Beruflich Dokumente
Kultur Dokumente
EDITORS
VIBHA DHAWAN
PM GANAPATHY
DK KHURANA
• PUBLISHED BY
IDRC - TIFNET
© IDRC -
IDRC
CRDI
CuD
CANADA
South Asia Regional Office 11 Jor Bagh, New Delhi 110 003
CONTENTS
Pages
Foreword V
Acknowledgements vii
Summary of Discussion and Recommendations ix
Annexure I 95
List-of participants
Annexure II 99
Workshop Schedule.
FOREWORD
This is the first publication of the TIFNET (see inside back cover)
and it is sincerely hoped that it will enhance the usefulness of this informal
network.
PM Ganapathy
Regional Forestry Coordinator (IDRC)
Cherla B. Sastry
Principal Program Officer (Forestry)IDRC,
ACKNOWLEDGEMENTS
These proceedings would not have come in its present form without
the contribution of all participants who attended this workshop. We
extend our thanks to them. The help of Mr Dharmendar Rawat in typing
this volume is gratefully acknowledged.
SUMMARY OF DISCUSSIONS AND
RECOMMENDATIONS
ix
plants will remain stable in the future. It is estimated that the market for
tissue culture plants is enormous and upto ten times the present
production level can be accomodated in the international market.
x
Final Recommendations
Participants felt that such meetings should be organised at yearly
interval so as to have interaction among scientists involved in tree tissue
culture. In this field, at present there are far too many failures which
unfortunately are not reported in the research publications. What is
published is largely the successes and the problems encountered are not
discussed. For example, for Dalbergia sissoo, over half a dozen papers are
listed in the literature describing propagation both from juvenile and adult
explants. But in practical terms, it is seen that in vitro formed shpots fail
to multiply and thus from each nodal explant, one can, at best produce one
plant. This problem is common in many other tree species and basic
research is required to study the role of mother explant. Rooting is a severe
constraint for many tree species. Even species which root fairly easily in
vivo fail to root under in vitro conditions. Bamboo falls in this category
and many laboratories round the world are working on developing
techniques for bamboo tissue culture. The success is restricted to few
genera only. It is seen that the rooting of shoots from explants is much
difficult and varies between 20%-40%. Such protocols are not of much use
for large scale propagation.
xi
A. TISSUE CULTURE TECHNIQUES
In Vitro Propagation of Grape (Vitis Spp.):
Establishment, Proliferation and Development
of Shoot-tip Cultures on Defined Media
The paper reports multiplication methods for the three cultivars of grape
(Vitis vinifera L. cv. perleue, Pusa seedless and hybrid, 4-3). The
investigation reveals that growth and differentiation of shoot tip is cultivar
dependent and the rooting percentage declines with repeated sub culturing
(optimum during first five sub culture3).
INTRODUCTION
Grape (Vitis spp) is conventionally propagated vegetatively. This
Lechrnque has come to stay because it is economical and efficient. The
basic drawback of the system, however, is that it does not allow rapid
production of vines that may be available in large number for commercial
production for the release of new varieties. It is highly desirable to define
the procedure needed for a fast rate of propagation in vitro. Shoot tip
culture of grapes have been studied in many laboratories. However, there
is still lack of published data on the survival of explants on initiation
medium and the effect of cytokixiins on proliferation and growth. This
3
paper reports survival and subsequent growth of explants of three
genotypes of grape on culture media. The influences of cytokinin / auxin
on shoot proliferation and rooting of shoot, are also described.
Shoot tips (10mm in length) were removed from eight year old field
grown plants of grape (Vitis vinifera L. cv. Perlette', Pusa Seedless' and
hybrid, 'W 4-3'). Shoot tips were surface sterilized with 0.1 per cent (W/V)
mercuric chloride solution containing 0.01 per cent Tween 20 wetting
agent for seven minutes and rinsed seven times in sterile distilled water.
Isolated shoot tips were individually transferred in flasks containing 75
ml of MS (Murashige and Skoog) medium. Different concentrations of
NAA, IBA and BAP were tested for establishment of shoot tips, shoot
proliferation and rooting of in vitro raised shoots. The pH of the media
was adjusted to 5.8 prior to sterilization at 15 lbs for 15 minutes. All
media were gelled with 8 g 11 agar. Cultures were maintained at 26°C
with 16 h light (3500 lux).
RESULTS
4
As regard to time requirement, Perlette 'and 'Pusa Seedless 'have
similar time requirement (20-21 days) for establishment in the culture
medium.
New growth was visible after one and half weeks of culture but the
shoot tips were considered established only when the new growth spread
approximately to 2 cm diameter. Cultures attained this phase within
20-21 days. Bud cultures appeared fresh green healthy leaves with
reduced lamina. Slow growing shoot tips were frequently subcultured to
accelerate their growth. In contrast, hybrid W 4-3' genotype took 32-36
days for establishment in the same medium.
5
b) Shoot Multiplication
Effect of different concentrations of BAP (5,10,15 and 2 ji. M) on in
vitro shoot multiplication was studied. After 21 days of culture in
establishment medium, shoot tips were transferred on to 16 different
proliferation medium. Highest number of shoots (13) were recorded
within 3 weeks of transfer in proliferation medium containing 10 p.M BAP.
Shoot multiplication was further increased (18 folds) by keeping these
shoots into the same medium for another three weeks. 'Pusa Seedless' and
'Perlette' performed equally well as regard to shoot multiplication.
However, shoot production per initial explant in hybrid 'W 4-3' was low
(9 shoots per explant) under similar cultural conditions and duration
(Table 2). During the process of shoot multiplication fungal and bacterial
contamination was noticed, apparently with no detrimental effect on
growth during subcultures.
c) In vitro Rooting
Nodal explants (a portion of stem each with one node) were prepared
from each in vitro raised plant and transferred individually onto rooting
medium. Different concentrations of IBA (1,5,10,15,20 jiM) were tried for
6
induction of root. Primary root development was lowest at IBA
concentrations of 1 and 20 jiM. Number of primary roots increased in all
the three genotypes upto 10 jiM IBA in 7 days. Maximum number of
primary roots were formed at 10 p.M IBA in 7 days of culture period.
Maximum number of primary roots were formed at 10 p. M IBA in 7 days
of culture period. Maximum number of primary roots in all the three
genotypes were formed at 10 p. M IBA after 7 days. Number of primary
root ranged from 4.5 to 5.6 and the root system developed normally with
secondary and tertiary branching whereas at high concentration of IBA
i.e. 20 jiM shoots remained stunted.
DISCUSSION
The results of the present investigation reveal that the growth and
differentiation of explants (shoot tips) of grapevine are under strong
hormonal control. This is in conformity with the finding of Novak and
Juvova (1983). The essentiality of NAA and BAP for shoot tip
establishment have also been investigated by Chee et al. (1984) with Vitis
spp. The effect of cytokinins on shoot multiplication of grape have been
confirmed by various workers (Pool and Powell, 1975; Jone and Webb,
1978; Muffins and Srinivasan, 1976; Novak and Juvova, 1983 and Chee
et al., 1984). Adventitious shoot formation is enhanced considerably by
arresting the apical dominance of shoot. Cytokinin is known to eliminate
the apical dominance (Vasil, 1985). Barless and Skene (1980) reported
that the in vitro shoot multiplication is a function of BAP concentration.
Under present study 10 p. M of BAP proved its efficiency by giving
maximum number of shoots per initial explants. The same trend was
followed in all the three genotypes tested. However, at lower
concentration of BAP,( less than 10 jiM) multiple shoot formation declined
drastically. In contrast, the higher dose of BAP i.e. 20 p. M produced large
number of weak shoots which did not root properly and died after 15-20
days.
7
optimum rooting was recorded in the very first subculture of the nodal
explant.
REFERENCES
8
In Vitro Propagation of Albizia lebbek Using
Axifiary and Apical Buds
An in vitro propagation protocol has been worked out for axillary and apical
bud multiplication of Albizia lebbek L. . The buds (2-3 mm long) excised
from off-shoots of elite Albizia trees (10-15 years old) were placed on MS
medium supplemented with BAP (1.0 mg F1) and IAA (0.1 mg F1).
Established shoots were multiplied on the MS medium supplemented with
BAP (2.0mg F1) and IAA (0.5 mg F1). The highest degree of rhizogenesis
was achieved on medium supplemented with IBA (0.1 mg 1k).
INTRODUCTION
9
is the best alternative. On Albizia nñcropropagation there are only two
reports available (Gharyal and Maheshwari, 1983; Rao, 1985 personal
communication). No commercially viable protocol is available for
true-to-type Albizia plantlet propagation. In the present paper, the
results of studies on axillary and apical bud regenerated plantlets, which
are true-to-type are presented.
10
revealed that phytohormones are essential for establishment of the
culture. In a series of trials the MS medium was supplemented with
combination of cytokimns and auxms. The results are summarized in
table 1. The concentration of three cytokinins viz, BAP, Kn and 2iP were
kept at 0.5, 1.0,2.0 mg 1.1 with combination of IAA or NAA at 0.1 mgF1.
11
The effect of Kn and 2iP with any auxin combination was less as compared
to BAP. It was found that highest percentage (96.9%) of bud sprouting
occurred in the medium supplemented with 1 mg 1.1 BAP.
Effect of three cytokinins and two auxins were studied for their
efficiency on multiple shoot formation from sprouted buds. It has been
previously noted that on hormone -free medium no multiple shoots were
formed. Table 2 shows multiple shoot formation from sprouted buds at
various concentrations of cytolcinins. In general, with increase in
concentration of BAP, multiple shoot formation was increased. Maximum
number of shoots per explant (2.7) were obtained with 5.0 mg BAP.
Increasing the BAP concentration to 10.0 mg F1 did not result in an
increase in the number of shoots. Compared to BAP, Kn and 2iP were less
efficient in inducing bud break and multiple shoot formation.
An experiment was conducted with BAP (2.0, 5.0 and 10.0 m g 11)
and LAA (0.1, 0.5 and 1.0 mg F') in order to determine the optimum
concentration of cytokinin and auxin needed for shoot formation.
Maximum number of shoots (3.8) were produced on BAP at 2.0mg 11 and
LAA at 0.5 mg F' (Table 4). It was observed that for multiple shoot
formation, a combination of BAP (2-10 mg 11) and IAA (0.5 mg 11) was
optimal.
12
explants. Both apical bud and axillary buds produced equal number of
shoots and no difference was observed in shoot proliferation.
15
Table 6: Induction of root formation on 4-7 cm long leafy shoots.
In each treatment 25-30 macro-shoots were treated for
rhizogenesis. Rooting was recorded at the end of 6 weeks
ACKNOWLEDGEMENTS
This research has been financed by a grant (No. 3/(5)581-NES/564)
from the Department of Non-Conventional Energy Sources (DNES),
Government of India.
16
REFERENCES
Gharyal P.K. and Maheshwari S.C., 1983. In vitro differentiation of
plantlets from tissue cultures of Albizia lebbek L. Plant Cell
Tissue. Organ Cult., 2:49-53.
Murashige T. and Skoog F., 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures Physiol Plant., 15:
473-497.
Rao P.V.L., 1985. (Personal Communication) unpublished Ph.D. thesis
I.T.T., Kharagpur.
17
Forest Tree Tissue Culture: Current Status and
Future Prospects
G. Lakshmi Sita
Department of Microbiology and Cell Biology
Indian Institute of Science
Bangalore 560 012, India
In this paper the need for tree biotechnology and the goals set and achieved
at Indian Institute of Science (I.I.Sc.) are discussed. Species selected are
commercially important either for the oil (sandalwood), timber (rosewood)
or multiple uses such as eucalypts. The cultures are initiated from the adult
trees marked for the desired characters. The plantlets are successfully
transferred to the soil. The paper also describes the main hurdle in
commercialisation of tissue culture techniques for tree species. It is
important that industry feels the requirement of high quality planting
material. At the same time attempts should be made to reduce the cost of
plantiet production. The research results of the author are still at the testing
stage and the clonal fidelity will become available with the planting material
attaining maturity. However, in eucalypts, tissue culture raised plants are
out performing the seed raised plantation.
INTRODUCTION
18
the perennial crops. Also plants of high economic value such as
ornamentals and horticultural plants received more attention. General
interest in tissue culture propagation arose with its grand success with
orchids. In fact, all commercial orchid growers have a tissue culture
laboratory attached to their nurseries. In the past two decades, the
technique has expanded to include many other ornamentals and some
horticultural species. In comparison, tissue culture of forest tree species
is comparatively at a developing stage. In the recent past, in addition to
the forest biologists in government, industries and universities, molecular
biologists are also getting involved in tree tissue culture research. A
retrospect of the scenario of tree tissue culture clearly shows that major
advances have been made in tree tissue culture including genetic
transformation of trees in the last decade. Genetic transformation has
successfully been achieved in hybrid poplar (Fillate et al., 1987) and
Douglas fir (Dandekar et al., 1987). However, there is only one example
of genetic transformation of a forest tree for a potentially commercially
important trait (Sederoffet al., 1986).
19
increasing. To meet the increasing demands and severe shortages,
currently practiced tree improvement programmes are not adequate.
Production of biomass is critical for over 5,76,900 villages in India, not
only for the supply of fuel and fodder, but also to meet the wood needs of
the country for timber, pulp and fibre. On one hand the forest cover is
gradually shrinking, and on the other hand, the gap between supply and
demand of wood based material is widening resulting in near crisis
situation. Hence there is an urgent need to improve the quality and
quantity of forest trees to meet the demand.
Forest trees have been attractive model systems to work, not only
because of the challenge they pose, but also because of their economic
importance. In the Department of Microbiology at the Indian Institute of
Science, our aim has been to study the basic and applied aspects of
economically important trees with the long term goal of improvement by
genetic manipulation, using cell and tissue culture technology and
recombinant DNA technology. During the last 15 years we have
developed tissue culture techniques for mass propagation of sandalwood
(Lakshmi Sita, 1979, 1986a, 1987, 1993a; Lakshmi Sita et al., 1979,
20
1980a,b, 1991); eucalypts (Lakshmi Sita 1979, 1981, 1986b; Lakshini Sita
& Vaidyanathan, 1979; Lakshmi Sita & Chattopadhyay, 1988; Lakshmi
Sita et aL, 1986); rosewood (Lakshmi Sita and Ravindaran, 1991;
Lakshmi Sita and Raghava Swaniy, 1992; Raghava Swamy et aL, 1992);
mulberry (Lakshmi Sita and Ravindaran, 1989; Lakshmi Sita and
Sreenatha, 1992), red sandalwood (Lakshmi Sita and Sreenatha, 1992)
and cashew (Lakshmi Sita, 1989) etc. Some of these results will be
discussed.
21
and multiplication by tissue culture methods to reduce the harvesting
period to half or even less.
Protoplast Cultures
Isolation of protoplasts, and regeneration of plants is a prerequisite
for any study on genetic manipulation. We have isolated protoplasts from
mesophyll tissue, suspension cultures of cliploid and triploid cultures.
Isolation and culture of protoplasts was better from the cells of suspension
cultures. Various parameters such as pH, enzyme concentrations,
concentration of the osmoticum were studied in relation to, protoplast
yield. Isolated protoplasts divided and formed colonies and callus
subsequently. Isolated protoplasts were observed to develop into
plantlets.
22
embryos was obtained as a result of gibberellic acid (GA). The
mechanism of regulation mediated by any phytohormone is far from clear.
They are thought to predominantly control gene expression at both
transcriptional and translation levels. Hormones exerting
transcriptional control, enhance preferential synthesis of particular gene
transcripts, ultimately leading to enzyme induction. a-amylase thus
induced was studied in detail (Mridula, unpublished). A progressive
increase in the a-amylase activity was seen throughout the development
of somatic embryos. A 40-fold induction of a -amylase specific activity
was seen in the fully mature embryos.
23
in the range of 66% to 55% at the nucleotide level. At the amino acid level
homology of 43% has been obtained with a barley gene. These parameters
are significant to indicate that PSarnR represents an a-amylase gene
from sandalwood. Work is in progress to sequence the complete
a-amylase gene from sandalwood (paper communicated).
24
for rapid propagation of elite trees. E. citriodora is important for its
essential oil. In order to multiply high essential oil containing species
tissue culture methods were used. Upto 100 shoots can be obtained from
one tube. Shoots can be separated and rooted out successfully. Eucalyptus.
grandis and E. tereticornis are largely used in the paper industry for pulp.
In seedling raised conventional plantations variation from plant to plant
is very high. In order to multiply plants with larger girth and straight
bole, tissue culture methods were developed. Excised shoots could be
successfully rooted and planted out.
25
which have commercial use and the potential for patents. Unless the
need for the development is felt by the industry, research cannot progress
further. Also, unless the cost of plantlet production by tissue culture
techniques is brought down considerably to match with or less than the
conventional methods of propagation, the efforts are not worthy enough
to justify. Data of 40 year old vegetatively propagated white pine
established from rooted cuttings out produced trees of seed origin.
Further the clonal fidelity of forest trees particularly conifers, will become
available as present experimental plantings mature.
The eucalypt tissue cultured tree plantings are doing very well and
performing better than the seed raised plants. Even sandalwood tissue
culture raised trees (Lakshmi Sita, 1993a) are performing better. Clonal
fidelity in trees micropropagated by organogenesis has not been
adequately tested with many species. However, the uniformity observed
so far in the plantings is encouraging. In rosewood also we found that 5
year old plants obtained by organogenesis are doing better than the root
sucker, or the seed raised plants. Commercial application of tissue
culture is still limited to micropropagation of few trees. However, this is
changing in India as Department of Biotechnology has sponsored two pilot
scale facilities for the mass cloning of forest tree species. Many forest trees
are under trial.
26
of somatic embryogenesis now as compared to what it was fifteen years
ago. Forest tree tissue culture has now reached a stage where routine
micropropagation will soon become part of silviculturist tool. It can only
reproduce a specific genotype which cannot result in genetic improvement
per Se. Research on micropropagation will stress on mass propagation of
mature trees and reduction of costs. Research on micropropagation by
somatic embryogenesis has to be intensified to reach the ultimate goal of
mass propagation since it also allows automation which in turn would
reduce the cost per propagule. Tree improvement in the true sense can
only be achieved by somaclonal/gametoclonal variation and other
strategies. Organogenesis based on adventitious shoot bud formation
from callus cultures is now the only available method for obtaining
somaclonal variation in most forest trees. However, the improvement of
techniques in future will allow the exploitation of cell culture techniques
(cell suspensions and protoplasts) for obtaining somaclones. This type of
research will take many years because of long life span of trees.
REFERENCES
Bonga J.M. and Durzan, D., 1987. Cell and Tissue Culture in
Forestry, Vol.1,2,3. Martinus Nijhoff., Dordrecht.
DandekarA.M., Gupta P.K., Durzan D.J. and Knauf V., 1987.
Transformation and foreign gene expression in micropropagated
Douglas fir (Pseudotsuga menziessii). Biotech.5: 587-590.
Fillate J.J., McCown M., Sellmer J., Haissig B. and Comai L., 1987.
Agrobacterium mediated transformation and regeneration of
poplar. Mol. Gen. Genet., 206: 192-196.
27
Haissig B.E., Nelson N.D., and Kidd G.H., 1987. Trends in the use of
tissue culture in forest tree improvement. Biotech. 5: 52-59.
Kedharnath S., 1988. Involving high yielding strains in forest tree
species. In Strategies for Forest Genetics and Tree Improvement
:
Research in India.
Krishnamurty A.V.R.G., 1988. Genetics and tree improvement research
in India: Present status and future research strategy. In:
Strategies for Forest Genetics and Tree Improvement Research in
India.
Lakshmi Sita G., 1979. Morphogenesis and plant regeneration from
cotyledonary cultures of Eucalyptus. Plant Sci. Lett., 14: 61-68.
Lakshmi Sita G., 1981. Tissue culture of Eucalyptus species. In:
R.C. Umaly et al. (editors), Tissue Culture of Economically
Important Plants. Proc. International Symposium held in
Singapore, April 28-30, COSTED and ANBS, pp. 180-184
Lakshmi Sita G., 1986a. Sandalwood (Santalum album). In: Y.P.S.
Bajaj (editor), Biotechnology in Agriculture and Forestry. Vol.2,
Springer Verlag, Berlin, pp. 363-374.
Lakshmi Sita G., 1986b. Progress towards clonal propagation of
Eucalyptus grandis. In : Withers, L.A. and Alderson, P.B.,
(editors), Plant Tissue Culture and its Agricultural Applications.
London, pp. 159-167.
Lakshmi Sita G., 1987. Triploids. In: J .M. Bonga and D. Durzan
(editors), Cell and Tissue Culture in Forestry, Vol.2. Martinus
Nijboff, Dordrecht, pp. 285-304.
Lakshmi Sita G., 1989. Differentiation of embryos and leafy shoots from
callus cultures of cashew (Anacardium occidentale L.). In : Proc.
Plant Tissue Culture, Conference 1989, Shillong (In Press).
Lakshmi Sita G., 1993a. Tissue cultured plants of sandalwood
(Santalum album). Curr. Sci., (In press).
Lakshmi Sita G., 1993b. Eucalyptus. In: M.R. Ahuja (editor)
Micropropagation of Woody Trees. Martinus Nijhoff Publication
Dordrecht.
Lakshmi Sita G. and Chattopadhyay S., 1988. Improvement of forest
regeneration from shoot callus of rosewood (Dalbergia latifolia
Roxb.). Plant Cell Reports, 5: 266-268.
28
Lakshmi Sita G. and Raghava Swainy B.V., 1992. Application of cell
and tissue culture technology for mass propagation of elite trees
with special reference to rosewood (Dalbergia latifolia). Indian
For., 118:36-47.
Lakshmi Sita G. and Ravindran S., 1989. Micropropagation of
difficult-to-root elite cultivars and induction of embryogenesis in
mulberry (Morus spp.). Proc. Plant Tissue Culture, Conference
1989, Shillong, (In Press).
Lakshmi Sita G. and Ravindran S., 1991. Gynogemc plants from ovary
cultures of mulberry. In: J. Prakash and R.LM. Pierik (editors),
Horticulture : New Technologies and Application. Kiuwer
Academic publishers, pp. 225-229.
Lakshini Sita G. and Sreenatha K.S., 1992. Plantlet production from
shoot tip cultures of red sandalwood. Curr. Sci. (In Press).
Lakshmi Sita G. and Vaidyanathan C.S., 1979. Multiplication of
Eucalyptus by multiple shoot production. Curr. Sci., 48: 350.
Lakshmi Sita G., Chattopadhyay S. and Tejavati H.J., 1986. Plant trees
by tissue culture. In: G.M. Reddy (editor), Plant Cell and Tissue
Culture of Economically Important Plants. Proceedings of the
National Symposium, pp. 195-198.
Lakshmi Sita G., Mridula S. and Gopinathan K.P., 1991. Growth
hormone regulated gene expression during somatic
embryogenesis in sandalwood (Santalum album). Proc.
Workshop on: Gene Structure and Expression. uS, Bangalore,
Dec. 11-13, 1991.
Lakshmi Sita G., Raghava Rain N.y., and Vaidyanathan C.S., 1979
Differentiation of embryoids and plantlets from shoot cultures of
sandalwood. Plant Sci. Lett., 15: 265-271.
Lakshmi Sita G., Raghava Rain N.y., and Vaidyanathan C.S., 1980,
Triploids from endosperm of sandalwood by
experimental embryogenesis. Plant Sci. Lett., 20:63-69.
Lakshmi Sita G., Shobha J. and Vaidyanathan C.S., 1980.
Regeneration of whole plants from suspension cultures of
sandalwood. Curr. Sci., 49: 196-198.
Lakshmi Sita G., Vaidyanathan C.S. and Ramakrishnan T., 1982.
Applied aspects of tissue culture with special reference to tree
improvement. Curr. Sci., 51:88-92.
29
Mohan Kumar P., Rajasekaran P. and Haridas P., 1993. Mass
Propagation of Eucalyptus spp. (E.grandis andE. globulus). In V.
Dhawan, PM Ganapathy and DK Khurana (editors). Tissue
Culture of Forest Tree Species : Recent Researches in India.
IDRC-TIFNET. New Delhi: pp
Raghava Swamy B.V., Himabindu K. and Lakshmi Sita G.,1992. In vitro
micropropagation of elite rosewood (Dalbergia latifolia). Plant
Cell Reports, (In Press).
Sederoff R., Stomp A.M., Chilton W.S. and Morre L.W., 1986. Gene
transfer into loblolly pine by Agrobacterium tumefaciens.
Biotech, 4:647-649.
Winton L., 1978. Morphogenesis in clonal propagation of woody Plants.
In : T. A. Thorpe (editor) Frontiers of Plant Tissue Culture.
University of Calgary Press, Canada, pp 419-426
30
Mass Propagation of Eucalyptus Spp. (E. grandis
and E. globulus) Through Tissue Culture
INTRODUCTION
31
A fundamental technique for improving the yields of fuelwood from
man-made plantations is to improve the genetic stock. However,
conventional breeding methods when applied to tree species take several
years for the development of improved lines. The selection of elites from
existing stands followed by their in vitro culture appears to be an
eminently practical solution to the problem. Micropropagation is now
recognised as a viable alternative to conventional vegetative and seed
propagation methods. The advantages of this method are fast
multiplication under aseptic conditions in short time and space. Thus,
starting from limited planting stock, large number of plants identical to
the mother plant can be produced which will give enhanced biomass
production capacity.
32
at breast height and height ranging from 170 cm to 300 cm and 30 to 40
m, respectively. Axillary buds from 6-8 month old juvenile branches
(coppiced shoots) of old trees felled for fuel were found to be the ideal
explant.
The protocols for large scale production ofE. grandis andE. globulus
plants through tissue culture have been standardised (Rajasekaran et al.,
1991). Each explant undergoes 4 main phases - initiation, multiplicaton,
rooting and establishment. The first phase of initiation takes 5-6 months
for establishment of cultures and then multiplication is carried out in a
medium containing higher cytokinin content. When subcultured to low
cytokinin medium, the multiplication rate increases and in 25-30 days
each culture can be multiplied 3 to 4 times to produce over 200 tiny shoots.
During the process of multiplication and subculture, the sizeable shoots
are transferred to a rooting medium. Rooting takes place within 10 to 15
days and plantlets can be transplanted to nursery in 15 to 20 days. A
common medium has been formulated for both the species for
multiplication and rooting.
33
(1) The standardisation of a common medium for two species at
the multiplication and rooting stages saves considerable cost
and time.
(3) Direct transfer of these plants to soil in poly pots also reduces
time and cost of production.
(4) Almost 95% of the shoots mE. grandis and 85% mE. globulus
develop roots.
(5) Over 95% E. grandis and 80% E. glob ulus plantlets survive in
the nursery.
34
The data pertaining to some of the areas shosw that tissue cultured
plants are uniform and show very little variation. The table 2 gives the
summary of these data:
35
Table 3: Statistical analysis of girth measurements of some tissue
cultured and seedling plants
1988 5636 37.0 20.7 4.3 20.5 32.0 17.3 4.3 24.6
1988 4540 31.0 37.3 8.1 21.7 41.0 23.6 13.8 58.2
1989 5048 25.0 17.7 3.0 17.0 25.0 13.2 2.4 27.8
1989 4944 25.0 13.2 2.4 17.8 25;0 13.1 3.9 29.4
1989 4291 30.0 23.4 5.3 12.5 30.0 25.6 3.1 12.2
CONCLUSION
The rapidly widening demand-supply gap for fuelwood and
industrial wood is very evident from the table 4:
2000 225 47 — — — —
36
This wide gap between demand and recorded production is being
filled by over-exploitation of natural forests and illegal fellings. While the
government is rightly attempting to control deforestation by legislation,
it is worth recommending that simultaneous encouragement for the
creation of high-yielding energy plantations could be a constructive step
towards the solution of this problem. The plant biotechnological
techniques as demonstrated in this paper, have now attained a sufficient
degree of maturity and can make a genuine contribution towards
increasing the productivity of man-made forests in India.
REFERENCES
37
Factors Affecting Somatic Embryogenesis in
Four-year-old Callus of a Fabaceous Tree -
Albizia richardiana King & Pram
Bright green and compact embryogenic calli were raised from hypocotyl
explants of Albizia richardiana on B5 (Gamborg et al., 1968) supplemented
with 10 p.M RAP 0.9% agar and 3% sucrose. The calli maintained
morphogenic potential for nearly four years on MS basal medium. Addition
of 0.lp.M ABA to MS medium, enhanced the frequency of embryogenic
callus cultures by three-folds. Simultaneously, it also inhibited abnormal
proliferations and production of secondary embryos as well as
differentiation of shoot buds. Increasing concentrations of ABA enhanced
the browning of calli and decreased the percentage of caulogenic cultures.
On the other hand, BAP increased the pereentage of embryogenic calli as
well as promoted abnormal formation and proliferation of secondary
embryos. However, in combination at equimolar concentrations of 1 p.M
ABA and BAP, somatic embryogenesis did not occur, indicating their
antagonistic effects.
38
INTRODUCTION
Seeds were procured from a seed store at Dehra Dun and germinated
aseptically on modified Knoop's medium (Tomar and Gupta, 1988b). The
hypocotyl explants excised from 12-day-old seedlings were reared on B5 +
10 pM BAP medium to raise callus cultures as described earlier (Tomar
and Gupta, 1988a). With a view to evaluate the effects of ABA and its
interaction with BAP, on the morphogenic potential of bright green and
compact calli, they were subcultured on MS medium (Murashige and
Skoog, 1962) augmented with 0.01, 0.1, 1 and 10 pM ABA individually
as well as in combination with 1 pM BAP. Similarly, to assess the effects
of osmotic shock, callus masses were first kept at 30°C in sterilized 1 M
sucrose or mannitol solution for 45 and 90 minutes, and then washed with
sterilized distilled water before transferring them to MS basal medium.
The MS medium was gelled with 0.9% agar (Difco-Bacto, U.S.A.) and
supplemented with 2% sucrose (B.D.H., U.K.). The pH was adjusted to
5.8 with iN NaOH and iN HC1 before autoclaving. Filter-sterilized ABA
was added in different concentrations to autoclaved MS medium or the
39
same containing 1 p.M BAP in a laminar flow cabinet. Green globular
callus pieces of 200±50 mg (fresh wt) were inoculated in each tube.
Only such shoots which had leaf primordia and leaves on a stem axis
(1 mm or longer) and embryos with distinct plumule-radicle axis (2-5 mm
in length) were scored. The data were recorded 40 days after subculture.
The significance of differences in morphogenic responses was checked by
employing Chi-square test at 5% level.
RESULTS
40
The effect of ABA (0.01-1 tiM) was also evaluated in combination
with 1 BAP. Individually, the two hormones promoted embryogenesis
(except on 10 jiM ABA) but in combination, they interacted
antagonistically (Table 1). Besides the quantitative effect, both hormones
also influenced the quality of somatic embryos. ABA inhibited the
development and proliferation of secondary embryos, whereas BAP
promoted both of them.
41
the same superscript are not significantly different as determined by
Chi-squate test at 5% level.
42
DISCUSSION
The role of ABA in plant cell physiology are known to be varied and
several modes of action have already been suggested. It can alter the
permeability of membranes to ions (Raschke, 1979), water (Glinka and
Reinhold, 1971) and malate (van Kirk and Raschke, 1977), in various parts
of plants. Michler and Lineberger (1987) thought that red and blue light
spectra produced high levels of ABA in carrot cell suspensions, which in
turn, stimulate development of somatic embryos.
ACKNOWLEDGEMENTS
This work was supported by the research project sanctioned to SCG,
by the United States Department of Agriculture under the Cooperative
Agriculture Research Grant No. FG-In-6019. The senior author (U.K.T.)
is grateful to the Council of Scientific and Industrial Research, New Delhi,
for the award of a Senior Research Fellowship and subsequently a
Research Associateship.
REFERENCES
Ammirato P.V., 1973. Some effects of abscisic acid on the development
of embryos from caraway cells in suspension cultures. Am. J.
Bot., 160:22-23.
44
Animirato P.V., 1974. The effects of abscisic acid on the development of
somatic embryos from cells of caraway (Carum carvi L.). Bot.
Caz., 135:328-337.
AmmiratoP.V., 1977. Hormonal control of somatic embryos from cultured
cells of caraway: Interaction of abscisic acid, zeatin and
gibberellic acid. Plant Physiol., 59: 579-586.
Brown C., Brooks F.J., Pearson D. and Mathias R., 1989. Control of
embryogenesis and organogenesis in immature wheat embryo
callus using increased medium osmolarity and abscisic acid. J.
Plant Physiol., 133:727-733.
Collins J.C. and Kerrigan A.P., 1974. The effect of kinetin and abscisic
acid on water and ion transport in isolated maize roots. New
Phytol., 73: 309-314.
Gamborg O.L., Miller R.A. and Ojima K., 1968. Nutrient requirements
of suspension cultures of soybean root cells. Expt. Cell Res., 50:
151-158.
Gharyal P.K. and Maheshwari S.C., 1981. In vitro differentiation of
somatic embryoids in a leguminous tree -Albizia lebbek L.
Naturwissenschaften, 68: 379-380.
Glinka Z. and Reinhold L., 1971. Abscisic acid raises the permeability
of plant cells to water. Plant Physiol., 48: 103-105.
Henson I.E., 1984. Effect of atmospheric humidity on abscisic acid
accumulation and water status in leaves of rice (Oryza sativa L.).
Ann. Bot., 54:569-582.
Jones H., Leigh R.A., Tomos A.D. and Jones R.G.W., 1987. The effect
of abscisic acid on cell turgor pressure, solute content and growth
of wheat roots. Planta., 170:257-262.
Kochba J., Spiegel-Roy P., Neumann H. and Sadd S., 1978. Stimulation
of embryogenesis in Citrus ovular callus by ABA, ethephon, CCC
and alar and its suppression by GAS. Z. Pflanzenphysiol., 89:
427-432.
Michier C.H. and Lineberger R.D., 1987. Effect of light on somatic
embryo development and abscisic acid level in carrot suspension
cultures. P1. Cell Tissue Organ Cult., 11: 189-207.
45
Murashige T. and Skoog F., 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. P1., 15:
473-497.
Quatrano R.S., 1986. Regulation of gene expression by abscisic acid
during angiosperm embryo development. In: B.J. Miflin,
(editor), Oxford Survey ofPlant Molecular and Cell Biology. Vol.
3. Oxford Univ; Press, London., pp. 467-477.
Raschke K., 1979. Movements of stomata. In: W. Haupt and M.E.
Feinleib (editors), Encyclopedia of Plant Physiology. Vol. 7.
Springer-Verlag., Berlin, pp. 467-477.
Renger Z., 1986. Effect of abscisic acid on plant development from
Hordeum vulgare embryogenic callus. Biochem. Physiol.
Pflanzen., 18:605-610.
Roberts D. R., 1991. Abscisic acid and mannitol promote early
development, maturation and storage protein accumulation in
somatic embryos of interior spruce. Physiol. P1., 83: 247-254.
Skolmen R.G., 1986. Acacia (Acacia koa Gray). In: Y.P.S. Bajaj, (editor),
Biotechnology in Agriculture and Forestry. Vol. 1.
Springer-Verlag., Berlin, pp. 375-384.
Stiliwell W. and Hester P., 1984. Kinetin blocks abscisic acid
phosphatidylethanolamine channels in lipid bilayers. Z.
Pflanzenphysiol., 114: 65-76.
Ta! M. and Imber D., 1971. Abnormal stomatal behaviour and
hormonal imbalance in flacca, a wilty mutant of tomato. II.
Hormonal effects on water status in the plant. P1. Physiol., 47:
849-850.
Tomar U.K. and Guiita S.C., 1988a. Somatic embryogenesis and
organogenesis in a tree legume - Albizia richardiana King. P1.
Cell Rep., 7: 70-73.
Tomar U.K. and Gupta S.C., 1988b. In vitro regeneration of plants in
some leguminous tree (Albizia spp.). P1. Cell Rep., 7: 385-388.
Van Kirk C.A. and Raschke K., 1977. Stomata! aperture and malate
content of epidermis. Effects of chloride and abscisic acid. P1.
Physiol., 59 (Suppi.): 96.
46
Van Steveninck RF.M., 1974. Hormonal regulation of ion transport in
parenchyma tissue. In: U.K. Zimmermann and J. Dainty
(editors), Membrane Transport in Plants. Springer-Verlag.,
Berlin, pp. 450-456.
Van Stevenmck R.F.M. and Van Steveninck M.E., 1983. Abscisic acid
and membrane transport. In: F.T. Addicott, (editor): Abscisic
Acid. Praegar Publishers, New York, pp. 171-233.
47
B. COMMERCIAL ASPECTS OF
MICROPROPAGATION
Commercialization of Plant Tissue
Culture Research in India
India is one of the leading countries in tissue culture research with several
first to its credit. There is a large body of researchers in several institutions
in the country who have worked on a wide variety of plants. Inspite of this
impressive background, we have been unable to put this technology to
commercial use. This paper discusses some of the reasons for the lack of
progress in this regard. Although the issues discussed primarily relate to
tissue culture research in general, these apply even more to research on tree
species.
INTRODUCTION
51
Tissue culture research in India has a long history. There is
widespread interest in the country in research in this area and several
excellent laboratories have been established in the universities and other
national laboratories of the CSIR, ICAR, etc. India is reported to have one
of the largest corpus of plant tissue culture scientists in the world, actively
pursuing research in various areas. A wide variety of plants ranging from
pomegranate to papaya and oil palm to orchids are being worked on. There
have been several innovative works and many original findings which
rank amongst the best in the world. Techniques such as embryo rescue,
in vitro pollination and fertilization, and anther culture are amongst the
important achievements of Indian scientists. While the quality of research
in tissue culture has been of a high order, there has been very little impact
in terms of actual utilization or application (commercialization). Much
concern has been expressed at this wide and practically unbridged gap.
There is so little to show on the ground for the large investment made so
far on tissue culture research.
Research Priorities
In India, th e government has been the prime mover of research and
its direction through control of funding. Much of the research has been
what the government and scientists consider is needed by the country and
the people and not what is priority for the industry. What is generally
52
missed in such research strategies is whether the research is marketable.
The crops that have generaliy been emphasized are amongst the most
difficult ones from the tissue culture point of view. This is the result of an
insular rather than a global approach. Plants such as the ornamentals
with a high sales realization in the market (both Indian and global) have
not been focussed on. These have received the lowest research priority as
a direct function of their relevance to social well-being. Whereas the above
socially-based priorities are not wrong or misplaced, what was overlooked
was that by the present time, a strong industrial base in tissue culture
could have been created. The industry would have also matured to the
level when it could have ventured into more difficult areas. The fact that
with a couple of exceptions, companies in India (for that matter anywhere
in the world) have primarily gone into the ornamental field and certainly
not into cereals, legumes, forest trees, etc., is testimony to this. Profit being
the prime motive of industry, social consideration can only be secondary
after survival in the market-place is ensured. What is actually needed is
an even spread of research effort between what is required by the people
and what makes money for the industry and ensures its viability.
53
former have a clear mandate to develop technologies, a lot of talent is
available in the universities. The volume of output from the universities
is much higher than that from the national laboratories. The cost of
university undertaken research is also often much lower. They also have
a more creative environment. The universities, on the other hand do not
have a clear mandate for undertaking long-term research. Besides, the
research being largely Ph.D. oriented, there is the resultant problem of
discontinuity of emphasis on a particular research topic. Clearly, these
relationships will need to be further clarified if meaningful outputs are to
be obtained.
Incentives
The incentive for scientists is primarily through publishing of
research papers since these are an important measure of an individual's
ability and serve to get him national/international recognition. Papers
also enable him to obtain a better salary through promotion or selection
to a higher post. He therefore has little incentive in branching out to
applied research (especially that funded by industry) where little can be
published. If such work is to be undertaken by universities and other
research institutions, methods have to be devised by which the progress
of an individual can be measured.
54
The solution perhaps lies in keeping the best researchers in their
positions while providing more benefits to them in situ. This can be
through higher pay, additional research support, consultancy fees, etc.
Regulations through which the university takes away one-third of the
consultancy fees earned by individuals as overheads need to be modified
to 10 per cent in order to attract scientists towards industry-related work
and make them more out-going. Free enterprjse among scientists should
wherein teachers/scientists could exploit commercially their individual
capabilities. In other words, a new 'cadre' of 'academic enterpreneurs'
needs to be developed and encouraged. Scientists who develop commercial
techniques and technologies should be monetarily rewarded in proportion
to the pricing of the technology. They should also be eligible to receive a
similar proportion of the royalties.
55
Protocol Development
need to be clearly defined in order that a researcher
can assess whether a research methodology is still a technique, at what
level it becomes a technology and with what additional clarifications does
it become a packaged, industrially-usable technology.
CONCLUSION
In 1989, Asia produced 75 million tissue culture plants. The present
world market is nearly 900 million plants. Laboratories in Europe, United
States of America, and other developed countries are slowly outpricing
themselves out of the market. We still have an excellent chance to make
56
our mark in commercial tissue culture in the world due to the remarkable
pool of intelligent, hard-working people we have coupled with low wages.
We need to do this before the industry in the West goes through another
technological revolution and overcomes the problem of scarcity of labour
and high wages. If we move fast enough, we are sure to catch up with the
biotech revolution.
Recommendations
The principal suggestions made in this paper are summarized below:
57
12. Industry should adopt science department/research
laboratories, fund positions and provide research support.
13. Government should provide soft loans for research and
contribute 50 per cent to the cost of industry-sponsored
projects.
14. Benchmarks need to be clearly defined by industry for the
researcher to assess the status of his protocol.
15. Clear, implementable protocols should be prepared by R&D
units together with cost-calculation.
16. Research projects should have cost-benefit analysis as an
integral part.
58
Tissue Culture Pilot Scale Facilities for the Mass
Cloning of Forest Tree Species
Vibha Dhawan
Tata Energy Research Institute
90,JorBagh
New Delhi - 110 003
Tissue culture of tree species in the recent past had remained more of an
academic exercise. The research was largely restricted to developing
protocols and not many plants were taken to the field. To bridge this gap
between laboratory and the field, Department of Biotechnology,
Government of India has sponsored two pilot scale facilities for the mass
cloning of forest tree species. The project was sanctioned in 1989 and within
two years, tissue culture propagated plantlets were sent to the forest lands
for evaluations. These facilities are more of the nature of a national facility,
as the protocol developed in other researeh organisation will be translated
into production protocols for large scale production. The plantlets are given
to State Forest Departments for field evaluation and are closely monitored
for clonal uniformity and increase in productivity over the conventionally
raised plantation. The field evaluation results are awaited.
INTRODUCTION
59
The main reason which can be attributed to non-comniercialisation
of tissue culture technique for tree species are:
60
coupled with limited land resources, the only alternative is to increase
per unit biomass production.
61
While many protocols were developed by Indian scientists, the
research results were not supplemented with field evaluation studies.
This was mainly because of the lack of facilities with research institutions
/universities.
62
At NC L's pilot plant, Eucalyptus tereticornis, Dendrocalamus
strictus and Tectona grandis are being multiplied.
.3
Multiplication from the juvenile tissue is acceptable for those
species in which the aim is to increase the quality of the planting material
such as Anogeissus and bamboos. Anogeissus is a poor seed setter and
further the seed viability is as low as 1-2%. This is a very promising species
for the greening of Aravalli hills. With increased demands from paper
industry, bamboos are cut indiscriminately. Simultaneously, there is an
awareness about planting bamboos, especially those species which are
required by the paper industry. This, however, is proving to be difficult
because of inadequate planting material. Further for most bamboo species
there is no selection work done either. Therefore, it is immaterial whether
the explants are taken from the seedling or adult. Another advantage of
propagating bamboos through vegetative propagation is that the initial
growth is much faster. The first harvest can be taken in 2-3 years from
vegetatively propagated bamboos in contrast to 5-6 years for seedling
raised bamboos (A.N. Chaturvedi, pers. communication). However, the
vegetative cuttings sometimes retain their physiological age and plants
produced through cuttings flower with the mother clump. Thus, cuttings
for the vegetative propagation/tissue culture propagation should be
always taken from the clumps of the known age.
64
Tree breeding is very different from crop breeding. The flowers are
formed at a certain height and thus are more difficult to manoeuver.
Unlike crop plants, trials with tree hybrids take many years for
evaluation. Mass multiplication of the desirable hybrids again is a
difficult exercise. One viable alternative for immediate biomass increase
is to mass multiply the plus trees existing in nature, test them in different
agroclimatic areas and make further selections. The selected clones can
be further multiplied to bulk up the initial stock for conventional
propagation. Species in which the adult tissue proves recalcitrant, the
cultures could be initiated from a large number of seeds and a few plants
of each genotype tested in the field. The cultures of all the genotypes are
maintained in the tissue culture laboratory and the promising ones later
mass multiplied. Adopting this approach, at TERI we have developed
tissue culture methods for Leucaena hybrids combining faster growth of
L. leucocephala with other frostJpsyllid resistant leucaena species. About
1000 copies of the following hybrids are put in the field for evaluation at
the M.P. and Haryana forest land:
Leucaena retusa x L. shannoni
L. leucocephala x L. diuersifolia
L. divers ifolia x L. leucocephala
L. divers ifolia x (L. pallida x L. leucocephala)
L. leucocephala x L. esculenta
L. pulverulenta x L. leucocephala
L. leucocephala x L. pallida
65
method when the aim is cloning. It is not desirable however, to go beyond
10 subculture passages so as to avoid risk of mass multiplication of any
abnormal shoot which might originate during shoot multiplication.
1 Clonal uniformity
2 Biomass yields from the tissue culture raised plantation vs.
plantation raised from conventional methods (cuttings/seeds).
The initial data is for survival and depending on the species other
parameters such as height, girth at breast height, number of branches
etc. can be monitored. At the monitoring stage the silvicultural practices
followed become more relevant. Even the fully hardened tissue-culture
propagated plants require more care than plant raised from seeds and
somewhat more than plants raised from cuttings.
For the evaluation of plants raised at TCPP, the field design and the
monitoring details required by us are given along with the plants. It is
not possible to monitor the entire production but we are planning to have
trials in ten hectares area for each species during each season for collecting
the details.
66
REFERENCES
67
C. CONVENTIONALTECHNIQUES
OF IMPROVING FOREST YIELDS
A Brief Account of Forest Tree Improvement in
Uttar Pradesh, India
Padmini Shivkumar
Forest Geneticist
Forest Research Lab.
Kanpur - 208 024
INTRODUCTION
71
indicates the total range of genetic variation within a species and thus it
gives a clue as to the amount of improvement which may be expected from
more intensive breeding work.
72
good performance. throughout the seven year period and hence could be
marked as best provenances for the region.
73
Table 1: Plus trees of Dalbergia 8i8800
74
Tree improvement has really just started to make its
contribution to forest production. It is without any question one of the
best tools of silviculture.
75
Comparison of Tissue Culture Plants Against
Seedling in Tectona grandis
The seedlings and tissue culture raised plants of Tectona grandis were
planted simultaneously at similar sites in Chandrapur (Maharashtra) in
1983. The comparative study of their volume (in 9th years), on the basis of
their 't values' indicated that tissue culture raised plants from plus D-ees do
not show any superiority over planted seedlings. Plants raised by this
technique do not show uniformity in their growth too. Other limitations of
Hssue culture technique are also discussed in this study.
INTRODUCTION
Propagation of plants has been fundamental occupation of mankind
since the dawn of civilization. For raising plantations of forest tree
species, there was little emphasis placed on genetics. In case of teak which
is an important timber species, however, importance of seed origin was
realised early in the second quarter of this century and provenance trials
of teak were laid at six different locations in India during 1928 to 1930.
76
The importance of selecting seeds from superior trees was soon realised.
On the other hand, vegetative propagation in some other species has a
long history. Poplars and willows, e.g. are traditionally raised by rooting
of cuttings.
From the selected plus trees of teak, the bud wood material was col-
lected and tissue culture plants were raised at National Chemical
77
Table 1:Height and girth data of plants raised through tissue
culture techniques and through seedlings
(Date of measurement - 22.2.92)
1. 1. 9.35 33 5.30 17
2. 2. 5.65 17 7.15 16
3. 3. 5.55 14 6.15 23
4. 4. CUT CUT 5.40 18
5. 5. 9.50 36 7.20 21
6. 6. 9.95 31 9.20 31
7. 7. 7.60 20 6.85 17
8. 8. 9.55 30 8.90 26
9. 9. 9.50 30 9.10 20
10. 10. 4.90 10 9.15 31
11. 11. 8.95 27 7.50 23
12. 12. 9.10 26 5.30 16
13. 13. 9.90 34 8.90 27
14. 14. 9.40 39 4.40 18
15. 15. 10.40 30 9.50 32
16. 16. 6.55 17 8.00 21
17. 17. 7.40 20 8.25 29
18. 18 10.40 25 4.25 14
19. 19. 6.75 14 10.05 31
20. 20. 8.85 21 9.05 35
21. 21. 6.90 26 4.15 21
22. 22 8.90 34 8.20 25
78
Laboratory (NCL) Pune. About 22 tissue culture plants were planted
during the rains of 1983 in Comptt. No. 480 (Section I) of West Chanda
Forest Project Division of Forest Development Corporation of
Maharashtra. Simultaneously, same number of seedlings raised by seeds,
79
were also planted in the same area. Their growth data were recorded and
analyzed (Table 1 and 2). The GBH, a good indicator of volume was used
for comparison purposes. The results so obtained, were further analyzed
for their 't' value.
Xl-X2
t value for volume =
S.E.21 + S.E. 22
0.65 - 0.47
(0.09)2 + (0.07)2
= 1.58
80
Thus, there was no significant difference at 95% probability in the
mean volumes of teak poles raised through seed and tissue culture at 9
years of age. As is evident from table 2, there was no uniformity in the
growth in the tissue culture raised plants. The seeds collected from
ordinary trees for raising seedling plants also did not exhibit any inferior
growth over tissue culture raised plants.
81
expensive than nursery raised seedlings. In tissue culture technique, the
large initial investment for laboratory, equipments, chemicals, etc. is at
times ignored. The extra recurring cost makes it more expensive because
of extra skill which results in the additional costs etc. According to rough
estimates the cost of producing plants by tissue culture exceeds that of
nursery grown seedlings by about 10 to 30 times, (Chaturvedi, 1987).
CONCLUSION
At the present status of research, tissue culture plantlets,
especially of Tectona grandis cannot be considered reliable planting
material for afforestation programme.
Tissue culture as such is not a bad technology but for forestry species,
it is still in its infancy and more research is needed to develop with respect
to cost-effective and viable protocols, especially their hardening and field
planting. Collection of material from superior trees, progeny testing at
different sites and maintaining the broad based genetic diversity within
the species is very essential. At present, in comparison to tissue culture,
alternative methods of clonal planting such as cuttings, layering, budding,
grafting, etc. are available and need to be refined further for raising large
plantations.
ACKNOWLEDGEMENT
Authors express their sincere gratitude to Shri A.N. Chaturvedi, IFS
(Retd.), Senior Fellow, Tata Energy Research Institute, for his guidance,
suggestions and critically going through this manuscript.
REFERENCES
82
Khoshoo T.N., 1989. Forestry in India. Problems and prospects. In: V.
Dhawan (editor). Application of Biotechnology in Forestry and
Horticulture.. Plenum Press, New York.
Mascarenhas A.F., 1989. Biotechnological applications of plant tissue
culture to forestry in India. In: V. Dhawan (editor). Application
ofBiotechnology in Forestry and Horticulture Plenum Press, New
York.
Wright J.W., 1976. Introduction to Forest Genetics. Academic Press,
New York.
83
Biomass Enhancement of Tree Legumes by
Rhizobium and VescicuJar Arbuscular Mycorrhizae
Field trials were conducted to evaluate the beneficial role of Rhizobiwn and
Vesicular Arbuscular Mycorrhizae (VAM) in biomass production of tree
legumes (Leucaena leucocephala, Prosopisjuliflora and Acacia nilorica).
Both indigenous and exotic strains of Rhizobium and VAM isolates were
used as inoculum. Nitrogenase activity in root nodules of Leucaena
leucocephala andAcacia nilotica showed the seasonal effect but in Prosopis
juliflora the nitrogenase activity increased in 4 month old plants, remained
constant at 8 month but decreased at 12 month. The introduced Rhizobium
isolates survived with high frequencies in field. In nursery more than 85%
nodule of A. nilotica were formed by inoculated isolates while after 12
months of transplantation more than 65% renodulation by introduced strains
was observed.
In all three tree species the total dry matter yield was higher in inoculated
plants than uninoculated plants. In P. juliflora the Rhizobium isolate
TAL600 showed 88% higher biomass yield over the control, while in A.
nilotica, Rhizobium isolate USDA 3325 showed two fold increase in total
dry matter yield. In all three tree species there was no correlation between
root colonization by VAM and biomass enhancement.
84
INTRODUCTION
85
MATERIAL AND METHODS
Source of Rhizobium
Strains ofRhizobium used in this study were USDA 3325, AB 3, AD
4, A 1, A 3, NGR 8, P 5 and Tal 600, Strain USDA 3325 for Acacia was
obtained from USDA culture collection while NGR 8 for Leucaena and Tal
600 for Prosopis were from NIFTAL Hawaii. Strains AB 3 and Al) 4 were
isolated from the root nodules of A. nhlotica growing at Barkha and
Dhanwas in North India. A 1 and A 3 were isolated from root nodules of
L. leucocephala and P 5 was isolated from P. juliflora growing at Gwal
Pahari (TERI's Research Station). VAM isolates were isolated from the soil
of the experimental site. Strain USDA 3325 was tetracycline resistant (25
jig m11) while isolates AB 3 and AD 4 were nalidixic acid (300 jig mi1) and
penicillin (400 jig m11) resistant respectively. The antibiotic resistance
were stable in these isolates over one year during storage. Antibodies were
developed against these strains in white rabbits. Cross reactivity among
these strains were observed by microimmunodiffusion technique (Ball
1990).
Field Plots
One year field trial of A. nilotica, and two years field trials of L..
leucocephala and P. juliflora were conducted at Gwal Pahari (TERI's
Research Station). The experimental design was a complete randomised
block design with 100 plants in each treatment. Plot of each treatment
was 5x5 metre, the distance between each replicate of treatment being 2
metre. The soil of experiment site was sandy loam, the total soil nitrogen
was 0.020-0.024% and available phosphorus was 2-5 ppm with the pH 8.6.
The indigenous Rhizobium population was one Rhizobium per gram dry
soil and this land was not under any agricultural practices for the last 10
years.
86
acetylene was added. Samples (1 ml) of gas were removed after 30 and 60
minutes of incubation by gas tight syringe and ethylene production
determined using a Perkin Elmer Gas Chromatograph equipped with a
flame ionization detector and 2 metre stainles steel column ( 2 mm
diameter) filled with porapark N(80-100 mesh).
Shoot, root and nodule fresh weight and nodules number were
recorded. The whole plants were dried to constant weight at 70°C, milled
to fine powder and samples digested for Kjeldahl determination of total
nitrogen (Milton and Walters, 1948). Mycorrhizal infection was
determined by the procedure of Kormanik et al. (1982).
Nodule Occupancy
Nodules (100) from each treatment were collected randomly at zero
time and 12 months old plants.Nodules were surface sterilized and plated
on selective and non selective plates containing YEMA medium. Nodules
of uninoculated plants were also plated on selective and non selective
plates. The strains were identified utilizing their antibiotic resistant
character and microimmunodiffusion technique.
87
a
C.)
0
a) Q)
CO
(0
Q)
a)
an 0
0 L
z
z
Months Months
0 month
12 months
100
>
C.)
(Ii
75
a) 10
o CO
-c 50 a)
0 an
I
z 05
25 z
0
— ,.)
0 + < + ÷
(.0 (-
z < Z
0 0
0
0
Z
0(I)
:3
Fig. 3: Percent nodule occupancy of introduced Fig. 4 : Effect of VAM on nitrogenase activity
Rhizobium isolates in A.niolica at zero time in root nodules of L. leucocephala in 12 month
(during transplanting to field) and 12 month old plants. Values are mean of 4 replicates.
old plants.
renodulation by the introduced strains of Rhizobium in field trials with
Acacia since this was initiated one year later than both Leucaena and
Prosopis.
89
transplanting (Fig.6). The colonization potential of G. calospora was not
enhanced by inoculum of any Rhizobium isolates but the per cent
colonization by G. caledonius and indigenous mycorrhizae increased due
to inoculation with Rhizobium strain TAL 600. In L. leucocephala the rate
of colonization by G. mosseae was enhanced by Rhizobium isolate A3 but
no effect on colonization with G. caledonius and indigenous mycorrhizae
was observed (Fig.7). mA. nilotica the percent colonization by Gigaspora
sp. and G. fasciculatum were not enhanced by any of Rhizobium isolates
used as inocula. However Rhizobium isolate AB3 stimulates per cent
colonization of indigenous mycorrhizae and recorded 15% higher
colonization (Fig.8).
Biomass
In P. juliflora the maximum dry weight yield at 12 months was
recorded in plants inoculated with TAL 600 (Fig.9) followed by plants
inoculated by indigenous mycorrhizae and G. caledonius respectively.
Indigenous mycorrhizae enhanced the nitrogenase activity of native
rhizobia but not with introduced Rhizobium isolates and thus enhanced
dry matter accumulation in indigenous mycorrhizae inoculated plants. In
L. leucocephala the total dry matter yield was higher with dual inoculation
of Rhizobium isolates A3 and NGR 8 with G. caledonius and indigenous
mycorrhizae respectively (Fig.10). This could be because of enhanced
nitrogenase activity of Rhizobium isolate A3 with G. caledonius. In A.
nilotica the higher dry matter production was recorded in plants
inoculated with Rhizobium strains USDA 3325, AB3 and dual inoculation
of AB3 with G. fasciculatum, respectively (Fig. 11).
90
regulated by temperature. In all the three tree legumes the onset of winter
caused senescence of the nodule and renodulation was observed with the
arrival of spring. Leucaena, a surface rooter renodulated more profusely
than the deep rooters Prosopis and Acacia. However the extent of
renodulation caused by the introduced isolate was very high in all the
three tree legumes indicating their survival and renodulation potential in
these ecosystems.
ACKNOWLEDGEMENTS
Authors are thankful to the Director, Tata Energy Research
Institute for providing infrastructure to carry out this work and Mr. Karan
Singh for typing the manuscript.
REFERENCES
Ball E.M, 1990. Agar double diffusion plates. In: H. Hampton, E. Ball
and S. De Boor, (editors). Serological Methods for Detection and
Identification of Viral and Bacterial Plant Pat hogens. The
American Phytopathological Society, St. Paul, Minnesota, USA,
pp. 111-120.
Basak M.K and Goyal S.K., 1975. Studies on tree legumes: 1. Nodulation
pattern and characterisation of the symbiont. Ann. Arid Zone.,
14:367-370.
Cornet F. and Diem H.G., 1982. Etude comparative de'efficaite et effet
de la double symbiose Rhizobium - Glomus mosseae sur la
croissanced d'Acacia holosericea etAcacia raddiana. Bois. For.
Trop., 198: 3-15.
Dreyfus B. and Dommergues Y., 1981a. Nodulation of Acacia species by
fast and slow growing tropical strains of Rhizobium. Appi.
Environ. Microbiol. 41 : 97-99.
Dreyfus B. and Dommergues Y., 1981 b. Relationship between rhizobia
of Leucaena and Acacia Spp. Leucaena Res. Rep., 2:43-44.
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Habish H.A. and Khairi S.M., 1970. Nodulation of legumes in the Sudan
II. Rhizobium strains and cross inoculation of Acacia spp. Exp.
Agric., 6: 171-176.
Hogberg P. and Kvarnstrom M., 1982. Nitrogen fixation by woody
legume Leucaena leucocephala in Tanzania. Plant Soil, 66:
2 1-28.
Kormanik P.P., Schultz R.C. and Bryan W.C., 1982. The influence of
vesicular arbuscular mycorrhizae on the growth and
development of eight hardwood species. Forest Sd., 20:531-539.
Milton R.F. and Walters W.A., 1948, Methods of Quantitative
Microanalysis. Arnold London.
National Academy of Sciences, 1979. In Tropical Legumes Resources
for the Future.. National Academy of Sciences, Washington, D.C.,
pp.123-163.
National Academy of Sciences, 1977. Leucaena: Promising Forage and
Tree Crop for the Tropics. National Academy of Sciences,
Washington D.C., ppll5.
Roskoski J.P., Petter I. and Pardo E., 1986. Inoculation of legnminous
trees with rhizobia and VA mycorrbizal fungi. For. Ecol. Manage,
6:57-68.
Thaper H.S., Kaniala V and Rawat D.S., 1990. Effect of VAM and
Rhizobium on growth of Acacia nilotica in saline and forest soil.
In: D.J. Bagyaraj and A. Manjunath. (editors) Mycorrhizal
Symbiosis and Plant Growth. Department of Agricultural
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Campus, Bangalore, pp 107-108.
94
ANNEXURE I
List of Participants
DrG.LakshmiSita
Dr SC Gupta
Department of Microbiology and
Department of Botany
Cell Biology
University of Delhi
Indian Institute of science
Delhi 110007
Banglore 560 012
Dr V Jagannathan
DrBanwariLal
19, Kale Park
Tata Energy Research Institute
15-A Someshwadi
158, Jor Bagh
Pune 411008
NewDethi 110003
95
Dr P Mohan Kumar Dr RD Iyer
R&D Department Division of Crop Improvement
Tata Tea Limited Central Plantation Crops Reseaith Institute
Munnar ICAR
Kerala685 612 Kasargod67O 124
Kerala
Dr NS Rangaswamy
Department of Botany Dr IV Ramanuja Rao
University of Delhi Khoday Biotek
Delhi 110 003 7th Mile
Kanakapura Road
Ms Archana Nair
Bangalore 560 062
271-5, Schucht Village
University of Florida Dr Ashis Tarn Roy
Gainesville Unicorn Biotek Ltd.
Florida 32603-2224 2nd Floor
USA Tirumala Complex
SD Road
Dr Kanan Nanda
Secunderabad 500003
Department of Botany
Andhra Pradesh
University of Delhi
Delhi 110007 Mr Rupinder Singh
Pinjore
Mr Biswajit Pal
District Ambala
Tata Energy Research Institute
Haryana
Tissue Culture Pilot Plant
Gual Pahari Campus Mr Sanjay Saxena
District Gurgaon, Haryana Tata Energy Research Institute
90, Jor Bagh
Mr Vijay G. Pande
New Delhi 110 003
IDRC
1l,JorBagh MrHLSharma
New Delhi 110003 Department of Non-conventional
96
Dr Padmini Shivkumar Dr Renu Swarup
Forest Geneticist Department of Biotechnology
Forest Research Laboratory Block II
Kanpur 208 024 CGO Complex
Lodhi Road
Dr AK Singh
New Delhi 110 003
Tata Energy Research Institute
Tissue Culture Pilot Plant Dr UK Tomar
Gual Pahari Campus Tata Energy Research Institute
Haryana Tissue Culture Pilot Plant
Gual Pahari campus
Dr AK Singh
Distt. Gurgaon, Haryana
Tata Energy Research Institute
90, Jor Bagh Dr ilK Srivastava
New Delhi 110003 Department of Biotechnology
Government of India
DrYS Sodhi
Block II
Tata Energy Research Institute
CGO Complex
90, Jor Bagh
Lodhi Road
New Delhi 110003
New Delhi 110003
Mr Sandeep Sood
Deciduous Forest Research Institute
P0 Regional Forest Res. Centre
Mandla Road
Jabalpur 482 001, MP
97
ANNEXURE II
WORKSHOP SCHEDULE
&30-9.30 AM Registration
9.30-10.30 AM Inauguration
Dr RK Pachauri Welcome Address
Dr S. Ramachandran Key Note Address
Mr Vijay G. Pande Address by Regional Director,
IDRC
Dr V. Jagannathan Overview of Tree Tissue Culture
Dr Vibha Dhawan Vote of Thanks
Tea Break
Lunch Break
99
SESSION I: CONTD.
Tea Break
Discussion
Panelists:
Dr V Jagannathan
Dr Kanan Nanda
Dr HK Srivastava
Dr PK Ghosh
Prof NS Rangaswamy
Dr Deepak Pental
Prof SC Gupta
100
March 17, 1992
Venue: Tissue Culture Pilot Plant, Gual Pahari (Distt. Gurgaon)
Lunch
Discussion
Panelists:
Dr SS Bhojwani
Dr IV Ramanuja Rao
Dr Renu Swarup
101
SESSION Ith CONVENTIONAL TECHNIQUES OF IMPROVING
FOREST YIELDS
Chairperson: Mr ON Kaul
Rapporteur: Sandeep Sood
Discussion
Panelists:
Mr ON Kaul
Dr HL Sharma
Dr KK Joshi
Tea
102
IDRC-TIFNET
1. Paulownia (China)
2. Fuelwood (China)
3. Tissue Culture (India)
4. Multipurpose Trees (India)
5. Silvipasture (India)
6. Agroforestry (India)
7. Poplar Improvement (India)
8. Farm Forestry (Nepal)
9. Paulownia (Pakistan)
10. Farm Forestry (China)
11. Fruit Trees (India)
12. Coastal Agroforestry (India)
13. Himalaya Eco-rehabilitation
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