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SUMMARY AND CONCLUSIONS
SUMMARY AND CONCLUSIONS
Forests play key role in the life of people inhabiting mountains or planes as they fulfill
their basic requirements such as oxygen, water and energy. Variety of trees, shrubs and
herbs provide wood for construction of big and small dwellings, fire wood, fiber,
vegetable oil and food additives. Noteworthy tropical trees are those that yield timber
Tectona grandis, Pterocarpus marsupium, Adina cordifolia, Gmelina arborea; food
additives Buchanania /anzan, tamarindus indica; fiber Bombax malabaricum; color or
marking ink Butea monospermum, Semecarpus anacardium; oil Shorea robusta,
Madhuca longifolia, etc. However, the most impressive is their potential to keep us in
good health. India has a long tradition of using plants as medicines. The great source of
infonnation is contained in the 'Vedas'. The 'Rig Vedas' is oldest (4500 - 1200 BC)
consists of 67 plants, 'Yajur Veda' (2000 BC) mentions 81 plants and 'Atharva Veda
(1200 BC) contains 289 plants with description and their healing properties. The books
on ayurvedic medicine Charka Samhita and Susruta Samhita refer to the use of more than
700 herbs. Since then significant advancements has taken place in drugs and cl•ug
development. However, the plants continue to play pivotal role in modern medicine, and
even today more than one quarter of our drugs are either derived from plants or contain
plant extracts. The most popu1ar analgesic, aspirin, was originally derived from the
species of Salix and Spiraea and some of the most valuable anti-cancer agents such as
paclitaxel and vinblastine are derived solely from plant sources. Thus, wild plants are
source of medicine from ancient times and continue to provide even today.
About 70-80% of people worldwide believe on traditional, largely herbal medicine to
meet their primary healthcare needs. There is no reliable figure for the total number of
medicinal plants on Earth, and number and percentages for countries and regions vary
greatly. Estimates for the number of species used medicinally include; 35,000- 70,000 or
53,000 world wide. Estimates reveal that about 11146 species are used medicinally in
China, 7500 species in India, 2572 in North America and 2237 in Mexico. The great
majority of species of medicinal plants are used only as etlmo medicines. Traditional
Medicinal System of China commonly employs 500-600 species, 1250-1400 in
237
Ayurveda, 342 in Unani and 328 in Shiddha. In USA, 121 drugs in current usc are
derived from 95 plant sp~-cies.
There is much trade in Medicinal and Aromatic Plants (MAPs) on scales ranging from
local to the international levels. Much of this is unrecorded in official statistics or poorly
documented because there is very less awareness among the decision-makers of the
significance of the trade to the healthcare and economies of their people, or about
problems of un-sustainability and sometimes deleterious impacts of wild collection on
natural habitat. China's production of medicinal plants from cultivated and wild
harvested sources, considered together, was calculated at 1.6 million tones in 1996, with
a total value in terms of finished products of US$ 3.7 billion. The reported annual imports
of MAPs materials into all countries during the 1990s amounted to an average of 400,000
tonnes, valued at US$ 1.2 billion, showing a I 00% rise between 1991 and 1997 . The
three leading exporting countries are China (Ca 140,000 tons per year over 1991-1997},
India (about one - third of the Chinese amount) and then Germany. There are at least
2000 species of MAPs marketed in Europe, these originating from over 120 countries. It
is guessed that the total number of MAPs in international trade may be about 2500
species.
The retail sales of pharmaceutical products were estimated at US$ 80-90 billion globally
in 1997, with medicinal plants contributing very significantly. A study of the 25 best
selling pharmaceutical drugs in 1997 found that II of them (42%) were either
biologicals, natural products or entities derived from natural products, with a total value
of US$ 17.5 billion. The total sales' value of drugs (such as Taxol) derived from just one
plant species (Taxus baccata) was US$ 2.3 billion in 2000. The world market for herbal
remedies in 1999 was calculated to be worth US$ 19.4 billion, with Europe in the lead,
followed by Asia (US$ 5.1 billion), North America (US$ 4.0 billion), Japan ( US$ 2.2
billion), and then the rest of the world (US$ 1.4 billion).
Some of the plant drugs of Ayurveda exported to other countries, their common name
and market price ( 1995-96) of the crude plant drugs in rupees are Abies, Aconitum,
Acarus, Antheurn, Asparagus, Bergenia, Boerhaavia, Cal/carpa, Centella, Cissampelos,
Embelia, Furnaria, Hedychiurn, Holarrhena, Inu/a racemosa, Juniperus, Leptadenia,
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Munma, Nardostachys, Picorhiw, Piper, Rauwoljia, Rubia, Saraca, Swertia, Valeriana,
Withania, Zanthoxylum and Zingiher.
More than 95% of the 400 plant spL"Cies used in preparing medicine by various industries
are harvested from wild population in India (Uniyal, 2000). Tropical trees play pivotal
role in preparation of medicines. They are used to cure diseases and serve as tonics. The
list of medicinal trees is large. Some of the commoly used medicinal trees are Acacia
catechu, A. sinuate, Aegle marmelos, Anacardium occidentale, Azadirachta indica,
Buchanania /anzan, Holerrhenaantidysentrica, Madhuca /ongifolia, Morinda pubescens,
Oroxylum indicum, Phyllanthus emblica, Pongamia pinnata, Pterocarpus marsupium,
Santa/urn album, Saraca asoka, Semecarpus anacardium, Stereospermum personarum, S,
suaveo/ens,Strychnos nux-vomica, Terminalia arjuna, T. bellirica and T. chebula.
Harvesting medicinal plants for commercial use, coupled with the destruction of
underground parts of slow reproducing, slow growing and habitat-specific species, are
the crucial factors in meeting the goal of sustainability. In Dashmula species, the
destruction is severe due to total uprooting of plants since the roots are the source of
active components. Furthermore, rising demand with shrinking habitats may lead to the
local extinction of many medicinal plant species. The renewed global interest in herbal
medicines is also a factor in indiscriminate falling of medicinal trees and destruction of
forests. Hence, research towards either genetically improving the species for higher
production of the active components or increasing the production of the compounds
under in vitro culture conditions is essential.
Despite major advances in synthetic drug chemistry including combinational
chemistry, plants continue to be the major source of most of the global drug demand.
Besides curatives, a large number of plants have been sourced for bio-pesticides, anti
microbial compounds, pharmaceuticals etc. Considering the enormous significance of
these compounds and utilities, a number of bio-resource rich countries worldwide have
strived to intensively mine for such compounds.
The biotechnological tools are important to select, multiply and conserve the
critical genotypes of medicinal plants. In vitro regeneration holds tremendous potential
239
lor the production of high 4uality plant based medicine. Tissue culture is useful for
multiplying and conserving the species, the species which are difficult to regenerate
through conventional methods and save them from extinction. Tissue culture raised plants
arc superior, uniform, season independent and require smaller space as compared to the
seed grown plants. However, application of in vitro technique in tree is less frequent than
shrubs and herbs.
Micropropagation of tree species offers a rapid means of producing clonal planting stock
for afforestation, woody biomass and conservation of elite gennplasm. The tissue culture
of forest trees shown promise in obtaining regenerants and clonal multiplication for
domestication of wild populations, afforestation and economically important trees that
have been cultivated for generations.
Considering the importance of medicinal trees for medicinal uses and for pharmaceuticals
and industries, these under utilized tree species can be exploited at the commercial level.
The medicinal value indicate that the use of various parts (leaves, roots, bark etc) of these
tree species as plant extracts may be helpful in overcoming the disorders/disease
predominant in the many rural areas of the country. The Genus Stereospermum include
several important species. S. personatum, S. neuranthum, S. suaveolens, S. frimbiatum, S.
glandulosum, S. wallichii, S. xylocarpum etc are some of the known species of the Genus
Sterereospermum distributed all over world .
Stereospermum personatum and Stereospermum suaveolens are among those
species, which are having medicinal properties and known for timber wood quality.
Stereospermum personatum (Hassk.)
Stereospermum personatum D.C belongs to family bignoniaceae. The vernacular name of
Stereospermum personatum are - parrel/pader (Hindi), atcapali (Bengali), k:irsel
(Marathi), ambuvagini (Tamil) and tagada (Telegu)(Ambasta 1986). Padar is a medium
sized medicinal tree , frequently used in Ayurvedic system of medicine. Its fruits cure
migraine; and bark is used to treat piles. It is one of the ingredients used in the
preparation of Chyavanprash, an Ayurvedic tonic. It has antimicrobial, anti protozoal, and
240
anti-intlammatory properties. Free radical swvenging and xanthine oxidase inhibitory
wnstitucnts have been obtained from extracts of both stem and stem-bark of S.
personarum, which is of high thcrap~'lltic value. Decoction of roots used in asthama,
cough and excessive thirst. Leaves are used in chronic dyspepsia. Ethanolic extract of
aerial parts showed activity against lymphocytic leukemia in mice.
Stereospermum suaveo/ens (DC.)
Stereospermum suaveo/ens (Roxb.) D.C belongs to family Bignoniaceae. The vernacular
name of Stereospermum suaveo/ens are - parallpadaria (Hindi), parlu (Bengali), padal
(Marathi), padri (Tamil) and godadalipulusu (Telegu). It has analgesic and wound healing
property. It's leaf paste used to cure wound and seed paste is used to cure for migraine. It
is antidyspeptic, astringent and a liver stimulant. It is one of the important ingredient of
Antiox capsules (It is a unique herbal formula that protects cells against free radical
oxidative damage) available in Bazaar of India (www.shamanshop.net). It is one of the
important tree species used in the preparation of ayurvedic formulation known as
Dashrnula. In Dashmula tree species, where the roots are used in preparation, of
ayurvedic formulations, the destruction of plants is severe and planting of seedling for
this species is almost negligible. Research activities are negligible in this species. The
application of in vitro culture for propagation of Dashmula tree species will help in
sustainable availability of propagules and plant products. Root bark is an ingredient of
Dasmoola. Decoction of roots used for fevers, inflammatory chest affections and
affections of brain. Stem bark used as diuretic and tonic. Flowers are given with honey to
stop hiccup. Ethanolic extract showed activity against Ranikhet disease,also showed
hypoglycemic action in rats and anticancer activity against human epidermoid carcinoma
of nasopharynx in tissue culture.
In nature, regeneration of Stereospermum personatum S. suaveo/ens occurs through seeds
that produce heterogeneous tree populations. Vegetative propagation is unknown in these
species. Development of in vitro plant regeneration procedure would facilitate production
and molecular characterization of clones of these useful medicinal tree species.
241
Objectives:
The major objectives of the present study were as follows:
A. Stereospermum personatum (Hassk.)
• To study the effects of different media on establishment of cotyledonary,
epicotyledonary node and mature explants of Stereospermum personatum.
• To study the effects of different plant growth regulators and additives on in vitro
shoot multiplication from nodal segments derived from cotyledonary and
epicotyledonary node explants of Stereospermum persona tum.
• To determine shoot multiplication potential of nodal segments during subculture.
• To find out rooting potential of micro shoots originated from different types of
explants.
• To study the effect of different plant growth regulators on callus formation from
seeds, cotyledon, epicotyledonary inter node, hypocotyl and petiole explants.
• To study the effect of different plant growth regulators on differentiation of
callus.
• To study the effects of different substrates on survival and growth of tissue
culture plants during acclimatization.
242
8. Stereo.\permum suaveolens (DC.)
• To study the effects of different media on establishment of cotyledonary,
epicotyledonary node and mature ex plants of Stereolpermum suaveolens.
• To study the effects of different plant growth regulators and adjuvants on in vitro
shoot multiplication from nodal segments derived from cotyledonary and
epicotyledonary node explants of Stereolpermum suaveo/ens.
• To determine shoot multiplication potential of nodal segments during subculture.
• To find out rooting potential of micro shoots originated from different types of
explants.
• To study the effect of different plant growth regulators on callus formation from
seeds, cotyledon, epicotyledonary inter node, hypocotyls and petiole explants.
• To study the effect of different plant growth regulators on differentiation of
callus.
• To study the effects of different substrates on survival and growth during
acclimatization of tissue culture raised plants.
243
MATERIAL AND METHODS:
A. Stereo.\permum personatum(Hassk.)
The fruits of Stereosperrnurn persona/urn were collected from Pithora forest, about
I 00 km away from Raipur (C. G). The seedlings were planted in the nursery beds of
departmental garden. Nodal segments and apical buds of field grown one and two
year-old plants of Stereosrerrnurn persona/urn were used as explants source for shoot
bud proliferation.
The seeds extracted from mature fruits of Stereosrermurn personatum were de
winged, soaked in lukewarm water overnight, and wrapped in moist cotton bed for
four days. The germinated seeds were transferred to protray for seedling growth. Rate
of germination was tested during various seasons. The seedlings attained 2-3 node
growth within 21 days. The shoots excised from 21-days-old seedlings were washed
gently under running tap water and with labolene to remove dust particles. The
cotyledonary and epicotyledonary nodes were cut from healthy seedlings, surface
sterilized with 0.2% (w/v) aqueous mercuric chloride for 5 minutes and washed 3-4
times with double distilled water.
The following experiments were planned to study explants establishment, shoot
multiplication, subculture, rooting and acclimatization.
o Effect ofMS (Murashige and Skoog), WPM (Llyod and McCown) and SH
(Schenk and Hildebrandt) media supplemented with 4.44 J.1M BAP on
establishment of cotyledonary and epicotyledonary nodes.
o Nodes of developed shoots were placed on MS medium and WPM both
supplemented with different concentrations (0.0, 0.04, 0.44& 4.44 J.!M) of BAP
to study the effect of BAP on shoot proliferation.
o Nodes of developed shoots were placed on MS medium and WPM both
supplemented with different concentrations (0.0, 0.04, 0.46 & 4.65 J.!M) of
Kinetin to study the effect of Kinetin on shoot proliferation.
244
, ' Individual nodes were pla~ed on MS medium containing 4.44 11M BAP or 4.65
~1M Kinetin or combination of BAP (2.221!M) and Kinetin (2.321!M) to study the
cllccts of combination of BAP and Kinetin on shoot prolifemtion.
o The eiTcct of different concentrations of PVP {0, 250, 500 & I 000 mg/1) in MS
medium supplemented with 0.44 11M was investigated on in vitro shoot
proliferation.
o The effect of different concentrations of citric acid (0, 25, 50 & I 00 mg/1) in
MS medium supplemented with 0.44 11M was investigated on in vitro shoot
proliferation.
o The effect of different concentrations of casein hydrosylate (0, 25, 50 & I 00
mg/1) in MS medium supplemented with 0.44 11M was investigated on in vitro
shoot proliferation
o The effect of different concentrations of adenine sulfate (0, 10, 20, & 40 mg/1)
in MS medium supplemented with 0.44 11M was investigated on in vitro shoot
proliferation.
o The effect of different concentrations of activated charcoal (I 00, 200 & 400
mg/1) in MS medium supplemented with 0.44 11M was investigated on in vitro
shoot proliferation.
o The nodal segments derived from epicotyledonary nodes were inoculated in
liquid medium (MS + 0.44 11M BAP + 250 mgll PVP) to study the effect of
liquid medium on in vitro shoot proliferation.
o Nodes derived from cotyledonary and epicotyledonary nodes were subcultured
on fresh MS medium supplemented with 0.44 11M and 250 mg/1 PVP every four
weeks to study the effect of subculture on shoot multiplication potential nodes.
o Effects of IBA (0.0, 2.46, 4.90 and 9.80 flM ), lAA (0.0, 2.85, 5.70 and 11.40
11M) and NAA (0.0, 2.69, 5.37 and 10.74 11M) on in vitro rooting of micro
shoots derived from cotyledonary and epicotyledonary node explants were
studied.
o In vitro rooted plantlets were removed carefully from culture medium and
washed under running tap water and transferred to net pots containing coco peat
and mixture of sand, soil and farm yard manure. After 4 weeks plants were
245
shilled into nursery containing mixture of sand, soil and manure (I: I: I) for
secondary hardening.
The following experiments were planned to study callusing in seeds, epicotyl,
hypocotyls, cotyledons and petiole ex plants of S. personutum and dilTerentiation of
callus.
o Effects of different concentrations of 2, 4-D (0.0, 2.26, 4.52 and 9.04 !JM),
NAA (0.0, 2.69, 5.37 and 10.74 !JM) and SAP (0.0, 2.22, 4.44 and 8.88 j.lM) in
MS medium on callus initiation from different explants were studied.
o Effect of 2, 4-D, NAA and SAP were studied on differentiation of callus.
B. Stereospermum suaveolens (DC.)
The fruits of Stereospermum suaveolens were collected from Pithora forest, about I 00
km away from Raipur (C.G). The seedlings were planted in the medicinal plant
garden of Pt. Ravishankar Shukla University. Cotyledonary nodes and nodal
segments from field grown seedlings and terminal twigs of one and two-years-old
plants of Stereosrermum suaveolens were used as explants and established by using
the methods described below.
The responses of cultures derived from each explants were studied separately at
establishment stage, shoot proliferation stage, rooting stage and acclimatization stage.
The methods and plan of work employed during these studies were as follows:
The seeds extracted from mature fruits of Stereosrermum suaveolens were de-winged,
soaked in lukewarm water overnight, and wrapped in moist cotton bed for four days.
The germinated seeds were transferred to protray for seedling growth. Rate of
germination was tested during various seasons and during the month of April and
June it showed maximum (45%) seed germination. The seedlings attained 2-3 node
growth within 21 days. The shoots excised from 21-days-old seedlings were washed
gently under running tap water and with labolene to remove dust particles. The nodes
were cut from healthy seedlings, surface sterilized with 0.2% ( w/v) aqueous mercuric
246
~hloride li>r 5 minutes ami washed 3-4 times with double distilled water. The
slerilizc'(lnodes were inoculated on e-stablishment medium.
The li>llowing experiments were planned to study cxplants establishment, shoot
multiplication, subculture, rooting and acclimatization.
o Effects of MS (Murashige and Skoog), WPM (Liyod and McCown) and SH
(Schenk and Hildebrandt) media supplemented with 4.44 J.LM BAP on
establishment of cotyledonary and epicotyledonary nodes.
o Nodes of developed shoots were placed on MS medium and WPM both
supplemented with different concentrations (0.0, 0.04, 0.44& 4.44 J.IM) of
BAP to study the effect of BAP on shoot proliferation.
o Nodes of developed shoots were placed on MS medium and WPM both
supplemented with different concentrations (0.0, 0.04, 0.46 & 4.65 J.LM) of
Kinetin to study the effect of Kinetin on shoot proliferation.
o Individual micro nodes were placed on MS medium containing 4.44 J.lM BAP
or 4.65 J.LM Kinetin or combination of BAP (2.22 J.IM) and Kinetin (2.32 J.IM) to
study the effects of combination of BAP and Kinetin on shoot proliferation.
o The effect of different concentration of PVP (0, 250, 500 & 1000 mg/1) in MS
medium supplemented with 0.44 J.lM on in vitro shoot proliferation.
o The effect of different concentration of citric acid (0, 25, 50 & 100 mg/1) in
MS medium supplemented with 0.44 J.lM was investigated on in vitro shoot
proliferation was investigated.
o The effect of different concentration of casein hydrosylate (0, 25, 50 & I 00
mg/1) in MS medium supplemented with 0.44 fJM on in vitro shoot
proliferation was investigated.
o The effect of different concentration of adenine sulfate (0, I 0, 20, & 40 mg/1)
in MS medium supplemented with 0.44 J.lM on in vitro shoot proliferation was
investigated.
247
o The etlt:ct of ditlcrent wnccntration of activated charcoal (I 00, 200 & 400
mg/1) in MS medium supplemented with 0.44 ~ on in vitro shoot
proliferation was investigated.
o The nodal segments derived from epicotyledonary nodes were inoculated in
liquid medium (MS + 0.44 f!M BAP + 25 mg/1 AS) to study the effects of
liquid medium on in vitro shoot proliferation.
o Nodes derived from cotyledonary and epicotyledonary nodes were
subcultured on fresh MS medium supplemented with 0.44 ~and 25 mg/1 AS
every four weeks to study the effect of subculture on shoot multiplication
potential of nodes.
o Effects of IBA (0.0, 2.46, 4.90 and 9.80 f!M), IAA (0.0, 2.85, 5.70 and 11.40
f!M) and NAA (0.0, 2.69, 5.37 and 10.74 ~) on in vitro rooting of
microshoots derived from cotyledonary and epicotyledonary nodes were
studied.
o In vitro rooted plantlets were removed carefully from culture medium and
washed under running tap water and transferred to net pots containing coco
peat and mixture of sand, soil and farm yard manure. After 4 weeks plants
were shifted into nursery bags containing mixture of sand, soil and manure
( 1 : 1 : 1) for secondary hardening.
The following experiments were planned to study callusing in seed, epicotyl,
hypocotyl, cotyledon and petiole explants of S. suaveole ns and differentiation of
callus.
o Effects of different concentrations of 2, 4-D (0.0, 2.26, 4.52 and 9.04 !JM),
NAA (0.0, 2.69, 5.37 and 10.74 f!M) and BAP (0.0, 2.22, 4.44 and 8.88 11M)
in MS medium on callus initiation from different explants were studied.
o Effect of 2, 4-D, NAA and BAP were studied on differentiation of callus.
248
Results:
A. Stereospermum per.~o11atum (llassk.)
Seed germination:
The seeds were extracted !rom mature truits of Stereospermum personatwn, de
winged, soaked in lukewarm water overnight, and placed between moist cotton layers
for four days. The germinated seeds when transferred to protray (plastic trays with
cavities) grew into seedlings and within 21 days attained 5 - 7 em height with 2-3
nodes.
• The rate of germination was studied during January-March, April-June, July
September and October-December. The seeds showed maximum (55%)
germination during April to June.
Axillary shoot proliferation:
Cotyledonary node explants
Explants establishment
Ten-days-old seedlings of S. personatum were washed gently under running tap water
and then with 1% labolene to remove dust particles. The cotyledonary node were cut
from healthy seedlings, surface sterilized with 0.2% (w/v) aqueous mercuric chloride
for 5 minutes and washed 3-4 times with double distilled water. The sterilized nodes
were inoculated on MS, WPM and SH medium each supplemented with 4.44 [1M
BAP to study the effect of medium on explants establishment.
• The cotyledonary nodes showed better establishment on MS medium than WPM
and SH media. Shoot bud elongation occurred in 80% nodal explants placed on
MS medium supplemented with 4.44 [1M BAP. The explants produced 1.0 ± 0.0
shoots per cotyledonary node, 22.0 ± 0.82 mm length per developed shoot and
number of nodes per shoot 2.6 ± 0.13. MS medium was found to be the best
medium for explants establishment.
249
Shoot proliferation
The nodal segments of shoots developed on MS medium supplemented with 4.44
11M BAP were used for shoot proliferation experiments.
• The nodes were cut and explanted on MS medium supplemented with ditlercnt
concentrations of BAP. The explants placed on MS medium supplemented with
0.44 11M BAP showed maximum 2.0 ± 0.16 shoots per node, 24.6 ± 0.92 mm
length per developed shoot and 4.0 ± 0.23 nodes per developed shoot.
• Different concentrations of activated charcoal, adenine sulphate, casein
hydrolysate, citric acid and PVP were added singly in MS medium supplemented
with 0.44 IJM, BAP to find their effects on shoot proliferation. The cotyledonary
nodes placed on MS supplemented with 0.44 11M BAP and 250.0 mg/1 PVP
showed maximum 2.2 ± 0.11 shoots per node, 26.6 ± 0.55 mm length per
developed shoot and 3.2 ± 0.11 nodes per developed shoot.
Subculture
• The nodal segments were placed on MS medium containing 0.44 11M BAP and
250.0 mg/1 PVP for five consecutive subcultures. Gradual increase in shoot
number was observed up to 3'd cycle and during this subculture maximum shoot
number, length (mm) per developed shoot and number of nodes per shoot were
2.6 ± 0.21, 27.8 ± 1.01 and 4.06 ± 0.22, respectively. It is estimated that 10.55
nodal propagules can be obtained from a single cotyledonary node of S.
personatum after 3nt cycle.
Root initiation
The micro shoots produced during 3'd subculture were used for rooting
experiments.
• The micro shoots placed on MS medium supplemented with and 2.46 IJM IDA
showed maximum (100%) root induction with 5.06 ± 0.26 roots per micro node,
and root length (mm) 17.0 ± 1.36.
250
•:plcotyledonary node ex plants
Establishment of epicotyledonary node ex plants
The shoots were excised from 21-days-old S. personatum seedlings, washed
gently under running tap water and with I% labolene to remove dust particles.
The I" and 2"d epicotyledonary nodes (nodes above cotyledonary node) were cut
from healthy seedlings, surface sterilized with 0.2% (w/v) aqueous mercuric
chloride for 5 minutes and washed 3-4 times with double distilled water. The
sterilized nodes were inoculated on establishment medium MS, WPM and SH
medium each supplemented with 4.44 flM BAP.
• Epicotyledonary nodes (I '1 and 2"d) were cut to a final size of I 0-12 mm and
placed on different media. Best (86.6%) shoot bud elongation occurred in
explants placed on establishment medium MS medium supplemented with 4.44
~M BAP followed by WPM. SH medium failed to induce shoot bud elongation.
The epicotyledonary nodes on MS medium with 4.44 flM BAP produced 1.4 ±
0.13 shoots per node, 26.04 ± 1.84 mm length per developed shoot and 4.0 ± 0.33
nodes per developed shoot. Thus, MS medium supplemented with 4.44 ~ BAP
was found to be the most suitable medium for establishment of epicotyledonary
nodal explants of S. persona/Urn.
Shoot proliferation
The shoot proliferation experiments were performed with MS medium. The
shoots developed from original epicotyledonary nodes on MS supplemented with
4.44 f!M, BAP were used as source of nodal segments for shoot proliferation
experiments.
• The nodal segments were placed on MS medium with different concentrations of
BAP for four weeks. BAP at 0.44 flM concentration induced better shoot
proliferation than 4.44 and 0.04 flM concentrations. At 0.44 f!M, BAP induced
251
maximum 2.4 ± 0.1 g shoots per node, 29.0 ± 0.41 mm length per developed shoot
and 3.5 ± 0.14 nodes per shoot.
• Different concentrations of activated charcoal, adenine sulphate, casein
hydrolysate, citric acid and PYP were added singly in MS medium supplemented
with 0.44 f.!M, BAP to lind their efl'ects on shoot proliferation. PYP was found more
suitable than other additives used. The nodal segments placed on MS medium
supplemented with 0.44 f.!M BAP and 250.0 mg/1 PVP produced maximum 2.78 ±
0.14 shoots per node, 36.0 ± 1.0 mm long developed shoots and 3.24 ± 0.16 nodes
per shoot. Thus, MS medium supplemented with 0.44 f.!M BAP and 250.0 mg/1
PVP was found to be the most suitable medium for shoot proliferation from
epicotyledonary nodal explants of S. personatum.
Subculture
• The nodal segments were placed on MS medium containing 0.44 f.!M BAP and
250.0 mgll PVP for seven consecutive subcultures. Gradual increase in shoot
number occurred up to 5th cycle. During fifth subculture shoot number, length
(mm) per developed shoot and number of nodes per shoot were 5.2 ± 0.15, 37.26
± 1.29 and 5.06 ± 0.15, respectively. It is estimated that 26.31 nodal propagules
can be obtained from a single epicotyledonary node of S. personatum after 5th
cycle.
Liquid medium
• The nodes were placed on semisolid medium and liquid media for shoot
proliferation. The number of shoots and nodes produced on semisolid and liquid
media did not show any significant difference. The nodes placed on semisolid
medium showed shoot number 2.2 ± 0.19, length (mm) per developed shoot 30.8
± 0.98 and number of nodes per shoot 3.8 ± 0.19 . The shoot number, shoot length
(mm) and number of nodes per shoot in liquid medium were 2.4 ± 0.13, 35.6 ±
0.62 and 4.06 ± 0.11, respectively. Moreover, shoots also were hyper hydrated in
liquid medium. This shows that semisolid medium is suitable for in vitro culture
of epicotyledonary nodes.
252
Cycle of liquid and semisolid media
• Suqnisingly, in the third subculture the shoots transferred after 15 days from
liquid medium with high concentration 8.88 11M BAP to semisolid MS medium
with low concentration 1.11 ~~M BAP showed enhanced shoot proliferation 15.2 :!:
0.85 shoots per node 26.8 :!: 0.51 mm length per developed shoot and 3.8 :!: 0.19
nodes per shoot.
Root initiation
The micro shoots produced during 5th subculture were used for rooting
experiments.
• The micro shoots placed on MS medium supplemented with 2.46 11M IBA showed
I 00% rooting with 4.4:!: 0.80 roots per shoot and 12.78 ± 0.84 mm root length.
Hardening of plantlets
In vitro rooted plants were washed and planted in pots. These pots were placed in
greenhouse for primary and secondary hardening.
• The tissue culture raised plants were transferred to net pots filled with coco-peat
and placed in green house at 70% humidity. These plants showed 83.3% survival
after 30 days of transfer. Secondary hardening was done in the nursery bags filled
with soil mixture (sand, soil and farmyard manure in I: I: I ratio). In this stage,
plants showed 80% survival.
Apical bud explants
Apical buds were cut from the field grown 1-2 year old field grown plants. The
leaves were removed and apical bud portion was washed in running tap water and
I% labolene to remove dust particles. The explants were surface sterilized with
.0.2% mercuric chloride for 10 minutes under aseptic condition and washed 3-4
with sterile water to remove traces of mercuric chloride. The explants were cut to
a final size of I 0-12 mm and inoculated on different media supplemented with
253
different plant growth regulators and adjuvants. However, the explants did not
respond to any combination.
Callus Initiation
• Callus initiation occurred in 60% petiole explants placed on MS medium
supplemented with 4.52 or 9.04 !1M 2, 4-D. The callus areas (mm2) on 4.52 or
9.04 !1M 2, 4-D werel8.6 ± 2.05 and 20.93 ± 2.55, respectively. However, the
callus dried after 4 weeks of subculture.
• Callus initiation occurred in 80% petiole explants placed on MS medium
supplemented with 2.69 or 5.37 !1M NAA. The callus areas (mm2) on 2.69 and
5.37 11M NAA were 35.73 ± 2.61 and 38.06 ± 2.14, respectively. The callus
showed rhizogenesis when transferred to second medium combined with 4.44 !1M
BAP. After 4 week, the callus subcultured on the same medium exhibited increase
in area to 36.06 ± 2.44 and 38.6 ± 2.12, respectively.
• Callus initiation occurred in 80% petiole explants placed on MS medium
augmented with 4.44 or 8.88 11M BAP. The callus areas (mm2) on 4.44 and 8.88
!1M BAP were 22.86 ± 2.56 and 28.46 ± 2.91, respectively. The callus transferred
to second medium increased in area to 25.73 ± 2.35 and 32.73 ± 2.36,
respectively, after 4 week.
• Callus induction occurred in 80% petiole explants placed on MS medium with
2.69 !1M NAA + 8.88 !1M BAP. The callus areas (mm2) on MS with 2.69 11M NAA
+ 8.88 11M BAP were 38.46 ± 2.12(Callus with roots) and 46.6 ± 2.27,
respectively. However, the callus transferred to second medium (MS with 2.69
!1M NAA + 17.76 !1M BAP) did not differentiate into organs but a bulbous
structure was observed. The callus areas (mm2) on MS medium with 2.69 !1M
NAA +17.76 !1M BAP was 65.73 ± 3.33 with bulbous structure.
• The petiole segments placed on MS medium supplemented with 5.37 1JM NAA +
4.44 !1M BAP showed percent callus induction 100% and callus area (mm2) 45.33
± 2.62 and on medium with 5.37 !1M NAA + 8.88 11M BAP callus induction
percent and callus area (mm2)were 100% and 55.06 ± 3.51, respectively.
254
However, the callus transtcJTed to second medium MS with 5.37 11M NAA +
17.76 11M BAP did not ditlcrentiate into organs but a bulbous structure was
observed in the callus. The callus areas (mm2) on MS medium with 5.37 11M NAA
+8.88~tM BAP, and 5.371!M NAA + 17.761!M BAP were 57.33 ± 3.71 and 75.0 ±
3.59 with bulbous structure, respectively.
• Callus initiation occurred in I 00% and 800/o epicotyl ex plants placed on MS
medium with 0.0 or 2.26 11M 2, 4-D. The callus areas (mm2) on 0.0 and 2.2611M 2,
4-D were 49.2 ± 3.93 and 39.06 ± 4.56, respectively. However, the callus dried
after 4 weeks of subculture.
• Callus initiation occurred in I 00%, 80%, 60% and 20% cotyledon explants placed
on the MS medium supplemented with 0.0, 2.26, 4.52 and 9.04 11M 2, 4-D. The
callus areas (mm2) on 0.0, 2.26, 4.52 and 9.0411M 2, 4-D were 63.93 ± 3.96, 53.86
± 3.72, 38.13 ± 1.51 and 28.53 ± 1.61 respectively. However, the callus dried
after 4 weeks of subculture.
B. Sterospermum suaveolens (DC.)
Seed germination
The seeds extracted from mature fruits of Stereospermum suaveolens were de
winged, soaked in lukewarm water overnight, and placed between moist cotton
layers for four days. The germinated seeds when transferred to protray (plastic
trays with cavities) grew into seedlings and within 21 days attained 5 - 7 em
height with 2-3 nodes. The germination of seeds was studied during January
March, April-June, July-September and October-December.
• The seeds showed best (45%) germination during April-June.
255
Alillury Shoot Proliferation:
Cotyledonary node ex plants
Ex plants establishment
The I 0-days-old seedlings of S. suaveolens were washed gently under running tap
water and with I% labolene to remove dust particles. The cotyledonary node
explants were cut from healthy seedlings, surface sterilized with 0.2% (w/v)
aqueous mercuric chloride for 5 minutes and washed 3-4 times with double
distilled water. The sterilized nodes were inoculated on MS, WPM and SH
medium each containing 4.44 J.1M BAP to study the effects of different media on
explants
• Maximum 80% nodal explants showed shoot bud elongation on MS medium with
4.44 ~ BAP. On this medium the explants showed maximum 1.2 ± 0.11 shoots
per node, 22.4 ± 0.59 length (mm) per developed shoot and 3.2 ± 0.19 nodes per
developed shoot.
Shoot proliferation
•
•
The nodal segments of shoots developed on MS medium supplemented with 4.44
~ BAP were used for shoot proliferation experiments.
The explants placed on MS medium supplemented with 0.44 ~ BAP showed
maximum 1.13 ± 0.09 shoots per node, 27.2 ± 0.51 length (mm) per developed
shoot and 3.8 ± 0.19 nodes per shoot.
The cotyledonary nodes placed on MS supplemented with 0.44 ~ BAP and 25.0
mg/1 AS showed 2.6 ± 0.21 shoots per node, 28.4 ± 0.39 length (mm) per
developed shoot and 4.1 ± 0.21 nodes per developed shoot.
256
Subculture
• The shoot number, maximum shoot length (mm) and number of nodes per shoot
in 4th subculture were 3.4 ± 0.13, 33.0 ± 0.51 and 4.4 ±0.13. It is estimated that
14.96 nodal propagules can be obtained from a single cotyledonary node of S.
suaveo/ens after 3'd cycle.
Root initiation
• The micro shoots placed on MS medium supplemented with 4.90 11M IBA showed
maximum (I 00%) root initiation with 4.2 ± 0.19 roots per micro shoot and 23.0 ±
0.65 rnm root length.
Epicotyledonary node explants
The shoots excised from 21-days-o1d seedlings of S. suaveolens and were washed
gently under running tap water and with 1% labolene to remove dust particles. The I'' and 2"d epicotyledonary nodes were cut from healthy seedlings, surface sterilized with
0.2% (w/v) aqueous mercuric chloride for 5 minutes and washed 3-4 times with
double distilled water. The sterilized nodes were inoculated on establishment medium
MS, WPM and SH each supplemented with 4.44 11M BAP.
Explants establishment
Epicotyledonary nodes I'' and 2"d were cut to a final size of I 0-12 rnm and placed on
MS, WPM and SH media each supplemented with 4.44 11M BAP for explants
establishment. Maximum (73.3%) nodal explants showed shoot bud elongation on
followed by WPM and SH media. The epicotyledonary nodes explanted on MS
medium with 4.44 ~ BAP produced 1.4 ± 0.13 shoots per node, 19.6 ± 0.99 rnm
length per developed shoot and 2.8 ± 0.19 nodes per developed shoot.
257
Shoot proliferation:
The shoot proliferation e)(pcrimcnts were perfonned with MS medium. The
shoots developed from original cpicotylcdonary nodes on MS supplemented with
4.44 iJM, BAP were used as source of nodal segments for shoot proliferation
C)(periments.
• Effect of different concentrations of BAP on shoot proliferation was studied.
BAP at 0.44 11M concentration in MS medium was found more suitable for shoot
proliferation than 4.44 and 0.04 iJM concentrations. At 0.44 iJM, BAP induced
rn8)(irnum 1.4 ± 0.13 shoots per node, 23.6 ± 0.52 mm length per developed
shoot and 3.6 ± 0.13 nodes per shoot.
• Activated charcoal, adenine sulphate, casein hydrolysate, citric acid and PVP
were used as adjuvants in the shoot proliferation medium. Adenine sulphate
was found more suitable for shoot multiplication than all other additives. The
nodal segments derived from epicotyledonary node explants placed on MS
medium supplemented with 0.44 iJM BAP and 25.0 mg/1 adenine sulphate
produced maximum 2.4 ± 0.13 shoots per node, 26.8 ± 0.31 mm length per
developed shoot and 4.5 ± 0.13 nodes per shoot.
• Comparison of number of propagules obtained from nodal segments of
cotyledonary nodes and epicotyledonary-nodal segments of S. personatum and S.
suaveolens revealed the following:
(a) Nodes derived from cotyledonary nodes and epicotyledonary nodal segments
of both the species S. personatum and S. suaveolens showed best shoot
proliferation response when placed on MS medium fortified with 0.44 jlM BAP.
(b) Nodes derived from epicotyledonary nodes of both S. personatum and S.
suaveolens produced more propagules than nodes derived from cotyledonay
nodes.
(c) Nodes derived from epicotyledonary nodes of S. personatum produced more
propagules than those of S. suaveolens.
(d) In S. personatum, nodes derived from cotyledonary nodes produced 8.0
propagules and nodes derived from epicotyledonary segments produced 8.4
258
'I
', .. ,
propagules; whereas in S. suaveolens, nodes derived from cotyledonary nodes
produced 4.29 propagulcs and nodes derived from epicotyledonary segments
produced 5.04 propagules.
Liquid medium
• The nodes were placed on semisolid medium and liquid media for shoot
proliferation. The number of shoots developed from the nodes and the number of
nodes on the developed shoots did not show any significant variation. The micro
node of S. suaveo/ens placed on semisolid medium showed 2.4 ± 0.13 shoots per
node, 25.4 ± 0.13 length (mm) per developed shoot and 4.4 ± 0.13 nodes per
shoot.
Semisolid liquid medium cycle
• The micro shoots sub cultured on semi solid medium MS with low ( 1.11 !JM)
concentration ofBAP when transferred from liquid medium with high (8.88 !JM)
concentration of BAP showed sudden burst of shoot proliferation within four
weeks. The shoots per node, shoot length (mm) and nodes per shoot were 9.2 ±
0.26, 22.0 ± 0.47 and 2.8 ± 0.23, respectively.
Subculture
• Sub culturing of nodes up to 5th cycle was done to study shoot proliferation
potential of nodal segments. Gradual increase in shoot number occurred up to 3'd
cycle. During this subculture maximum shoot number, maximum length (mm) per
developed shoot and number of nodes per shoot were 3.0 ± 0.16, 29.06 ± 0.51 and
4.6 ± 0.13, respectively. It is estimated that 13.8 nodal propagules can be obtained
from a single Epicotyledonary node of S. suaveo/ens after 3'd cycle.
Root initiation
• Root initiation occurred in I 00% micro shoots placed on MS medium
supplemented with 4.90 !JM IBA. Roots per micro shoot and root length (mm)
were 4.0 ± 0.23 and 28.6 ± 0.52, respectively.
259
A~~limatization of plantlets
• The tissue culture plants were washed in running tap water to remove agar-agar
and planted on net pots filled with coco peat. The pots were placed in green house
at 70% humidity where 86.6% of the plantlets survived after 30 days. After
primary hardening, the plants were transferred to polyethylene bags filled with
soil mixture, where they showed 84.6% survival.
Apical bud explants
Apical buds were cut from the field grown l-2 year old field grown plants. The
leaves were removed and apical bud portion was washed in running tap water and
I% labolene to remove dust particles. The explants were surface sterilized with
.0.2% mercuric chloride for I 0 minutes under aseptic condition and washed 3-4
with sterile water to remove traces of mercuric chloride. The explants were cut to
a final size of l 0-12 mm and inoculated on different media supplemented with
different plant growth regulators and adjuvants. However, the explants did not
respond to any combination.
Callus initiation
• No Callus initiation occurred when seeds, epicotyl, hypocotyl and cotyledons
were inoculated on MS medium supplemented with 2, 4 - D (0.0, 2.26, 4.52 and
9.04 f!M).
• Only swelling occurred when the petiole explants were placed on MS with 4.52
and 9.04 jlM 2,4 -D. Other media failed to induce callus formation.
260
Conclusions:
,_ Stereospermum personatum
Seed Gennination
The seeds of S. personatum showed gradual decrease in gennination with increase
in duration of seed storage. The seed gennination percentages during April-June,
July- September, Oct-December and Jan-March were 55.0, 40.0, 30.0 and 15.0,
respectively.
Cotyledonary node explants
• MS medium with 4.44 J.1M BAP was found to be most suitable medium for
establishment of explants. It induced shoot elongation in 80% of cotyledonary
explants.
• The cytokinin BAP at 0.44 J.1M in MS medium was found suitable for in vitro
shoot proliferation. On this medium, it produced 2.0 ± 0.16 shoots per node, 24.6
± 0.92 (mm) long shoot and 4.0 ± 0.23 nodes per developed shoot.
• The nodes placed on MS supplemented with 0.44 J.1M BAP and 250.0 mg/1 PVP
enhanced shoot proliferation with maximum 2.2 ± 0.11 shoots per node, 26.6 ±
0.55 (mm) long shoot and 3.2 ± 0.11 nodes per shoot.
• Sub culturing up to 3'd cycle was beneficial for in vitro shoot proliferation, as
gradual increase in shoot number occurred during these subcultures. During this
subculture it induced 2.6 ± 0.21 shoots per node, 27.8 ± 1.0 I mm long shoot and
4. 06 ± 0.22 nodes per shoot.
• The micro shoots placed on MS medium with 2.46 J.1M IBA showed maximum
(100%) root induction with 5.06 ± 0.26 roots per micro shoot and root length
(mm) 17.0± 1.36.
• Coco peat was found suitable substrate for primary and secondary hardening.
261
Epiwtylcdonary node cxplants
• MS medium supplemented with 4.44J1M BAP was better than WPM medium and
SH medium for establishment of explants. It induced shoot elongation in 86.6%
nodal explants.
• MS medium supplemented with 0.44J1M BAP was found suitable for in vitro
shoot proliferation. It induced 2.4 ± 0.18 shoots per node, 29.0 ± 0.41 mm long
shoots and 3.5 ± 0.14 nodes per shoot.
• Addition of PVP enhanced shoot proliferation. MS medium supplemented with
0.44 J1M BAP and 250.0 mg/1 PVP induced 2.78 ± 0.14 shoots per node, 36.0 ±
1.0 length (mm) per developed shoot and 3.24 ± 0.16 nodes per shoot.
• Sub culturing up to 5th cycle was beneficial for in vitro shoot proliferation, as
gradual increase in shoot number occurred during these subcultures. During this
subculture maximum 5.2 ± 0.15 shoots per node, 37.26 ± 1.29 mm long shoots
and 5.06 ± 0.15 nodes per shoot were observed.
• In liquid medium, the shoot length increased but the shoots were hyper-hydrated.
• Cycling of liquid medium with high concentration of BAP (8.88 J.IM) and semi
solid medium with lower concentration of BAP (0 1.11 J.IM) showed tremendous
increase in number of shoots and is suitable for in vitro shoot proliferation in S.
personatum.
• Root initiation occurred in I 00% micro shoots placed on MS medium with 2.46
J.IMIBA.
Apical bud explants
• The apical buds from 1-2 year old field grown plants of S. personatum inoculated
on different media supplemented with different plant growth regulators and
adjuvants did not respond in culture.
Callus culture
• Callus formation from epicotyl, cotyledon and petiole tissues of S. personarum
can be achieved on variety of media. A variety of media were tested for
262
di!Terentiation but none of them could induce caulogenesis or somatic
embryogenesis.
Stereospermum suaveolens
Seed germination:
The seeds of S. suaveolens showed decrease in germination percentage with increase in
duration of seed collection. The percent seed germination during April-June, July
September, Oct-December and Jan-March was 45.0, 30.0, 20.0 and 20.0, respectively.
Axillary shoot proliferation
Cotyledonary node explants
• MS medium with 4.44 11M BAP was found to be best medium for explants
establishment. It induced shoot elongation in 80% of cotyledonary explants.
• The cytokinin BAP 0.44 11M in MS medium was found better than kinetin and
other levels of BAP for in vitro shoot proliferation. At this level, it induced 1.13
± 0.09 shoots per node, 27.2 ± 0.51 (mm) long shoots and 3.8 ± 0.19 nodes per
shoot.
• Addition of 25.0 mg/1 adenine sulfate in MS supplemented with 0.44 j.!M BAP
and enhanced shoot proliferation in nodes derived from cotyledonary nodes.
The nodes placed on this medium produced 2.6 ± 0.21 shoots per node, 28.4 ±
0.39 (mm) long shoots and 4.1 ± 0.21 nodes per shoot.
• Sub culturing up to 4th cycle was beneficial for in vitro shoot proliferation, as
gradual increase in shoot number occurred during these subcultures. During 4th
subculture nodes produced 3.4 ± 0.13 shoots per node, 33.0 ± 0.51 (mm) long
shoots and 4.4 ±0.13 number of nodes per shoot.
• The micro shoots showed I 00% root induction when placed on MS medium
with 4.90 j.!M IBA.
• Coco peat is suitable substrate for primary as well as secondary hardening.
263
Ep•wtyk-donary node explants
• MS medium supplemented with 4.44 flM BAP was better than WPM medium
and SH medium for establishment of explants. It induced shoot elongation in
73.3% nodal explants.
• MS medium supplemented with 0.44 f1M BAP showed better result for shoot
proliferation. It induced 1.4 ± 0.13 shoots per node, 23.6 ± 0.52 (mm) long
shoots and 3.6 ± 0.13 nodes per shoot.
• Addition of adenine sulfate enhanced shoot proliferation. MS supplemented
with 0.44 11M BAP and 25.0 mg/1 AS induced maximum 2.4 ± 0.13 shoots per
node, 26.8 ± 0.31 length (mm) per developed shoot and 4.5 ± 0.13 nodes per
shoot.
• Sub culturing up to 3'd cycle was beneficial for in vitro shoot proliferation, as
gradual increase in shoot number occurred during these subcultures. During this
subculture it induced maximum 3.0 ± 0.16 shoots per node, 29.06 ± 0.51 mm
long shoots and 4.6 ± 0.13 nodes per developed shoot.
• Semi solid medium was found more suitable than liquid medium. However,
cycling of nodal segments on liquid medium with high concentration of BAP
(8.88 !l.M) and semi solid medium with lower concentration of BAP (1.11 11M )
showed tremendous increase in number of shoots and is suitable for in vitro
shoot proliferation inS. suaveolens.
• The micro shoots placed on MS medium with 4.90 !lM ffiA showed 100% root
initiation.
Apical bud explants
• The apical buds from 1-2 year old field grown plants of S. suaveolens inoculated
on different media supplemented with different plant growth regulators and
adjuvants did not respond in culture.
Callus culture
• Callus initiation could not be achieved from seeds, epicotyl, cotyledon, hypocotyl
and petiole tissues of S. suaveolens on any of the media tested.
264
