Chapter - 1

19

CHAPTER - 1

In vitro cultures from leaf and stem

explants of Oxystelma secamone (L)

Karst and Tragia involucrata L.

Chapter - 1

20

INTRODUCTION:

Plants are the important source of food for all living organisms. In the

beginning man depend on plant kingdom for his basic needs like food, clothing and

shelter. Subsequent exploitation of plants for curing ailments and leads to establish

herbal medicine. From earliest times itself, plants were used for treatment of disease

without knowledge about the compounds present and their mode of action. Over the

centuries societies around the world have developed their own tradition to make sense

of injuries medicinal plants and their uses. The wide spread use of herbal remedies

and health care preparations obtained from commonly used traditional herbs and

medicinal plants have been raised due to the occurrence of natural products with

medicinal properties (Thenmozhi & Rajeshwari sivaraj, 2010).

Despite the increasing use of medicinal plants, their future, seemingly, is being

threatened by complacency concerning their conservation. Reserves of herbs and

stocks of medicinal plants in developing countries are diminishing and in danger of

extinction as a result of growing trade demands for cheaper healthcare products and

new plant based therapeutic markets in preference to more expensive target-specific

drugs and biopharmaceuticals (Hoareau & DaSilva, 1999). Threatened medicinal

plant species have become the focus of world attention because they represent

vanishing and decreasing flora in need of protection and conservation and because of

their role as an essential commodity of healthcare (Gustafsson, 2002; Kala, 2002).

To avoid genetic loss of valuable medicinal plant conservation practices are

carried out globally by conserving them in ex situ method and this may include

growing the whole plants in botanical gardens or by seed storage for long time to

conserve diversity. Ex situ method of conservation or propagation of medicinal plants

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21

involves long duration and come across lot of sexual barriers. To overcome these

problems a branch of biology emerged during past two decade with the set of

experimental tools known as plant tissue culture.

Plant tissue culture is the technique of growing plant cells, tissues and organs

in an artificial prepared medium both static and liquid, under aseptic conditions. It has

extended the knowledge of fundamental botany, especially in the field of agriculture,

horticulture, plant breeding, forestry, somatic cell hybridization, phytopathology and

secondary metabolite production. It was discovered that this new area of plant

biology has commercial and practical applications in field of plant propagations as

well as in conservation of valuable germplasm (Razdan, 2008).

From the last two decades tissue culture which is becoming quite popular with

many species particularly those propagated vegetatively and plants considered as

threatened or endangered (Natesh, 2000; Rajasekharan and Ganeshan, 2002) because

its advantage then exsitu method of conservation. Thus the modern technologies

developed through tissue culture for conservation of medicinal plants is well

employed in both ex situ and in situ conservation practices. Thus, conservation of

medicinal plant genetic resources will lead to better conservation and utilization of

these important for better human well being and health (Rao & Arora, 2004).

In general concept of tissue culture is linked with the cell theory proposed by

Schlieden and Schwann. Based on this concept German botanist Haberlandt (1902)

developed the concept of in vitro culture by culturing the isolated mesophyll,

epidermal, stomatal guard cells and hair cells on artificial medium and though he

failed in his goal but put forth the concept of cellular totipotency that refers to the

potential of an individual cell to regenerate a whole plant. Hannig (1904) initiated

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22

culture of embryogenic tissue which becomes popular after Laibach (1925, 1929)

raised zygotic embryos isolated from non viable seeds. A major milestone in in vitro

culture was use of meristematic cells as explants for culture by Kotte and Robbins

(1922) independently. The root tips of pea and maize were cultured in an artificial

medium and maintained for longer period by sub culturing but they were ultimately

lost and they showed that meristematic cells are the best explants source for in vitro

culture.

Skoog & Miller (1955) discovered a cytokinin called ‘kinetin’, this was a

landmark finding in the history of plant tissue culture and also they proposed the

concept o hormonal control of organ formation that the ratio between cytokinin and

auxin induced organogenesis either caulogenesis or rhizogenesis. This landmark

finding lead to development of micropropagation in many plants particularly in cereal

and horticultural crops.

In vitro culture of plant tissues are mainly governed by the culture media. First

in vitro culture of plant tissue was first undertaken in the Knop’s solution enriched

with glucose. Hannig (1904) cultured excised embryos on mineral salts and sugar

solution rich media. After several predictions an important breakthrough came from

White (1934, 1937) used yeast extract along with in organic salts and sucrose later

yeast extract was replaced by Vitamin B such as pyridoxine, thiamine and nicotinic

acid. It was proved as one of the basic media for variety of cell and tissue cultures.

Several types of media were formulated on the basis of specific requirements of a

particular culture. Most popular culture media used are Murashige and Skoog;s (1962)

and Linsmaier and Skoog’s (1965) are generally used to induce organogenesis and

regeneration of plants. Likewise Gamborg’s B5 medium (1968) for cell suspension or

Chapter - 1

23

callus cultures and Nitsch and Nitsch medium (1969) is frequently used for the anther

culture.

Micropropagation can be achieved in a short time and space. It is the quick

and easy method of deriving plants with identical genetic constitution. It needs

minimum requirement of plant material for initiation of culture and gives raise plenty

of identical clones irrespective of season and with good health status. It was initiated

by Morel (1960), who propagated orchids through micropropagation since then many

crop species have been micropropagated. It can be extended in the field of

floriculture, agricultural crops and also in the management o disease resistant

varieties. Further it also plays a role in the production of plants with non viable seeds,

threatened and endangered species.

Many success achievements in micropropagation from several explants under

in vitro conditions the researchers focused on single cell cultures. Muir (1953)

demonstrated that when callus tissues were transferred to liquid medium and

subjected to shaking, callus tissues broke into single cells. Steward et al. (1958)

established somatic embryos in cell suspension culture of carrot and from semi solid

cultures of carrot by Reinert, (1959) demonstrated the cellular potency proposed by

Haberlandt. In supportive to this Vasil & Hildebradt (1965) regenerated whole plant

from the isolated single cell of tobacco plants. The concept of totipotency not only

applies the vegetative cells but it also holds true in reproductive tissues as shown by

Guha & Maheshwari (1966), isolated haploid somatic embryos from the microspore

culture of Datura. Later Bourgin and Nitsch (1967) obtained complete haploid plants

Nicotiana tobaccum this finding applies a tremendous impact on the plant breeding

Chapter - 1

24

because doubling of chromosome numbers in haploid plants readily yields

homozygous diploids.

Cell culture was an important finding in the history of plant tissue culture, it

provides valuable information on morphogenesis and plant development. Studies on

molecular, biochemical and physiological aspects of cells in culture have contributed

to an in depth understanding of cytodifferntiation and organogenesis and somatic

embryogenesis (Fukuda, 1997; Thomson & Thorpe, 1997; Zhou et al.1996).

From 1970s, tissue culture headed towards a new research area nothing but

cell engineering technologies means production of secondary metabolites from

cultured cells. This was quite disappointed in the first step as demonstrated by Nickel

during the period 1950-1960. But later rapid progress in these investigations attracted

many researchers and successful achievements were obtained in many plant species

(Veerporte et al.1998). The technology is now commercialized for many plants either

through callus culture or cell culture for eg; Pyrroquinozoline alkaloid from Adhatoda

zeylanica (Jayapaul et al., 2005), cardiac glycosides from Digitalis purpurea (Seitz

&Gartner 1994). Secondary metabolites are largely explored from the hairy root

cultures, which are infected with bacterium Agrobacterium rhizogenesis.

Initially the tissue culture techniques are concentrated on understanding the

various aspects of plant growth and development. When it was introduced the people

concentrated only on in vitro regeneration of plants i.e micropropogation and several

important results were also obtained that helpful in propagating the plants where seeds

are nonviable, woody species, conservation of endangered plants. After many

successive micropropagation process the plant biologist diverted their research

towards practical applications if plant tissue culture from morphological aspects.

Chapter - 1

25

Now it became an integral part of plant biotechnology. It is useful in the

improvement of useful crop plants and it forms an alternative conventional plant

propagation method. Plant tissue culture currently with vast applications helped the

mankind.

REVIEW OF LITERATURE:

In India alone among total number of medicinal plants traded in the country

only less than 10% are cultivated and remaining 90% are collected from the wild,

very often in a destructive and unsustainable manner (Natesh, 2000). It was estimated

by various calculations as nearly 4160 or 10,000 medicinal plants that are globally

threaten (Vorhies, 2000; Scippmann et al. 2002).

Micropropagation has played a very important role in the conservation of

depleting medicinal plants and also in other group of plants like cereals, vegetable

crops, legumes, oil seeds, floriculture, horticulture crops, and forest trees.

Micropropagation have been applied to many kind of medicinal plants and it

has been reviewed periodically by many researchers like Murashige (1974, 1978),

Evans et al., (1981), Ammirato (1983), Flick et al., (1983), Hussey (1983, 1986),

Bajaj (1986). Recently many others like Pence (1999), Gonzalez-Benito et al.,

(1999), Nalawade et al., (2003), Nalawade & Tsay (2004), Chaturvedi et al. (2007),

Paunescu (2009), Rai MK (2010), Sharma et al., (2010), Yadav et al. (2012) and

Najar et al. (2012) discussed the efficiency of in vitro propagation technique in

conservation of number of medicinal plants.

Pence (1999) reported the application of in vitro propagation methods to over

170 endangered plants derived from 60 families. Out of 170 plants many of them have

Chapter - 1

26

medicinal value such as Hedeoma multiflorum, Woodfordia fruticosa, Valerian

wallichii. Similarly, Gonzalez-Benito et al. (1999) reported the application of

micropropagation to over 35 rare and endangered medicinal plant species from 13

different families of endemic to Spain and some of the endangered medicinal plants

are also medicinal.

Nalawade et al. (2003) have reviewed the tissue culture studies of 80 Chinese

medicinal plants of Taiwan and also standardize the protocols of in vitro propagation

for the important Chinese medicinal plants establish a protocol for the

micropropagation of some medicinal plants like Limonium wrightii, Adenophora

triphylla, Gentiana davidii var. formosana, Anoectochilus formosanus, Scrophularia

yoshimurae, Pinellia ternata, Bupleurum falcatum, Zingiber zerumbet, Dendrobium

linawianum, and Fritillaria hupehensis through shoot morphogenesis, and Angelica

sinensis and Corydalis yanhusuo through somatic embryogenesis.

Chaturvedi et al. (2007) have reviewed eleven medicinal plants and reported

the importance of tissue culture work over the conventional breeding in the

propagation of medicinal plants which are required in large quantity for the drug

development like Dioscorea floribunda, Rauvolfia serpentine, Withania somnifera,

Aloe vera, Solanum khasianum, Podophyllum peltatum .. They emphasized the

importance of in vitro cloning of medicinal plants for the large scale production to

conserve them from the destruction.

Paunescu (2009) reported the importance of Biotechnological tools like in

vitro culture, cryopreservation, and molecular markers in the conservation of plant

genetic resources and summarizes the progress in establishing in vitro germplasm

collections of Romanian flora they are Artemisia tschernieviana, Astragalus

Chapter - 1

27

pseudopurpureus, Cerastium transsilvanicum, Dianthus callizonus, D. spiculifolius,

D.tenuifolius, Erigeron nanus, Hieracium pojoritense, and Marsilea quadrifolia.

In vitro studies in many medicinal plants of family Euphorbiaceae has been

carried out with different explants via direct and in direct organogenesis. Roy and

Jinnah (2001) studied the in vitro propagation of poinse Euphorbia pulcherrima. The

hormonal control of triterpinols synthesis in Euphorbia characias calli was studied by

Ferriera et al., (1992). Few studies available on the tissue culture of Phyllanthus

species are on callus culture of P. emblica, P. urinaria, P. amarus, P. abnormis, P.

caroliniensis, P. tenellus, P. niruri and on transformed root cultures of P. niruri

(Khanna and Nag, 1973; Unander, 1991; Ishimaru et al., 1992; Santos et al., 1994). In

vitro micropropagation of Baliospermum axillare was recorded by Singh and

Sudarshana (2003). High frequency regeneration from various explants of Jatropha

integerrima has been reported (Sujatha and Dhingra, 1993). Sujatha and

Mukta.(1996) worked on Jatropha curcas to study the morphogenesis and plant

regeneration from tissues. Propagation system for Manihot esculenta based on nodal

explants and axillary bud-derived meristems

Rai MK (2010) high lightened the biotechnological tools like in vitro culture,

micropropagation, mycorrhization, genetic transformation and development of DNA

banks as new methods for conservation of rare and endangered medicinal plants.

These are imperative and important alternatives for the conservation of rare and

endangered medicinal plants. Thus these biotechnological strategies would open up

new vistas in the field of conservation.

Sharma et al., (2010) emposizeds the importance of tissue culture technology

in the field of biodiversity conservation and also reported tissue culture protocols have

Chapter - 1

28

been developed for a wide range of medicinal plants, which includes endangered, rare

and threatened plant species. Some of these medicinal plants are Saussaurea lappa,

Picorrhiza kurroa, Ginkgo biloba, Swertia chirata, Gymnema sylvestre, Tinospora

cordifolia, Salaca oblonga, Holostemma, Celastrus paniculata,, Oroxylum indicum,

Glycyrrhiza glabra, Tylophora indica,Bacopa mooniera, Rauwolfia serpentina.

Yadav et al., (2012) reported that in vitro regeneration of plants offers a

tremendous potential solution for the propagation of endangered and superior

genotypes of medicinal plants which could be released to their natural habitat or

cultivated on a large scale for the pharmaceutical product of interest. They establish

protocols for micropropagation of some of the endangered and valuable medicinal

plants species of India such as, Aegle marmelos, Acorus calamus, Celastrus

paniculatus, Commiphora mukul, Peganum harmala, Prosopis cineraria, Simmondsia

chinensis, Spilanthes acmella, Stevia rebaudiana, Sapindus mukorossi.

Najar et al., (2012) reported that an ever increasing demand of uniform

medicinal plants based medicines leads to depletion of plants in natural condition and

this has to overcome by plant tisiue culture. Thus they gave importance to mass

propagation of medicinal plants to conserve biodiversity and reported the tissue

culture protocols developed for some medicinal plants like Oroxylum indicum,

Asparagus racemosus, Costus speciosus and Chlorophytum borivilianum.

Explant source

Micropropagation was achieved by using a piece of plant part known as

explant, which can be leaf, stem, root, shoot buds, cotyledons, seedlings, anthers,

Chapter - 1

29

immature embryos, nodal region, and a single cell. In this explants many successful

attempts have been made to in vitro regeneration of medicinal plants.

The protocols for leaf explants was successfully established in the plants like

Allium sativum (Havranek & Novek, 1973), Aristolochia indica (Manjula et al 1997),

Bacopa monniera (Tiwari et al 2000, 2001), Decalepis hamiltonii (Giridhar et al

2004), Delonix elata (Sudarshana et al., 1992), Desmodium oojeinense

(Vasanthakumari & Shivanna, 2005), Embelia ribes (Shankaramurthy et al., 2004),

Enicostemma littorale (Nagarathnamma et al., 2010), Flaveria trinervia (Sudarshana

and Shanthamma, 1991), Grindelia robusta (Fraternale, 2008), Lagerstroemia indica

(Niranjan et al., 2007), Nymphiodes cristatum (Niranjan et al., 2008), Piper colubrinum

(Yusuf et al., 2001), Thapsia garganica (Makunga et al., 2003), Zizyphus jujuba (Gu

& Zhang, (2005), Withania somnifera (Kulkarni et al 2000), and in Vetiveria

zizanioides (Muccarelli et al 1993),

Similarly in stem parts like nodal and intermodal segments Azadiracta indica

(Sharma et al 2002), Bacopa monniera (Mohapatra & Rath 2005), Baliospermum

axillare (Singh & Sudarshana, 2003), Astragalus melilotoides (Hou & Jia, 2004),

Baliospermum montanum (Johnson & Manickam, 2003), Asparagus racemosus

(Krishna & sanu, 2009), Aristolochia indica (Manjula et al 1997), Decalepis.

Araylpathra (Sudha et al 2005), Celastrus paniculatus (Sood & Chouhan 2009),

Cedrela fissilis (Nunes et al, 2002), Ceropegia candelabrum (Beena et al., 2003),

Emilia zeylanica (Robinson et al., 2009), Enicostemma littorale (Nagarathnamma et al.,

2010), Hemidesmus indicus (Siddique et al 2003), Holostemma ada-kodien (Martin,

2000), Kigelia pinnata (Thomas and Puthur, 2004), Wattakaka volubilis

(Arulanandam et al 2011), Tinospora cordifolia (Gururaj et al 2007), Tylophora

Chapter - 1

30

indica (Faisal et al 2007), Spilanthes acmella (Yadav & Singh, 2010), and in

Simmondsia chinensis (Kumar et al., 2010).

Micropropagation was also achieved with the help shoot tip explants in plants

such as Adhatoda beddomei (Sudha & Seeni 1994), Adhatoda vasica (Azad et al,

2003), Aegle marmelos (Das et al., 2008), Cryptolepis buchanani (Prasad et al, 2004),

Lippia alba (Gupta et al., 2001), Calophyllum apetalum (Nair & Seeni, 2003),

Orthosiphon spiralis (Elangomathavan et al., 2003), Withania somnifera (Kulkarni et

al 2000), and in Saussurea lappa (Johnson et al 2007).

In Boerhaavia diffusa (Sudarshana & Shanthamma, 1988), Bowiea volubilis

(Hannweg et al., 1996), Curcuma longa (Salvi et al, 2000), Embelia ribes (

Shankaramurthy & Krishna, 2006), Limonum cavanillenssii (Amo-Marco & Ibanez,

1998), and in Nymphiodes cristatum (Niranjan & Sudarshana, 2000) inflorescence

explants was used.

Role of growth hormones in in vitro regeneration of medicinal plants:

Haberlandt (1902) exploits the totipotency in plant cells and this was

unequivocally demonstrated for the first time in plants by Steward et al. (1964). After

discovery of kinetin by Miller et al. (1955), the major work on in vitro regeneration

has been centered on Nicotiana tabacum L. and followed by Daucus carota L. after

the demonstration of auxin, cytokinin ratio in the differentiation of shoots or roots or

both by Skoog and Miller (1957). It leads to the concept of totipotency of plant cell

with the regeneration of complete flowering plant of carrot from its phloem cells

(Steward et al., 1964). Thus, the micropropagation of medicinal plants remained

neglected till complete plants of Rauwolfia serpentine (L.) Benth., were produced

from its somatic callus tissue (Mitra and Chaturvedi,1970).

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31

The field performance of these tissue cultured plants depends on the selection

of the initial material, media composition, growth regulators, cultivar and

environmental factors (Chang et al., 1994). The effects of auxins and cytokinins on

shoot multiplication of various medicinal plants have been reported by Skirvin et al.

(1990). Lal and Ahuja (1996) observed a rapid proliferation rate in Picorhiza kurroa

using kinetin at 1.0–5.0 mg/l. Barna and Wakhlu (1998) has indicated that the

production of multiple shoots is higher in Plantago ovata on a medium having kinetin

along with NAA. Faria and Illg (1995) have also shown that the number of shoots per

explant depends on concentrations of the growth regulators and the particular

genotypes. The nature and condition of explants has also been shown to have a

significant influence on the multiplication rate. Mao et al. (1995) reported that the

actively growing materials were more responsive to shoot induction than dormant

buds in Clerodendrum colebrookianum. Also BAP was proved superior to 6- purine

(2ip) and TDZ for multiple shoot induction. In direct regeneration via callus culture

was achieved in many medicinal plants. Martin (2002) reported shoot regeneration

from callus culture of Holostemma ada-kodien in MS medium fortified with 1.5 mg/l

BAP. Pande et al. (2002) have reported the successful in-vitro regeneration of

Lepidium sativum from various explants on MS medium supplemented with 4.0 mg/l

BAP and NAA. The role of auxin and cytokinin ratio was in the callogenesis was alos

emphasized by the several authors like Kumar and Singh (2009) in Stevia rebaudiana,

Goel and Singh (2009) in Peganum harmala, Kumar and Singh (2009) in Prosopis

cineraria, Lal and Singh (2010) in Celastrus paniculatus, Yadav and Singh (2010) in

Spilanthes acmella and Aegle marmelos (2011) . Out of various cytokinins tested,

BAP was the most effective for inducing bud break. Effectiveness of BAP was also

observed in Leucaena leucocephala (Singh and Lal, 2007), Prosopis cineraria

Chapter - 1

32

(Kumar and Singh, 2009), Splianthes acmella (Yadav and Singh, 2010) and Acorus

calamus (Yadav et al., 2011).

Somatic embryogenesis is one of the methods of in vitro regeneration of

plants. It is a process of formation of embryo like structure from somatic tissue. The

somatic embryo may be produced either directly on the explants or indirectly from

callus or cell suspension culture. 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. Plant regeneration via somatic embryogenesis from

single cells, that can be induced to produce an embryo and then a complete plant, has

been demonstrated in many medicinal plant species (Tripathi and Tripathi, 2003).

Arumugam and Bhojwani (1990) noted the development of somatic embryos from

zygotic embryos of Podophyllum hexandrum on MS medium containing BAP and

IAA. Efficient development and germination of somatic embryos are prerequisites for

commercial plantlet production. Chand and Sahrawat (2002) reported the somatic

embryogenesis of Psoralea corylifolia L. from root explants on medium

supplemented with NAA and BAP. Rooting of shoots was best achieved using

different concentrations of auxins. In Aegle marmelos, MS half strength medium

supplemented with IAA proved better (Yadav and Singh, 2011). In Prosopis cineraria,

rooting was achieved on half strength MS medium supplemented with 3.0 mg/l IBA

(Kumar and Singh, 2009), while in Leucaena leucocephala, NAA resulted in better

root formation (Singh and Lal, 2007).

The present study aims to establish a protocol for the regeneration of selected

medicinal plants.

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33

MATERIALS AND METHODS:

Explant source and collection:

Oxystelma secamone (L) Karst. (Asclepiadaceae) is a slender laticiferous

climber growing on the banks of Kukkarahalli lake of University campus, Mysore.

Different parts like seeds, leaves, internode, and nodal region were collected and used

as explants.

Tragia involucrata (Euphorbiaceae) is a perennial evergreen twiner with

hispid, stinging bristles growing in the University campus and botanical garden,

maintained by department of Botany. Leaves and internodes were collected as

explants.

Culture media

Murashige & Skoog’s (MS) medium (1962), Gamborg’s B-5 (B-5) medium

(1968) & whites (WM) medium (1943 a & b) were used to test the response of

selected explants in different combinations of growth regulators. For rhizogenesis MS

half strength medium tested. The composition of media, growth regulators and

adjuvants are depicted in Table.1& 2.

Stock solutions of known strength of inorganic and organic components of the

medium and growth regulators were prepared and stored at 4⁰C. For all three media

sucrose (3% w/v) was used as carbohydrate source and gelled with 0.8% w/v

bacteriological grade agar. The pH of the medium was adjusted between 5.6 to 5.8

with 0.1N NaOH or 0.1N HCl prior to homogenization of the medium. The

homogenized medium was dispensed into culture tubes and conical flasks (about

Chapter - 1

34

15ml/tube and 25-30ml/conical flask) and plugged with non absorbent cotton. The

culture vessels were autoclaved at 121⁰ C for 15 minutes with 15 lb pressure. The

culture tubes were kept slantly for solidification. After solidification the sterilized

culture wares were transferred to the inoculation chamber.

Surface sterilization, inoculation of explants and incubation of

cultures

Selected explants of both the plants were thoroughly washed in running tap

water for 30 minutes followed by soaking in 5% (v/v) liquid detergent labolene for 5

minutes and finally the excess traces of detergents were removed by washing under

running tap water. Then, the explants were surface sterilized with 0.1%(v/v)aqueous

mercuric chloride for 1-5 minutes and finally the material was washed aseptically in

sterilized, cooled distilled water for 5-6 times to remove the traces of sterilant and

then they were cut into required size.

In O. secamone, mature pods were collected from an elite vine growing at

Kukkarahalli Lake, Mysore. The seeds were washed in running tap water for 30 min

followed by soaking in 5% (v/v) liquid detergent labolene for 5 min and washed

thoroughly under running tap water. The seeds were surface strlized with 0.1% HgCl2

for 10 minutes and finally rinsed with 5-6 changes of sterilized cooled distilled water.

Then they were germinated on MS basal medium.

After seed germination in O. secamone, 20-30 day seedlings of height 8-10 cm

were used as source of explants. Leaves, nodes and internodes were selected and were

inoculated in the culture vessels.

Chapter - 1

35

The culture media and the instruments required for the inoculation were

transferred to the inoculation chamber and exposed to ultra violet rays for 30 minutes

prior to inoculation. The surface sterilized, washed explant pieces were transferred to

culture vessels under the flame. In each culture tubes 1-2 segments while in each

culture flask 3-5 fragments were placed. After inoculation the cultures were incubated

and maintained in incubation chamber at 25±2⁰C under 16/8 hr photoperiod of 1000-

2000lux light intensity provided by white, fluorescent tubes with 60-80% relative

humidity.

Control of polyphenolic exudation:

The explants or callus after three to four days of inoculation turned completely

brown colour which indicates the presence of polyphenols in T. involucrata. It was

confirmed by a test described by Gibbs (1974) & Fiegel (1960). Adding 0.5 ml of

ferric chloride to the 1 ml extract of explants, the solution turned olive brown colour

indication presence of polyphenols.

The release of polyphenols in culture was neutralized by soaking the explants

in autoclaved water for at least one to two hours before inoculation or initial

incubation of explants under dark for 48 hrs after inoculation or transfer of this callus

to fresh medium at an interval of 4 days (Ghosh and Bannerjee, 2003) or addition of

activated charcoal to the medium at 2.0-10.0 gm/l or addition of antioxidants like

ascorbic acid and citric acid to the medium.

Chapter - 1

36

Study on effect of growth regulators on callogenesis and shoot

regeneration:

The growth regulators as mentioned in table 2 are used at different

concentrations. The auxins like 2.4-D (1.0-10.0 mg/l) , NAA (0.5-3.0 mg/l), IAA

(0.5-3.0 mg/l) and IBA (0.5-3.0 mg/l) and the cytokinins like BAP (1.0-10.0 mg/l)

and Kn (1.0-10.0 mg/l) were tested individually and also in various concentrations

and combinations of auxins and cytokinins to determine the effect on callogenesis and

shoot regeneration from selected explants.

For growth studies effect of auxins and cytokinins individually on the growth

rate of callus of both the species has been studied. The callus induced on 2, 4-D

containing medium was subcultured on the same medium but with different

concentrations of 2,4-D, BAP, NAA, IAA, Kn and TDZ (0.5-5.0 mg/l). After 30 days

fresh weight of callus mass was recorded and the same was dried at 60⁰C for

overnight and dry weight was recorded. The results were showed in Table 3.

Root induction in in vitro regenerated shoots:

The shoots regenerated during subculture were excised aseptically and

transferred to half strength MS basal liquid medium supplemented with auxins such as

IAA, IBA and NAA at different concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/l.

The shoots of 4-5 cm height were excised and inserted into the sterile filter paper

bridge that acts as supporting medium for shoots and also absorbing medium. The

filter paper bridge with shoots are transferred into the culture vessels and kept for

incubation. The date of root initiation and number were recorded and root length was

measured 30days after the date of root initiation.

Chapter - 1

37

Acclimatization/Hardening:

After 4-5 weeks in rooting medium, the well rooted in vitro shoots were

removed from the culture medium and the roots were washed in sterile distilled water

to remove the medium. The plantlets were then transferred to plastic pots containing

garden soil mixed with vermiculite and sand (1:1:1) and maintained in the hardening

room under 25±2⁰C with 80-90% relative humidity for two to three weeks. The

hardened plants were transferred from incubation chamber to green house with

ambient temperature (27-30⁰ C) and 70% humidity. The number of plants survived

was recorded.

Chapter - 1

38

Table 1: Composition of synthetic culture media used for the in vitro

propagation studies

CONSTITUENTS WM MS B5

Macronutrients Mg/l Mg/l Mg/l

KNO3 80 1900 2500

NH4NO3 - 1650 -

KH2PO4 - 170 -

NaH2PO4. H2O 19 - 150

MgSO4. 7H2O 750 370 250

(NH4)2.SO4 - - 134

CaCl2. 2H2O - 440 150

Micronutrients

H3BO3 1.5 6.2 3.0

MnSO4. 4H2O 5.0 22.3 -

MnSO4. H2O - - 10.0

ZnSO4. 7H2O 3.0 8.6 2.0

Na2MoO4. 2H2O - 0.25 0.25

CuSO4. 5H2O 0.01 0.025 0.025

CoCl2. 6H2O - 0.025 0.025

KI 0.75 0.83 0.75

FeSO4. 7H2O - 27.8 -

Na2EDTA. 2H2O - 37.3 -

EDTA Na Ferric salt - - 43.0

Organic supplements

Vitamins

Thiamine HCl 0.01 0.5 10

Pyridoxine HCl 0.01 0.5 10

Nicotinic acid 0.05 0.5 1.0

Myoinositol - 100 100

Others

Glycine 3.0 2.0 -

Sucrose 30gm/l 30gm/l 30gm/l

Chapter - 1

39

Table 2. Growth regulators and adjuvant employed for the in vitro propagation

studies

GROWTH REGULATOR SOLVENT USED TO DISSOLVE

Auxins

2,4 Dichloro phenoxy acetic acid (2,4-D) Absolute alcohol

Naphthalene acetic acid (NAA) Absolute alcohol

Indole acetic acid (IAA) Absolute alcohol

Indole butyric acid (IBA) Absolute alcohol

Cytokinins

Benzyl amino purine NaOH (0.1N)

Kinetin (Kn) NaOH (0.1N)

Thidiazuron (TDZ) NaOH (0.1N)

Gibberllin

Gibberellic acid (GA3) Absolute alcohol

Adjuvants

Coconut milk -

Casein hydrolysate -

Activated charcoal -

Ascorbic acid -

Chapter - 1

40

RESULTS

1. Leaf Culture:

1.1: Influence on different culture media

Morphogenetic potentiality of both the plant leaf explants are tested by using

the MS dmedium, B5 medium & White’s medium supplemented with growth

regulators like 2,4-D (0.5-5.0 mg/l), Kn (1.0-10.0 mg/l) and medium without growth

regulators are used as control.

The explants of T. involucrata and O. secamone showed better response on the

MS medium than other media. Hence MS medium was selected for further studies and

the percentage of callus formation on all the three media is presented in table 1.1.

1.2: Influence of explants region

Young and small leaf segments showed better and fast response than the

mature leaf explants. Leaf explants were excised into posterior region, middle region

and anterior region and these segments were used as explants source. Different types

of responses were observed when these segments were cultured in the medium. The

basal segment produced callus within 10-12 days of incubation whereas in the middle

segments callus formation appeared within 12-14 days but in apical region it took

more than 15 days and production was meager.

1.3: Neutralization of browning

In T. involucrata exudation of phenolics and browning of the explants (Fig

1.1) is one of the major problems in establishing in vitro cultures. After inoculation

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41

the explants became brown coloured within two days and ultimately necrosis of

explants occurred within five to six days.

Pretreatment of explants in autoclaved water for one to two hours prior to

inoculation did not show any changes in culture, the explants under goes browning

after 5-6 days of incubation. Incubation of cultures immediately after inoculation in

dark chamber for two to three days also has no any marked effect on exudation.

The activated charcoal (2.0-5.0mg/l) significantly reduced the phenol content

in culture media but fail to induce callus even after transfer to fresh medium. MS

medium supplemented with ascorbic acid (1.0-5.0 mg/l) to the basal medium showed

marked effect on callogenesis even from the brown explants. Lower concentration of

ascorbic acid (1.0 mg/l) no effect on control of exudation and increase in the

concentration upto 3.0- 4.0mg/l the explants remains brown for long culture and

transfer to fresh medium induce callogenesis. Ascorbic acid (2.0 mg/l) found to be

better concentration in inducing callus (Fig.1.2) from the explants and the survival

rate was about 85%. Initially the explants show browning but after six days bulging of

explants lead to development of callus.

Citric acid (1.0-5.0 mg/l) also exhibit same kind of results but explants

cultured in the medium supplemented with 1.0 mg/l induce callus after transfer to

fresh medium and shows nearly 60% survival rate . Higher concentration of citric acid

exhibit only 40% of survival rate of explants. In the present study ascorbic acid was

found as better antioxidant to minimize the exudation and to maintain culture.

Chapter - 1

42

1.4: Influence of growth regulators

Plant growth regulators play an important role in the morphogenetic

potentiality of explants cultured. It influences the formation and nature of callus.

Different types of auxins (2, 4-D, NAA & IAA) and cytokinins (BAP & Kn) were

tested for callogenesis.

Among the auxins, 2, 4-D found to be the best for callus induction (Fig.1.) in

T. involucrata compare to NAA and IAA (Table: 1.2). In cytokinins (Table: 1.3) the

leaf become brownish with in 24 hrs and produce little dark yellow mass of tissue

(Fig.1.3). However in O. secamone, medium fortified with 2,4-D ((Fig.1.4) and Kn

(Table; 1.4 & 1.5). individually showed better callogenesis.

Explants cultured on hormone-free basal medium (control) did not produce

any callus, and incubated explants senesced after a few days.

Effect of 2, 4-D

The leaf explants of T. involucrata produced proliferated callus in the medium

supplemented with 2, 4-D (1.0-5.0 mg/l). It was found that MS medium supplemented

with 2.0mg/l produced luxuriant callus and it was noticed that, high concentration of

hormone decreases callogenic potentiality of explants. The morphogenic nature of

callus was exhibited in table 1.2.

In O. secamone, leaf explants showed callusing at lower concentrations of 2,

4-D (0.5 – 2.0 mg/l), 75% cultures formed callus. When 2, 4-D concentration was

increased from 2.0 to 3.0 mg/ l almost all the cultures showed callusing. At still

higher concentration (4.0 & 5.0 mg/l) there is decline in the production of callus. The

callus was faint yellow friable at higher concentrations while in 3.0mg/l concentration

Chapter - 1

43

it was green or dark green in colour and friable appeared on all surface of explants

(Table: 1.4).

Effect of NAA

The explants of T. involucrata were cultured on MS medium supplemented

with different concentrations of NAA (1.0-5.0 mg/l). The callus formed at all

concentrations was yellow and friable (Fig.1.5) in nature At lower concentration (0.5

mg/l) and at higher concentration (5.0 mg/l) the leaf explants became brown and no

callus formation but at concentrations (2.0-3.0 mg/l) the brown coloured explants

produced scanty callus, it was soft and yellowish (Table: 1.2).

In O. secamone, the explants produced callus on MS medium supplemented

with NAA (1.0-5.0 mg/l). The explants at lower concentration of NAA (1.0 mg/l)

produced scanty callus and moderate amount of callus formation was observed in MS

medium fortified with 3.0 mg/l NAA, in both the cases whitish friable callus was

noticed. At higher concentration, the explants remained greenish for few days and

dried.

Effect of IAA

Different concentration of IAA (1.0-5.0 mg/l) was supplemented for both the

plants. In T .involucrata at lower concentrations (1.0 & 3.0 mg/l) little amount of

brown friable callus was induced and undergoes necrosis while at higher

concentration (5.0 mg/l) brown friable callus produced root initials in long culture.

Leaf explants of O. secamone produced callus on the medium containing IAA

(1.0-5.0 mg/l). Maximum amount of callus was noticed in the medium fortified with

1.0 mg/l IAA (Fig.1.6). The callus was whitish, compact and hard.

Chapter - 1

44

The morphogenic nature of callus of both the plants induced on MS medium

supplemented with 2, 4-D, NAA and IAA was shown in table 1.2 &1.4.

Effect of BAP

The explants of T. involucrata did not show any response on MS medium

supplemented with BAP. The explants undergo browning and produce little dark

yellow mass of tissue. The callus remains as such even it was transferred to fresh

medium (Fig.1.3).

Various concentrations of BAP (1.0-5.0 mg/l) played a significant role in the

induction of callus from leaf explants of O. secamone. At 1.0 mg/l BAP explants

showed curling at their margins but no callusing. Significant amount of callus was

produced on the medium supplemented with 2.0 mg/l BAP compared to 3.0 and

4.0mg/l BAP and callus was pale brownish compact and nodular (Fig.1.7)) but shoot

bud formation was absent. At higher concentrations callus was scanty and light

yellowish friable and turned brown (Table. 1.5).

Effect of Kn

The explants of T. involucrata did not show any response on MS medium

supplemented with Kn due to browning.

The explants of O. secamone showed callogenesis in the MS medium

supplemented with Kn (1.0-5.0mg/l). After two weeks of incubation explants induced

callus. At lower concentrations (1.0-2.0 mg/l) callus formed was scanty and also at

5.0 mg/l concentration. Maximum amount of callus was formed on the medium

fortified with 3.0 mg/l Kn (Fig.1.8) and the callus was whitish hard proliferative and

juicy.

Chapter - 1

45

1.5: Influence of Growth regulators in combination

Morphogenic potentiality of leaf explants of O. secamone were tested in the

MS medium supplemented with various combinations of cytokinins (BAP & Kn) and

auxins (2, 4-D, NAA & IAA).

Effect BAP and auxins

BAP in combination with different auxins like 2, 4-D, NAA and IAA were

tested and data was recorded at the end of fourth week

Medium supplemented with different combinations of BAP (1.0-5.0 mg/l) + 2,

4-D (0.5-4.0 mg/l), BAP (1.0-5.0 mg/l) + NAA (0.5-4.0 mg/l) and BAP (1.0-5.0 mg/l)

+ IAA (0.5-4.0 mg/l) were tried and results were recorded (Table 1.6).

MS medium supplemented with BAP in combination with 2, 4- D was found

to be better callus induction medium. At lower concentrations of BAP and 2, 4-D the

amount of callus formed was very scanty and it was found that BAP (3.0 mg/l) and 2,

4-D (1.0 mg/l) is a better concentration for callogenesis (Fig.1.9). The callus was pale

greenish and nodular. Higher concentration of BAP and 2, 4- D also induce callus but

it was in negligible amounts. Shoot buds were not induced.

Among the combinations of BAP and NAA, best callogenesis was seen in the

combination of BAP at 4.0 mg/l and NAA at 2.0 mg/l. The calli was light yellowish

and soft. Other combinations yield little amount of callus.

Leaf explants showed rhizogenesis alongwith green compact callus after 8-10

days of incubation in the combination of BAP (3.0 mg/l) and IAA (1.0mg/l). In other

combinations only light green coloured callus formation occured in little amount.

Chapter - 1

46

Effect Kn and auxins

Morphogenic potentiality of leaf explants of O. secamone were tested in the

MS medium fortified with Kn along with different auxins like 2, 4-D, NAA and IAA.

The results were similar to that of BAP. Callus formation takes place, in the

medium supplemented with Kn (1.0mg/l-5.0mg/l) and 2, 4-D (1.0 -3.0 mg/l). Among

the tested combinations 3.0mg/l Kn alongwith 1.5mg/l 2, 4-D induced whitish,

smooth and non nodular callus. The callus proliferated luxuriantly but shoots never

induced. As the concentration of Kn or 2, 4-D increased beyond the optimum

concentrations callus proliferation gradually decreased.

The leaf explants did not show much significant response on Kn and NAA

supplemented medium. However media containing Kn (1.0-5.0mg/l) with (1.0-3.0

mg/l) NAA produces little amount of callus at the cut edges and did not show any

proliferation and they remained green for 3-4 weeks and became yellow.

Rhizogenesis was noticed after 15 days of culture in the medium fortified with

Kn (2.0mg/l) and IAA (1.0 mg/l). The number of roots produced after callogenesis

and increase in the concentration of IAA leads development of root primordia with

scanty callus.

1.6: Role of subculture

1.6a: Subculture of T. involucrata callus

The leaf explants after neutralization of phenolics induce white greenish or

light yellow, translucent, granular, friable or soft callus (Fig.1.10). The primary callus

was subcultured on the same medium which was used for initiation of callus showed

Chapter - 1

47

only proliferation but after 30 days of culture instead of somatic embryogenesis callus

turned brown and it was reduced by transfer of this callus to fresh medium

supplemented with different growth regulators.

The callus obtained from primary culture was also transferred to the medium

fortified with different concentrations of (i) BAP (1.0-10 mg/l) alone or in

combination with NAA (0.5-3.0 mg/l) or IAA (0.5-3.0 mg/l) or IBA (0.5-3.0 mg/l),

(ii) Kn (1.0-10 mg/l) alone or in combination with NAA (0.5-3.0 mg/l) or IAA (0.5-

3.0 mg/l) or IBA (0.5-3.0 mg/l), (iii) coconut water (5-15%), (iv) IAA (2.5 - 10.0

mg/l) alone and (v) IBA (2.5-10.0 mg/l) alone. Different morphogenic responses were

tabulated in table 1.9.

MS medium supplemented with BAP (1.0-10 mg/l) produced yellow nodular

callus. At lower concentration (1.0-3.0 mg/l) the callus was soft but in higher

concentrations (5.0 & 10.0 mg/l) brown, friable as well as soft callus (Fig.1.11) was

noticed and at maximum proliferation was observed in BAP (3.0 mg/l) when compare

to lower and higher concentration.

The medium supplemented with NAA (0.5-1.5 mg/l) keeping BAP (3.0 mg/l)

constant to evaluate the potentiality of callus showed only the proliferation of

luxuriant yellow friable soft callus (Fig.1.12). Higher concentration of callus produces

huge amount of callus.

The callus did not show much significance response except proliferation on

the medium supplemented with Kn alone or combinations with auxins.

The callus subcultured on IAA and IBA supplemented medium showed well

proliferated yellow, nodular and friable compact callus and after 10 days of culture

Chapter - 1

48

root initials were produced. The number of roots were more in the IBA (2.5 & 5.0

mg/l) fortified medium than IAA supplemented medium. Higher concentrations (10.0

mg/l) of both the auxins gave raise to very negligible amount of callus.

The callus was also subcultured on MS medium supplemented with growth

adjuvant like coconut water and casein hydrolysate to test the morphogenic

potentiality of callus tissue. Casein hydrolysate supplemented medium showed no

response but in coconut water the callus became white, translucent, friable compact

and proliferated (Fig.1.13) well in the medium fortified with 10% of coconut water.

The leaf callus transformed to MS medium supplemented with various growth

hormones as mentioned above shows only the luxuriant proliferation and fails to

induce shoot buds but rhizogenesis (Fig.1.14). was noticed in cultures fortified with

IAA and IBA.

1.6b: Subculture of O. secamone callus

The primary callus obtained from the standard medium induced shoot buds

during sub cultures. The callus was subcultured on MS medium fortified with

different concentrations of BAP (1.0-10 mg/l) alone or in combination with NAA

(0.5-1.0 mg/l) or IAA (0.5-1.0 mg/l), and Kn (1.0-10 mg/l) alone or in combination

with NAA (0.5-1.0 mg/l) or IAA (0.5-1.0 mg/) for somatic embryogenesis. The

morphogenic responses were tabulated in table 1.11.

High percentage of shoot buds was observed in BAP fortified medium than

Kn. The leaf callus when cultured on MS medium supplemented with BAP (1.0-10

mg/l) produces green nodular hard callus. The same callus after two weeks of

subculturing on the same medium with NAA (0.5-1.0 mg/l) induces creamy white

Chapter - 1

49

sectors with well developed embryoids that later turned into shoot buds. MS medium

with BAP (3.0 mg/l) + NAA (1.0 mg/l) showed maximum number of shoot buds

(Fig.1.15). The number of buds decreased at higher concentrations of NAA, so

keeping NAA constant BAP in lower concentration (1.0-3.0 mg/l) produces more

number of shoots than higher concentration (5.0 & 10.0 mg/l) brown, friable callus

was noticed and at maximum proliferation callus was observed in BAP (3.0 mg/l)

containing medium. When compared to other concentration.

Subculturing the callus on the medium having BAP with various

concentrations of IAA did not showed much morphogenic response and also the

subcultured callus on Kn containing medium with different concentrations of NAA or

IAA showed further proliferation of hard whitish callus but differentiation of shoots

or roots was not observed. The callus proliferated on IAA containing medium turned

brown and produced roots at later stages.

Effect of ½ strength medium

Callus which was initiated from the standard medium is subcultured to the half

strength MS medium with same concentrations of hormones as that of standard

media. The response is very poor compared to the standard media.

Effect of different pH treatment

Callus from induction medium was subcultured on the same medium with

varying pH levels to know the effects of pH on the proliferation of callus and also for

the induction of embryogenic callus. The range of pH used being 2-10. The medium

fails to solidify at pH 2 and 4 but at pH 6, 8 and 10 the media solidification takes

Chapter - 1

50

place. The callus at pH 6, 8 and 10 did not show any morphological behavior and

remained as such.

The callus proficiency was also tested at the pH levels ranged from 5.0-6.5. At

pH 5.0 and 6.5 the proliferation was very scanty. At pH 5.5 and 6.5 moderate

proliferation of callus noticed while luxuriant proliferated callus tissue was observed

in the medium maintained at pH 5.8

Influence of coconut milk

Callus initiated on standard medium was subcultured on MS medium

supplemented with 3 mg/l BAP + 1 mg/l NAA and 5 mg/l BAP + 1 mg/l NAA

supplemented with various percentage of coconut milk ranging from 5%-25%.

Very slow growth callus was noticed in 5, 10 and 15% CM in media

supplemented with 3 mg/l BAP + 1 mg/l NAA where luxuriant callus was observed in

20% CM but in the medium supplemented with 25% shows moderate amount of

callus proliferation

All cultures showed induction of shoot buds (Fig. 1.16). The percentage of

shoot bud formation was more in 20% coconut milk supplemented standard medium

while low concentration (5%) coconut milk did not play any role in increase in the

number of shoots.

2. Stem culture:

2.1: Influence of different culture media

The explants such as nodal, and intermodal segments were inoculated in MS

medium, B5 medium & White’s medium supplemented with growth regulators like

Chapter - 1

51

2,4-D(0.5-5.0 mg/l), Kn (1.0-10.0 mg/l) and medium without growth regulators are

used as control.

The explants showed maximum response on MS medium as observed in leaf

cultures. Hence MS medium was selected for further studies and the percentage of

callus formation on all the three media is presented in table 1.7 and 1.8.

2.2: Neutralization of Browning

The same methods dexcribed in the leaf culture were employed to neutralize

the phenolics in stem explants. Like leaf explants the stem segments respond well in

the medium supplemented with ascorbic acid (2 mg/l) and it was found to be better

antioxidant in checking phenolics and induces callus from the explants.

2.3: Response of different explants type

Nodal explants of T. involucrata when cultured on MS medium supplemented

with 2,4-D or Kn or BAP alone or in combination with different auxins does not play

any role in the induction of multiple shoots. Initially the explants undergo browning

after three days of inoculation and finally senesce within five days of culture.

Ascorbic acid controls the death of explants but fail to induce callogenesis. Medium

supplemented with 2, 4-D in higher concentration (5.0 mg/l) induce scanty amount of

brownish callus after three weeks of culture which later necrosed.

Callogenesis was noticed on the internode explants of T. involucrata when

cultured on MS medium supplemented with 2, 4 –D or Kn. Presence of ascorbic acid

in the medium neutralizes browning of explants. The explants cultured on MS + 2, 4-

D (1.0, 2.0 & 5.0mg/l) or MS + kinetin (1.0, 2.0, 5.0 & 10.0 mg/l) showed bulging

towards the cut ends after seven days of culture and initiate callus all over the surface

Chapter - 1

52

with in 15 -20 days (Table.1.7 and Graph. 1.4).

The percentage of callus formation was more (91.6) in the 2.0 mg/l 2, 4-D

fortified medium (Fig. 1.17). Whereas decrease or increase in the concentration

showed decline in the proliferation of callus and the callus tissue was light green and

friable in nature. Luxuriant pale yellowish nodular compact callusing was observed in

Kn (2.0 mg/l) supplemented medium and also increasing the concentration showed

meager callus formation (Fig. 1.18).

The callus obtained from primary culture were subcultured on the medium

fortified with different concentration of (i) BAP (1.0-10 mg/l) alone or in combination

with NAA (0.5-3.0 mg/l) or IAA (0.5-3.0 mg/l) or IBA (0.5-3.0 mg/l), (ii) Kn (1.0-10

mg/l) alone or in combination with NAA (0.5-3.0 mg/l) or IAA (0.5-3.0 mg/l) or IBA

(0.5-3.0 mg/l), (iii) coconut water (5-15%), (iv) IAA (2.5 - 10.0 mg/l) and (v) IBA

(2.5-10.0 mg/l).

Like leaf culture shoot buds never observed. During subculture luxuriant

proliferation of callus (Fig. 1.19 and 1.20) only noticed in all kinds of tested

combinations of growth regulators but the callus subcultured on BAP or Kn along

with IAA induces number of root primordial along with proliferation.

Stem segments both nodes and internodes of O. secamone were cultured on

MS medium supplemented with 2, 4-D (1.0-10.0 mg/l) or BAP (1.0-5.0 mg/l) or Kn

(1.0-5.0 mg/l) alone exhibit different kinds of responses and the results were tabulated

in table 1.8 and represented in graph 1.5).

The nodal explants in 2, 4-D supplemented medium showed only callusing at

all concentrations (Fig. 1.21). The hormone fails to induce elongation of axillary buds

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53

even after six weeks of culture. The callus was greenish nodular compact and

maximum proliferation was noticed in 1.5 mg/l concentration.

Elongation of axillary buds was observed only in BAP (3.0 mg/l) containing

medium besides proliferated callus. The explants initially produce white, soft callus

but after three weeks of culture the axillary buds sprouted and gave rise to one to two

shoots (Fig. 1.22). the shoots were excised after it reached to 4-5 cm length and

transferred to half strength liquid medium supplemented with IAA or IBA for

rhizogenesis. Increase in the concentration of BAP (5.0 mg/l) induces multiple shoot

buds without callusing (Fig. 1.23) but they fails to elongate in the same concentration

and elongation was achieved in subculture.

Among the tested concentration of Kn on nodal segments it was observed that

formation of yellowish callus (Fig. 1.24) in scanty amount and the tissue fails to

proliferate even after four weeks of culture and there was no sign of multiple

induction.

The internodes when cultured on different concentrations of growth regulators

exhibit only callogenesis and morphologically it similar with nodal callus. Among the

tested concentration of growth hormones the maximum proliferation of callus tissue

was noticed in 2, 4-D (1.5 mg/l) BAP (3.0 mg/l) and Kn (2.0 mg/l)

2.4: Influence of subculture in induction of somatic embryogenesis

The primary that was induced from stem explants on medium supplemented

with 2, 4-D was subcultured on the MS medium supplemented with combinations of

auxins and cytokinins in different combinations to achieve embryogenesis. the tested

combinations were (i) BAP (1.0-5.0 mg/l) alone or in combination with NAA (0.5-

Chapter - 1

54

1.0 mg/l) or IAA (0.5-1.0 mg/l), and Kn (1.0-5.0 mg/l) alone or in combination with

NAA (0.5-1.0 mg/l) or IAA (0.5-1.0 mg/l), and also with growth adjuvants like

coconut water (5-15%) and casein hydrolysate (50 & 100 mg/l) individually and the

influence of growth regulators on plant regeneration was recorded.

Friable callus was subcultured on MS medium supplemented with either BAP

or Kn alone. Within 4 weeks of culture adventitious shoot buds were differentiated

from the surface of the callus. Shoot buds were elongated within 5 weeks of culture.

The callus after subculture onto the MS medium, having various concentrations of

BAP (1.0-5.0 mg/l) showed that highest shoot regeneration frequency (70%) per

callus mass was obtained at 2.0 mg/l BAP and increase in the concentration of BAP

was not beneficial as callusing increased gradually with suppression of shoot

formation (Table. 1.12).

The results indicate that NAA or IAA in combination with BAP had a

synergistic effect on multiple shoot induction (Table. 1.13). It was found that NAA

(1.0mg/l) in combination with BAP (3.0 mg/l) resulted multiple shoots (Fig. 1.25) in

nearly 85% of explants whereas only 65% of explants induce multiple shoots in the

medium supplemented NAA (1.0 mg/) and BAP (2.0mg/l). Increase in the

concentration of BAP (5 mg/l) produced clumps of highly-reduced shoots with

smaller leaves where such abnormal shoots were grown normally when subcultured

on low concentrations of BAP. Highest number of shoots (64.8 ± 0.74) were recorded

in the BAP (3.0mg/l) and NAA (1.0mg/l) concentration and it also favours the length

of shoots (4.50 ± 0.12cm). The shoot bud organogenesis was also observed in

medium fortified with combination of BAP and IAA supplemented medium in the

range of 0.5-1.0 mg/l (Table. 1.14). Compare to NAA, the number of multiple shoots

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55

per callus gradually decreased with increasing in concentration of BAP. After eight

weeks culture the best result (45%) was achieved at 2.0mg/l BAP+ 1.0mg/l IAA,

expressed more number of shoots (4.0±0.47) and these buds fail to elongate in the

same concentration, subculture of shoot buds on NAA (1.0mg/l)+ BAP (3.0mg/l)

showed increase in the formation of shoots along with elongation.

Among various concentrations of Kn tested, the highest shoot regeneration

frequency (55%) was recorded at 2.0 mg l-1 concentration (Table. 1.15). Increasing

the concentrations of Kn from 4.0-5.0 mg l-1 resulted in a decrease (15-20%) in the

rate of shoot regeneration capacity while in combination with auxins better response

in multiplication was achieved. Keeping NAA (1.0 mg/l) constant, Kn (2.0mg/l)

induces shoots in 33-34% of cultures with mean length of 2.25 ± 0.95 cm while

increase in the concentration (3.0mg/l) exhibit a slight increase (41-42%) in the

frequency of shoot formation (Fig. 1.27) whereas decreases the length (2.00±0.81) and

number (11.0±1.82) of shoots (Table. 1.16).. Further the explants inoculated at higher

concentrations of Kn (5 mg/l) proliferation of callus was observed. Supplementation

of IAA instead of NAA along with Kn was also not beneficial for induction of

multiple shoots. In Kn (2.0 mg/l) + IAA (1.0 mg/l) supplemented medium nearly 30%

of the cultures induce multiple shoots with short internodes.

The tested complex extracts such as CM (10 & 15%) & CH (50 & 100 mg l-1)

did not improve the regeneration ability, shoot number and shoot length. The extracts

increasing the proliferation of callus and suppress shoot bud formation.

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56

2.5: In vitro rooting of micro shoots

In vitro differentiated shoots from both stem derived callus and adventitious

bud elongation of O. secamone were excised aseptically and transferred on to half and

full strength MS liquid medium supplemented with different concentrations of IAA,

IBA and NAA (0.5-2.0mg/l) respectively. After two to three weeks of culture high

frequency of rooting was observed in half strength medium whereas full strength

medium shows low frequency and induces multiple shoots on NAA (2.0mg/l)

supplemented medium (Fig. 1.27).

Among the auxins tested IAA found to be most effective (Table. in induction

of rooting (Table. 1.17) from the cut ends of shoots after 15 days of culture and

produced maximum number (3.6±0.12) of roots in 80% of cultures fortified with

1.0mg/l IAA. (Fig. 1.28 and 1.29).

2.6: Hardening of regenerated plants

After three weeks of incubation, the well grown shoots with roots were

separated and transferred into pots containing garden soil mixed with vermiculite and

sand (1:1:1) and maintained in the hardening room under 25±2⁰C with 80-90%

relative humidity for 15-20 days (Fig. 1.30). After two weeks of hardening, the

hardened plants were transferred from incubation chamber to green house for further

acclimatization and finally to garden soil. It was observed that the plants show nearly

80% survival capacity.

Chapter - 1

57

DISCUSSION

Tissue culture techniques have been increasingly applied for micropropagation

and conservation of medicinal plants through direct or indirect regeneration methods.

In vitro plant regeneration is influenced by many factors like media composition,

exogenous growth hormones, nature of explants, genotypes and cultural

environments. The present study reveals the importance of mentioned factors in the

regeneration of selected medicinal plants.

Culture medium is one of the important factors in establishing in vitro culture

because it is specific to a particular tissue. In the present study it was found that MS

medium was an effective medium for the growth and maintenance of callus as well as

for plant regeneration as described in several workers since from Murashige & Skoog,

Sudarshana & Shanthamma (1991), Mathur (1992), Komalavalli & Rao (2000),

Madhavan & Joseph (2001), Catapan et al ( 2002), Jain et al (2003), Vasanthakumari

& Shivanna (2005), Shankaraamurthy & Krishna (2006), Tawfikk & Mohamed

(2007), Vijaya et al (2008), Robinson et al., (2009), Patel & Shah (2010), Sharanappa

& Rai (2011), Mehta et al (2012), Pattar & Jayaraj (2012) ..

Any part of the plant body acts as explants for in vitro culture that may

directly or indirectly induce somatic embryogenesis and it has also been showed that

the suitability of different basal media depends on the genotype, age and type of

explants (Franclet, 1991). As mentioned in literature survey many workers

successfully regenerate plants by different explants on different types of basal media

with growth regulators. The plant parts such as leaf segments, internode segments,

nodal regions with axillary buds, root, ovule, anther, pollen, embryo, and endosperm

act as explant source.

Chapter - 1

58

The present study revealed that exogenously supplied growth hormones are

essential for the induction of callus from all the tested explants and no callus was

induced by MS basal medium alone. Nin et al.(1996) reported no callogenic response

from leaf explants on hormone free medium and explant died after few days. The

hormonal content of a culture medium is crucial for any sustained growth of the

culture and The growth and development of higher tissues in vitro, especially during

callogenesis in various concentration mixtures, is controlled by gradients of

endogenous plant growth substances (Narayanaswamy, 2005).

Leaves are usually considered as efficient explants for high frequency

propagation in vitro. In order to develop an efficient regeneration system for T.

involucrata, leaf and stem as explants was used and attempted to adjust hormone

types and concentrations in the induction medium but failed to achieve the described

results because of heavy browning and non differentiation of explants. Browning of

explants and subsequent death is a common problem in tissue culture of plant species

containing phenolic compounds and extra steps are usually incorporated in the culture

protocol to overcome this problem.

Addition of antioxidants like ascorbic acid and citric acid favors the control of

browning and leads to organogenesis. Among the two antioxidant tested in T.

involucrata ascorbic acid found to be beneficial as reported in Tylophora indica

(Neelam & Chandel, 1992) and in Gymnema elegans (Komalavalli & Rao.1997)

where as citric acid found best in cultures of Gymnema sylvestre (Komalavalli & Rao,

2000) and Prosopis cineraria (Shekhawat et al., 1993).

Activated charcoal is known to be a very effective compound to absorb

potentially harmful phenolics in culture media, produced by the tissues (Weatherhead

Chapter - 1

59

et al. 1979). Fridbourg et al. (1978) suppose the absorption of substances by AC,

which are necessary for cell growth. Weatherhead et al. (1978, 1979) found the

absorption of growth regulators (auxins and cytocinins) and of thiamine, nicotinic

acid and inositol. Addition of Activated charcoal (2.0-5.0mg/l) significantly reduced

the phenol content in present culture media but fail to induce callus or shoot as

reported in Vitis spp. (Reustle & Natter,1994), Nyctanthes arbor-tristis (Reeth &

Shivamurthy,2005). & Gymnema sylvestre (Komalavalli & Rao, 2000). The negative

effect of AC on plating efficiency could be due to a direct inhibitory effect on the one

hand and to the absorption of substances which are essential for growth on the other

hand. So, using the described culture system, AC has to be considered as an

unsuitable additive to support present culture but in many cases it is reported to have

beneficial effects in Eucalyptus tereticornis (Das & Mitra, 1990), Sesbania sesban

(Shanker & Mohan ram, 1990) and in Strelitzia reginae (North et al. 2011).

In culture, Cell proliferation, which started at the injured part of explants, may

have been due to the accumulation of auxins at the point of injury, which stimulated

cell proliferation in the presence of growth regulators. Depending upon the auxin type

the percentage of callus formation varies. In the majority of plant species the synthetic

hormone 2, 4-D interacted with endogenous hormones of the explants and stimulated

the cells to proliferate into callus mass (Narayanaswamy, 1994). As observed in the

culture of Tylophora indica (Faisal & Anis, 2003), Guizotia abyssinica (Kumar et

al,2000), Embelia ribes (Shankaramurthy et al,2004), Desmodium oojeinense

(Vasanthakumari & Shivanna, 2005), Flaveria trinervia (Sudarshana & Shanthamma,

1991), Rauwolfia serpentina (Perveen & Ilahi, 1978), Epilobium parviflorum

(Akbudak & Babaoglu 2005). In present investigation also 2, 4-D plays a major and

distinctive role in the induction of callus from different explants of both the species.

Chapter - 1

60

In T. involucrata, at lower concentration of 2, 4-D (1.0mg/l), 75% of leaf

cultures showed callus formation the frequency was increased up to 83.3% at 2.0mg/l

of 2, 4-D and the callus was white greenish granular in nature. The color of callus

changes to light yellow with gradual decrease in the formation of callus with

increased concentration. Similarly in O. secamone the highest frequency (98%) was

noticed in same concentration and at higher concentration a decline in the percentage

of callus was noticed and the callus was Light green/green friable or compact in

nature. The callus was highly proliferative in both the cases at 2.0mg/l concentration.

Therefore MS medium containing 2, 4-D (2.0 mg/l) was considered as optimum

concentration for induction and for proliferation of callus in both the plants. The same

type of results were found in plants like Tylophora indica (Faisal and Anis, 2003),

Withania somnifera (Rani et al., 2003), Ruta graveolens (Ahmed et al. 2010), Ananas

comosus (Amin et al., 2005) and in Epilobium parviflorum (Akbudak & Babaoglu,

2005).

Initially the explants produce white greenish or light yellow, translucent,

granular or friable soft callus. But, after 30 days of culture callus turns brown, this

may be due to synthesis of phenolics and frequent transfer of these cultures to fresh

medium reduced browning. This approach has been used in many species like Rubus

fruticosus (Broome and Zimmermann, 1978), Kalmia latifolia (Lloyd and McCown,

1980), Euphorbia lathyris (Ripley and Prece, 1986), Hevea brasiliensis (Senevirathne

& Wijeskara, 1996) Pimpinella anisum (Chand et al., 1997), Pisonia alba

(Jagadishchandra et al, 1999), Rauwolfia tetraphylla (Ghosh and Bannerjee, 2003),

Sida rhombifolia (Guha et al, 2006) and in Datura metel (Ravikumar and

Krishnamoorthy, 2010). By this method, here also the problem was overcome and

produced luxuriant proliferated callus through subculturing of callus on MS medium

Chapter - 1

61

supplemented with different concentrations and combinations of growth regulators

such as BAP, NAA, CW, IAA and IBA at 15-20 days interval for callus proliferation

and also for induce somatic embryogenesis.

The calli were kept on all tested media for approximately one month and

showed no differentiation of shoot but shows high frequency of callus multiplication

and luxuriant proliferation. Sokmen and Gurel (2001) stated that during subculturing,

changing subculture number and volume of plant growth regulators may cause

formation of shoot or root in plants because some cells might differentiate. Such

types of indirect organogenesis was reported in many plant species including Cassia

obtusifolia (Hasan et al. 2008), Triticum aestivum (Rahman et al. 2008), Agave

amanuensis (Andrijany et al. 1999), Rotula aquatica (Martin, 2003), Phellodendron

amurense (Azad et al. 2005) and Acmella calva (Senthilkumar et al. 2007). But in T.

involucrata, no plant regeneration was achieved though out the period of subculture

but calli continued their growth normally. Our investigation related with the report

of Guha et al (2006) in Sida rhombifolia, achieved different morphological

characters of the callus by serial sub culturing on fresh medium but it fails to induce

somatic embryos and also in Epilobium parviflorum (Akbudak & Babaoglu, 2005)

no regeneration was achieved when primary culture was subcultured on different

concentration of growth regulator for nearly one month. The observation of Sethi et

al. (1990), is significant in this regard which indicated that the addition of hormones

at times not only induce proliferation of cells resulting in increased growth but also

may change characteristics of cells making them neoplastic and incapable of

differentiation.

Chapter - 1

62

Rhizogenesis was achieved by sub culturing callus on medium containing IAA

or IBA.IBA (2.0 & 5.0mg/l) induced a mat of white roots. On IAA (2.0 & 4.0mg/l)

supplemented medium a few roots were induced.IBA induced roots efficiently in case

of Swaisonia formosa (Jusaitis, 1997) and Cunila galoides (Fracro and

Echiverrigarray, 2001).

In vitro regeneration can be obtained by embryogenesis or organogenesis. In

O. secamone regeneration has been obtained by organogenesis from embryogenic

calli drived form leaf and stem explants. There are many reports on the regeneration

of various medicinal plants like Asparagus cooperi (Ghosh and Sen, 1994) , Flaveria

trinervia ( Sudarshana and Shanthamma, 1991), Lagerstroemia indica (Niranjan et al.

2007), Plumbago zeylanica (Das and Rout, 2002); Holostema ada-kodien (Martin,

2003); Gloriosa superba (Sivakumar and Krishnamurthy 2003); Abrus precatorius

(Biswas et al., 2007); Scoparia dulcis (Hassan et al., 2008); Plumbago rosea

(Gopalakrishnan et al., 2009); Desmodium oojeinense (Vasanthakumari and

Shivanna, 2005) via callus culture and also organogenesis is one of the efficient

methods for mass multiplication of medicinal plants Direct organogenesis in many

plants like Ruta graveolens ( Faisal et al. 2005), Pineapple (Hamasaki et al. 2005),

Citrulus lanatus (Sultana and Bari, 2003), Irvingia gabonensis (Fagimi et al. 2007),

Albizia lebbeck (Mia and Rao, 1996).

In many protocols for somatic embryogenesis a strong auxin such as 2, 4-D or

auxin/cytokinin concentration is used in the primary culture medium to support both

cell proliferation and induction of embryogenesis (Ananthakrishnan et al. 1999;

Cardoza and D’ Souza, 2000: Mathew et al, 2000). Because the proembryogenic mass

developed in culture media containing auxins is generally believed to synthesize all

Chapter - 1

63

the genes necessary to complete the globular stage of embryogenesis (Zimmerman,

1993). But often if embryos are exposed to too much auxin during development, they

fail to accumulate storage proteins and germinate at a lower frequency (Stuart et al.

1985). In O. secamone the highly proliferative green and compact calli derived from

MS medium supplemented with 2, 4-D were subjected to subculture on MS medium

supplemented different combination and concentration of auxins and cytokinins

showed regeneration of shoots and proliferation of callus. This results supports the

opinion of Ananthakrishnan et al. (1999), D’ Souza and Cardoza (2000), Mathew et

al. (2000) and also Stuart et al. (1985).

Growth regulators especially cytokinins and auxins provide a mechanism for the

regulation of organogenesis (Thorpe, 1980). Type and concentration of cytokinins

used remarkable influenced in the regeneration of shoots form callus tissue.

In the present investigation among BAP and Kn, BAP was found effective in

the induction of shoot buds from callus tissue. Highest percentage of shoot (70%)

were observed in the stem derived calli on 2.0mg.l BAP while medium supplemented

with Kn (2.0mg/l) recorded high percentage (55%) and reduced number of shoots

with shorter internodes. Similar response was also observed in the propagation of

Asclepias (Chi Won and John, 1985) and Gymnema sylvestre (Reddy et al., 1998).But

in leaf derived calli, the cytokinins does not play any role in the shoot budinduction.

The report indicates that the cytokinin BAP at most of the concentration was

comparatively more effective in inducing shoot buds from callus and shoot

multiplication whilst Kn was considerabaly less effective. Superiority of BAP over

other cytokinin in producing shoot has also been confirmed in many medicinal plants

like Atropa belladonna (Benjamin et al. 1987), Piper nigrum (Mathews and Rao

Chapter - 1

64

1984), P. longum (Bhat et al. 1992), P. colubrium (Kelakr et al. 1996), Hybanthus

enneaspermum (Natarajan et al 1999), Psoralea Corylifolia (Jeyakumar and

Jayabalan, 2001), Adhatoda vasica (Azad et a. 2003) Holorrhena

antidysentrica.(Kumar et al. 2005), Gardenia gummifera (Gajakosh et al. 2011).

It has been our study lower concentration of both the cytokinins enhances shoot

induction and increase in the concentration above the optimum concentration

(2.0mg/l) leads decrease in the shoot formation, indicating that lower concentration of

cytokinins favoured shoot induction than higher concentration. The same kind of

result were shown in plants like Ruta graveolens (Faisal et al. 2006), Coleus forskholii

(Reddy et al. 2001), Tylophora indica (Faisal and Anis, 2005), Anthurium andraeanu

(Jahan et al. 2009) Colocasia esculenta (Chand et al.1999), turmeric and ginger

(Balachandran et al. 1990). In contrast to these reports Vasanthakumari and Shivanna

(2005) and Sudarshana and Shanthamma (1991) reported highest concentration of

BAP induces multiple shoot from callus cultures of Desmodium oojeinense and

Flaveria trinervia respectively.

The presence of cytokinin along with auxin is necessary for indirect shoot

induction as noted by Skoog and Miller (1957). In the present study addition of auxins

such as IAA/NAA to the culture medium in combination with cytokinin, BAP

enhances the frequency of shoot initiation and shoot number. These results are in

agreement with those in Rawolfia tetraphylla (Ghosh and Banerjee, 2003) and

Withania somnifera (Kannan et al 2005) where NAA in combination with cytokinin

has shown to promote shoot bud differentiation. The stem derived callus tissues was

transferred to different shoot bud induction medium become nodular and were found

highly competent for shoot bud initiation. Shoot buds generally arose as cluster within

Chapter - 1

65

the callus as well as abaxial surface of callus. It was observed that callus clumps

cultured on BAP (3.0 mg/l)+ NAA (1.0mg/l) produced maximum number of shoots

than BAP+IAA combination media. It indicates NAA showed the synergistic effect

with BAP and enhanced the induction of shoot buds from callus and increased the

shoot morphogenic response as compared to the combination of other auxin (IAA)

with BAP. A similar observation also reported in Petasites hybridus (Wldi et al.

1998), Eucalyptus grandis (Luis et al. 1999), Coleus forskholii (Reddy et al. 2001),

Saussurea medusa (Zhao et al. 2001), Rauvolfia tetraphylla (Faisal and Anis, 2002),

Rotula aquatica (Martin, 2003), Tylophora indica (Faisal and Anis, 2003), Psoralea

corylifolia (Anis and Faisal. 2005); Ruta graveolens (Faisal et al. 2006), and in

Solanum nigrum (Sridhar and Naidu, 2011).

In against to our finding Sharma and Vimala devi (2006) reported IAA is better

than NAA along with cytokinin (BA) for induction of multiple shoots in Peristrophe

bicalyculata. This view was supported by Erdei et al. (1981) in Digitalis lanata,

Mustafa et al. (1997) in Kaemperia rotunda; Rout et al. (1999) in Plumbago

zeylanica, Kaur et al. (1999) in Valeriana jatamansi and Solanum trilobatum

(Arokiasamy et al. 2002).

The leaf derived callus tissues was transferred to different shoot bud induction

medium shows proliferation of callus in all concentrations of BAP. Highest

proliferation was observed in 2.0 mg/l concentration. Along with NAA, the cytokinin

exhibited shoot buds. Like stem calli, NAA and BAP combination perform well than

IAA+BAP combination. Maximum percentage of shoot regeneration was noticed in

BAP (3.0mg/l) +NAA (1.0mg/l) combination. The addition of low concentration of

auxin along with cytokinin had an added advantage in intiation of more number of

Chapter - 1

66

shoot buds. The higher requirement of cytokinin for shoot proliferation in callus

cultures has been reported by many workers (Flick et al, 1983; Baburaj et al., 1994;

Dodds and Roberts, 1985; Dornenberg and Knorr, 1995; Begum et al, 1995; Rout et

al. 1996; Abo.1997; Haque et al. 1997; Rahman et al. 2004; senthilkumar et al.2007;

Ravikumar and Krishnamoorthy, 2010). Incorporation of a balanced ratio of growth

supplements could therefore bring out cellular differentiation leading to organ

formation in callus tissue. Shoot induction was very low or absent at different

concentrations of Kn alone or in combination with NAA or IAA as reported in

Artemisia absinthium (Zia et al. 2007), Withania coagulans (Valizadeh and

Valizadeh, 2009) and in Withania somnifera ( De Silva and Senarath, 2009).

Axillary bud development has proven to be the most applied and reliable

system for true-to-type in vitro propagation in general. Nodal explants as the best

source of multiple shoot induction has reported in many medicinal plants such as

Rauvolfia serpentina (Roy et al.1995), Emblica officinalis (Rahaman et al. 1999),

Holorrhena antisysenterica (Ahmed et al. 2001) and Enicostemma hyssopifolium

(Seetharam et al.2002). In O. secamone, multiple shoots were achieved from the nodal

explants cultured on the medium supplemented with BAP alone. The nodal explants

initially induce callus and later the bud breaking takesplace in 3.0 mg/l BAP and but

in 5.0mg/l induces average four to five shoots without intervening of callus. On the

other hand Kn was not effective in induction of multiple shoot buds as has been

reported by Chandra and Gowda (2003) in mulberry, Sudha et al. (1988) in

Holostemma annulare. In present study also Kn was not so effective as BAP in

multiple shoot induction. These findings support the report of George (1993),

cytokinins especially BAP, overcome the apical dominance, release the lateal buds

from dormancy and promote shoot formation. Multiple shoot formation was also

Chapter - 1

67

reported in other medicnal plants like, Phyllanthus corolininsis (Catapan etal. 2000),

Celastrus paniculatus (Lakshmi and Seeni. 2001), Ruta graveolens (Faisal et

al.2005), Passiflora foetida (Anand et al. 2012) and Sida cordifolia (Sivanesen and

Byoung, 2007).

Formation callus along with bud breaking in nodal expalnts is in agreement

with the report of Martin (2002). He noticed callus formation from nodal explants of

Holostemma ada-kodien and suggested that the induction of callus and the

regeneration of multiple shoots from nodal explants may be due to the presence of

some internal components from the preexisting axillary buds that are essential for

induction of caulogenesis. This view was supported by Karnawat et al (2011) after

inducing high multiple shoots form nodes of Verbesima encelioides. Similar studies

on Sida cordifolia (Pattar and Jajaraj, 2012), Leptadenia reticulate (Farzin et al. 2007)

and Hypericum maculatum (Bacila loan and Ana Coste, 2010) reported indirect

organogenesis from nodal explants.

After determining the optimum cytokinins and auxin levels for shoot

regeneration, synergistic effect of complex extracts was studied to improve the

frequency of regeneration. CM (10 & 15%) & CH (50 & 100 mg l-1) did not

significantly improve the regeneration ability. Treatment with CM & CH resulted in

callusing and suppression of shoot bud proliferation. The result correlated with the

finding of Komalavalli and Rao (2000) in Gymnema sylvestre, reported suppression of

shoot bud formation by CM and CH. In contrast to this same authors reported

beneficial effect of CM in shoot sprouting of G. elegans and similar report was given

by Gras and Calvo (1996) in Lavandula latifolia.

Chapter - 1

68

Excised healthy shoots obtained directly and in directly were cultured on half

strength or full strength MS liquid medium supplemented with different

concentrations o IAA, IBA and NAA individually at the range of (0.5-2.0mg/l). the

effects of these auxins on root induction as well as the length of the roots were

examined after one two weeks of culture. No rooting was observed in MS basal

medium an dulls strength medium with auxins showed very poor response in rooting,

but half strength MS medium comes out with high frequency roots . Half strength

MS medium was found better in many medicinal plants such as Ruta graveolens

(Faisal et al.2006), Lagerstroemia indica (Niranjan et al. 2007), Lavendula vera

(Andrade et al. 1999), Ocimum kilimandscharicum (Saha et al. 2010). The rational

behind the favorable effect of reduced macronutrient concentration is that the

concentration of nitrogen ions needed for root formation is much lower than for shoot

formation and growth (Driver and Suttle, 1987).

Among the auxins tested IAA found to be most effective in induction of

rooting from the cut ends of shoots after 15 days of culture and produced maximum

number (3.6±0.12) of roots in 80% of cultures fortified with 1.0mg/l IAA. Similar

observation were made by Gopi et al. (2006) in Ocimim gratissimum; Sharma and

Vimaladevi (2006) in Peristrophe bicalyculata; Sudarshana and Shanthamma (1991)

in Flaveria trinervia; Ahmed et al. (2005) Phyla nudiflora, Nikam et al. (2009)

Momordica cymbalaria; Rizvan et al. (2010) in Artemisia vulgaris.

Other auxins like IBA or NAA are also had beneficiary effect in inducing

roots from culture shoot in many medicinal plants like, Aloe polyphylla (Abrie and

Staden, 2001), Tylophora indica (Faisal and Anis, 2003), Crataeva magna

(Benniamin et al. 2004), Ruta graveolens (Faisal et al. 2006).

Chapter - 1

69

In vitro derived plantlets of O. secamone were morphologically similar to in

vivo plants and nearly 75-80% of regenerated plants were successfully acclimatized.

The present investigation on in vitro studies could be useful for the production

of plantlets of O.secamone throughout the season and also provide alternative source

to intact plant that is callus development to for the extraction of drug in both the

plants. Though micropropagation the medicinal plants are conserved from the line if

extinction.

Chapter - 1

71

Table 1.3: Morphogenic nature of leaf explants of T. involucrata on MS medium

supplemented with cytokinins individually.

MS

Medium

Concentration

(mg/l)

Nature of Response

Basal - -

BAP

1.0

Scanty yellowish callus

explants turned browning and senesced

2.0

3.0

4.0

5.0

Kn

1.0

Scanty yellowish callus

explants turned browning and senesced

2.0

3.0

4.0

5.0

Table 1.4: Morphogenic nature of leaf explants of O. secamone on MS medium

supplemented with various auxins individually

MS

Medium

Concentration

(mg/l)

% of

Response Nature of Response

Basal - - -

2.4-D

0.5 65 Light green compact proliferation

1.0 75

2.0 98 Whitish green compact luxuriant proliferation

3.0 75

4.0 55 Whitish green compact decline in proliferation

5.0 45

NAA

1.0 50 Whitish friable less proliferated callus

2.0 65

3.0 81 Whitish friable proliferated callus

4.0 - Explant remain green & dried up

No callus 5.0 -

IAA

1.0 50 White, compact & hard well proliferative callus

2.0 55

Whitish, hard proliferative callus 3.0 65

4.0 48

5.0 50

Chapter - 1

72

Table 1.5: Morphogenic nature of leaf explants of O. secamone on MS

medium supplemented with various Cytokinins individually

MS Media Concentration

(mg/l) % Response Nature of Response

BAP

1.0 - Curling of Explants, No callus

2.0 75 Pale brown compact highly

proliferative callus

3.0 42 Pale brown, compact, hard, less

proliferative 4.0 35

5.0 35 Light yellowish, less proliferative,

turns brown as it ages

Kn

1.0 68 whitish scanty, no proliferation

2.0 65 whitish, no proliferation

3.0 75 whitish hard proliferative juicy

4.0 60 whitish turn brown

5.0 45 Faint yellwoish turns brown

Chapter - 1

73

Table 1.6: Morphogenic nature of leaf explants of O. secamone on MS

medium supplemented with combination of Growth regulators

MS Medium Concentration

(mg/l) Nature of response

Basal - -

BAP + 2.4-D

(1.0 - 5.0 )

+

(0.5 - 4.0)

Pale greenish nodular callus induced and

supported better proliferation

BAP (3.0) +2.4-D (1.0) shows better

callogenesis. No shoot buds

BAP + NAA

(1.0- 5.0 )

+

(0.5 - 4.0)

Light yellowish soft calli induce from cut

edges.

BAP (4.0) + NAA (2.0) induced high

amount of callus. No shoot buds

BAP + IAA

(1.0- 5.0 )

+

(0.5 - 4.0)

Light green/green compact callus

BAP (3.0) +IAA (1.0) callus with roots

No shoot buds

Kn + 2.4-D

(1.0- 5.0 )

+

(1.0 - 3.0)

Whitish smooth & non nodular callus.

Better proliferation of callus.

No shoot buds

Kn + NAA

(1.0- 5.0 )

+

(1.0 - 3.0)

Greenish scanty callus appeared at cut edges.

No proliferaton & become yellow as it ages.

No shoot regeneration

Kn + IAA

(1.0- 5.0 )

+

(1.0 - 3.0)

Kn (2.0) + IAA (1.0) shows rhizogenesis

along with callus

Chapter - 1

74

Table 1.7: Morphogenic response of stem explants of T. involucrata on MS

medium supplemented with different growth regulators

Growth Regulator Concentration % of Response Nature of callus

2.4 D

1.0 66.6 Callus appeared at cut edges

& gradually covered all over the surface.

Light yellow, friable

transulent

2.0 91.6

3.0 75.3

4.0 60.0

5.0 58.3

Kn

1.0 91.6

Greenish nodular

compactcallus

2.0 91.6

3.0 86.0

4.0 84.3

5.0 83.1

10.0 58.3

Table 1.8: Morphogenic response of stem explants of O. secamone on MS

medium supplemented with different growth regulators

Growth Regulator Concentration

(mg/l) % of Response

Nature of response

2.4-D

0.5 63.0

Greenish compact nodular callus.

No shoot formation

1.0 70.0

1.5 94.0

2.0 88.0

3.0 67.0

5.0 45.0

8.0 32.0

10.0 20.0

BAP

1.0 51.0 White soft

transulent callus 2.0 73.0

3.0 75.0 Callus with shoot

4.0 60.0 Proliferated callus

5.0 23.0 Multiple shoot without callus

Kn

1.0 45.0 Yellowish juice callus

Less proliferative

No multiple shoots

2.0 50.0

3.0 60.0

4.0 40.0

5.0 27.0

Chapter - 1

75

Table 1.9: Effect of sub culture on T. involucrata callus cultured on MS medium

with various supplements

Growth

Regulator

Concentration

(mg/l)

Organogenesis

(Shoot/Root)

Growth of

Callus Nature of Callus

BAP

1.0 - Moderate growth Yellow nodular

compact soft.

Non embryogenic

2.0 - Moderate growth

3.0 - Profuse mass

5.0 - Profuse mass Brown,friable,&soft.

Non embryogenic 10.0 - Moderate growth

BAP +

NAA

3.0 + 0.5 - Profuse mass Light yellow friable

nodular & soft 3.0 + 1.0 - Profuse mass

3.0 + 1.5 - Luxuriant mass

Kn

1.0 - Profuse mass

Pale greenish friable,

Non embryogenic

2.0 - Luxuriant mass

3.0 - Moderate

5.0 - Moderate

10.0 - Slow

Kn + NAA

2.0 + 0.5 Root Profuse mass Light green compact

callus 2.0 + 1.0 - Luxuriant mass

2.0 + 1.5 - Profuse mass

IAA

2.5 Root Profuse mass Yellow, nodular &

friable compact hard

callus 5.0 Root Profuse mass

IBA

2.5 Root Profuse mass Yellow, nodular &

friable compact hard

callus 5.0 Root Profuse mass

Chapter - 1

76

Table 1.10: Effect of sub culture on T. involucrata callus on MS medium

supplemented with growth adjuvants

Growth

adjuvant

Concentration

( % ) Organogenesis

Growth

of Callus Nature of Callus

Coconut water

5 - Slow

growth

White,transuluscent,

friable and compact 10 -

Luxuriant

growth

15 - Profuse

growth

Casein

hydrolysate

5 - Slow

growth

Friable & compact

nature 10 -

Slow

growth

15 - Slow

growth

Table 1.11: Induction of multiple shoots from O. secamone leaf callus on MS

medium supplemented with BAP and NAA

*: % of callus proliferation #: no shoot formation

Plant growth regulator Frequency of

shoot regeneration

(%)

No.of shoots/explant

Mean ±S.D

Shoot length (cm)

Mean ±S.D BAP NAA

1.0 - 41* # #

2.0 - 75* # #

3.0 - 66* # #

5.0 - 39* # #

10.0 - 22* # #

1.0 0.5 39 24.49±0.49 2.05±0.41

2.0 0.5 75 46.43±0.49 3.94±0.86

3.0 0.5 63 34.50±0.51 3.78±0.14

5.0 0.5 25 22.58±0.49 1.80±0.07

10.0 0.5 17 10.45±0.51 1.05±0.06

1.0 1.0 55 21.45±0.51 2.87±0.07

2.0 1.0 65 42.10±0.81 3.12±0.28

3.0 1.0 80 52.19±0.92 4.80±0.83

5.0 1.0 40 20.45±0.51 1.15±0.05

10.0 1.0 23 8.10±1.25 0.76±0.16

Chapter - 1

77

Table 1.12: Induction of multiple shoots from O. secamone stem callus on MS

medium supplemented with BAP

Plant growth regulator

BAP

Frequency of shoot

regeneration (%)

No. of Shoots/explant

Mean ±S.D

Shoot length (cm)

Mean ±S.D

1.0 14 18.0±14.1 1.74 ± 0.14

2.0 70 44.5 ± 2.41 3.92 ± 0.21

3.0 55 20.8 ± 1.05 3.28 ± 0.09

4.0 53 18.0±� 2.75±�

5.0 40 17.2±�� 1.38±�

Table 1.13: Induction of multiple shoots from O. secamone stem callus on MS

medium supplemented with BAP and NAA

Plant growth regulator

BAP NAA

Frequency of

shoot regeneration (%)

No. of Shoots/explant

Mean ±S.D

Shoot length (cm)

Mean ±S.D

1.0 0.5 44 9.45±0.51 1.08±0.35

2.0 0.5 60 15.1±0.87 1.32±0.32

3.0 0.5 75 44.5±0.50 3.10±1.20

4.0 0.5 35 24.0±0.81 2.10±1.20

5.0 0.5 40 19.2±0.78 0.99±0.32

1.0 1.0 55 17.5±0.51 1.15±0.27

2.0 1.0 65 37.4±1.32 3.58 ± 0.06

3.0 1.0 85 64.8±0.74 4.50 ± 0.12

4.0 1.0 70 28.0±1.43 3.01±1.40

5.0 1.0 40 24.2±0.72 0.70±1.40

Chapter - 1

78

Table 1.14: Induction of multiple shoots from O. secamone stem callus on MS

medium supplemented with BAP and IAA

Plant growth regulator

BAP IAA

Frequency of shoot

regeneration (%)

No. of shoots/explants

Mean ±S.D

Shoot length (cm)

Mean ±S.D

1.0 0.5 30 1.25±0.43

Shoots fail to

elongation

2.0 0.5 40 1.81±0.44

3.0 0.5 40 1.80±0.41

4.0 0.5 35 2.75±0.78

5.0 0.5 20 2.66±0.84

1.0 1.0 19 1.24±0.37

2.0 1.0 42 2.23±0.49

3.0 1.0 38 3.01±1.02

4.0 1.0 35 4.60±0.52

5.0 1.0 20 2.60±1.51

Table 1.15: Induction of multiple shoots from O. secamone stem callus on MS

medium supplemented with Kn

Plant growth regulator

Kn

Frequency of shoot

regeneration (%)

No. of shoots/explant

Mean ±S.D

Shoot length (cm)

Mean ±S.D

1.0 20 18.0 ± 1.41 1.74 ± 0.14

2.0 55 30.6 ± 1.20 2.98 ± 0.11

3.0 17 25.0 ± 0.70 2.46 ± 0.05

4.0 15 10.1±0.62 1.66±0.35

5.0 20 7.7±0.40 0.98±0.05

Chapter - 1

79

Table 1.16: Induction of multiple shoots from O. secamone stem callus on MS

medium supplemented with Kn and NAA

Plant growth regulator

Kn NAA

Frequency of shoot

regeneration (%)

No. of shoots/explant

Mean ±S.D

Shoot length (cm)

Mean ±S.D

1.0 0.5 29 5.6±1.14 1.15±0.10

2.0 0.5 22 9.0±2.64 1.20±0.60

3.0 0.5 17 7.03±0.25 1.60±0.22

4.0 0.5 25 10.0±0.8 1.30±0.12

5.0 0.5 15 8.42±0.71 0.77±0.10

1.0 1.0 32 7.0±0.92 1.95±0.95

2.0 1.0 33.6 21.5 ± 2.36 2.25 ± 0.95

3.0 1.0 41.6 11.0 ± 1.82 2.00 ± 0.81

4.0 1.0 35.4 9.0±0.61 1.15±0.10

5.0 1.0 10.9 6.02±0.61 0.55±0.04

Table 1.17: Table 3. Effect of IAA on root induction in ½ MS basal medium

Concentration (mg/l) % response Average No.of roots/shoot

Mean ±S.D

0.5 60 2.0±0.02

1.0 80 3.6±0.12

1.5 50 2.8±0.24

2.0 30 1.5±0.08

Chapter - 1

80

Fig.1.1 Leaf explants of T. involucrata on MS+ 2,4-D (2.0mg/l) medium without

Ascorbic acid

Fig. 1.2. Leaf explants of T. involucrata on MS+ 2,4-D (2.0mg/l) medium with Ascorbic

acid (2.0 mg/l)

Chapter - 1

81

Fig. 1.3. Leaf explants of T. involucrata on MS+ BAP (2.0mg/l) medium with Ascorbic

acid (2.0 mg/l)

Fig. 1.4. Leaf explants of O.secamone on MS+ 2,4-D(2.0mg/l) medium showing callusing

Chapter - 1

82

Fig.1.5.Leaf explants of T. involucrata on MS + NAA (1.0 mg/l) showing callusing

Fig. 1.6. Leaf explants of O.secamone on MS+ IAA (1.0mg/l)medium showing callusing

Chapter - 1

83

Fig. 1.7. Leaf explants of O.secamone on MS+ BAP (2.0mg/l) medium showing callusing

Fig. 1.8. Leaf explants of O.secamone on MS+ Kn (3.0mg/l) medium showing callusing

Chapter - 1

84

Fig.1.9. Leaf explants of O.secamone on MS+ BAP (3.0 mg/l) + 2, 4-D (1.0 mg/l)

medium showing callusing

Fig.1.10: Proliferation of T. involucrata leaf callus after transferred to fresh MS

medium + 2,4-D (2.0 mg/l)

Chapter - 1

85

Fig . 1.11. Proliferation of T. involucrata leaf calli on MS + BAP (5.0 mg/l) during

subculture

Fig . 1.12. Proliferation of T. involucrata leaf calli on MS + BAP (3.0 mg/l) + NAA (1,5

mg/l) during subculture

Chapter - 1

86

Fig . 1.13. Proliferation of T. involucrata leaf calli on MS + Coconut water during

subculture

Fig. 1.14. Developed root mass from the primary callus of T. involucrata on MS + IBA

(5.0 mg/l) medium

Chapter - 1

87

Fig. 1.15a Fig. 1.15b

Fig. 1.15c

Fig. 1.15 (a, b and c). Induction of multiple shoots from primary leaf callus of

O.secamone on MS+ BAP (3.0 mg/l) + NAA (1.0 mg/l)

Chapter - 1

88

Fig. 1.16. Induction of multiple shoots from primary leaf callus of O.secamone on MS+

coconut water (20%)

Fig.1.17. Callus formation on stem segments of T. involucrata on MS+ 2,4-D(2.0mg/l)

Chapter - 1

89

Fig.1.18. Callus formation on stem segments of T. involucrata on MS+ Kn(2.0mg/l)

Fig.1.19. Proliferation of T. involucrata stem callus on MS + BAP (3.0 mg/l) + NAA (1,0

mg/l) during subculture

Chapter - 1

90

Fig.1.20. Proliferation of T. involucrata stem callus on MS + Kn (2.0 mg/l) during

subculture

Fig.1.21. Callus formation on stem segments of O. secamone on MS+ 2,4-D(1.5 mg/l)

Chapter - 1

91

Fig.1.22 (a and b). Callus formation along with multiple shoots from nodal explants of

O. secamone on MS+ BAP (3.0mg/l).

1.22a

1.22b

Chapter - 1

92

Fig.1.23. Induction of multiple shoots from nodal explants of O. secamone on MS+ BAP

(5.0mg/l).

Fig.1.24. Callus formation on stem explants of O. secamone on MS+ Kn (2.0mg/l

Chapter - 1

93

Fig 1.25a..Induction of shoot buds on stem callus of O.secamone subcultured on

MS+BAP (3.0mg/l) +NAA (1.0 mg/l)

Fig 1.25b. Elongated multiple shoots of O.secamone

Chapter - 1

94

Fig.1.26. Induction of shoot buds on stem callus of O.secamone subcultured on MS+Kn

(2.0mg/l) + NAA (10 mg/l).

Fig.1.27. Induction of Multiple shoot along with rooting in the excised shoots of O.

secamone on MS full strength liquid medium supplemented with IAA (2.0 mg/l)

Chapter - 1

95

Fig.1.28. Induction of rooting in the excised shoots of O. secamone on MS halfstrength

liquid medium supplemented with IAA (1.0 mg/l)

Fig.1.29. O. secamone microshoot with bunch of roots

Fig.1.30. Hardened In vitro regenerated shoot of O. secamone

Chapter - 1

70

Table 1.1: Response of leaf explants on different types of medium

Plant Basal Murashige &

Skoog’s (MS)

Gamborg’s

B5 medium

White’s

Medium (WM)

Oxystelma secamone NR 90% 65% 35%

Tragia involucrata NR 90% 45% 30%

Table 1.2: Morphogenic nature of leaf explants of T. involucrata on MS

medium supplemented with different auxins individually.

Growth

Regulator

Concentration

(mg/l)

% of

Response Nature of Response

Basal - - -

2.4 D

1.0 75 White greenish Granular

2.0 83.3 White greenish Granular

3.0 70.5 White greenish Granular

4.0 54 Light yellow Granular

5.0 41.6 Light yellow Granular

NAA

1.0 00 No Callus

2.0 40 Explant become browing &

produce yellow, friable Callus 3.0 44

4.0 31

5.0 00 No Callus

IAA

1.0 30

Brown friable callus formed at cut

edges. No root formation

2.0 44

3.0 50

4.0 38

5.0 40 Brown friable callus with root

initials