Chapter – I

INTRODUCTION AND REVIEW OF LITERATURE

INTRODUCTION

The Western Ghats, extending along the west coast of India, covers

an area of 180,000 square kilometers. The Western Ghats comprises the

major portion of the Western Ghats and Sri Lanka Hotspot, one of 34

global biodiversity hotspots for conservation and one of the two on the

Indian subcontinent. The area is extraordinarily rich in biodiversity.

Although the total area is less than 6 percent of the land area of India, the

Western Ghats contains more than 30 percent of all plant, fish,

herpetofauna, bird, and mammal species found in India. Like other

hotspots, the Western Ghats has a high proportion of endemic species.

The region also has a spectacular assemblage of large mammals and is

home to several nationally significant wildlife sanctuaries, tiger reserves,

and national parks. The Western Ghats contains numerous medicinal

plants and important genetic resources such as the wild relatives of grains

(rice, barley, Eleucine coracana), fruits (mango, garcinias, banana,

jackfruit), and spices (black pepper, cinnamon, cardamom, and nutmeg).

Biodiversity in the Western Ghats is threatened by a variety of

human pressures. Of the approximately 180,000-square-kilometer area in

the Western Ghats region, only one-third is under natural vegetation.

Moreover, the existing forests are highly fragmented and facing the

prospect of increasing degradation.

1

Vegetation in the Western Ghats

According to a recent study conducted by the Indian Institute of

Remote Sensing (IIRS), incorporating both field-based analysis of

vegetation communities as well as satellite image interpretation, there are

four major forest types in the Western Ghats: evergreen, semi-evergreen,

moist deciduous, and dry deciduous. Together the forests cover

approximately 20 percent of the total area of the Western Ghats. Among

the four broad vegetation types, moist deciduous forests occupy the

largest area followed by semi evergreen, dry deciduous, and finally

evergreen.

The majority of the area under moist forest types falls within the

southern states of Kerala and Karnataka. Together they account for 80

percent of the evergreen forest and 66 percent of the moist deciduous

forests in the Western Ghats (IIRS 2002).

Evergreen forests

The highest levels of endemism are found in the evergreen forests.

These forests occur within a 200-1,500-meter elevational range and, 500-

to 5,000-millimeter rainfall range. They vary widely along the length and

breadth of the Western Ghats. A broad distinction can be made between

the northern evergreen forests and the southern evergreen forests. The

Wayanad evergreen forests of Kerala represent a transition zone from the

moist Cullenia-dominated forests in the south Western Ghats to the

northern drier dipterocarp forests (Rodgers and Panwar 1988).

The habitat types of the southern Western Ghats tropical evergreen

forests also include the wet montane evergreen forests and shola-rassland

complexes in the higher elevations (1,900-2,200 meters). The montane

evergreen forests are diverse, multistoried and rich in epiphytes, with a

2

low canopy at 15 to 20 meters (Puri et al. 1989; Ganesh et al. 1996).

More than half the tree species found in these forests are endemic,

especially among the families Dipterocarpaceae and Ebenaceae. The

majority of the fifty endemic plant genera are also monotypic. The

distribution of richness and endemism is not uniform within this forest

type, with some areas having higher concentrations of endemics than

others.

Semi-evergreen forests

Semi-evergreen forests occur primarily in the states of Maharashtra, Goa,

and Karnataka in the Western Ghats, within an elevational range of about

300-900 meters (IIRS 2002). This forest type includes secondary

evergreen dipterocarp forests, lateritic semievergreen forests, bamboo

brakes, and riparian forests as described by Champion and Seth (1968).

The structure and composition of these forests varies widely from north

to south and especially from east to west. The dominant species include:

Terminalia paniculata, Aporusa lindleyana, Olea dioica, Syzygium spp,

Mesua ferrea, Vateria indica, Elaeocarpus tuberculatus, Celtis

timorensis, Hopea parviflora, Lagerstroemia microcarpa, Holigarna

arnottiana, Hydnocarpus laurina, Memcylon umbellatum, and Careya

arborea. These forests also tend to have high levels of tree diversity and

endemism (IIRS 2002).

Moist deciduous forests

The moist deciduous forest type occupies the largest area within

the Western Ghats. It occurs within an elevational range of 500-900

meters in areas with mean annual rainfall of 2,500-3,500 millimeters. The

swath of moist deciduous forests is very narrow on the steeper, windward

side of the mountain range, where the southwest monsoon rains promote

3

wet evergreen forests. On the less steep leeward side, the drier conditions

caused by the rain shadow result in a broader, uneven swath of moist

deciduous forests that extend further into the Deccan Plateau. Rainfall on

the leeward side is influenced by complex landforms, with some areas

receiving less than one-fifth of the 3,000 millimeters or more of annual

precipitation that is deposited higher in the mountains.

Dry deciduous forests

The dry deciduous forests occur on the leeward side of the Western

Ghats Mountain Range within an elevational range of 300-900 meters in

areas of 900-2,000 millimeters mean annual rainfall. They extend across

the southern Indian states of Karnataka and Tamil Nadu. The tall Western

Ghats mountain range intercepts the moisture from the southwest

monsoon, so that the eastern slopes and the Deccan Plateau receive

relatively little rainfall, from 900 to 1,500 millimeters. The undulating

hillsides have very shallow soils. Thorny plants become more common in

areas where grazing pressure is high Kamal et al. (2007).

Plant species in Western Ghats It is estimated that there are four thousand species of flowering

plants known from the Western Ghats and 1,500 (nearly 38 percent) of

these are endemic (Nair and Daniel 1986). Approximately 63 percent of

India’s woody evergreen taxa are endemic to the Western Ghats

(Johnsingh 2001). Of the nearly 650 tree species found in the Western

Ghats, 352 (54 percent) are endemic (Daniels, 2001). The tree genera

endemic to the Western Ghats include Blepharistemma, Erinocarpus,

Meteromyrtus, Otenophelium, Poeciloneuron, and Pseudoglochidion.

Other plant genera endemic to the Western Ghats include Adenoon,

Griffithella, Willisia, Meineckia, Baeolepis, Nanothamnus, Wagatea,

4

Campbellia, and Calacanthus (Nair 1991). The grass family Gramineae

(Poaceae) has the highest number of endemic genera and the genus

Nilgirianthus has the maximum number of endemic species (20) across

all genera in this family (Nair 1991).

There are several centers of plant endemism and species richness

within the Western Ghats. For instance, of the 280 woody endemic

species found south of Karnataka, 70 species are endemic to the

southernmost Travancore region (Nair 1991). Herbaceous species

richness is the highest in the stretch of hills to the south of Kodagu

district in Karnataka (Nair 1991). The Nilgiri Mountains are one of the

most important centers of speciation for flowering plants in the Western

Ghats, with 82 species restricted to this area alone (Daniels 2001).

Several species are endemic to the Agastyamalai-Nilgiri Hills and

the Sri Lankan highlands, including Abarema subcoriacea, Biophytum

nudum, Chrysoglossum maculatum, Eugenia rotundata, Fahrenheitia

zeylanica, Filicium decipens or fern tree, Pavetta zeylanica, and Rubus

micropetalus or wild aspberry. The flora of the Agastyamalai Hills bears

a remarkable similarity to that of Sri Lanka’s southwestern wet zone not

just in terms of shared taxa, but also with respect to the remarkably high

incidence of highly localized “point” endemics (Ramesh & Pascal, 1997).

Tree species endemism is the highest in the southern Western Ghats,

while herb species endemism appears to be highest in the north (Daniels

2001).

The Western Ghats comprise the mountain range that runs along

the western coast of India, from the Vindhya-Satpura ranges in the north

to the southern tip. The ecosystems of the Western Ghats are located

mainly in the following regions, the tropical wet evergreen forests in

5

Amboli and Radhanagari; the Montane evergreen forests in

Mahabaleshwar and Bhimashanker; moist deciduous forests in Mulsi and

the scrub forest in Mundunthurai.

There is a great variety of vegetation all along the Ghats, scrub

jungles, grassland along the lower altitudes, dry and moist deciduous

forests, and semi-evergreen and evergreen forests. There are two main

centres of diversity, the Agashyamalai hills and the Silent Valley. The

complex topography and the heavy rainfall have made certain areas

inaccessible and have helped the region retain its diversity.

Among the floristic diversity, medicinal plants are an important

source which have been used all over the world. India’s vast resource

base of medicinal plants is well known to the world over. In India, the

medicinal plants are widely used by all sections of the population, and

country is richly endowed with a wide variety of plants of medicinal

value, which represents the great national resource (Alok, 1991). It is

estimated that atleast 70.00 per cent of country’s population relay on

herbal medicines for primary health care in the country (Holley and

Williams, 1996). In India, different classical medicinal systems such as

Ayurveda, Siddha and Unani are being practiced in the country and in

addition to this; innumerable local folk medicinal traditions exist. In all

over 8000 plant species are in medicinal use. Across the country, this

constitutes 45.00 per cent of 17,500 known flowering plant species of

India (Ved et al., 2000). This rich medicinal wealth is mainly distributed

in two hot spots diversity that is north eastern region and western ghat.

The Western Ghat comprises of a hill range running about 1500

km long the western edge of Indian sub-continent. Although it covers a

mere 5.00 per cent of the country’s total land area in the country, it is

6

believed to be more than 27.00 per cent of country’s plant species

remarkably high level of endemism ranging from 25.00 to 60.00 per cent

of recorded species in various taxa (Pascal, 1992). Sixty three per cent of

Indian ever green tree species are believed to be endemic to that area. In

addition to species richness, it is endemic of biodiversity of the regions.

Sixty per cent of amphibians, 50.00 per cent of reptiles and 25.00 per cent

of plants of Western Ghats are endemic (Vijay, 2006). Sixty three per

cent of India’s ever green tree sp. is believed to be endemic to this area.

Medicinal trees are important components of the biodiversity of the

Western Ghats. The high anthropogenic pressures and associated

Fragmentation of the landscapes has resulted in loss of habitat and

species. Medicinal plants are also under constant threat due to over

exploitation from natural habitats in the absence of cultivation.

Biogeographically, the Western Ghats have long been isolated from the

vast south-east Asian humid forest tract and thus protect a relict pocket of

evolutionarily distinct biota. Geology, soil and climate also contribute to

promote high biodiversity in three forests. This region so many palnt

species are critically endangered or extinct of the Western Ghats one of

the species conserves here Abutilon ranadei Woodr & Stapf. some

important information of the genus Abutioln and their family Malvaceae.

The genus Abutilon Mill. Belongs to family Malvaceae and is

represented by about 150 species. India is home to 12 species, 2

subspecies and 5 varieties Paul, (1993), Woodrow (1897) of these two

species and 4 varieties are endemic to India Tetali et al. (2004). The

genus is distributed mostly in the tropical or subtropical parts of the

world. Many species are commercially important as they are highly

ornamental.

7

Family Malvaceae

Family Malvaceae is cosmopolitan in distribution. This plant family

comprises 88 genera and 2300 species. In Pakistan it is represented by 19

genera with 94 specific and intraspecific taxa e.g Abutilon, Hibiscus,

Abelmoscus, Gossypium etc. The members of this family are mostly

annual to perennial herbs to shrubs or small trees Paul, (1993).

Importance of the family Malvaceae (Bhattacharyya, 1971 & Singh,

1983). Popular plants of the family Malvaceae are generally valued for

commercial cotton, gorgeous spring blossoms and some for their

colourful foliage. Some plants are grown for decoration (Hibiscus).

Additionally they have medicinal value as well. Marsh mallow (Althaea

officinalis) has demulcent properties. Sometimes it is used to treat

inflammations and irritations of the mucous membranes such as the

alimentary canal, the urinary and the respiratory organs. The root is used

for peptic ulceration and gastritis. Hollyhock, (Althaea rosea) has uses

similar to marsh mallow. Its flowers are used medicinally for their

emollient, demulcent and diuretic properties. Roots of marsh mallow are

used medicinally for cough. Salmalia malabarica gum is good for curing

kidney troubles, leucorrhoea and tuberculosis. Flowers and barks of the

same plant have ability to cure conjunctivitis and cutaneous infections. It

is also expectorant, laxative and suppurative. Hibiscus rosa is

aphrodisiac. Leaves are good for curing boils. Flowers are laxative.

Abelmoschus esculentus improves and increases sperm count. Hibiscus

cannabinus treats guinea worm sores. Hibiscus sabdariffa is good to treat

high blood pressure. Economically Malvaceae is very important. The

commercial cotton is derived from the dense hairy seeds of Gossypium.

Cotton fibres have been traced to the Indus valley civilization. Gossypium

8

hirsutum cultivated in the USA, yields long staple cotton, G. arboretum

and G. herbaceum which yield short staple cotton, are cultivated in Asia.

Cotton seeds are rich in fat and yield edible oil called the cotton seed oil.

It is also used in manufacturing soaps and lubricants; the oil cake is used

as a cattle feed. Hibiscus cannabinus is another plant which yields fiber,

used widely for cordage, ropes etc; fatty oil used in the manufacture of

linoleum, paints and varnishes and also as edible oil and oil cake is used

as a cattle feed. Other fiber yielding plants of minor importance are

Abutilon indicum, A. persicum and A. theophrasti (China jute), Sida

cordifolia, S. acuta, Urena lobata and many others. Hibiscus sabdariffa

(commonly called rosella) is a shrub, native to the West Indies; its

epicalyx and calyx are fleshy, rich in acids and pectin and used in

preparation of jellies and confectionery. Many species of Hibiscus such

as H. mutabilis, H. rosasinensis, H. schizopetalous are cultivated as

ornamentals; H. elatus (blue mahoe) is the national flower of Jamaica, H.

syriacus growing in eastern Mediterranean has large flowers of pinkish

colour and is commonly called ‘rose of sharon’.

The fruits of Abelmoscus esculentus or lady finger are used as a

vegetable. Roots of Althaea officinalis or marshmallow are used

medicinally, considered to be a very useful herbal remedy for cough. A.

rosea or holly hock is a cultivated garden ornamental. The mucilaginous

roots of Pavonia hirsua from Zimbabwe are added to milk to hasten

butter production Dutta, (1988). The mucilaginous substance obtained

from the stem of Kydia calycina is used for clarifying sugars.

Genus Abutilon Mill.

Annual or perennial herbs, undeshrubs or shrubs. Leaves simple,

entire or lobed, mostly cordate at base, acute or acuminate at apex,

9

palminerved without nectarines. Flowers axillary, solitary, sometimes in

lax panicles by reduction or dercrescence of upper leaves, rarely in lax

corymbose racemes; pedicels jointed in the upper half. Epicalyx absent.

Calyx usually campanulate; lobes 5, divided to the middle or below.

Corolla usually yellow, orange, whit or pink , rarely with a dark purple

centre, rotate, campanu-late. Staminal column shorter than petals, much

widened at base. Carpels and style-branches 5-40, styles filiform ;to

clavate, capitate stigmatose at apex. Schizocarps globular, campanulate,

rarely discoid; mericarps 50-40 , dehiscent, follicular, flattened-reniform,

round, acuminate or biaristate at apex, often falling leaving a slender

truncate, columella. Seeds 2-9 in each mericarp, reniform to subreniform,

upper ones ascending; lower ones pendulous or horizontal , finally falling

out of mericarp. In tropical and subtropical regions of the World , ca

150species; 12 in India. Abutilon Parker, (1921) is a large genus of about

150 species of broadleaf evergreens in the mallow family (Malvaceae).

The genus includes annuals, perennials, shrubs, and are conspicuous, with

five petals, mostly red, pink, orange, yellow or white. Small trees from 1-

10 m tall, and is found in the tropical and subtropical regions of all

continents.

Importance of genus Abutilon

Valuable fibers are drawn from different species of Abutilon e.g.

Abutilon indicum, A. polyandrum and A. asiaticum Nasir, (1979). the

fibers obtained from these plants are used for making ropes cordage’s ,

jute dyes, drugs, rugs , wrapping cloth, tissue papers, for making coarse

cloth, cigarette paper, rubber, tyre, fabrics, shoe polishes ,etc. Its leaves

are applied in ulcers and gonorrhoea. Leaves of A. indicum yields

mucilage used in pectorial troubles. Abutilon avicennae provides a useful

10

fiber named Chinese jute. Seeds of Abutilon specie are laxative and

demulcent Sammia (2008).

Abutilon ranadei Woodr.& Stapf. was first collected by N.B.

Ranade, ex-keeper of the herbarium at the College of Science, Pune

Woodrow & Stapf. (1894) described it as a new species and named it

after Ranade. It is an endemic known so for only from four district of

Maharashtra State. According to Cooke (1901), it is a rare plant due to its

narrow range of distribution and extreme rarity the species has been

declared as endangered Nayar and Sastry, (1987), Venkanna & Das Das

(2000) or even presumed extinct Ahmedullah and Nayar, (1986).

However it was recollected from its type locality after a lapse of almost

95 years (Mistry& Almeida, 1989; Almeida, 1996; Walter and Gillett,

(1997). Since then the species has been collected from eight new

localities in Pune, Satara, Kolhapur and Ratnagiri district.

It is an interesting species of Abutilon, restricted to hill slopes in

main crest of Sahyadri ranging from Amba ghat in south to Junnar-

Harischandragad region in north. The individuals are sparsely distributed

in the region and known only form few localities showing (Table-1 and

Photo plate No. 1).

It is critically endangered species of Western Ghats. It has flowers

of ornamental value. The seed setting is very low and there is a need for

restoration of natural populations of these species. This species is intended

to be recovered by conventional and tissue culture method.

The immediate major is reintroduction at the historical sites of

occurrence. As per Primik (2004) 10,000 individuals in 20, 000 Sq. k.m.

are to be planted for viable population. Although it is a immediate measure

11

to save the species from being extinct, the real cause for its course of

extinction is to be figured out.

In view of this it is intended to study the species thoroughly which

is expected to pin point the cause of extinction in view of this following

parameters were studied viz.

a) Morphology

b) Anatomy

c) Polynology

d) Floral biology

e) Cytology

f) Seed germination and viability

g) Soil microflora

h) Phytochemistry

h) Tissue culture protocol

i) AFLP Marker

12

REVIEW OF LITERATURE

Abutilon ranadei, commonly know as Shonghanta, is perhaps the

critically endangered species in Western Ghats. Endemic and Threatened

Flowering Plants of Maharashtra. Botanical Survey of India (Ahmedullah

& Nayar 1986; Nayar & Sastry, 1987; Mistry & Almeida, 1989 and

Mishra & Singh, 2001). Morphological studies of Abtuilon ranadei

reported in some Floras and articles (Woodrow, & Stapf, 1894; Cooke,

1901; Paul, 1993 Bachulkar & Yadav, 1997; and Punekar et al. 2001).

Taxonomical and some related work of other species of genus

Abutilon. Pollen morphology of Abutilon Mill., from Pakistan (Siddiqui,

et al. 1984). Effects of shade on reproduction and some morphological

characteristics of Abutilon theophrasti and Leaf margin serration and

taxonomy in the genus Abutilon Benvenuti and Macchia (1994); Bhat

(1999). Pollination of three species of Abutilon (Malvaceae) intermediate

between bat and hummingbird flower syndromes (Buzato, et al. 1994).

Circumscription of Malvaceae (Malvales) as determined by a preliminary

cladistic analysis of morphological, anatomical, palynological, and

chemical characters (Alter and Steven, 1997). Genus Abutilon reported

in anatomical and dermatological studies of some other species, Root,

Stem, Leaf of Abutilon theophrasti (Ysegul, et al. 2003). Intraspecific

variation in morphological characteristics and growth habit of newly and

accidentally introduced velvetleaf (Abutilon theophrasti Medik.) into

Japan. (Kurokawa et. al 2003b). Taxonomic Revision of Genus Abutilon

Mill. in Saudi Arabia General View of Malvaceae Juss. (Wafaa 2009).

Implication of foliar epidermal features in the taxonomy of Abutilon Mill.

(Malvaceae) and Pollen morphology of 14 species of Abutilon and

Hibiscus of the family Malvaceae (Nighat et al. 2009).

13

The area under Abutilon ranadei is decreasing day by day due to its

critically endangered species in Western Ghats its potential for better

adaptation to diversified soil and climatic conditions. There is few

localities of the Maharashtra State because poor germination, slow

growth of rootstock seedlings, lack of information on season and age of

rootstocks for grafting and micropropagation protocol has rendered the

clonal multiplication process more difficult to produce large scale. The

available information on various aspects of seed dormancy, softwood

grafting and micropropagation in Abutilon ranadei is very meager, the

relevant literature on other Abutilon species, grown under similar

situations have been reviewed. For better understanding of the subject the

information is organized under the following headings. a. Different seed

treatments on breaking dormancy. Propagation of stem cutting by

softwood grafting. Micropropagation.

When Haberlandt attempted the first cell culture studies, his

intention was to develop a versatile tool to explore morphogenesis and to

demonstrate totipotency of plant cells (Haberlandt, 1902). The first report

of viable callus culture was reported by Gautheret (1939) and White

(1939) in tobacco and carrot, respectively. The relative concentration of

auxin and cytokinin decides the plant morphogenesis in vitro (Skoog and

Miller, 1957). Effect of Auxins and Kinetin on in-Vitro Regeneration in

Tissue Cultures of Abutilon-Indicum (Nataraja and Patil 1980).

Hardseededness and germinability of velvetleaf (Abutilon theophrasti) as

affected by temperature and moisture. (Horowitz and Taylorson 1984).

Abscisic Acid and Sucrose Control of Velvetleaf (Abutilon tbeopbrasti)

Ovule Development In Vitro (Laura et al. 1984). Abscisic acid and

14

sucrose control of velvetleaf (Abutilon theophrasti) ovule development in

vitro and The in vitro culture of excised ovules from velvetleaf (Abutilon

theophrasti). (Thompson, et al. 1984). Responses of Isolated Floral Buds

and Anthers of Abutilon indicum in-Vitro. (Nataraja and Patil 1984).

Micropropagation has a great commercial potential due to the speed of

propagation, decreased production space requirement and the ability to

multiply elite clones exhibiting superior growth and enhanced stress

tolerance (Garton and Mosses, 1985; Kane et al., 1989).

Intrapopulation variation in Abutilon theophrasti seed mass and its

relationship to seed germinability (Baloch, et al. 2001). The most widely

used cytokinins are kinetin, BAP and 2-IP, auxins or cytokinins alone

cannot show organogenesis through cell division or callus in vitro. Since

many fruit crops are sensitive to photoperiod requirement these are

cultured in vitro under day night regime and a light cycle of 16 hrs

followed 8 hrs of darkness is the most preferred one (Conger, 1987). The

optimum growth of tissue cultures has been observed at a temperature

regime 24-26°C (Pierik, 1987). Establishment of culture explant Pierik

(1987) reported that genotype, plant age, physiological state (vegetative

or regenerative), stage and age of explants, general health of plant,

position of explant within plant, size of the explant, method of

inoculation etc. affects the growth of explant in vitro. Effect of shade on

velvetleaf (Abutilon theophrasti) growth, seed production, and dormancy

(Bello, et al., 1995). Temporal changes in velvetleaf (Abutilon

theophrasti) seed dormancy (Cardina and Sparrow, 1997).

Record of Abutilon ranadei Woodrow & Stapf in an area other

than the type locality. (Bachulkar & Yadav 1997). Showed that different

15

treatments like hot water treatment at 700C for 10 minutes followed by

concentrated H2SO4 scarification for five minutes was most effective in

breaking the dormancy which was imposed by seed coat in Abutilon

indicum. (Gupta et al. 2001). Techniques to remove hard seededness in

the wild medicinal plant Abutilon indicum. Journal of Medicinal and

Aromatic Plant Sciences (Gupta, 2001). Ecological and conservation

studies of Abutilon ranadei Woodr. et. Stapf. (Tetali et al. 2004).

Micropropagation also can be used to establish and maintain virus-free

plant stock. This is done by culturing the plant's apical meristem, which

typically is not virus-infected. Once new plants are developed from the

apical meristem, they can be maintained and sold as virus-free plants.

The major branch of biotechnology which has become

commercially viable is micropropagation. Micropropagation has been

defined as ‘in vitro’ regeneration of plants from organs, tissues, cells or

protoplasts (Beversdorf, 1990) and as ‘the true to type propagation of a

selected genotypes using in vitro culture techniques’ (Debergh and Read,

1991). True-totype propagation has advantages for heterozygous fruit

crops such as aonla, pomegranate, papaya etc. The concept of totipotency

is the basis for micropropagation. Micropropagation has been

successfully employed for rapid production of uniform and superior

quality planting material. Rapid and large scale clonal propagation of

many fruit crops is now possible tissue culture. The regeneration can take

place as extension or proliferation of shoot tip or axillary buds, direct

production of organs from explant i.e. organogenesis or through the

production of somatic embryos i.e. embryogenesis. An in vitro

propagation involving an unorganized callus phase may produce variant

16

plants i.e. somaclonal variants, clonal in vitro propagation is achieved by

using meristems and axillary buds (Kumar and Kumar, 1998). In tissue

culture studies, one has to make use of plant growth regulators in addition

to the supply of other salts, mineral, vitamins and sugars to obtain the

desirable response to achieve the set of objectives. Auxins are a class of

plant growth regulators which cause cell elongation, apical dominance

and root initiation. The most frequently used auxins are 2, 4-D, NAA,

IAA and IBA of which IAA occurs naturally in plants. NAA and 2, 4-D

are the most effective auxins to initiate callus. Cytokinins are

phytohormones which promote cell division, proliferation of tissues.

While selecting a suitable explant for micropropagation, these factors

should be taken into consideration. Muralikrishna (1988) reported highest

percentage of shoot establishment in explants of 10 mm size followed by

7.5 mm. As the size of explant increased the shoot establishment

percentage of also increased upto 10 mm (56.3%) and later decreases as

size increased. He also stated that the rapid adventitious shoot initiation

was found only in axillary bud (9 shoots/culture) than shoot tip explants.

The later were able to produce fewer (2.17) shoots per culture in

pomegranate. In Jackfruit, Rajmohan and Mohankumaran (1988) studied

the influence of explant source on the in vitro propagation. They reported

maximum response from the seedling explants (17.4 shoots) followed by

the fresh stem sprouts from five year old trees (4.50 shoots). A drastic

reduction was observed from explants excised from ten to thirty year old

trees (2.80 and 2.09 shoots, respectively). Maximum number (15-20) of

shoots were induced in shoot tip closely followed by cotyledonary node

(10-15), nodal section (8-10) and hypocotyls (6-8) of ber (Mathur et al.,

1993). Nodal segment explants of 1-2 cm length when cultured from mid

17

portion of the indeterminate shoots of mature tree of aonla cv. NA-7 in

the month of April-July to August- November gave better bud induction

(Mishra et al., 1999). Singh et al. (2002) studied the in vitro propagation

of grapes. They obtained better culture establishment with nodal explants

than the shoot tips. Studies on in vitro propagation of papaya cv. CO-5

revealed that highest callusability per cent within 25 days on MS medium

supplemented with 1.0 mg/l IAA and 4.5 mg/l BA in case of shoot tip

culture than explants like leaf bits and nodal segments (Suthamathi et al.,

2002). Micropropagation studies of explants from mature trees Aonla

Verma and Kant (1996) cultured nodal segment explants of aonla on

modified MS medium supplemented with BA (3-5 mg/l) and NAA (0.5

mg/l) during February-April and August-October. The bud break

observed in only 8-10 per cent of explants. Leaching of phenolic

compounds resulted in the loss of almost 90 per cent in cultures. The

regenerated shoots were elongated on hormone free medium and

subsequently rooted on half strength MS medium supplemented with IBA

(3.0 mg/l) and sucrose (1.5%). In aonla cv. NA-7, Mishra et al. (1998)

reported heavy inborn contamination and phenol exudation were the

major problem in establishing the in vitro cultures of aonla. Dipping of

explants in bavistin (1%) and cholramphenicol (0.1%) for 60-90 minutes

followed by treatment with HgCl2 (0.1%) for 8 minutes significantly

reduced the explant contaminationn. The application of paraffin wax on

the cut end of explants reduced browning by 20 per cent and increased

the survival rate to 80 per cent as compared to non-waxed explants with

or without antioxidant. Non-waxed explants treated with PVP showed the

50.30 per cent survival and browning to an extent of 47.73 per cent. In

aonla, Kant et al. (1999) reported 3-4 multiple shoots from nodal explants

18

from mature tree on MS medium supplemented with BAP (5 mg/l) and

IAA (0.5 mg/l) along with antioxidants. These shoots were elongated on

hormone free MS medium. Rooting of the shoots was achieved on half

strength MS medium fortified with IBA (2 mg/l) and sucrose (1.5%).

Micropropagation studies in aonla were carried out from nodal

explants by Mishra et al. (1999) to develop the protocol for mass

multiplication of true-to-type plants. The better shoot proliferation (33-

37.5%) on modified MS medium supplemented with kin 0.4 mg/l + GA

1.0 mg/l than in WPM. Higher GA3 concentration (3 mg/l) caused

complete defoliation and dropping of determinate shoots. Regenerated

shoots from 3 week old cultures in MS media supplemented with growth

regulators and antioxidants failed to produce roots. Mishra and Pathak

(2001) studied the effect of nodal position and seasonal influence on in

vitro shoot proliferation of aonla. The highest bud induction rate

(76.40%) and longest indeterminate shoot (0.53 cm) was obtained with

explants excised from the 10th to the 15th node during August-November

in MS medium supplemented with kin (0.4 mg/l) and GA3 (0.4 mg/l).

Other fruit crops Amin and Jaiswal (1987) developed a rapid clonal

propagation through in vitro shoot proliferation from nodal explant of

guava. The best response was reported on MS medium supplemented

with 4.5 µM BA alone. The response of in vitro shoot nodal segments

was better in comparison to the field grown trees. Maximum response

with minimum contamination and browning of explant and medium was

obtained during April-June. Muralikrishna (1988) reported the strategies

of micropropagation of pomegranate. To control browning in culture

medium he claimed that the subsequent transfer of explants on fresh

19

medium as well as soaking of explants in 0.1 per cent ascorbic acid for 1-

2 hours in the solution. In micropropagation studies, the axillary buds and

shoot tip explants were regenerated on basal MS medium supplemented

with BAP 0.5 µM. Growth of axillary bud was slower than shoot tips, but

axillary bud produce about 9.5 buds per explant as compared to 2.33 buds

per explant by shoot tip. Rooting occurred when shootlets were soaked in

IBA 100 mg/l solution for one hour before subculture. In jackfruit,

Rajmohan and Mohankumaran (1988) reported that problem of

polyphenol interference was minimized by incorporation of activated

charcoal (1%) and GA3 1 mg/l in the establishment medium, insoluble

PVP in the proliferation medium and by frequent subculturing.

They recorded cent per cent survival and production of healthy,

growing cultures of shoot apices in MS medium supplemented with GA3

1 mg/l along with AC (1%). In woody plants, no tissue lacks phenolic

compounds including growing cell. Oxidation of phenolic compounds

released from the cut ends of explants by phenol oxidase, peroxidase

cause lethal browning of plant and culture medium. This problem can be

overcome by sealing the cut ends of explants with liquid paraffin wax

(Bhat and Chandel, 1991). Shoot organogenesis in cv. Nana, a dwarf

variety pomegranate was studied by Zhang and Stoltz (1991). Each

culture produced a mean of 5.2 shoots with BA 1 µm+ NAA 2 µM while

6.6 shoots produced with BA 2 µM + NAA 1 µM. Study indicated that

auxin (NAA) at 1 µM was optimum for in vitro shoot formation and that

BA levels more than 2 µM inhibits shoot formation. Amin and Jaiswal

(1993) described a method of shoot tip culture for rapid multiplication of

mature jackfruit trees. BA and kinetin (4.5 to 9 µM) either alone or in

20

combination, supported shoot proliferation. The ideal season for

collection of apical budswas during November to January. Shoots rooted

with 60 per cent to 80 per cent success using half strength MS salts and

10 µM IBA or NAA. In Zizypus mauritiana, Mathur et al. (1995)

reported that stem explants from mature tree when grown on modified

MS medium with 3800 mg/l potassium nitrate, 2475 mg/l ammonium

nitrate, 11 µM BA produced 15-20 shoots per inoculation. Rooting was

induced by pretreatment with 50 µM IBA or NAA for 24 hours followed

by transfer to auxin free whites medium. Roy and Roy (1996) reported

that maximum shoots proliferation from shoot tips and nodal segments

was induced in jackfruit on MS medium containing BA 2.5 mg/l and

NAA 0.5 mg/l, with an average of 10.3 shoot/culture after 28 days.

Rooting of excised shoots was achieved on half strength MS medium

containing NAA and IBA each at 1.0 mg/l. Seventy five per cent of the

plantlets survived after transplanting into the open field. Roy et al. (1996)

reported that multiple shoots were obtained from nodal explants of S.

cumini on MS medium supplemented with kinetin 2.5 mg/l. Repeated

subculture resulted in rapid shoot multiplication. Addition of 100 mg/l

urea to media increased number of shoots rooted in half strength MS

media containing 0.5 mg/l each of IBA, NAA and IAA. After

transplanting in field, 75 per cent plantlets were survived with uniform

growth, in tamarind Balakrishnamurthy and Ganga (1997) reported the

seasonal influence of explant collection on induction of multiple shoots

from axillary buds. The monthwise collection and culture of axillary buds

in MS medium fortified with six per cent sucrose, BA 0.5 mg/l and GA3

0.5 mg/l indicated that best results with respect to per cent bud break

(82.5%), number of days taken for bud break (9.33 days), per cent

21

response to multiple shoot induction (22.50%), number of shoots per

culture (2.17) and length of micro shoots (3.21 cm) were recorded by the

axillary buds collected and cultured during the month of May. The

axillary buds collected and cultured during the months December,

January, February, March, September and October did not record any

response. Kantharajah et al. (1998) observed high frequency adventitious

bud formation in pomegranate, when nodal cuttings of cv. Wonderful

were cultured on half MS medium supplemented with BAP 0.5 mg/l +

NAA 0.1 mg/l. NAA was most effective than BAP in root elongation and

the highest shoot length occurred on media containing NAA 0.1 mg/l.

Roots were induced after 12 days on WPM with NAA 2 mg/l. Mehta et

al. (2000) developed a protocol for in vitro regeneration of plants via

adventitious bud formation from mature embryo axis of tamarind.

Induction of adventitious shoot buds was achieved in the cut surface of

the axis of tamarind. Induction of adventitious shoot bud was achieved in

the cut surface of the axis when cultured on medium containing zeatin

0.91 µM, calcium panthothenats 0.41 µm and biotin 0.40 µM supported

the differentiation of buds to form elongated shoots. Rooting occurred in

half strength MS medium with 2 per cent sucrose following a 72 hrs

treatment with auxin mixture in dark. Suthamathi et al. (2002) carried out

in vitro studies on papaya cv. CO-5. They obtained highest callusability

in case of shoot tips cultured on MS medium supplemented with 1.0 mg/l

IAA and 4.5 mg/l BA. Development of shoots was observed from shoot

tip callus cultured on MS medium supplemented with BA 4-5 mg/l

compared to kinetin. Pattepur (2003) did the tissue culture studies in

tamarind. He obtained multiple shoots when cotyledonary explants were

cultured on MS medium supplemented with BAP 0.5 mg/l + coconut

22

water (CW) 10 per cent. Auxillary buds collected from mature tree gave

highest per cent bud break and response to multiple shoot induction on

medium containing BAP 2.0 mg/l + CW 10 per cent was noticed.

Biotechnology for Endangered Plant Conservation (Anca Paunescu

2009). In vitro Micropropagation of Abutilon indicum L. through Leaf

Explants. Plant Tissue (Jyoti et al. 2009). In Vitro Propagation for

Conservation of Rare and Threatened Plants of India –A Review (Vera,

2010), Biswas (2007).

Abutilon ranadei is most important parameter of the genetic

diversity because the extinct of the species survey of the literature related

to the genus or family. Using protein electrophoresis to investigate the

phylogeny of velvetleaf (Abutilon theophrasti) (Stegink and Spencer

1988). Localization of Abutilon mosaic virus DNA within leaf tissue by

in-situ hybridization (Horns and Jeske 1991).Abutilon mosaic

geminivirus double-stranded DNA is packed into minichromosomes

(Pilartz and Jeske 1992). Complementation and recombination between

mutants of complementary sense genes of DNA A of Abutilon mosaic

virus (Evans and Jeske 1993). DNA forms indicate rolling circle and

recombination dependent replication of Abutilon mosaic virus (Holger et

al. 2001). Molecular and morphological differentiation between the crop

and weedy types in velvetleaf (Abutilon theophrasti Medik.) using a

chloroplast DNA marker, seed source of the present invasive velvetleaf

(Kurokawa et al. 2004). AFLP mediated genetic diversity of malvaceae

species (Nighat et al. 2010).

Most important factor of the Chemical investigations on Abutilon

species were undertaken as early as the 1929; when Kosaka, (1929);

23

Zaheera, (1990); some chemicals phytochemical investigation are

reported, Pentacyclic Triterpene and phytochemical investigation of

Abutilon Pakistanicum Ahmed et al. (1990); Ahmed et al.(1991); Ahmed

et al. (1993); Ahmed et al. (2006); reported the presence of anthocyanins

in Abutilon avecenae, however real chemical research on Abutilon

species began only in 1956 with the isolation of Rutin from Abutilon

avicenae. Developments after 1960 progressed more rapidly mainly

because of the invention of more refined techniques of separation and

structure elucidation. Since then large variety of compounds have been

isolated from genus Abutilon and majorities of them are flavonoids,

terpenoids and tetraketones various author Yemets and Steblyuk, (1994);

Sauar Ali (2009), Mabry (1970), Bagi (1985).

This study will provide valuable information about basic

information and restoration of the species point of view. This is the initial

step towards conservation of species.

AIMS AND OBJECTIVES

The present work was initiated for the doctoral dissertation, which

entails the morphology, anatomy, floral biology, cytology, seed

germination, vegetative propagation, soil microflora, phytochemistry,

tissue culture and AFLP marker of Abutilon ranadei the critically

endangered species in Western Ghats of family Malvaceae. In order to

correlate the need of above parameters with the active ingredients, the

research plan was chalked out as detailed below.

24

PLAN OF WORK

The plan of work of these studies had six phases.

Phase I: Collection for the minimum 10 localities

1. Collection of the plant materials (herbarium specimen, stem

cutting, floral buds, seeds and explants).

2. Preservation of the plant material for anatomical and other

experimental work.

Phase II: Morphology and Anatomy

1. Study of morphological characters like vegetative characters and floral

characters

2. Study of anatomical characters different parts like Root, Stem, Petiole

and Leaf

Phase III: Floral biology and Cytology

1. Study of floral biology

2. Chromosomal study (Meiosis)

Phase IV: Propagation by conventional methods

1. Vegetative propagation by stem cutting in different treatments

2. Seed Germination by various treatments

Phase V: Photochemistry

1. Preparation of plant material for the phytochemical study

2. Extraction of aerial parts and roots in different solvents.

25

3. Preliminary phytochemical study like Tanin, Saponine, Akaloides,

Protein, Ash, Total Ash, Reducing sugar, non-reducing sugar,

carbohydrates etc.

4. Antibacterial activity

Phase VII: Micropropagation

1. In vitro studies in Imature seed

2. Callus Induction for different explants like (Root, Stem, Petiole, Leaf,

Floral buds and Anther).

3. Multiplication by nodal segments and immature seeds

Phase VIII: AFLP Marker

1. Study of the genetic diversity in minimum five localities by different

primers

26