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REVIEW OF LITERATURE 11

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2. REVIEW OF LITERATURE ' -3.t .. ~ - .._ ,,::> -'

2 ' 2.1 Conservation attempts on Adenia hondala and ~ a l i ~ j i r ~ i l i i ~ - i i i o n t a n u m .

Attempts have been made to conserve Adenia hondala and Baliospermum

montanzrm through in vitro propagation and tq~ restore them in the natural habitat. Nodal

segments were used as explants for in virro multiplication in Adenia hondala

(Antonisamy and Manickam, 1997).

Micropropagation of Baliospermum inonranum was achieved on Murashige and

Skoog's (MS) medium augmented with Bf\P using nodal segements. The maximum

number of shoots (3.4+0.25) was found in MS fortified with BAP (3.10pM). In vitro

raised shoots were rooted at half strength MS medium augmented with various

concentrations and combinations of auxin: namely IAA, IBA, NAA. The maximum

number of roots was observed on half strength MS medium fortified with IBA (9.84

pM) combined with NAA (5.37 pM) (Johnson and Manickam, 2003).

The ethanolic extract, chlorofonn s~>luble and insoluble fractions of the roots of

Baliospermum moiztanum showed significantly effective analgesic activity (Nayak et

al., 2003).

2 . 2 Physiochemical studies

Phytochemical studies were conducted on Baliospermum nloiztanum. When

screened for the presence of steroids, terpenoids, alkaloids, flavanoids and saponins,

leaves showed the presence of steroids, terpenoids and saponins (Mukerjee and Roy,

1980).

Tetraphyllin B and Epi-tetraphy1,in - two cyanogenic glycosides have been

isolated from the Kenyan plant, Adenia volkensii. The structure was established by

NMR spectra and GLC (Gondwe et al., 1978).

REVIEW OF LITERATURE

Modeccin, the toxin of Adenia digitata, was purified from the roots of this plant

by affinity chromatography on Sepharose 4 B. Modeccin inhibits protein synthesis in in

vitro in a lysate of rabbit reticulocytes and in Ehrlich ascites cells, and its effect on cells

is decreased in the presence of Lactose (Anna Gasperi-campani, 1978).

A fatty acid, found as a minor component in the seed oil of Baliospermum

axillare is shown to be 11,13 diydroxytetracos-trans-9-enoic acid (axillarinic acid) by

the studies of Shahid Hussain et al. (1980).

2 .3 Conservation of Rare and Endangered Plants using in vitro methods.

Nodal segments and shoot tips of Dicfyospennum ovalifolium (Commelinaceae)

were used as explants for in vitro culture and establishment of this rare endemic or

threatened plant species. The best results of shoot proliferation were obtained on MS

medium supplemented with 3 r n g ~ - ' BAP. The in vitro rooting of shoots was induced

after 4 weeks on MS medium supplemented with 1 m g ~ - ' IAA and 2 m g ~ . ' IBA.

Rooted plants were successfully transferred to their original habitat (Thoyajaksha and

Ravishankar, 2001).

Multiple shoots were induced from 6-8 week old shoot of Calophyllum

aperallum, an endemic medicinal tree of Western Ghats in MS medium containing 8.8

pM BAP. The shoot cultures were transferred to half MS basal medium for 4 weeks to

induce the elongation of shoots. Rooting of the microshoots was achieved when

cultured in quarter strength MS medium supplemented with 9.8 pM IBA for 4 weeks

followed by transfer to quarter MS basal medium for 4 weeks. The rooted plants were

transferred to clay pots filled with soil, sand and fannyard manure (1:l:l) watered in a

mist chamber at a relative humidity of 80-90% (Lakshmi and Seeni, 2003).

Drosera indicrr L. is a threatened and endangered insectivorous plant. First

successful protocol for D. indica L. was established using stem segments as explants.


Multiple shoots were obtained in MS supp,emented with 5x10" M BAP and 2x10.~

NAA. Rooting was obtained in 2x10.~ M NAA (Nalini and Murali, 2002).

An efficient protocol was developed in a rare and medicinal plant Psoralea

corylifolia for inducing callus and plant regeneration using petiole explants. Maximum

percentage of shoot bud formation (82.2%) was obtained on MS medium fortified with

the combination of BAP (3 mgL-') and IAA (1.0 mgL-') 85% rooting was noticed in 1

mgL.' IBA (Jeyakumar and Jayabalan, 2001).

In Agave-victoriae-regina, an endmgered species, somatic embryos were

produced through a callus phase from seedling stem segments cultured on MS with BA.

Embryos and shoots developed on MS medium. A total of 92% survival was observed

(Aaligendro Martinez - Palacios et al., 2003 I.

A micropropagation method has been developed for multiplication and

conservation of Atropa acuminata by indwtion of axillary shoot proliferation from

shoot tips and nodal explants using Murashige and Skoog (MS) medium supplemented

with BAP (1 rng~- ' ) and IBA (1 rngLe'). Rooting became high in full strength RT

(Revised tobacco) medium containing IBP, (1 mgL.') In vitro raised plantlets were

hardened and transferred to soil. (Ashok Ahuja et al., 2002).

Eremostachys superba Royle ex Bt:nth is an endangered species of potential

ornamental value. A protocol has been rleveloped to micropropagate E. superba.

Excised embryos could be germinated on MS basal medium. Excision of roots was

necessary to promote growth of plumule. lvlultiple shoots could be induced in cultures

of shoot tips as well as nodal segments on IVIS medium containing BAP and IAA. The

shoot were rooted in IBA and plantlets were hardened and transferred to soil in the open

(Sunnichan and Shivanna, 1998).


Rapid micropropagation of Trichopus zeylanicus Gaertn. sub sp. travencoricus,

a rare ethnomedicinal herb endemic to Western Ghats of southern India was achieved

by culturing shoot tips (0.3 - 0.5 cm) of two months old axenic seedling on woody plant

medium. Only BAP induced callus free multiple shoot bud formation. The shoots

developed from bud cultures were rooted in '/z WPM medium with 0.5 m g ~ - ' each of

NAA and IBA and 90 -100% of the rooted plants were established in the field after

hardening (Krishanan et al., 1995).

On MS medium with a combination of 13.2 pM BAP and 2.68 pM NAA, shoot

tips and single node explants from young shoots of RauvoZjia micrantha Hook., a rare

medicinal plant, induced shoots at high frequency (77%). Up to 3 shoots were obtained

from each node in 8 weeks. The excised shoots rooted on medium containing 2.6 pM

NAA, were hardened and established in pots (Sudha and Seeni, 1996).

Joshi and Dhar (2003) reported micro propagation protocol for Saussurea

obvallata (DC), a rare, threatened and near endemic medicinal herb of the Indian

Himalayan region. Multiple shoots were formed from epicotyl explants on MS medium

supplemented with 1.0 pM Kn and 0.25 pM NAA. Rooting was observed in half MS

supplemented with 2.5 pM IBA. The plantlets were transferred to ex-vitro conditions.

Following 15 days in vitro rooting period and 12 day of ex-vitro acclimatization 66.7%

of the plantlets had established in the field.

Efficient protocols of axillary bud multiplication and indirect organogenesis

were established for Holostemma ada-kodien Schult. (Asclepiadaceae) in MS medium

supplemented with 2 m g ~ . ' BAP and 0.5 m g ~ . ' IBA. An average of eight shoots per

node were induced and it was the best for axillary bud proliferation. More than 15

shoots differentiated from callus developed from nodal explants in MS medium fortified

with 1.5 m g ~ . ' BAP. Half strength solid MS or Liquid medium with 0.05 m g ~ ~ ' IBA


exhibited the best in vitro rooting. 90% oi the rooted shoots survived in the field.

(Martin, 2002)

Nepenthes khasiana (Nepenthaceae), a carnivoms pitcher plant from northern

India has been successfully propagated from lateral buds at the north Eastern Hill

University in India. Two thousand plantlets have been reintroduced to natural habitats

(Tandon et al., 1990).

2.4 Plant Tissue Culture

2.4.1 Culture medium

Whites medium is one of the earliest plant tissue culture media originally

formulated for root culture. To induce organogenesis and regeneration of plants in

cultured tissue MS (Murashige and Skooj:, 1962) and SH (Schenk and Hildebrandt

1972) media containing the desired salt copcentration are widely used. B5 medium was

proved valuable for cell suspension or c~ l lus cultures. Nitsch medium (Nitsch and

Nitsch, 1969) is frequently used for anther culture and culture of soybean, red clover

and other legume species. Nutritional coniponent of this medium supplemented rapid

growth of cells for embryogenesis and in protoplast culture. Success in employing these

various media in all probability lies in the fact that the ratios as well as the

concentration of nutrients nearly matter the optimum requirements with regard to the

growth and differentiation of respective cell or tissue systems. The percentage of bud

breaks and shoot multiplication were generally higher in MS medium compared to

several other media. Chen et al. (1995) 1,eported that the multiplication of Eucommia

colmoides was higher in MS basal medium than in WPM (Woody Plant Medium).

2.4.2 Role of Plant Growth Regulators

The influence of plant growth regulators and their interactions on

micropropagation of different plant species have been discussed in detail by Mor and


Zieslin (1987). Rout et al. (1988) and Rout and Das (1997 a, b). Rout et al. (2000)

reported the effect of auxins and cytokinins on the shoot multiplication of various

medicinal plants. Cytokinin levels were shown to be the most critical for the

multiplication of many medicinal plants (Rout et al., 2000).

Cytokinins promote cell division and cell expansion in plant tissue culture.

Growth is stimulated by adding a cytokinin to a proliferation medium for the

micropropagation of temperate fruit trees (Kadota and Nimmi, 2003).

BA at lower concentration was found to promote shoot differentiation at higher

concentration (>I.OpM) resulted in the callus proliferation and inhibition of shoot

differentiation (Kumar et al., 1999). Vasanth et al. (2002) reported that MS medium

containing BAP (0.3 mgL-') and Kn (0.2 mgL-') combination was found to be the best

for the induction of multiple shoots in Panicum sumatrence.

A synergistic effect of BA in combination with an auxin has been demonstrated

in many medicinal plants of Asclepiadceae viz., Holostemma annulare (Sudha et al.,

1998), Hemidesmus indicus (Sreekumar et al., 2000) and Holostemma ada-kodien

(Martin, 2002).

Shoot multiplIcatIon was a function of cytokinin activity but sustained growth of

shoots depended on a synergistic balance between BAP and NAA. Bonger and Aderkas

(1992) reported the increased activity of BAP compared to Kn. The combination of

BAP (0.5 m g ~ . ' ) and NAA (0.01 mgL-') for shoot initiation and multiplication

corresponds well with the findings of Razdan (1990), where NAA was considered more

effective than IAA due to its better stability.

Benjamin et al. (1998) reported that BA at high concentration (1-5ppm)

stimulated the development of the axillary meristems and shoot tips of Atropa

hellado~lnu. Lal er ul. (1988) reported a rapid proliferation rate in Picrorhiza kurroa


using Kn at 1.0-5.0 mgL". B m a and Walchlu (1988) indicated that the production of

multiple shoots was higher in Plantago ovata on a medium having 4-6 pM Kn along

with 0.05 pM NAA.

Jeyakumar and Jayabalan (2001) have reported BAP as the most effective

cytokinins for shoot bud regeneration in Psoralea cosylifolia. The maximum percentage

of shoot bud formation (82.2%) was obtained on MS medium fortified with the

combination of BAP (3.0 mgL-') and &4 (1.0 mg~.'). Das et al. (2001) reported

multiple shoot induction from nodal explants of Plumbago rosea L on MS medium

supplemented with 1.5 m g ~ . ' BAP, 50 mgL1 adenine sulphate and 3% sucrose.

Saxena et al. (1998) reported an average of 3-5 fold multiplication in Psoralea

corylifolia when axillaq shoots were allowed to continue in primary cultures for 8

weeks on MS medium supplemented with 2.5 mgL-' BA+ 1.0 mgL-' NAA + 5.0 m g ~ . '

adenine sulphate.

The addition of adenine sulphate in the culture medium influence rapid shoot

proliferation of shoot multiplication of (Zurcuma longa. The frequency of shoot

multiplication increased 4-fold at every Cweek interval culture on MS medium

supplemented with 4.0 m g ~ . ' BA, 1.0 mgL-' IAA, and 100 m g ~ . ' adenine sulphate

(Rout et al., 1995).

Gibberellic acid at 0.1 - 0.5 mgL-' and adenine sulphate at 50 - 100 mgL-' had a

promising effect on shoot proliferation and elongation. The rates of multiplication of

Zingiber oficinale were higher in a medium containing BAP (4.0 - 6.0 mgL-'), IAA

(1.0 - 1.5 rng~. ' ) and 100 - mgL-' adenine sulphate. They also reported that the number

of shootslexplants depended on concenb.ltions of the growth regulators and the

particular genotypes (Palai et al., 1997).

2 .4 .3 Additives

Synthetic phenyl urea derivatives, especially TDZ, have activities higher than

adenine derivatives like Kn and BAP (Mok et al., 1982). TDZ has been used to

micropropagate a wide array of woody species because of its great ability to stimulate

shoot proliferation and regeneration (Hutteman and Preece, 1993 and Lu, 1993) TDZ

has produced good results for shoot regeneration (Pawlicki and Welander, 1994) and

shoot proliferation with Pome fruits trees compared with IBA.

Thidiazuron (N-phenyl-N'-1, 2.3- thiadiazol-5-ylurea; TDZ), a substituted urea,

with cytokinins- like activity, stimulated shoot proliferation in chickpea (Cicer

arierinum L.) TDZ induced high frequency of shoot formation as compared to BAP and

also minor salts of MS medium played an important role in increasing the number of

shoots. Roots could be induced in these shoots in MS medium supplemented with

0.5pM IBA (Rajendar et al., 2002). TDZ facilitates multiple shoot proliferation of many

plant species (Huetteman and Preece, 1993; Lu, 1993; Murthy et al., 1998). It has been

found to be less susceptible to plants degrading enzymes than endogenous cytokin~ns. It

is active at lower concentrations than the amino purine cytokinins (Mok et al., 1987)

Moreover, plant regeneration can be stimulated through exposure to TDZ for a

relatively short time (Visser, 1992). TDZ has exhibited a strong cytokinin activity in

several culture systems (Thomas and Katterman, 1986; Mok et al., 1987). TDZ has

been reported to induce multiple shoot formation in various dicots. It has been shown to

promote shoot regeneration at a much lower concentration than other cytokinins, and

shoot regenerated with comparable or greater efficiency than with other cytokinins (Lu,

1993).

Nair and Seeni, (2003) described plantlet development from in vitro culture of

Cosciniurn ,fenestratum (Gaertn.). Embryonal axis excised from mature seeds cultured

on Murshige and Skoog (MS) basal medium with 0.005- mgL.' thidiazuron (TDZ) or


0.5 mgL.' BAP produced axillary shoots at cotyledonary and lor stem nodes. Shoots

production was greatest (2.2) in germinated embryos on MS medium with 0.005 mgL.'

TDZ.

Frequent addition of a low concentration of auxin or a second cytokinin to a

medium containing TDZ can significantly tahance shoot proliferation (Chalupa, 1987).

Multiple shoots of switch grass failed to be induced when TDZ was used alone. A low

concentration of 2,4-D in a TDZ containing medium induced multiple shoot clumps

from intact seedlings (Gupta and Conger, 1098).

Addition of 2,4-D to the medium did not enhance multiple shoot formation. But

resulted in a reduction in multiple shoot regeneration. With higher 2,4-D concentrations,

the number of explants that responded and produced multiple shoots was reduced since

more explants produced callus. Multiple shoot formation did not increase at higher

concentration of TDZ (Srivantanukul et al., 2000).

High concentrations of BA resulted in callus formation from the shoot apical

meristem. TDZ could stimulate multiple slloot formation in Kenaf without the addition

of auxin or other cytokinin into the medium. The effective use of TDZ as the sole plant

growth regulator to induce regeneration has been reported for many species (Malik and

Saxena, 1998; Murthy and Saxena, 1995).

More number of shoots was differentiated from the callus grown on MS medium

supplemented with BA 2.0 m g ~ - ' and NAA 0.5 mgL-'. Further multiplication was

achieved by repeatedly subculturing the nodal segments on MS medium containing BA

0.5 m g ~ - ' and NAA 0.1 mgL-'. About 95% of the in virro shoots developed roots after

they were transferred to half strength MS medium containing IBA 1.0 m g ~ - ' . 95% of

the plantlets were successfully acclimatized and established in the field (Saritha et a[.,

2003)


2 .4 .4 Explant

The level of explant competence for in vitro adventitious bud and shoot

formation was dependent on several factors such as physiological conditions, sensitivity

to seasonal changes and endogenous level of growth regulators. The endogenous

content of natural auxins and cytokinins in callus tissue may play an important role for

the induction of organogenesis (Chaulpa, 1975)

Many authors have described Micropropagation of various plant species,

including many medicinal plants, during the last two decades (Murashigel978; Withers

and Anderson, 1986; Skirvin et al., 1990). From a practical and pharmaceutical point of

view, propagation from meristems is not technologically difficult, and it yields plants

that are genetically identical with the donor plants (Hu and Wang, 1983).

2.4.5 Axillary Bud Multiplication

A key step in the improvement of a crop is the development of regeneration

technology for in vitro culture (Ananthakrishnan, 2003).

There are three ways by which micropropagation can be achieved. These

enhance axillary bud breaking production of adventitious buds directly or indirectly via

callus and somatic embryogenesis directly or indirectly via callus and somatic

embryogenesis directly or indirectly on explants (Murashige, 1974). Axillary bud

breaking produces the smallest number of plantlets, as the number of shoots produced is

limited by the multiplication of axillary buds placed in each culture. Adventitious bud

formation has a greater potential for multiplication, as shoots may arise from any part of

the inoculum. Somatic embryogenesis has the potential for producing the greatest

number of plantlets, but unfortunately, to date, it can be induced in the fewest number

of species.


Micropropagation of medicinal plants has been achieved through rapid

proliferation of shoot tips and axillary buds in culture. Numerous factors are reported to

influence the success of in vitro propagation of different medicinal plants and therefore,

it is unwise to define any particular reason for the general micropropagation of

medicinal plants. The factors that influence micropropagation of medicinal and

aromatic plants have been reviewed by Murshige (1977). Hussey (1980, 1983), Hu and

Wang (1983), Bhagylaskshmi and Singh (1988), and Short and Roberts (1991).

In vitro propagation by meristem c~~ltures or axillary buds is a standard practice

in elite species requiring uniformity. The major attraction of this approach is that it

ensures rapid and large-scale multiplication, free from pathogens either of bacterial or

viral origin (Rajender Rao et al., 2002).

Mao et al. (1995) observed that the nature and condition of Clerodendron

colebrookianum explants had a significant influence on the multiplication rate; Actively

growing materials were more responsive to shoot induction than dormant buds. Explant

density in the culture vessels influenced the rate of multiplication of Clerodendron

colebrookianum. Single shoot cultures gave higher initial shoot numbers (5, 10 and 15

shoots/culture). They also proved that BA proved superior to 2iP and TDZ for multiple

shoot induction.

Seasonal variations exercised a profound influence on regeneration. Explants of

the December and April batch of Solanunr ruberosum were highly tuberogenic or May-

Nov showed poor response (Narayanaswamy, 1990).

A rapid rate of propagation depeuds on the subculturing of proliferating shoot

cultures. Subculture also decreases the el'fect of the competition of developing shoots

for nutrients (Rout et al., 2000).


Upadhyay et al. (1989) reported a gradually improvement in shoot number with

the increase in subcultures. They proposed that the effect reflected a time-dependant

adaptation of the explants to in vitro condition, which is essentially completed during

the first few subcultures. Rout et al. (1999) demonstrated a significant improvement in

shoot multiplication rate by subculturing Plumbago zeylanica at 4-week intervals.

Shweta et al. (1999) reported that the maximum multiple shoot production in

Scutellaria discolors was obtained in MS medium containing 1 mgL-' BAP and 0.1

rng~. ' NAA. 15-20 shoots were obtained from one explant with in a period of ten

weeks.

Eva (1999) reported successful plant production of Sycamore maple using TDZ.

The best proliferation capacity occurred with 0.04pM TDZ and 1 . 0 ~ BA, both shoots

and callus. This combination also showed a stimulatory effect on the length of the

newly formed shoots.

Usha Mukundan et al. (2002) reported maximum multiple shoot formation from

nodel explants in Tylophora asthmatica on MS medium supplemented with 1.5 mgL.'

BAP and in llraria picta when BAP 1.0 m g ~ . ' was used. The in vitro grown shoots

were rooted in half strength MS basal medium supplanted with 0.5 mgL-' IBA. Survival

rate was 96%' and 100% for T. asthmatica and Urariapicra respectively.

Multiple shoots were produced from nodal explants of cassava (Manihor

rscrrlenta Crantz.) by a two-step procedure in which, a 6 to 8 day exposure to 0.11-

0.22pM Thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by

culture on agar-solidified MS medium supplemented with 2.2pM Gbenzyladenine (BA)

and 1.6pM Gibberellic acid (GA3).

2.5 Callus culture and Plant Regeneration.

REVEW OF LITERATURE

Organogenesis can be brought about in callus by the controlled initiation of an

organ primordium through the manipulation of the nutrient and the hormonal

constituents in the culture media. The phenomenon is also dependent upon a number of

other factors such s source of origin of thi: callus, its genotype and age, endogenous

hormone levels and various physical factors (Narayanaswarny, 1990).

The induction of callus growth and subsequent differentiation and organogenesis

was accomplished by the differential application of growth regulators and the control of

conditions in the culture medium. With the stimulus of endogenous growth substances

or by the addition of exogenous growth regulators to the nutrient medium, cell division,

cell growth and tissue differentiation are induced (Rout et al,. 2000). Several reports are

available on the regeneration of various metlicinal plants via callus culture

Callus stages are normally avoided where possible when dealing with rare

species. It proved impossible to obtain plantlets of Lavatera oblongijiolia (Malvaceae)

without passing through a callus stage, and plantlets were subsequently regenerated

from the callus cultures. Weaned plants were assessed for genetic integrity.-using

isozyme, cytogenetic, and image analysis techniques and shown to be uniform for the

characters examined (Irionodo and Perez, 1990).

Saxena et al. (1997) reported plant regeneration via organogenesis from callus

cultures derived from mature leaves, stems, petioles and roots of young seedlings of

Psoralea corylifolia. The calli differentiated into green nodular structures, which

developed into dark green, shoot buds in lhe media supplemented with 2.5 m g ~ ~ ' BA

and 1.0 rng~. ' NAA. The addition of 5.0- rng~.' -adenine sulphate (Ads) in the culture

medium resulted in quick growth of shoot t~uds with in 4 weeks of culture.

Rout and Das (1997a) described in vitro organogenesis of Zingiber oficinale via

callus culture. Shoot regeneration was marimal on media supplemented with 5.0 m g ~ ~ '


I3A, 1.0 m g ~ - ' IAA and 100 m g ~ - ' adenine sulphate. They also reported that shoot bud

regeneration was the highest under continuous illumination. Further, Shasany et al.

(1998) reported the influence of different growth regulators on high frequency plant

regeneration from intemodal explants of Mentha arvensis.

In vitro differentiation of shoot buds directly from explants of medicinal plants

or via callus cultures has been demonstrated. Tsay et al. (1989) reported the direct

regeneration of shoot buds from primary explants of Pinellia ternate, whereas Satheesh

and Bhavanandan (1988) reported the regeneration of shoots from callus of Plumbago

rosea using appropriate concentrations of axuins and cytokinins.

Auxins play an important role in many aspects of growth and dfferentiation.

The process influenced by auxins includes cell enlargement, cell division, vascular

differentiation, apical dominance, and root formation. Both exogenous and endogenous

auxin are closely involved in the process of somatic embryogenesis (Michalczuck et al.,

1992)

Auxins and cytokinins do not enhance cell division in callus when applied

separately. Added simultaneously, however, they have a positive effect. This positive

effect on the growth is explained by an enhanced RNA synthesis. The applied

combinathn of auxins and cytokinins provokes a first peak of RNA synthesis after two

days of culture proceeding cell divisions in the calli. After seven days of culture, a

second peak of RNA synthesis occurs. This peak is associated with the appearance of

the first bud primordial, thus conditioning bud formation (Rehman et al., 2001).

Sucrose is reported to be the most effective carbon source for somatic

embryogenesis (Ammarito, 1983) and its concentration in the medium can influence

embryo initiation and differentiation (Meijer and Brown, 1987). Sucrose utilization as

both a substrate and effector of morphological development involves a specific set of


genes, which are modulated in response to changes in sugar levels (Koch, 1996; Koch et

al., 2000).

Carbohydrate biosynthesis and metabolism are important aspects of bulb growth

and propagation in geophytes. Starch is the major plant storage carbohydrate in several

genotypes and its concentration varies among species and the type of storage tissues

(Miller, 1992).

2.6 in vitro Root Induction

In vitro induction of roots from growing shoots has been achieved in standard

media containing auxin and in media in tne absence of auxin depending on plant

genotype (Rout et al., 1988). There is a marked variation in the rooting potential of

different plant species and systematic trials are often needed to define the conditions

required for root induction. Moderate to high concentrations of all cytokinins inhibit

rooting. Rooting improved in many woody and herbaceous species when the

concentration of macro salts was lowered to half or less and the concentration of

sucrose was lowered from 2 or 3% to 0.5% (Webb and Street, 1977).

The microshoots of various medicinal plants rooted only on MS medium

without growth regulators (Cristina er al., 1390; Mao et al., 1995; Saxena et al., 1998).

Addition of IAA, IBA or NAA to the MS medium produced rooting (Jha and Jha, 1989;

Hossain et al., 1993; Faria and Illg, 1995; Palai et al., 1997). With the increase in the

concentration of IBA, root formation was inhibited (Hossain et al., 1993). Rout et al.,

(1999) reported the induction of rooting in microshoots of Plumbago zeylanica on half

strength MS medium supplemented with 0.25 m g ~ - ' IBA with 2 % sucrose.

Prolific rooting of in vitro grown microshoots is critical for the successful

establishment of these shoots in the green l~ouse and field. For a long time, rooting was

considered as a single phase process, but numerous studies led to the division of the


process of adventitious root formation into several successive interdependent phases

(Jarvis, 1986: Gaspar et al., 1992, 1994), and Moncousin (1987) identified three phases

referred to as induction, initiation and expression.

2.7 Somatic Embryogenesis

Large-scale production of elite plants in vitro will be advantageous for the

conservation of the germplasm of valuable endangered medicinal plants. Somatic

embryogenesis has gained attention as an important plant propagation method for large-

scale micropropagation (Manjula er al., 2000)

Somatic embryogenesis has proved to be useful for micropropagation and the

production of mutants, artificial seeds and materials for use in plant genetic engineering

(Pavingerova et al., 1994) Somatic embryogenesis generally occurs through two

different pathways; namely, directly from the explant and indirectly following callus

formation from explants. Direct embryogenesis, ultimately from single cells taken

directly from explant tissue, may help to retain clonal fidelity (Tanaka et al., 2000).

Distinct stages in somatic embryogenesis have been described: induction, early

growth, embryo maturation and germination or conversion of plantlet. These stages

often require sequential changes in medium composition. Auxins and cytokinins at

various concentrations and combinations have been used for initiation of somatic

emhryogenesis (Kipnis et al., 1994).

Most frequently, induction and early embryo development as well as the

maintenance of a proliferating proembryogenic mass are promoted by a relatively high

concentration of 2,4-D or other auxin. However, embryo maturation is usually

associated with a reduction or the omission of auxin from the medium (Ammarito,

1987).


Regeneration of plants in vitro via somatic embryogenesis has some distinct

features such as single cell origin, the consequent low frequency of chimeras and the

production of a high number of regeneranis. (Ammirato, 1983; Sato, 1993) Somatic

embryogenesis has several other advantages including the efficiency of process and the

morphological and cytological uniformity of the plantlets (Vasil and Vasil, 1986).

Availability of an efficient plant regeneration system via continuous production of

somatic embryos will be useful in micropropagation, germplasm conservation and

genetic transformation studies (Sahrawat ant1 Chand, 2001).

The ability of somatic plant cells in culture to regenerate entire plants by

somatic embryogenesis is a remarkable 1)iological phenomenon. The transition of

somatic cells into cells that are capable of forming an embryo is the most important

process of somatic embryogenesis and also :he area that is least understood (De Jong et

al., 1993).

Direct embryogenesis from tissue :;ections, without the callus phase, seemed

more suitable for mass propagation bec:ause genetic rearrangement was limited

compared with the embryogenesis via calli. which often showed aberrant chromosome

numbers during culture (Tanaka et al., 2000). Direct embryo differentiation offers the

best possibility of avoiding passage through callus and recovery of clones of genetically

uniform plants (Maheswaran and Willian~s, 1989). Moreover single cell origin of

embryos from epidermal cell may make the system highly amenable to genetic

transformation.

Distinct stages in somatic embryogt:nesis have been described: induction, early

growth, embryo maturation and germination or conversion of plantlets. These stages

often require sequential changes in med.um composition. Auxin and cytokinin at

various concentrations and combinations have been used for initiation of somatic


embryogenesis. Most frequently, induction and early embryo development as well as

the maintenance of a proliferating proembryogenic mass are promoted by a relatively

high concentration of 2,4-D or other auxin. However, embryo maturation is usually

associated with a reduction or the omission of auxin from the medium (Ammaritto,

1987).

Ammirato and Steward (1971) suggested the high osmotic potential of MS

medium containing high auxin concentrations of sucrose fostered embryo development

and maturation, but restricted embryo growth.

The high potential of 2,4-D in the induction of somatic embryos and the effect

of reduced salt levels in the successive development and conversion of somatic embryos

has been documented in many plants including the members of Asclepiadaceae such as

Tylophora indica (Rao and Narayanaswamy, 1972) and Hemidesmus indicus (Sarasan

rt al., 1994).

Halperin and Wetherell (1964) first recognized the importance of auxin for

somatic embryogenesis. Since then several studies have shown that the process of

embryogenesis takes place with the induction of cells with embryogenesis competence

in the presence of high concentration of auxin, and the development of embryogenic

competence cells into embryos takes place in the absence of or in the presence of low

concentration of auxin.

Somatic embryogenesis was observed upon the addition of high concentrations

of IAA (57.08 WM). IAA was especially able to induce embryogenesis at a high

frequency (58%). In contrast IBA, 2,4-D has no effect on inducing embryogenesis

(Tanaka ef a/. , 2000).

Somatic embryogenesis in carrot shows that the presence of auxin in the

medium plays important roles both in the induction of embryo development in culture


and in the subsequent elaboration of prop:~r morphogenesis in embryo development

(Schiavone and Cooke 1987; Michalezuk et rrl., 1992).

Most protocols for somatic embryogi:nesis use a strong auxin such as 2,4-D or a

combination of auxin cytokinin concentratic'n in the primary culture medium to support

both cell proliferation and induction of embryogenesis (Chaulpa 1987).

The removal of auxin from the cultures is considered to inactivate several genes

or synthesize new gene products for i:he completion of embryo development

(Zimmermann, 1993). Thus the level of auxm available to cultures is generally believed

to be crucial for somatic embryogenesis.

According to Zimmermann (1993) the removal of auxins from the culture

medium is a prerequisite to inactivate sever.d genes or to synthesize new gene products

necessary for the successful completion of embryo development.

Cytokinins have been reported to induce somatic embryogenesis in species

Trifolium, Brassica napus, Baccopa moneir~z (Tiwari et al., 1998).

Zhou et al., (1994) reported that th dizuron (TDZ) has a positive effect on the

induction of somatic embryogenesis in cell: from Cayratia japonica. A medium having

2.4-D and TDZ induced high frequency sonatic embryogenesis. Cytokinins in general

are known to foster somatic embryo development (Fujimura and Komamine, 1975);

higher cytokinin levels are believed to counteract residual auxin effects to prevent

inhibition of somatic embryogenesis (Wilson er al., 1996).

Choi et al. (1997) reported that the cytokinin treatment suppressed the normal

growth of embryos, but stimulated the st:condary somatic embryogenesis from the

surfaces of primary embryos.

In Psoralea corylifolia there was an enhancement in the production of

cotyledorlary stage embryos on ABA enriched media. ABA inhibited precocious


germination of embryos and promoted a normal course of ontogeny and maturation of

embryos (Arnmirato, 1974).

The somatic embryos regenerated from tissues grown in vitro have been

reported for many plants (Bajaj, 1995; Brown er al. 1995). Kochba and Button (1974)

reported that in Citrus maintenance of the cultures with out subculture for prolonged

periods of time induces expression of somatic embryogenesis. Rout et al. (1995b) noted

that factors like photoperiod and incubation conditions (light intensity, photoperiod and

temperature) controlled in the proliferation of somatic embryos in Acacia catechu.

2.8 Somatic Embryogenesis - Germination

Efficient development and gemination of somatic embryos are prerequisites for

commercial plantlet production. Omission or lowering of growth regulator

concentrations in culture media has improved embryo development and germination of

many medicinal plants (Arumugam and Bhojwani, 1990; Kumar, 1992, Wakhlu et al.,

1990).

Germination of somatic embryos was also achieved on MS medium with out the

~rowth regulator (Purohit e f al., 1994, Hirai e f al, 1997, Choi et al., 1997).

However, Arumugham and Bhojwani (1990) noted that the inclusion of BA

(2pM) and GA3 (2.8wM) in the medium stimulated embryo development of

Podophyllum hexandrum, although 75% of the embryos did germinate on basal MS

medium devoid of growth regulator. Wakhlu et al. (1990) reported that the somatic

embryos of Bunicum persicum matured and germinated on the basal medium

supplemented with 1.0 m g ~ - ' Kn. Kunitake and Mii (1990) reported that 30-40 % of

somatic embryos of Asparagus of/icinalis germinated after being treated with distilled

water for a week: they were subsequently transferred to half strength MS medium

supplemented with 1.0 m g ~ ~ ' IBA, 1.0 m g ~ - ' GA3 and 1% sucrose.


2.9 Acclimatization and Field Establishment . - Acclimatization of micro propagated plants in the green house under field

conditions was reviewed by Preece and !;utter (1991). Satheesh and Bahvanandan

(1988) reported that when micropropagated plants of Plumbago rosea were transferred

to pots containing a 1: 1 sand and soil mixture under green house conditions, about 60%

of the plants survived.

Jha and Jha (1989) noted that the nighest survival of Cephaelis ipecacuanha

when the plants were maintained for a week period in liquid medium and then

transferred to green house conditions. Approximately 60% of the rooted plants of

Centella asiatica survived in pots containing a 1:l:l mixture of soil, sand and well-

rotted cow dung (Patra et al., 1998). The plants were supplied with MS inorganic salts

twice a week before the transfer to the green house (Palai et al., 1997).

Rout et al. (1999) reported that allout 95% of micropropagated plantlets of

Plumbago zeylanica were established in the green house within 2-3 weeks of transfer

under 85 % relative humidity.

2.10 Histological Studies

Histological techniques are widely used in many areas of research. Structural

analysis is an important step in the study of the organisation and changes in the plant

body, and it is an extremely useful approach in the study of plant morphogenesis

(Wetmore and Wardlaw, 1951).

Different histological methods have contributed significantly to our

understanding of in vitro culture systems. A good histological study based on

histological changes provides additional irsight into the cellular process and provides


clues and allows for the proposal of hypotheses for further experimentation (Yeung,

1999).

Histogenesis of shoot bud differentiation in somatic callus tissues of Rosmarinus

[email protected] has been studied by Misra (2002). Studies showed that shoot meristems

mostly differentiated towards the outer edges of callus but occasionally produced

internally. In endogenous shoot bud differentiation, meristemoid developed in the

subsurface callus surrounded by vacuolated cells from outside.

A histological study of the developmental pattern of multiple shoot formation in

Hibiscus canabinus, demonstrated the de nova regeneration of adventitious shoot from

the enlarged shoot apex on a medium with TDZ (Srivatanukul, 2000).

Histologically, the primordial showed a typical embryo shape with a well -

developed vascular bundle between the shoot and the root primordial. Embryos had

both stomata1 cells and a root system with polarity. Longitudinal sections of somatic

embryos in Dendrunthemum grandiflorum were observed microscopically. An embryo

at the early cotyledonary stage showed a central vascular bundle between a shoot

primordium and a root primordium and a well-developed vascular bundle was observed

in an embryo to middle cotyledonary stage. On the other hand, no vascular connection

was observed between a somatic embryo and surface cells of a ray floret tissue. These

results indicate that the regenerants were properly derived from genuine somatic

embryos (Tanaka er a/.. 2000).

Couillerot er al. (1978) found that Cichorium inrybus callus develops from

vasculaf parenchyma cells that start proliferation from the third day of culture. Towards

the sixth day. meristematic initials can be observed in the newly formed callus tissue.

Only superficial meristematic initials or nodules differentiate into apical meristems.


Growth regulators are able to direct organogenesis in the direction of root or shoot

formation (Schoof and Langhe, 1988).

2.11 Histochemical and Histoenzymologit:al Studies

Armda et al. (2000) noticed that during callus morphogenesis in Eucalyptus

urophyllus, an increase in calcium concen~ration gave higher total protein and sugar

contents, an increase in peroxidase specific activity and changes in the histological

characteristics.

Studies on the calcium-mediated cc~nversion of sucrose to starch in relation to

the activities of amylases and sucrose-met;~bolizing enzymes in sorghum grains raised

through liquid culture, suggested that the assimilation of Ca++ by grain is essential for

maintaining high activity of a-amylase to generate starch primers required for the

conversion of sucrose to starch during grain filling in starch (Bhatia and Singh, 2000).

Malik and Usha (1977) reported histochemical localization of metabolic

substances and enzymes during root initiation, differentiation and maturity in the stem

and adventitious roots of Impatiens balsarnin. Root initials had high accumulation of

metabolic reserves in between the vascular bundles. Storage starch was gradually

depleted in the enlarged root initials and used in wall building material of substrate for

the respiration. The role of peroxidase in lignin biosynthesis during dedifferentiation

was brought out.

Studies on the in vitro cultures of Ipomea species by Zink and Velky (1979)

showed that the levels and the developmental patterns of the two acid phosphatases in

were influenced by the pH of the medium.

2.12 Biochemical Studies

Total proteins and free amino acids were evaluated in four leaves of day-neutral

Abelnloschrls, the first and second leaf bea;.ing dormant vegetative axillary buds and the


third and fourth leaf bearing reproductive axillary buds, during six chronologically

comparable stages of development. The pattern of changes in the concentration of

protein and free amino acids varied among the four leaves and the flower buds (Nabeesa

and Neelakandan, 1987).

Changes in the level of total lipid, soluble sugar, starch and proteins were

valuated during protocom formation of in vitro grown embryos of the terrestrial orchid

species, Spathoglottis plicata. The results indicated during initial stages of protocorm

formation, the stored lipids and proteins were used and at later stages, the accumulated

starch was used for organogenesis (Krishnan et al., 1993).

Studies on the starch turnover in shoot forming tobacco callus by Thorpe et al.,

(1986) showed that, greater net synthesis of starch occurred in shoot-forming tissue and

the loss of starch began later in tissue culture in the presence of gibberellic acid. Newly

synthesized starch was not immediately utilized in the organogenic process, but its

utilization could be correlated with the shoot forming process.

2.13 Physiochemical Analysis

Physiochemical analysis was used to test the quality of the regenerated plant.

The study of foreign matter, total ash content, acid insoluble ash, alcohol soluble

extractive, water soluble extractive, volatile oil content etc. have a direct bearing on the

purity and strength of the medicinal plant (Anonymous, 2001).

Mukerjee and Ray (1980) screened seventy-four Indian plant species for the

presence of steroids, terpenoids, alkaloids, flavanoids and saponins.

Pharmacognostic studies have been carried out in Acacia catechu with a view to

bring out the numerical ash and extractive values (Mary et a[., 1989). Brindha et al.

(2000) studied the features of two botanically different but pharmaceutically similar


drugs by using physiochemical constants. Srivastava (2001) conducted

pharmacognostic studies on Curcuma angustifolia.

Preliminary Phytochemical screenilkg of the Alocacia indica was carried out

using TLC technique. TLC patterns of hexane, chloroform, alcohol extracts of the

tubers was analysed by Brinda et al. (1931). Thankamma et al. (1995) camed out

chromatographic method for the detection of pepperin (Piper nigrum) in Ayuwedic

preparations.

2.14 Isozyme Studies

Electrophoretic and isozymes techniques are powerful and useful1 tools in plant

regeneration investigations. Numerous publication and reports can be found in its

application. The technique has been employed more extensively on plant material

derived from regenerated plants. Isozymes are multiple molecular forms of an enzyme

with similar or identical substrates specificities occurring within the same organism.

Isozymes can be employed as effective makers particularly in studies on differentiation

(Scandalios,l974) and genetics (Jacobs 1975 a)

The phenomenon of enzyme multiplicity had been known in a few cases for

many years, but it was not until the develol~ment of the zymogram technique by Hunter

& Markert (1957) that the occurrence of iss3zymes came under extensive investigations.

This technique involves electrophoretic separation usually of crude tissue extract,

followed by the demonstration of zcnes of enzymes activity using specific

histochemical staining procedures applied directly to the electrophorectic medium (eg.

Agar, starch, acrylamide). The facility of the zymogram method had made possible the

rapid screening of a large umber of enzymes in relatively small amounts of tissue

extracts (Scandalios and Sorenson, 1977).


Thorpe and Gaspar (1975) has reported on the changes in peroxidase isozyme

pattern that occur when tobacco callus is induced to form shoots. Comparison of

peroxidase changes in callus and cell suspensions of Solanum melongena L. with

organized tissue demonstrated that there are differences (del Grosso and Alichio, 1981).

Since peroxidases is probably involved in IAA metabolism, the study of the enzyme is

of interest in growth and differentiation process in culture. The system demonstrates

dramatically however, the need for biochemistry and genetically defined systems for

such studies. Peroxidases almost certainly perf01111 a number of diverse functions in the

cell in addition to IAA oxidation and lignification (Harkin & Obst, 1973).

If the plants are sampled at comparable stages, a remarkable uniformity of

electrophoretic pattern can be obtained which was found to be highly reproducible

(Bassari, 1977). Comparative studies using a number of isozymes systems on

suspension culture of Phaseoulus vulgaris derived from different tissues of a single

seedling also indicate some differences ( b i s o n and Boll, 1975). Isoelectric focusing

patterens of esterase and peroxidase in Brassicoraphanus clearly showed that it was a

hybrid (Kato and Tokumasu, 1979).

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