. The first report of plantlet regeneration via in vitro somatic embryogenesis was in

Daucus carota (Reinert, 1958; Steward et al., 1958). This pathway has offered a great potential

for the production of plantlets and its biotechnological manipulation. In addition to the

development of somatic embryos from sporophytic cells, embryos have been induced from

generative cells such as in the classic work of Guha and Maheshwari (1964) with Datura innoxia

microspores.

Robbins (1922) seems to have been the first person to have successfully cultured

excised shoot tips on a medium containing sugar. Tip explants of between 1.75 and 3.75 mm

were taken from pea, corn and cotton, and placed in a liquid medium. For some reason the

cultures were maintained in the dark where they only produced shoots with small chlorotic

leaves and numerous roots. Although it is tempting to suppose that the potential of shoot culture

for plant propagation might have been appreciated at a much earlier date had the cultures been

transferred to the light, the rapid rate of shoot multiplication achieved in modern use of this

technique depends on later developments in plant science (George and Debergh, 2008).

Only very slow progress in shoot culture was made during the next 20 years. As part of

his pioneering work on plant tissue culture, White (1933) experimented with small meristem tips

(0.1 mm or less) of chickweed (Stellaria media), but they were only maintained in hanging drops

of nutrient solution. Leaf or flower primordia were observed to develop over a six-week period.

Shoot culture of a kind was also carried out by La Rue (1936). His explants largely consisted of

the basal and upper halves of seed embryos. Nevertheless, the apical plumular meristems of

several plants were grown to produce entire plants. Whole plants were also obtained from

axillary buds of the aquatic plant Radicula aquatica. Significant shoot growth from vegetative

14

shoot tip explants was first achieved by Loo, and reported in 1945 and 1946. Asparagus shoot

tips 5-10 mm in length were supported on glass wool over a liquid medium and later grown on a

solidified substrate.

Honours for establishing the principles of modern shoot culture must therefore be

shared between Loo and Ball. Ball (1946) was the first person to produce rooted shoots from

cultured shoot apices. His explants consisted of an apical meristem and 2–3 leaf primordia. There

was no shoot multiplication but plantlets of nasturtium (Tropaeolum majus) and white lupine

(Lupinus alba) were transferred to soil and grown successfully.

The two major developments which made shoot culture feasible were the development

of improved media for plant tissue culture (Murashige and Skoog, 1962) and the discovery of the

cytokinins as a class of plant growth regulators (Miller, 1961), with an ability to release lateral

buds from dormancy (Wickson and Thimann, 1958; Sachs and Thimann, 1964).

Haramaki (1971) described the rapid multiplication of Gloxinia by shoot culture and by

1972 several reports of successful micropropagation by this method had appeared (Adams, 1972;

Haramaki and Murashige, 1972). Since then the number of papers on shoot culture published

annually has increased dramatically and the method has been utilised increasingly for

commercial plant propagation (George and Debergh, 2008).

During dedifferentiation, storage products typically found in resting cells tend to

disappear. New meristems are formed in the tissue and these give rise to undifferentiated

parenchymatous cells without any of the structural order that was characteristic of the organ or

tissue from which they were derived. Although callus remains unorganised, as growth proceeds,

some kinds of specialised cells may again be formed. Such differentiation can appear to take

place at random, but may be associated with centres of morphogenesis, which can give rise to

organs such as roots, shoots and embryos. The de novo production of plants from unorganised

cultures is often referred to as plant regeneration (George, 2008).

15

Tissue culture technique has been used successfully for in vitro mass propagation of

various medicinal plants (Table 1).

Table 1. In vitro culture of some important medicinal plants.

Plant species Explants Nature of Response Reference

Bacopa monnieri Leaf explants &

Nodal segments

Mass propagation Mohapatra and Rath

(2005)

Calastrus paniculatus Nodal segments Shoot culture Sood & Chouhan

(2009)

Clitoria ternatea Linn Nodal segments Shoot culture Rout (2005)

Ginkgo biloba Apical & Nodal

segments

Shoot culture Tommasi &

Scaramuzzi (2004)

Glycyrrhiza glabra Nodal segments Axillary bud culture Vadodaria et al.

(2007)

Gymnema sylvestre Seeds Seed culture Komalavalli & Rao

(2000)

Holostemma ada-

kodien

Nodal segments Bud culture Martin (2002)

Oroxylum indicum Nodal segments Shoot culture Dalal & Rai (2004)

Picrorhiza kurroa Nodal segments Mass propagation Martin et al. (2006)

Saussurea lappa Shoot tip Shoot culture Johnson et al. (2007)

Swertia chirata Shoot tip Shoot culture Balaraju et al. (2009)

Tylophora indica Nodal segments Mass propagation Faisal et al. (2007),

Sharma & Chandel

(1992)

Tinospora cordifolia Nodal segments Mass propagation Gururaj et al (2007)

16

Although most experiments have been conducted with the tissues of higher plants, callus

cultures can be established from gymnosperms, ferns, mosses and thallophytes. Many parts of a

whole plant may have an ultimate potential to proliferate in vitro, but it is frequently found that

callus cultures are more easily established from some organs than others. Young meristematic

tissues are most suitable, but meristematic areas in older parts of a plant, such as the cambium,

can give rise to callus. The choice of tissues from which cultures can be started is greatest in

dicotyledonous species. A difference in the capacity of tissue to give rise to callus is particularly

apparent in monocotyledons. In most cereals, for example, callus growth can only be obtained

from organs such as zygotic embryos, germinating seeds, seed endosperm or the seedling

mesocotyl, and very young leaves or leaf sheaths, but so far never from mature leaf tissue (Green

and Phillips, 1975; Dunstan et al., 1978). In sugar cane, callus cultures can only be started from

young leaves or leaf bases, not from semi-mature or mature leaf blades (George, 2008).

Strains of callus differing in appearance, colour, degree of compaction and

morphogenetic potential commonly arise from a single explant. Sometimes the type of callus

obtained, its degree of cellular differentiation and its capacity to regenerate new plants, depend

upon the origin and age of the tissue chosen as an explant. Loosely packed or ‘friable’ callus is

usually selected for initiating suspension cultures (George, 2008).

Some of the differences between one strain of callus tissue and another can depend on

which genetic programme is functioning within the cells (epigenetic differences). Variability is

more likely when callus is derived from an explant composed of more than one kind of cell. For

this reason there is often merit in selecting small explants from only morphologically uniform

tissue, bearing in mind that a minimum size of explant is normally required to obtain callus

formation (George, 2008).

Auxin is produced by a large number of ectomycorrhizal fungi (Ek et al., 1983; Gay and

Debaud, 1987) and it has been hypothesized by Slankis as early as 1973 that fungal IAA could

be a signal molecule involved in mycorrhiza formation. Results obtained by Gea et al. (1994)

with IAA-overproducing mutants of Hebeloma cylindrosporum suggest that fungal IAA

17

modulates root morphogenesis during mycorrhiza formation. Considering that IAA

overproducing mutants form a hypertrophic Hartig net, these authors hypothesized that fungal

IAA favourably affects the physiology of the host root for the development of this interface,

which is essential for reciprocal nutrient exchange between plant and fungus.

The most commonly detected natural auxin is IAA; but endogenous occurrence of 4-

chloro-IAA (Engvild, 1985) and of indole-3-butyric acid (IBA) (Ludwig-Müller and Epstein,

1991) have also been demonstrated. Furthermore, the weak auxin phenylacetic acid (PAA)

occurs naturally in plants (Okamoto et al., 1967) and there are precursors and metabolites of IAA

present in plant tissues, like indole-3-pyruvic acid, tryptamine (Cooney and Nonhebel, 1991) or

tryptophol (Rayle and Purves, 1967; Percival et al., 1973).

Auxins exert an effect on DNA replication, while cytokinins seem to exert some control

over the events leading to mitosis (Jouanneau, 1971). Normal cell divisions require synchrony

between the S phase and cell division, suggesting that auxin and cytokinin levels in cultures need

to be carefully matched. Auxin starvation resulted in G2-arrest in tobacco cell suspension (Koens

et al., 1995). Activation of cell division is also coupled with activation of cdc 2, the main cell

cycle regulating kinase (John et al., 1993). Cells are thought not to enter mitosis unless cytokinin

is present (Machakova et al., 2008).

Auxins are known for their ability to promote adventitious root formation. This action is

definitely also coupled with stimulation of cell division – increased expression of cyclin B1 and

cdc 2 was observed well before the first cell division (Hemerly et al., 1993). Early stages of

lateral root formation are also regulated by polar auxin transport (Casimiro et al., 2001). Polar

transport of auxin is the decisive force of apical dominance (Cline, 1994).

Cells, which respond to auxin, revert to a dedifferentiated state and begin to divide. How

auxin brings about this reprogramming is understood only to a very limited extent. Lo Schiavo et

al. (1989) found that auxins cause DNA to become more methylated than usual and suggested

that this might be necessary for the re-programming of differentiated cells. Thus, tissue-specific

programmes specifically associated with differentiation would be eradicated by

18

hypermethylation, with perhaps a small fraction of the cells reaching an ultimate state of

dedifferentiation in which they become capable of morphogenesis, or embryogenesis (Terzi and

Lo Schiavo, 1990). A high rate of DNA methylation was found in the early somatic embryo

stage in cultures of Cucurbita pepo (Leljak-Levanic et al., 2004).

Irvine et al. (1983) reported having tested 79 potential regulants for their ability to initiate

callus from immature sugarcane leaf tissue. From the effective compounds, 96% had structures

known to be associated with auxin activity. Auxin-induced root formation is thought to require,

or induce, the promotion of polyamine synthesis (Friedman et al., 1985). Compared to callus

subcultured to media entirely free of auxin, even a low level of auxin delayed the appearance of

chlorophyll and shortened the period over which it accumulated (Sunderland and Wells, 1968).

Other workers have made similar observations. Increasing the concentration of IAA led to a

progressive reduction in chloroplast development within chicory callus (Wozny et al., 1973), but

in other tests IAA or NAA have been reckoned to be less inhibitory to chlorophyll formation

than 2,4-D (Davey et al., 1971).

Some investigators have employed mixtures of many different auxins (Blackmon et al.,

1981), but as the effect of individual compounds can vary in different genotypes, most

researchers prefer to use one or at most two compounds. However, a mixture of more than one

auxin can be particularly effective for root induction and a mixture of a synthetic auxin and IAA

has been found by many workers to be more effective than the synthetic compound on its own. A

mixture of 2,4-D (or 2,4,5- T) and IAA was found to promote embryogenic callus formation in

wheat, pearl millet and some varieties of rice (Nabors et al., 1983). Mixtures of auxins are also

more effective in inducing regeneration of wheat, barley and triticale (Przetakiewicz et al.,

2003). IAA and synthetic auxins such as NAA and 2,4-D are rapidly taken up into cultured

tissues. The compounds are subsequently absorbed into cells as whole molecules (via uptake

carrier or diffusion, see above), but dissociation then causes them to be retained within the cell,

because the plasmalemma is impermeable to auxin anions. IAA and NAA anions can be

exported only by the efflux carrier (Norris and Bukovak, 1972; Raven, 1979; Edwards and

Goldsmith, 1980; Minocha and Nissen, 1985; Minocha, 1987).

19

Different regeneration pathways such as direct, somatic embryos or callus-mediated shoot

regeneration were explored in different species to get optimum multiplication rate utilizing

different explants. Rooting and field establishment as a part of micropropagation protocol

development has been applied successfully without much difficulty. However, in Asparagus

recemosus in which in vitro rooting is difficult, a combination of several PGRs— NAA, KIN,

adenine sulfate, and phloroglucinol—has been included in one half strength MS medium to get

85% rooting (Nishritha and Sanjay 2008). However, the possibilities of pulse treatment with

auxins followed by in vitro or ex vitro rooting, as proven efficient in other recalcitrant species,

has not been well exploited in the medicinal plants of Western Ghats except in a few species

such as Celastrus paniculata (Gerald et al., 2006).

Although used in research, the natural cytokinins, 2-iP (6-((3-Methyl-2-

Butenyl)Amino)Purine) and zeatin are not used by commercial laboratories routinely, because of

their cost. Fortunately, several chemical analogues of natural cytokinins apart from KIN have

been prepared which are found to be highly active as cytokinins. Although they are chiefly N6-

substituted adenine derivatives, some other slightly less structurally-related compounds also

possess cytokinin activity, for example 4- alkylaminopteridines (Iwamura et al., 1980), and 6-

benzyloxypurines. Some of these analogues are reported to be more active than KIN or

benzyladenine (BA), and are particularly effective in promoting morphogenesis (Wilcox et al.,

1981). Although KIN is not yet accepted as a naturally occurring cytokinin, and thought to have

arisen in the original isolates by structural rearrangement (Hecht, 1980), many natural cytokinins

that are structurally related to KIN have been identified, either as free bases, as glucosides,

ribosides, or nucleotides (Entsch et al., 1980).

Dicotyledonous callus, or suspension cultures requiring auxin (e.g. 1 mg/l IAA) but not

cytokinin for growth, can be cultured for long periods without auxin when a high concentration

of cytokinin (e.g. 0.1-1 mg/l KIN) is added to the medium. At this level the cytokinin appears to

increase the natural auxin content of the tissues but not to cause auxinhabituation, because after a

prolonged period of culture, removal of the cytokinin again causes the cells to become auxin-

dependent (Syono and Furuya, 1972). In transformed tissue the expression of the gene coding for

iso-pentenyltransferase resulted in an increase of endogenous cytokinins and a parallel decrease

20

of endogenous IAA (Akiyoshi et al., 1983); similarly, the application of synthetic auxin NAA

led to a decrease of endogenous 2-iP and zeatin in tobacco cells (Vankova et al., 1992). In

contrast to this, reduction of the auxin concentration in the cultivation medium resulted in a very

significant increase of endogenous cytokinins, namely 2-iP and zeatin (Zazimalova et al., 1996).

It is apparent that not only auxins and cytokinins per se, but the levels of both hormones and

particularly the proportion of one to the other are determinants for cell cycle, cell division and

differentiation control.

In nearly all cases only low rates of cytokinin have been effective, for example, shoots of

sugar beet were rooted on MS medium containing 0.5 mg/l KIN and no auxin (Konwar and

Coutts, 1990). Boxus and Terzi (1988) advocated the addition of 0.5 mg/l KIN and auxin to the

rooting medium for strawberries and several woody plants, finding that at this concentration, the

cytokinin had a bacteriostatic effect and rooting was not impaired. Rosa hybrid ‘White Dream’

required the addition of 1 mg/l BA to IBA for root induction and development. BA promoted

axillary bud proliferation of Castanea in the experiments of Vieitez and Vieitez (1980), whereas

KIN was without effect. Elliott (1970) found KIN to be incapable of promoting the growth of

rose shoot tips. On the other hand, only 0.5-5 mg/l KIN (together with gibberellic acid) induced

the proliferation of potato shoots, and BA and 2-iP were not effective. BA gives a high rate of

shoot proliferation in Gerbera, but the best shoot quality is obtained using 5-10 mg/l KIN (Pierik

et al., 1982; Hempel et al., 1985). Benefits are often only noticed when adenine is administered

together with a cytokinin such as KIN, or BA. Adenosine and adenylic acid can sometimes act in

the same way as adenine (Skoog and Tsui, 1948; Nitsch et al., 1967) but they are generally even

less effective.

The addition of 0.1-50 mg/l ABA to media containing 2,4-D, adenine and KIN, increased

the number of embryos which grew from the globular stage to the heart-shaped stage in soybean

suspension and callus cultures (Phillips and Collins, 1981). KIN promoted ethylene evolution

because it prevented added IAA being converted rapidly into indoleacetylaspartic acid (Moshkov

et al., 2008). Thin cell layers of Nicotiana tabacum were found to produce 100 times more

ethylene on a root inducing medium (containing 10 mM IBA and 0.1 mM KIN) than tissues

cultured on other media (Moshkov et al., 2008).

21

Over-exposure to a regulant through excessive concentration, or prolonged treatment, can

result in a different developmental outcome to the one desired. The maximum number of

adventitious shoots from Datura innoxia internodes, was obtained when they were pre-incubated

with 1 M 2,4-D for 6 days (haploid plants) or 12 days (diploid plants), before transfer to a shoot

inducing medium (MS with 46.5 M KIN). Longer periods of pre-incubation resulted in less

shoots being produced: there were none at all after 36 days (Forche et al., 1981). Only two days

of incubation with 0.1-10 mg/l 2,4-D was necessary to increase the number of buds regenerated

from leaves of Prunus canescens (Antonelli and Druart, 1990).

A medium containing adenine sulphate in addition to KIN, which is conducive to shoot

formation in Nicotiana tabacum, was noted by Scott et al. (1964) to cause a marked increase in

the activities of two enzymes of the oxidative pentose phosphate pathway (glucose-6-phosphate

dehydrogenase and 6-phosphogluconate dehydrogenase), compared to their activities in a non-

shoot-forming medium. Ballade (1971) maintained that newly initiated root initials, arising from

single nodes of Nasturtium officinale, could be made to develop into shoot meristems by placing

a crystal of KIN on each explant which was then transferred to a medium containing 0.05%

glucose.

An observation by Murashige and Skoog (1962) that the presence of casein hydrolysate

allowed vigorous organ development over a broader range of IAA and KIN levels, may be of

significance. Pith phloem callus of tobacco proliferates on Zapata et al. (1983) MY1 medium

supplemented with 10–5 M IAA and 2.5 x 10–6 M KIN, but forms shoots on Murashige et al.

(1972) medium containing 10–5 M IAA and 10–5 M KIN. Tobacco pith callus grown on the

medium of Linsmaier and Skoog (1965) with l2 M IAA and 12 M KIN is wholly unorganised,

but if grown on the same medium supplemented with 600 M Ltyrosine (23), 80 M adenine

sulphate and 2.7 mM NaH2PO4.H2O, adventitious shoots are formed (Thorpe and Murashige,

1970; Murashige, 1961).

The growth of callus and the formation of adventitious organs from thin cell layers

excised from superficial tissues of the inflorescence rachis of Nicotiana, depended on the initial

22

pH of Linsmaier and Skoog (1965) medium containing 0.5 M IBA and 3 M KIN

(Mutaftschiev et al., 1987). Digby and Skoog (1966) discovered that normal callus cultures of

tobacco produced an adequate level of thiamine to support growth providing a relatively high

level of KIN (ca. 1 mg/l) was added to the medium, but the tissue failed to grow when moved to

a medium with less added KIN unless thiamine was provided. The use of 9.1 M dicamba

permitted the formation of wheat scutellar callus, which produced more somatic embryos in

conjunction with 2.6-4.7 M KIN, than that induced by the optimum rate of 2,4-D (3.6 M)

(Carman et al., 1988).

NPA prevented the growth of tobacco callus when incorporated into the medium at 200

M, but 2 - 20 M promoted growth in conjunction with IAA. The compound seemed to reduce

auxin activity or enhance that of cytokinin, because callus cultured with 200 M naptalam plus

12 M IAA and 2.5 M KIN initiated buds only when NPA was present (Feng and Linck, 1970).

TIBA alone improved callus formation and quality in cultures of pepper (Kaparakis and

Alderson, 2003).

Cultured without growth regulators, disks cut from the leaves of Begonia rex regenerated

a root at the base of the longest vein, but the presence of 5 mg/l KIN abolished the polarity of

organogenesis. Adventitious shoots then arose over the whole area of both sides of the leaf

(Schraudolf and Reinert, 1959). Auxin and cytokinin became essential additions to Linsmaier

and Skoog (1965) medium to produce bulblet, callus and root formation from explants cut from

the distal (and normally non-regenerative) part of Lilium longiflorum bulb scales (Dennis and

Ascher, 1976). Legrand (1972) noticed that the polarity of shoot regeneration from Endiva leaf

fragments was progressively reversed, the longer they were cultured in darkness.

Marcotrigiano and Stimart (1981) found that in the light, hypocotyls of Paulownia

required 3 mg/l IAA in the medium to produce shoots at the maximum rate, whereas under

continual darkness only 1 mg/l IAA was necessary. Cytokinin (KIN, 3 mg/l) was required at the

same concentration under both regimes.

23

Chemical synthesis of IAA

Chemical structure of IAA

IUPAC name: 2-(1H-indol-3-yl)acetic acid

The organic synthesis of IAA was submitted by Johnson and Crosby (1973). IAA has been

prepared by the Fischer indole synthesis, by hydrolysis of indoleacetonitrile, from the reaction of

gramine-type compounds with cyanide ion under conditions which hydrolyze the nitrile, by the

reaction of indole with ethyl diazoacetate followed by hydrolysis, through oxidation of

indolepyruvic acid, and by ultraviolet irradiation of tryptophan. In addition, the intermediate of

agrobacterial IAA biosynthesis, indole-3-acetamide, has been detected in plant tissues (Saotome

et al., 1993).

24

Commercially available preparations used for protoplast isolation are often mixtures of

enzymes from a fungal or bacterial source, and have pectinase, cellulase and/or hemicellulase

activity: they derive part of their effectiveness from being of mixed composition (Evans and

Cocking, 1977).

No chemical alternatives to the natural gibberellins or abscisic acid are available, but some

natural gibberellins are extracted from cultured fungi and are available for use as exogenous

regulants (Machakova et al., 2008). No plant appears to possess all of the gibberellins, some

have only been found in fungi and some only in higher plants; nor are the various gibberellins

equally active, some are precursors and some catabolites of active gibberellins (Moshkov et al.,

2008).

Most plant pathogenic bacteria produce IAA through indole-3-acetamide (IAM). In this

pathway, tryptophan is converted to IAM by tryptophan-2-monooxigenase (iaaM), and IAM is

metabolized to IAA by IAM-hydrolase (iaaH). The capacity to produce IAA through the IAM

pathway is associated with bacterial virulence and with gall formation. Cohen et al. (2002)

transformed two strains of Fusarium pathogenic to Orobanche by using the bacterial iaaM and

iaaH genes and showed that the transgenic isolates produced increased levels of IAA in axenic

cultures. Colletotrichum gloeosporioides f. sp. aeschynomene produces large amounts of IAA in

axenic cultures through the bacterial IAM pathway (Robinson et al., 1998).

The range of chemicals produced by endophytes is very diverse. Like their host plants,

they synthesis a wealth of secondary metabolites which are not directly involved in the

metabolism of the micro-organisms but play a role in the fitness and survival of themselves and

their hosts (Schulz et al., 2002; Tan and Zou, 2001). These functional metabolites include

alkaloids, terpenoids, steroids, quinones, isocoumarin derivatives, flavanoids, phenols and

phenolic acids, and peptides. Some species produce novel antimicrobial agents (e.g.

cryptocandin from Cryptosporiopsis quercina), other produce potent anti-cancer compounds

(e.g. taxol from Taxomyces andreanae) and yet others produce compounds that can be utilized

industrially, such as enzymes and solvents (Strobel et al., 2003). Mathew et al. (2010) reported

the presence of endophytic fungi as contaminant in plant tissue culture.

25

A successful tissue culture protocol starts with effective explant sterilization (Dodds and

Roberts, 1985). In this study a simple and fast protocol using commercial bleach (sodium

hypochlorite, NaOCl) was evaluated for explants sterilization and in vitro establishment in

comparison to mercuric chloride (HgCl2) which is mostly used in reported groundnut tissue

culture studies. Optimization of this protocol was an important aspect in any tissue culture

studies to ensure that large numbers of clean explants survived sterilization (Cheng et al., 1992;

Li et al., 1994; Kanyand et al., 1997; Venkatchalam et al., 1999; Sharma and Anjaiah, 2000).

The disinfectants widely used are sodium hypochlorite, calcium hypochlorite, ethanol,

mercuric chloride, hydrogen peroxide, silver nitrate and bromine water. Hypochlorite is known

to be a very effective killer of bacteria, even micromolar concentrations are enough to reduce

bacterial populations significantly. However, little is known about the exact mechanisms of its

bacteriocidal activity. When diluted in water the hypochlorite salts (NaOCl, Ca(OCl)2) lead to

the formation of HoCl whose concentration is correlated with bactericidal activity (Nakagarwara

et al., 1998). A balance between concentration and time must be determined empirically for each

type of explant because of phytotoxicity (Oyebanji et al., 2009).

Ethanol is a powerful sterilizing agent but also extremely phytotoxic. Therefore, the

explant is typically exposed to it for only a few seconds or minutes. Explants such as seeds or

dormant buds can be treated for longer periods of time since the tissue that will develop is

actually within the structure that is being surface sterilized (Oyebanji et al., 2009). To enhance

effectiveness in sterilization procedure, a surfactant like Tween 20 is frequently added to the

sterilizing solution (and in some laboratories a mild vacuum is applied during the procedure); in

general, the sterilizing solutions containing the explants are continously stirred during the

sterilization period (Oyebanji et al., 2009). Ethanol, in general, is used prior to treatment with

other compounds. The use of a two-step (two-source) sterilization procedure has proven

beneficial with certain species. Ethanol is usually combined with hypochlorite for effectiveness,

e.g. the use of 90 or 70% ethanol for 3 minute and sodium hypochlorite (3.5%) for about 30

minute (Oyebanji et al., 2009).

26

Despite the best timing and selection efforts it is almost impossible to eliminate

contamination from in vitro grown plants. In fact according to (Leifert et al., 1991) losses due to

contamination in vitro average between 3 and 15% at every subculture in the majority of

commercial and scientific plant tissue culture laboratories, the majority of which is caused by

fungal, yeast and bacterial contaminants (Leifert et al., 1994). Uninformed use of antimicrobial

chemicals (such as antibiotics) may cause phytotoxicity, retard explants growth and encourage

the build up of resistance. Furthermore, even though some antibiotics may give comparatively

high activity when tested on defined bacteriological media these results are not usually replicable

on the complex tissue culture media and so the expected results are usually elusive (Barrett and

Cassells 1994).

Plants growing in the external environment are invariably contaminated with micro-

organisms and pests. These contaminants are mainly confined to the outer surfaces of the plant,

although, some microbes and viruses may be systemic within the tissues (Cassells, 1997).

Explants are primarily taken from actively growing shoots from plants grown in protected

environments. There are tiny cracks and crevasses in plants in which fungal spores and bacteria

can exist (Preece, 2008).

Methods are available to free plants from specific virus diseases. Providing these

techniques are employed, or virus-tested material is used for initiating cultures, certified virus-

tested plants can be produced in large numbers. Terminology such as virus-free and bacteria-free

should not be used, as it is impossible to prove that a plant is free of all bacteria or viruses. One

can only prove that a plant has been freed from a specific contaminant provided the appropriate

diagnostic tools are available (George and Debergh, 2008).

Because they are started from small explants and must be grown on nutritive media that

are also favourable for the growth of microorganisms, plant tissue cultures must usually be

established and maintained in aseptic conditions. Most kinds of microbial organism, and in

particular bacteria and fungi, compete adversely with plant material growing in vitro. Therefore,

as far as possible, explants must be free from microbial contaminants when they are first placed

on a nutrient medium. This usually involves growing stock plants in ways that will minimise

27

infection, treating the plant material with disinfecting chemicals to kill superficial microbes, and

sterilising the tools used for dissection and the vessels and media in which cultures are grown

(Cassells and Doyle, 2005).

Success at this stage firstly requires that explants should be transferred to the cultural

environment, free from obvious microbial contaminants; and that this should be followed by

some kind of growth (e.g. growth of a shoot tip, or formation of callus). Usually a batch of

explants is transferred to culture at the same time. After a short period of incubation, any

container found to have contaminated explants or medium is discarded. Stage I would be

regarded as satisfactorily completed if an adequate number of explants had survived without

contamination, and was growing on. The objective is reproducibility, not 100% success (George

and Debergh, 2008). Using stem segments with dormant axillary buds of Pyrus calleryana, Rossi

et al. (1991) used a concentration of 3% sodium hypochlorite for 20 minutes and only had a 20%

rate of contamination.

Root cultures may also be used to grow beneficial mycorrhizal fungi, and to study the

process of root nodulation with nitrogen-fixing Rhizobium bacteria in leguminous plants. For the

latter purpose, various special adaptations of standard techniques have been adopted to allow

roots to become established in a nitrate-free medium (Raggio et al., 1957; Torrey, 1963).

Separated cells from leaf tissue of tobacco preinfected with Tobacco Mosaic Virus have been

used to study the formation of viral RNA’s in the infected cells, and for studies on the interaction

between leaf tissue cells and elicitor chemicals produced by fungal pathogens (Dow and Callow,

1979).

A major detriment to using dormant buds as explants is the occurrence of microbes

within the buds. The outer scales of dormant buds of Acer, Aesculus, Betula, Fagus, Populus,

Quercus, and Ulmus were shown to host populations of fungi and bacteria (Warren, 1976).

Contaminants grow more rapidly on media containing amino acids. Casein hydrolysate is

therefore sometimes added to the media for Stage I shoot cultures so that infected explants can

be rejected quickly (Schulze, 1988). The health of shoots grown from seedling shoot tips of

28

Feijoa (Acca) sellowiana was improved when 500 mg/l CH was added to Boxus (1974) medium

(which does not contain ammonium ions) (Bhojwani et al., 1987).

Damm et al. (2009) characterise known and new species and designate epitypes to

provide the basis for accurate identifications of Colletotrichum species. This goal has been

achieved for species with curved conidia from herbaceous hosts (Damm et al., 2009), which in

the past were mostly identified as Colletotrichum dematium. Multi-gene analyses and

morphological characterisation revealed several diverse and distantly related species, including

four new species. Seven species were epitypified, including Colletotrichum dematium and the

type species of the genus, Colletotrichum lineola. A second study confirmed most of the

previously recognised groups (Sreenivasaprasad and Talhinhas 2005) within the Colletotrichum

acutatum species complex. Most of these could be defined on the basis of type strains or strains

suitable for epitypification. Literature reports (Lubbe et al., 2004, Johnston et al., 2005) and

preliminary studies using ITS sequence data indicated that Colletotrichum boninense represents a

species complex as well. A multi-locus molecular phylogenetic analysis of strains previously

identified as Colletotrichum boninense resulted in clades that could be recognised as separate

species with differences in host range, distribution and morphology, including Colletotrichum

boninense sensu stricto, Glomerella phyllanthi, Colletotrichum hippeastri and several

presumably new species. Most of the species in the Colletotrichum boninense complex and some

in the Colletotrichum acutatum species complex form teleomorph states in culture (Damm et al.,

2009).

Fruit rots (anthracnose) were previously often attributed to Colletotrichum

gloeosporioides and Colletotrichum acutatum. Identifications were, however, based on

morphological characters or, if gene sequence data were used, comparisons were often made

with wrongly applied names. Colletotrichum gloeosporioides was epitypified (Cannon et al.,

2008) so that living cultures and sequence data are, available for comparison with fresh

collections. Analysis of sequence data of 25 isolates (selected from 140 obtained strains based on

diversity of host and morphology) from eight tropical fruits are compared with the

Colletotrichum gloeosporioides epitype. Contrary to previous assumptions, none of these isolates

from tropical fruits was Colletotrichum gloeosporioides sensu stricto (Phoulivong et al., 2010).

29

The five gene regions used in this study resolved Colletotrichum asianum, Colletotrichum

fructicola, Colletotrichum horii, Colletotrichum kahawae and Colletotrichum gloeosporioides in

the Colletotrichum gloeosporioides species complex as distinct phylogenetic lineages with high

statistical support. Many tested strains could not be assigned to any known taxa in this analysis.

They also reported Colletotrichum species from Amaryllidaceae, Orchidaceae, Cordyline

fruticosa and Jasminum sambac, with the latter including two new species (Wikee et al., 2010),

and updated the typifications of Colletotrichum coccodes, Colletotrichum falcatum and

Colletotrichum musae.

The genome analysis of Colletotrichum orbiculare is now going to be completed, which

allows comparisons of genomic data of other Colletotrichum species, Colletotrichum

gloeosporioides, Colletotrichum higginsianum, Colletotrichum graminicola and Colletotrichum

orbiculare that belong to different phylogenetic clades within the genus. These data will provide

comprehensive basis for studying the biology of the different Colletotrichum species (Damm et

al., 2010).

Colletotrichum graminicola is a destructive pathogen of maize, causing stalk rot and leaf

blight, while Colletotrichum higginsianum attacks many cultivated forms of Brassica as well as

Arabidopsis thaliana, providing a model pathosystem in which both partners can be genetically

manipulated (O’Connell et al., 2004). Colletotrichum orbiculare is an anthracnose fungus

which infects Cucurbitaceae (Damm et al., 2010).

Fusarium is a vast genus of 78 species of ubiquitous fungi which includes plant

pathogens, saprophytes, and endophytes. Fusarium lateritium Nees (Gibberella baccata (Wallr.)

Sacc.) is the main species in the section lateritium and has been reported on numerous hosts,

mainly woody and fruit trees as well as shrubs and plants, where it causes wilt, tip or branch

dieback, and cankers. Results obtained with pathogenicity tests of Fusarium lateritium (Belisario

and Santori, 2009, Santori and Belisario, 2008, Santori et al., 2010) supported the speculation

that isolates of the pathogen obtained from hazelnut twig cankers and from nut gray necrosis

(NGN)-infected fruit might represent a homogeneous morphogroup within Fusarium lateritium

that was adapted to the host. This speculation was rooted in the observation that Fusarium

30

lateritium isolates obtained from hazelnut caused the typical NGN symptoms when inoculated on

nuts coupled with an extremely abundant production of sporodochia on the colonized nuts.

Conversely, Fusarium lateritium isolates from other hosts were able to produce only a brown

necrosis on the shell of Corylus avellana nuts with a few sporodochia, without penetrating

inward (Belisario and Santori, 2009). Traditionally, distinctive features of conidia and

conidiation as well as morphology, growth rate, and color of colonies are informative for species

identification within the genus Fusarium (Clark et al., 1990). However, members of the genus

Fusarium are commonly accepted to be difficult to identify at the species level if simply relying

on morphological traits.

Molecular techniques have made a significant impact on fungal species identification as

well as on phylogenetic and taxonomic studies, including the differentiation of intraspecific

groupings (Mbofung et al., 2007, Nitschke et al., 2009) or between very closely related species

(Hong et al., 2006). These techniques include dominant and co-dominant highly variable

molecular markers such as random amplified polymorphic DNA (RAPD), inter-simple-sequence

repeats (ISSRs), and sequence data from a number of DNA regions. In addition, multiple gene

genealogies have been used and sequence analyses of nuclear ribosomal DNA (nrDNA), -

tubulin ( -tubulin), or translation elongation factor 1 (TEF-1 ) have been broadly considered

reliable in species or subspecies identification as well as in the vast Fusarium genus (Nitschke

et al., 2009, Summerell et al., 2003). Hence, the molecular data, when combined with

morphological and biological features, allow a more robust identification of unresolved taxa

(Alves et al., 2008, McKay et al., 2009, Nitschke et al., 2009).

Various studies refer to morphological and molecular characterizations of several

Fusarium sp. but limited information is available on Fusarium lateritium. This species has not

been fully resolved and it is considered to be a species complex which might contain several taxa

that need to be fully characterized. Previous characterizations based on RAPD and restriction

fragment length polymorphism (RFLP) of intergenic spacer (IGS) and internal transcribed spacer

(ITS) regions of nrDNA have revealed little genetic variation in isolates from sweet potato

infected in comparison with isolates from other hosts (Hyun and Clark, 1998). More recently,

Geiser et al. (2005) carried out a phylogenetic analysis within Fusarium section lateritium on

31

sequenced portions of -tubulin and TEF-1 genes to resolve Fusarium xylarioides (teleomorph

Gibberella xylarioides), the causal agent of coffee wilt, from Fusarium lateritium or Fusarium

stilboides. Similarly to Fusarium avenaceum, Fusarium lateritium can be considered to be a

multiple phylogenetic species for its cosmopolitan nature as well as for its diverse host range

(Nalim et al., 2009).

Gibberellic acid was found to be synthesized by the fungus Gibberella fujikuroi

(Fusarium moniliforme) (Borrow et al., 1995) as well as by Agaricus bisporus (Pegg, 1973),

Aspergillus ochraceus, Penicillium funiculosum (Selim et al., 1985), Sphaceloma monihoticola,

Sphaceloma menthae, Sphaceloma perseae, Sphaceloma rhois and Sphaceloma bidentis

(Rademacher, 1992) The metabolic parameters of Gibberella fujikuroi have been observed to

change at different growth temperatures. While this organism has been shown to have an

optimum of 28°C for growth (Pastrana et al., 1993), a closely related unidentified Fusarium has

been found to grow optimally at 35°C (Righelato et al., 1976). The optimal pH ranges reported

for the growth of white-rot fungi vary from species to species, and sometimes different pH

optima have been reported for the same species, some having a wide range of pH tolerance than

others. For instance, maximum growth has been observed at pH 7–7.5 in Penicillium

chrysogenum (Pirt and Callow 1960), while Gibberella fujikuroi has a wider pH range, pH 4–7

(Borrow et al., 1964). Studies related to pH dependence (besides other culture conditions such as

temperature medium, light, etc.) have shown that the production of plant growth hormones, such

as auxin, is favoured under alkaline conditions (Witztum et al., 1978, Nowak, 1979, Zieslin and

Geller 1983, Toyomasu et al., 1993, Sandberg et al., 1993).

Furthermore, the disease symptoms caused by some fungal pathogens are similar to the

symptoms caused by high IAA concentrations and include epinasty, tumor formation, and plant

organ deformation (Tudzynski and Sharon, 2002). CgOpt1, a putative oligopeptide transporter

from Colletotrichum gloeosporioides, that is involved in responses to IAA was identified and

characterized (Veronique et al., 2009). Patten and Glick (2002) suggest that bacterial IAA plays

a major role in the development of the host plant root system.

32

Colletotrichum graminicola strain M1.001 was collected from infected maize (Forgey et

al., 1978). This strain was selected for sequencing at Broad because it is the most commonly

used lab strain, and it is easily manipulated genetically. This strain is very aggressive on both

maize leaves and stalks. It can be transformed, and crosses as both a male and a female with

most other field strains tested (Vaillancourt and Hanau, 1991).

Colletotrichum graminicola strain M5.001 was collected from infected maize. The

genome of this strain was sequenced to about 1X coverage. M5.001 is similar to M1.001 in its

level of aggressiveness to maize leaves and stalks, and it can also be transformed relatively

easily. It is fertile as both a male and female in crosses with M1.001 (Vaillancourt and Hanau,

1991).

The Colletotrichum higginsianum isolate selected for sequencing is IMI 349063 from

the CABI (Centre for Agricultural Bioscience International) Culture Collection, which was

originally isolated from Brassica rapa (pak-choi). This isolate was chosen because a wealth of

genomic resources was already available for this genotype, including large EST collections,

proteomic data and random insertional mutants. This isolate appears to be asexual (O'Connell et

al., 2004).

Fungal taxonomy is traditionally based on comparative morphological features (Lodge et

al., 1996; Sette et al., 2006; Crous et al., 2007; Zhang et al., 2008). However, special caution

should be taken when closely related or morphologically similar endophytes are identified,

because the morphological characteristics of some fungi are medium dependent and cultural

conditions can substantially affect vegetative and sexual compatibility (Zhang et al., 2006; Hyde

and Soytong, 2007). Furthermore, the conventional methods cannot be applied for identifying

fungal isolates that fail to sporulate in culture, which are categorized as mycelia sterilia (Lacap et

al., 2003). Various optimization of growth conditions have been used to promote sporulation of

these fungi, such as different culture media, potato dextrose agar (PDA), malt extract agar

(MEA), corn meal agar (CMA), potato carrot agar (PCA), and water agar (WA), as well as the

inclusion of host tissues in plate cultures (Guo et al., 2000). Nevertheless, a large number of

33

fungi still do not sporulate in culture, and these mycelia sterilia are considerably frequent in the

endophyte studies (Lacap et al., 2003).

In contrast, molecular techniques exhibit high sensitivity and specificity for identifying

microorganisms and can be used for classifying microbial strains at diverse hierarchical

taxonomic levels (Sette et al., 2006). Several recent studies have shown that genetic methods can

be successfully used in the studies of endophytic fungi (Gao et al., 2005; Wang et al., 2005;

Arnold et al., 2007; Ligrone et al., 2007; Sanchez Marquez et al., 2007; Morakotkarn et al.,

2007). Most of the endophytic fungi were detected and identified by comparative analyses of the

ribosomal DNA sequences, especially the ITS region. For example, Harney et al. (1997)

identified arbuscular mycorrhizal fungi from Artemisia californica using the ITS region. Also

based on ribosomal DNA sequences, Guo et al. (2000) and Lacap et al. (2003) evaluated the

endophytic fungal ‘morphotype’ concept concerning mycelia sterilia. A high diversity of

endophytic fungal communities was revealed from either Heterosmilax japonica or Livistona

chinensis using a cultivation-independent approach by analyzing fungal DNA sequences

extracted from plant tissues (Guo et al., 2001). Ganley et al. (2004) studied morphologically

similar endophytes and parasites based on the ITS region, and found that the endophytic fungi in

west white pine were actually most closely related to, but distinct from, the parasites. Peintner et

al. (2003) first recorded ectomycorrhizal Cortinarius species from tropical India and established

their phylogenetic position using ITS sequences. Queloz et al. (2005) monitored the spatial and

temporal dynamics of the tree-root endophyte Phialocephala fortinii using the restriction

fragment length polymorphism (RFLP) analysis.

A more precise assessment of diversity and identification of fungi can be achieved using

the polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) of

the ITS of the fungal nuclear ribosomal DNA repeat (Egger, 1995). The PCR-RFLP technique

with specific oligonucleotide primers ITS 1 & ITS 4 (White et al., 1990) and restricting with

different endonucleases, has been successfully used to analyze regions of ribosomal DNA of

various groups of fungi (Gomes et al., 1999; Glen et al., 2001; Cai et al., 2006; Shenoy et al.,

2007; Hyde and Soytong, 2008) and can be considered as a useful tool. Sequence analysis of the

ITS region of nuclear ribosomal DNA has been widely used for molecular identification and

34

phylogenetic diversity studies of fungi. Their have also been some studies on the phylogeny and

diversity of endophytes isolated from inner bark and roots of medicinal plants using molecular

methods, especially in the identification of morphospecies (Lacap et al., 2003; Promputtha et al.,

2005). Earlier studies from our laboratory have shown that Pestalotiopsis is one of the most

dominant genera in Azadirachta indica, Holarrhena antidysenterica, Terminalia arjuna and

Terminalia chebula (Mahesh et al., 2005; Tejesvi et al., 2005, 2007).

These fungi occur in most parts of the world and survive for many years in the soil (Nasr

Esfahani and Ansari Pour, 2008). The isolation of pure DNA is crucial for the study of gene

expression in these filamentous fungi, because it is a pre-requisite for several molecular biology

techniques, including gene isolation by polymerase chain reaction (PCR), Southern blotting, and

the construction of genomic DNA libraries (Gonzalez-Mendoza et al., 2010). However, DNA

extraction from these filamentous fungi has been described as being rather complicated, because

most of the available protocols include the growth of mycelium in liquid culture, followed by

maceration in liquid nitrogen, and usually require additional lysis steps, such as mechanical

disruption or sonication, enzymatic digestion or use of toxic chemicals (Al-Samarrai and

Schmid, 2000; Alaey et al., 2005). Additionally, although some methods do not involve

maceration in liquid nitrogen, they are still time consuming and require special columns (Noor

Adila et al., 2007). A number of protocols have been established for fungal DNA. However,

many of these protocols are apparently suitable for certain groups or morphological forms of

fungi but may not be versatile and efficient for extracting nucleic acids from diverse groups of

filamentous fungi (Raeder and Broda, 1985; Bolano et al., 2001).

When the first large-scale phylogenetic studies based on 18S sequences were published -

first and foremost Field et al. (1988) phylogeny of the animal kingdom - the gene was celebrated

as the prime candidate for reconstructing the metazoan tree of life. And in fact, 18S sequences

later provided evidence for the splitting of Ecdysozoa and Lophotrochozoa (Aguinaldo et al.,

1997; Halanych, 2004), thus contributing to the most recent revolutionary change in our

understanding of metazoan relationships. Methodological innovation within the last years came

from the incorporation of secondary structure into phylogenetic analyses. In particular RNA

specific substitution models considering paired sites in rRNA genes have been shown to

35

outperform standard DNA models (Telford et al., 2005; von Reumont et al., 2009; Tsagkogeorga

et al., 2009; Jow et al., 2002; Dohrmann et al., 2008).

Although IAA production by several fungi was reported (Arshad and Frankenberger,

1991; Costacurta and Vanderleyden, 1995), there is no report on utilizing the fungal IAA in plant

cell cultures. The present study was therefore undertaken to study the effect of IAA extracted

from fungal contaminants and to investigate its effects on plant tissue culture of Alternanthera

sessilis and Scoparia dulcis. With this background, this study was also aimed for successful

propagation of Alternanthera sessilis and Scoparia dulcis a famine food plant under in vitro

conditions. Intensive studies for the occurrence of microbial contaminants are available only for

bacteria (Thomas et al., 2008; Luna et al., 2008), but diversity of fungal contaminants in plant

tissue culture has not been studied much. Furthermore, this study provides information roundly

on morphological identification and laid the foundation to further exploring molecular

techniques in fungi identification.

36

OBJECTIVES

i. In vitro callus induction and plant regeneration of Alternanthera sessilis and Scoparia

dulcis.

ii. To study the diversity of fungal contaminants from in vitro cultures.

iii. Screening, extraction and quantification of IAA from fungal contaminants and its

application in plant tissue cultures.