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PROCEEDINGS OF THE NATIONAL WORKSHOP ON MASS PROPAGATION OF TREE
SPECIES THROUGH IN VITRO METHODS'HELD AT NEW DELHI ON MARCH 16-17. 1992
TISSUE CULTURE OF FOREST TREE SPECIES : RECENT RESEARCHES IN
ARCHIV 97797
IDRC - TIFNET
IDRCL*
7
PROCEEDINGS OF THE NATIONAL WORKSHOP ON "MASS PROPAGATION OF TREE
SPECIES THROUGH IN VITRO METHODSHELD AT NEW DELHI ON MARCH 16-17w 1992
TISSUE CULTURE OF FOREST TREE SPECIES : RECENT RESEARCHES IN INDIA
EDITORS VIBHA DHAWAN PM GANAPATHY DK KHURANA
• PUBLISHED BY
IDRC - TIFNET
© IDRC -
IDRC CRDI CuD
CANADA
Through support for research, Canada's International Development Research Centre (IDRC) assists scientists in developing countries to identify long-term, workable solutions to pressing development prob- lems. Support is given directly to scientists working in universities, private government and non-profit organizations. Priority is givenio iesearch aimed at achieving equitable and sustain- able development Projects are designed to maximize the use of local materials and to strengthen human and institutional capacity. Led by the dedication and innovative approach of Third World scientists --often in collaboration with Canadian partners — IDRC-supported research is using science and technology to respond to a wide range of complex issuesin the developing world. IDRC is directed by an international Board of Governors and is funded by the Government of Canada.
South Asia Regional Office 11 Jor Bagh, New Delhi 110 003
CONTENTS
Pages
Foreword V
Acknowledgements vii
Summary of Discussion and Recommendations ix
A. TISSUE CULTURE TECHNIQUES 1. In vitro propagation of grape 3
(Vitis spp.): Establishment, proliferation and development of shoot-tip cultures on defined media
A K Singh, B.B. Sharma and R.M. Pandey
2. In Vitro propagation of Albizia lebbek 9 using axillary and apical buds*
Ashis Taru Roy
3. Forest tree tissue culture : Current 18 status and future prospects
G Lakshmi Sita 4. Mass propagation of Eucalyptus 31
spp. (E. grandis and E. globulus) through tissue culture
P. Mohan Kumar, P. Rajasekaran and P. Haridas 5. Factors affecting somatic embryogenesis 38
in four-year-old callus of a fabaceous tree - Albizia richardiana King & Pram
U.K Tomar and S.C. Gupta
B. COMMERCL&L ASPECTS OF MICROPROPAGATION
6. Commercialization of plant tissue 51 culture research in India
I.V. Ramanuja Rao
7. Tissue Culture Pilot scale facilities 59 for the mass cloning of forest tree species
Vibha Dhawan
C. CONVENTIONAL TECHNIQUES OF IMPROVING FOREST YIELDS
8. A brief account of forest tree 71 improvement in Uttar Pradesh, India
Padmini Shiukumar
9. Comparison of tissue culture plants 76 against seedling in Tectona grandis
R.M. Dayal, V.K Koul and Anmol Kumar
10. Biomass enhancement of tree legumes 84 by Rhizobium and vescicular arbuscular mycorrhizae
Sunil Khanna, Banwari Lal and Alok Adholeya
Annexure I 95
List-of participants
Annexure II 99
Workshop Schedule.
FOREWORD
The contribution of Plant Cell and Tissue Culture (PCTC) towards rapid multiplication of unique cultivars of herbaceous plants is widely acknowledged. Its role in mass propagation of woody perennials, however, has not been adequately studied. Its adoption in tree improvement programmes continue to be in its infancy due to intrinsic biological hazards. With the growing concern for sustainable development of forest resources and increasing emphasis on productivity of land; need for mass propagation of improved trees through genetic engineering is attracting global attention now.
The International Development Research Centre of Canada (IDRC) realised the importance of PCTC in alleviating the socio-economic status of third world countries, and thereby funded such researches on different taxa including the forest trees. These PCTC projects have made notable success in several countries. The Tissue Culture (India) project executed by the Tata Energy Research Institute (TERI) has played the pioneering role in India in the field of tissue culture propagation of forest tree species, earning national recognition to this Institute - as a centre for mass cloning of arboreal taxa.
With the objective of bringing together scientists of diverse background and experience in tissue culture of forestry species the workshop on "Mass Propagation of Tree Species through in vitro Methods" at New Delhi during March 1992 was organised. The papers presented in the workshop ( many of which are reproduced in the Proceedings) reflect the progress and advances achieved in tree tissue culture with a special reference to the development of viable regenerative protocols, scaling up feasibilities, networking of techniques and commercialization of processes. It is hoped that the deliberations of this workshop will provide thrust to micropropagation of forest tree species for attaining ecological security and economic sustainability.
The International Development Research Centre and Tata Energy Research Institute wish to place on record their deep appreciation to all those who contributed to the success of the workshop - organizers, resource persons and participants.
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.
New Delhi - 110 003 Dev. Khurana
September, 1993 TIFNET Coordinator
PM Ganapathy
Regional Forestry Coordinator (IDRC)
Cherla B. Sastry
Principal Program Officer (Forestry)IDRC,
ACKNOWLEDGEMENTS
These proceedings are an outcome of the workshop "Mass Propagation of Tree Species Through In vitro Methods" held at TIC, New Delhi on March 16-17, 1992.
International Development Research Centre (IDRC), Ottawa, Canada and Tata Energy Research Institute (TERI), New Delhi, India were cosponsors of this workshop. Thanks are due to all those who have contributed one way or the other in the organisation of this workshop from technical support to the secreterial assistance.
A large portion of the credit for arranging this workshop goes to Dr CB Sastry, Principal Program Officer (Forestry), and the Regional Director Mr VG Pande, IDRC, New Delhi for their invaluable leadership and guidance for making this workshop a success.
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
A. TISSUE CULTURE TECHNIQUES
Tissue culture is a well established technology for the ornamentals and some horticultural species. While in literature, protocols are listed for many tree species, very few can be applied for large scale propagation. In trees there are distinct juvenile and adult phases. In the juvenility phase, the tissues are more responsive to tissue culture techniques but in the adult phase, the tissues become recalcitrant. Unfortunately, the trees can only be evaluated for the desirable traits in the adult phase and there are no juvenile characters which can be taken as markers for the later growth.
The basic research in the field of tree tissue culture needs to be intensified. Methods ofintroducingjuvenility in the adult tree by spraying growth regulators, inducing coppicing, root suckers etc. should be worked out for individual species. Some species which are conventionally propagated through vegetative techniques of rooting of cuttings can also be multiplied through tissue culture. Whenever a new cultivar is introduced/developed for the initial bulking the regular practice should be to clone it by tissue culture techniques. Apart from the advantage of fast multiplication, the plants remain disease free.
B. COMMERCIAL ASP•ECTS OF MICROPROPAGATION
Tissue culture propagation is exploited on a commercial scale for many ornamental plants and few horticultural plants. It is a routine method of multiplication for orchids and many other ornamentals, especially in the developed countries. In last few years, tissue culture has emerged as a commercially viable venture for the developing countries. These laboratories, however, are thriving on the exports. Tissue culture propagation is labour intensive and wage rates being high in developed countries, it is expected that the demand of tissue culture propagated
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.
As industry is now interested in tissue culture, few problems have arisen in the tissue culture research and one must take a timely action to prevent good scientists being converted as administrators. It is important that immediate steps are taken so as to ensure availability of good scientists and managers in the years to come.
C. CONVENTIONAL TECHNIQUES OF IMPROVING FOREST YIELDS
The productivity of Indian forests is very low and there is considerable scope for the improvement At present there are large targets in terms of area to be planted with meager financial assistance. The result is that more emphasis is placed on the quantity and not on the quality. Even collection of seeds from phenotypically superior trees is not done either due to difficulty in collecting the seeds or since the amount collected is inadequate from all type of trees. For some economically important tree species seed orchards have been raised but the availability of seed from orchards are far too low to meet the demand of the planting material. Seeds are usually collected by the local casual labourers who pay more emphasis on the total quantity collected. They are paid for total weight of seeds collected on the basis of per kg seed.
The role of microorganisms in biomass production is again a neglected field. It is well documented that considerable gains in yields are achieved in crop plants by inoculating the efficient rhizobium strain. For tree species, this exercise has been done for few species such as pines. Conventionally the surface soil of pine plantation is mixed in the nursery soil to ensure good establishment (indirect way of ensuring availability of mycorrhizal strain). Perhaps studies in this direction would improve yields as it is practically impossible to give artificial fertilizer to the forest trees. Research in this direction should be intensffied especially to select rhizobium/mycorrhizal strains which can survive in harsh environment.
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.
The active participation of foresters in tree tissue culture work is essential for its success. Identification of superior material should be done carefully as any error in identifying the material will be reflected as multiple copies of the inferior selection. Tissue culture should be a part of the tree improvement programme.
xi
A. TISSUE CULTURE TECHNIQUES
In Vitro Propagation of Grape (Vitis Spp.): Establishment, Proliferation and Development
of Shoot-tip Cultures on Defined Media
A.K. B.B. Sharma** and R.M. Pandey Tata Energy Research Institute
90, Jor Bagh New Delhi- 110003
**Di•i of Fruits and Horticulture Technology Indian Agricultural Research Institute
NewDelhi, India.
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).
Key words: Vitis, in vitro multiplication, clonal propagation
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.
MATERIAL AND METhODS
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
a) Establishment of Shoot Tips inCulture Survival of shoot tips was better at 10 M BAP in combination with
0.5 p.M NAA (Table 1). Shoot tip survival of Pusa Seedless' was similar to that of Perlette'. However, survival of shoot tip of hybrid, 'W 4-3' was significantly low as compared to the other two genotypes. It is also clear from table 1 that increasing concentration otNAA from 0.5 p. M to 2.5 pM, the survival of shoot tips declined in all the three genotypes.
Growth of the shoot tips improved significantly at the lowest concentration of NAA (0.5pM) as compared to the 2.0 and 2.5 i.tM NAA. Similar trend was recorded in all the three genotypes. BAP 10 p.M was found to be most optimum which recorded maximum explant growth in all the three genotypes.
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.
Table 1 : Effect of NAA and BAP on per cent survival/growth of shoot tips in culture medium.
Growth regulators S urvival/grow th % Mean
Pusa Perlette Hybrid BAP NAA
(jiM) (jiM) seedless W 4-3
5 0.5
2.0
2.5
87/70
65/57
58/30
90/84
76/78
73/70
69/47
38/40
30/28
82.00/67.00
59.67/58.33
53.67/42.67
10 0.5
2.0
2.5
88/85
68/55
50/40
89/87
80/70
73/67
80/65
58/25
47/40
85.67/79.00
68.67/50.00
56.67/49.00
15 0.5
2.0
2.5
85/00
70/00
70/00
81/00
73/00
73/00
75/00
57/00
58/00
80.33/00
66.67/00.
67.00/00
Mean 71.22/50.16 78.60/76 36.88140.88
C.D. at 5% for means NAA and BAP
24.79 14.63
C.D. at 5% for any two genotypes 14.31
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.
Table 2 : Response of shoot tips on shoot production at different levels of BAP
Media
constitution
Genotypes Mean
Pusa Perlette Hybrid
seedless W 4-3
MS+BAP 5p.M 10 8 5 7.67
MS+BAP 10 p.M 18 16 9 14.33
MS+BAP 15 p.M 13 11 8 10.69
MS+BAP 13 12 6 10.33
Mean 13.50 11.75 7.00
C.D. at 5% for concentration means 2.33
C.D. at 5% for any two genotypes 2.02
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.
In vitro raised shoots did not vary significantly in their rooting potential. Excellent rooting was recorded with 10 p. M IBA. However, 0.1 p. M IBA have been reported to induce optimum rooting of shoots of various clones of grapevine (Novak and Juvova, 1983). Under present studies,
7
optimum rooting was recorded in the very first subculture of the nodal explant.
REFERENCES
Barless M. and Skene K.G.M., 1980. Studies of the fragmental apex of grapevine I. The regeneration capacity of leaf primordial fragments in vitro. J. Exp. Bot., 31:483-488.
Chee R., Pool R.M. and Bucher R., 1984. A method for large scale in vitro propagation of Vitis. New York Food and Life Sciences Bulletin, 109: 1984.
Jone R. and Webb K.J., 1978. Callus and axillary bud culture of Vitis vinifera. Sylvaner Riesling, Scientia Hortic., 9:55-60.
Mallins M.C. and Srinivasan C., 1976. Somatic embryos and plantlet from an ancient clone of the grapevire (cv. Cabernet -Sauvignon) by apomixis in vitro. J. Exp. Bot., 27 :1022-1030.
Novak F.J. and Juvova Z., 1983. Clonal propagation of grapevine through in vitro axillary bud culture. Sci. Hortic., 18:231-240.
Pool R.M. and Dowell L.E., 1975. The influence of cytokinin on in vitro shoot development of 'Concord' grape. J. Am. Soc. Hort. Sd., 100:200-202.
Vasil I.K., (editor), 1985. Cell Structure and Somatic Cell Genetics of Plants. Vol.2. Academic Press, New York, pp. 149-212.
8
In Vitro Propagation of Albizia lebbek Using Axifiary and Apical Buds
ASHIS TARU ROY Unicorn Biotek Ltd.
2nd Floor, Tirumála Complex, S.D. Road Secunderabad - 500 003, Andhra Pradesh
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).
Key words : Albizia lebbek., apical bud, tissue culture, axillary bud
INTRODUCTION
The author has conducted a critical study on in vitro propagation of Albizia lebbek L. (East Indian walnut) mainly because the species is fast growing and produces good quality fuel wood besides being an excellent fodder tree. Thus it is a highly desirable tree from sociai forestry point of view. The method of propagation involving a callusing phase has a built-in-uncertainty for clonal uniformity as callus cells are not stable during tissue culture. In order to circumvent any possibility of such variations, apical and axillary bud culture and their subsequent rooting
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.
MATERIAL AND METHODS
Off-shoots of elite Albizia lebbek (10-15 years old) trees growing in the fields of the Indian Institute of Technology, Kharagpur served as the source of explant material. Shoot tips (2.0-2.5 cm in length) or nodal segments (2.5 cm) containing lateral buds were surface disinfected in a solution of sodium hypochiorite (1%) with 0.1% Tween 20 as surfactant for 15 mm. After sterilization tissues were washed with sterile distilled water three times. Apical bud explants were obtained by removing two or three pairs of leaves and excising the terminal 3-5 mm of the shoot. To obtain lateral bud explants the leaf scale covering the bud was first removed. A shallow incision (1-2 mm) was made into the stem and the bud excised with a small portion of the adjacent stem tissue. These explants were inoculated in culture tubes.
The basic MS (Murashige and Skoog, 1962) medIum was used throughout the experiment. The effect of the cytokinins BAP, Kn, 2iP, and auxins NAA and IAA were tested either alone or in combination for bud establishment and multiplication. The pH of the medium was adjusted to 5.7 with 1 N KOH or 1 N HC1 prior to sterilisation. The medium was gelled with 0.8% agar and was dispensed as 25 ml aliquotes in 25 x 150 mm tubes and was autoclaved at 1.46 kg cm2. Media were cooled as slants of 450•
Cultures were maintained under low intensity illumination at 16 hr photoperiod. The cultures were incubated at 26 ± 2°C and at relative humidity of 50-60 per cent.
RESULTS AND DISCUSSION
The effect of different cytokinins and auxins on axillary and apical bud establishment were studied. A number of preliminary experiments
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.
1.0
2.0
0.5
1.0
2.0
KIN 0.5
1.0
2.0
0.5
1.0
2.0
2iP 0.5
1.0
2.0
0.5
1.0
2.0
LAA 0.1
0.1
0.1
NAAO.1
0.1
0.1
117
122
105
107
92
109
92
94
108
Cultures with sprouted buds (%)
70.4
96.1
64.7
64.1
85.0
56.0
66.4
54.1
38.4
59.0
43.9
31.3
46.7
31.5
17.4
36.9
21.3
8.3
TABLE 1: Effect of different cytokinins and auxins on axillary and apical bud for establishment after 4 weeks of culture on MS medium (16 hr photoperiod, 3000 lux, 26±2°C)
Cytokinin Auxin No. of Cultures
BAP 0.5 IAAO.1 108
0.1 104
0.1 119
NAAO.1
0.1 87
0.1 75
IAA 0.1
0.1 111
0.1 86
NAA 0.1
0.1 98
0.1 99
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.
When IAA was replaced with NAA, higher percentage of sprouting (85.0%) was observed. For bud break of the tested cytokinins, BAP is the best and for proper shoot growth, it should be supplemented with either JAA or NAA.
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.
Comparison of the results achieved on IAA and NAA supplemented medium indicated that low levels of IAA stimulated the highest number of multiple shoots, with 0.5 mgF1 giving the best result. IAA at 5.0 mg 11
and NAA at all tested concentrations resulted in a reduction in the number of shoots per culture (Table 3).
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.
Experiment on explant type (apical and axillary buds) on multiple shoot formation was tested (Table 5). Apical bud and axillary buds were cultured on a medium continuing 2.0 mg 1' BAP and 0.5 mg F' IAA to compare differences in shoot proliferation between these two
12
explants. Both apical bud and axillary buds produced equal number of shoots and no difference was observed in shoot proliferation.
Culture of established buds on a medium containing 2.0 BAP and 0.5 mgl1 IAA resulted in a 3.8 fold increase in 5 weeks. Shoots formed in vitro were recultured onto the same medium and shoot multiplication was determined after 2nd and 3rd subculture. About five fold multiplication was observed at both samples (4.9 ± 0.6 shoots per culture at 3rd subculture). However, 5th subculture onwards the cultures appeared to be visibly degenerating, as characterized mainly by increasing number of senescing leaves.
Table 2: Effect of different concentrations of cytokinins on established culture for multiple shoot formation, after 5 weeks of culture on MS medium (16 hr photoperiod, 3000 lux, 26±2 °C)
Cytokinin .
Conc.1 (mg l )
No. of cultures
Cultures with
No. of shoots per
multiple culture shoots(%)
BAP 0.5 17 35.3 1.1
1.0
2.0 5.0
10.0
22
19
25
26
40.9 63.2 72.0 46.2
1.3 ± 0.4 2.4 ± 0.4
2.7±0.6 1.4 ± 0.4
Kn 0.5
1.0
2.0 5.0
10.0
18
27
29
25
31
38.9 51.9 48.3 32.0 25.8
1.1±0.3 1.4 ± 0.4 1.5 ± 0.5
1.2 ± 0.3
1.0±0.1 2iP 0.5 30 23.3 0.8±0.1
1.0
2.0
5.0
29
24
29
31.0
25.0 17.2
1.3 ± 0.2
1.0 ± 0.2
1.0±0.1 10.0 21 14.3 1.0±0.1
13
Table 3: Eeffect of different concentrations of IAA and NAA on established cultures for multiple shoot formation, after 5 weeks of culture on MS medium (16 hr photoperiod, 3000 lux, 26±2°C)
Auxin Conc. (mg 11)
-
No. of cultures
Cultures with multiple shoots (%)
No. of shoots per culture
IAA 0.1 38 47.4 1.4 ± 0.3
0.5 36 63.9 2.1 ± 0.8
1.0 29 55.2 1.4 ± 0.2
• 2.0 22 45.5 1.1 ± 0.2
5.0 17 35.3 1.0 ± 0.2
NAA 0.1 25 40.0 1.1±0.4
0.5 31 51.7 1.7 ± 0.8
1.0 17 41.2 1.2 ± 0.3
2.0 30 30.0 1.0 ± 0.2
5.0 28 25.0 1.0±0.1
Many of the shoots which were regenerated did not undergo rhizogenesis. Only 10-15% of the shoots showed root formation on rooting medium. Leafy shoots of 4-7 ems length were placed in media with different concentrations of IAA or IBA (0.1, 0.5, 1.0, 2.0 6). It was observed that IBA was more effective in root induction as compared to IAA. In case of IAA, 0.5 mg 1.1 was the best concentration. The best response in IBA was obtained in 0.1 mg 1.1. After about 2-3 weeks when the plantlets had well developed roots, they were transferred to pots containing sterilized soil mixture (1 soil :2 peat :7 perlite) in a 10 x 40 cm plastic container, covered with a plastic bag to prevent desiccation and maintained in a growth chamber at 26± 2°C under high light intensity. After 3-4 weeks plantlets were transplanted into soil. The percentage of survival was 50-60 per cent.
14
Table 4: Effect of BAP (2.0,5.0,10.0 mg 1') and LAA (0.1,0.5,1.0 mg 1') on established cultures for multiple shoot formation, after 5 weeks of culture on MS medium (16 hr photoperiod, 3000 lux, 26± 2°C)
BAP (mg 11) IAA (mg 1.1) No. of c itures
Cultures with multiple shoots (%)
No. of shoots per culture
2.0 0.1 41 60.9 2.3 ± 0.9
5.0 0.1 29 65.6 2.1 ± 0.6
10.0 0.1 37 51.3 1.9±0.6 2.0 0.5 38 100.0 3.8 ± 1.0
5.0 0.5 34 85.2 3.0 ± 0.8
10.0 0.5 39 64.1 2.7 ± 0.4
2.0 1.0 25 56.0 1.9 ± 0.4
5.0 1.0 44 45.5 1.6 ± 0.4
10.0 1.0 39 38.5 1.4 ± 0.2
Table 5: Effect of explant type on multiple shoot formation, after 5 weeks of culture on MMS-C medium supplemented with BAY (2.0 mg and IAA (0.5 mg 1") (16 hr photoperiod, 3000 lux, 26±2°C)
Explant type No. of cultures Cultures with multiple shoots (%)
No. of shoots per culture
Apicalbud 44 95.5 3.8± 1.0
Axillary bud 49 100.0 3.7 ± 0.9
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
Growth regulator
Concentrati rooting (mg
oi1 for F )
Percentage ± SD
Control 0.0 18.2 ± 2.9
IBA 0.1
0.5
1.0
2.0
89.7±8.5 72.4 ± 6.2
61.4±5.3
54.5±4.3 IAA 0.1
0.5
1.0
2.0
32.2±3.4 57.6 ± 5.6
42.9 ±4.1
22.1±2.4 Gharyal and Maheshwari (1983) reported successful plant
production from meristem tip by using IAA. In our report it is found that IAA combined with BAP gives a high rate of multiplication.
The culture of apical and axillary buds onto the medium formulated from this research resulted in a 3-4 fold multiplication after first subculture. However, transfer of shoots which proliferated in vitro onto the same medium resulted in a 5-fold increase in the same length of time. No increase in shoot proliferation was achieved by prolonging the culture period beyond second subculture. In actual practice, with ideal laboratory and nursery facilities, the results can be further improved and thousands of uniform saplings can be supplied on demand.
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.
Key words: Eucalypts, genetic manipulation, micropropagation, rosewood, sandalwood, tree improvement.
INTRODUCTION
Forest tree tissue culture has witnessed remarkable advances during the last decade and half and has come of age with a bright future (Bonga and Durzan, 1987). In the early 70's very few scientists had ventured into this area in view of the inherent problems associated with
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).
According to Winton (1978) who reviewed tree tissue culture research, 2.5 billion trees are planted every year in the USA alone and at some point upto 10% might be required from aseptic cultures. That means we would have to come out with several million propagules each year. To achieve such large numbers, the only practical method in the long run is somatic embryogenesis in suspension cultures. The fact that this method has not been successful for trees should not deter us from making a beginning.. While it is still difficult to achieve somatic embryogenesis routinely in all the species, each year the risk seems smaller and each year our knowledge grows both from empirical and basic biochemical and now molecular studies. In this paper the need for trees biotechnology and the goals and achievements set and reached at Indian Institute of Science is discussed.
Problems of Conventional Tree Improvement Forest genetics and tree improvement research in India is hardly
three decades old (Krishnamurty, 1988). Tree improvement by conventional methods of propagation and breeding is slow and time consuming. Due to the increase in population, the demand for quality and quantity of firewood and wood based products is continuously
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.
Extensive work has been done on agricultural crops by conventional methods, but very little on tree improvement. Trees belonging to horticultural and plantation crops have been considerably improved over the centuries, since they-were brought under cultivation. Forests are often left to regenerate naturally or are artificially regenerated by seedling or planting seedlings. In either instance, forests receive minimum cultivation during their lives compared to agronomic crops which have undergone innumerable selective modifications. On the other hand forest trees suffer a 10,000 year deficiency in applying selection pressure targeted to human needs (Haissig et al., 1987). As a result, natural and artificial regeneration of forests has mostly occurred from seed produced from natural stands of wild type. Present day forest trees are quite heterozygous and are imitations of trees, nature designed for their needs. Breeding programmes of trees are hampered due to long life cycles, polyploidy, complex pollination mechanisms, polygenic control of desirable characters, self sterility favouring heterozygous state, lack of selection methods and natural barriers of interspecific crosses etc. (Kedharnath, 1988).
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.
Sandalwood (Santalum album) Sandalwood is commercially important for its essential oil and the
wood is used for carving handicrafts. The world requirement for sandalwood oil is 600 tonnes of which only 100 tonnes is met by natural resources. In addition, the production of sandalwood is going down since 1974. One of the causes, is the spike disease caused by mycoplasma like organisms (MLO). There are isolated patches of disease resistant plants in the forest areas where the surrounding area is infected. Hence, there is an urgent need to improve the natural resources to meet the world demand. In order to develop mass propagation methods for desirable qualities such as disease resistance and good heart wood containing plants, tissue culture methods were employed. It is more convenient to use germinating seedlings as explants for initiation of cultures as juvenile explants prove to be more responsive. However, for commercial application of the developed technology it is desirable to have selected superior phenotypes. In other words, when aim is cloning of superior genotypes one must initiate cultures from the tissue from the proven tree which is usually possible in the adult phase. However, we have successfully induced cultures from adult plant material and differentiation by somatic embryogenesis was obtained from callus cultures. Over the years, we have established techniques for routine propagation of sandalwood. Tissue cultured plants have been established on the ground. We had the opportunity to study the growth and establishment of trees reared by clonal methods (Lakshmi Sita, 1993). The oldest tree (8 years) has now grown to a height of 7-8 mts and 65 cm in girth. Trees have flowered and fruited. These trees were compared with the plus trees from which cultures were initiated. Normally sandalwood plants are cut around 40 years. It is possible by selection of elite materials
21
and multiplication by tissue culture methods to reduce the harvesting period to half or even less.
In addition to developing diploid plants, tissue culture methods for the multiplication of triploid plants using endosperm as the explant source were also developed. It is well known that triploid plants are fast growing as in aspen (Populus tremuloides). In diploids as well as triploids early developmental stages from globular embryos to fully developed plants were studied histologically. Observations from the suspension cultures showed the origin of the embryos from multicellular aggregates rather than from single cells.
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.
Gene Cloning and Expression Gene cloning and transfer have limited success in forest trees and
very little work is done among the tropical trees. For successful genetic manipulation in trees, an understanding of regeneration from cell/protoplast culture and genome organization is a prerequisite. Having successfully established regeneration system in sandalwood via somatic embryogenesis, we have selected this as a model system to understand the molecular basis of differentiation. In order to understand and realize the long term goals of cloning genes for disease resistance/yield or any other desirable characters, technique of gene cloning and gene transfer need to be established with known genes. In vitro somatic embryogenesis has been exploited to study the expression of a-amylase during differentiation from undifferentiated callus. Differentiation of somatic
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.
The induction of the enzyme .is clearly a result of GA action as embryos obtained on NAA and kinetin lack the induction of a-amylase and show activity comparable to the levels detected in the undifferentiated callus. The molecular weight of the a-amylase induced by GA was found to be 45 KD by Western blot analysis, using polyclonal antibodies raised against purified a-amylase from Aspergillus oryzae. It has been established unequivocally that GA induces the a-amylase gene expression. Amongst all the GA regulated genes, a-amylase is the only enzyme where significant progress has been made in understanding the molecular aspects, especially in barley. There are no reports on the a-amylase cloning from somatic embryos of sandalwood or any other in vitro system. Moreover, there is no information on these genes or the control of their expression in forest trees. Hence it was thought that the cloning of a-amylase from sandalwood would facilitate in understanding the gene cloning and organization in trees of a conserved gene family like a-amylase. As a preliminary step, to facilitate the study of a-amylase gene regulation and expression at molecular level, it was necessary to isolate an a-amylase eDNA clone. Since an induction of a-amylase specific transcripts to 25-fold was seen in GA treated tissue, wherein embryo's were induced, mRNA was isolated to prepare a cDNA library with the barley a-amylase clone P155.3 as a probe (Muthukrishanan, personal communication) several positive clones were identified. Of these one clone named PSamR, was characterized further. The physical map was constructed using Sanger's dideoxy method and found to contain 800 bp and thus represents a-amylase gene only partially. The sequence comparison of the partial cDNA clone with known a-amylase cDNA sequences accessed from the data base reveal homology
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).
Thus the technology developed will help in future genetic manipulation in sandalwood.
Rosewood (Dalbergia latifolia) Tree legumes are greatly under exploited. Rosewood is one of the
most valuable timbers belonging to the family and has been in the world market for centuries commanding high prices. These trees are slow growing and native to India but were once widespread in distributions. Their export value is more than the local market and is already beyond the reach of common man because of exorbitant prices. Rapid propagation of superior trees of good form, cylindrical bole, narrow crown and having disease resistance is of utmost importance. Conventional propagation by grafts and rooted cuttings is time consuming and not applicable for raising large number of plants. Seed propagation is not satisfactory as the percentage of germination is very low and seedlings are highly variable. With a view to develop tissue culture techniques for mass propagation of this species, two approaches were considered viz., 1) Induction of organogenesis and somatic embryogenesis (Lakshmi Sita and Raghava Swami, 1992), 2) Induction of multiple shoots (Lakshmi Sita and Ravindaran, 1991) by manipulation of cytokinins from axillary meristems and apical meristems. Plants have established in the ground and some have grown to a height of 30 ft in 5 years. The technology is ready for mass propagation. Detailed experimental techniques are available in the quoted published papers.
Eucalypts (Eucalyptus spp.)
Since the success with sandalwood our attention has turned to various forest trees of commercial value. Micropropagation methods from axillary and terminal meristems were developed in respect of three species ofEucalyptus namely E. citriodora, E. grandis and E. tereticornis
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.
Similarly in other economically important trees like red sandalwood, cashew, mulberry etc., tissue culture approaches have been used.
CONCLUSIONS AND FUTURE PROSPECTS
Work done in our laboratory as well as other national and international laboratories, clearly shows that biotechnology strategies have great promise for the improvement of trees but have not progressed much beyond micropropagation. Biotechnological applications of trees have lagged behind those of several herbaceous crop species, because adult tissues of many tree species from mature individuals are recalcitrant to tissue culture techniques. Trees usually respond much more like the recalcitrant cereals or grasses in culture. The present status of tree tissue culture, however, is adequate to initiate commercialization programmes in respect of a few species. Already commercialization has proved successful in case of Eucalyptus species (Mohan Kumar et. al., 1993). Technology in other trees like rosewood and poplar is ready. In trees, such as, hybrid poplar and loblolly pine and Douglas fir, transformation has already been reported, but only one example of commercially important trait introduced viz, herbicide resistance glyphosate into hybrid poplar. Preliminary studies have, however, shown that transformed plants have only elevated levels of tolerance, but not resistance.
Most of the tree tissue culture research has centered on methods, primarily the development of culture media and techniques to induce juvenility in trees for micropropagation. The thrust is towards methods,
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.
With the exception of sandalwood and Norway spruce, the conversion of embryoids into plantlets is difficult to achieve with high frequency. In addition, somatic embryogenesis in tree tissue culture is highly genotype specific. We have also observed the difference in some varieties of sandalwood, where differentiation has proved to be difficult. Somatic embryogenesis or putative embryogenesis has been observed by us in Eucalyptus species, teak, mulberry and cashew, wherein embryogenesis, observed is not physiologically similar to the zygotic embryos. In cashew and mulberry, precocious germination has been observed, resulting in only well developed root. Shoot formation was rudimentary. Since the potential of somatic embryogenesis is well documented by several authors there is an urgent need for further research to develop embryos which are physiologically similar to zygotic embryos and to develop successful somatic embryogenesis in most of the economically important trees. There is a clear and better understanding
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.
High frequency regeneration of forest tree species from leaf pieces (discs) will foster rapid progress on the application of recombinant DNA technology to achieve the goals of genetic manipulation for tree improvement. Research already done in conifers indicates that it is possible to insert commercially important genes. Increased frequency of regeneration from individual cells and protoplasts will allow progress in direct DNA insertion and organelles insertion and somatic cellular hybridizations to realize the hitherto difficult interspecific and intergeneric crosses.
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.
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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.
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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.
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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
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Mass Propagation of Eucalyptus Spp. (E. grandis and E. globulus) Through Tissue Culture
P. Mohan Kumar, P. Rajasekaran and P. Haridas R&D Department Tata Tea Limited
Munnar Kerala - 685 612
A complete lab-to-land procedure for the micropi-opagation of two species
of Eucalyptus viz: E. grandis and E. globulus has been established at an
industrial level for the first time in India. High rate of multiplication and
rooting, direct transfer of these plantlets to nursery with less than 5%
mortality are some of the salient features of the protocol. Over 1.45 lakh
plants were established in the field. Observations reveal better growth and
uniformity among the tissue cultured plants as compared to the seedlings
of the same age.
Key words: E.grandis, E. globulus, inicropropagation, tissue culture
INTRODUCTION
The Ministry of Environment and Forests, Government of India, had announced a new National Forest Policy in 1988 (Anonymous, 1988). Under the clause covering forestry research, there are certain broad priority areas of R&D which need special attention. The first such priority area identified their in for research is:
"Increasing the productivity of wood and other forest produce per unit area per unit time by the application of modern scientific and technological methods"
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.
Eucalyptus species have been found to be suitable as a fast growing short duration fuel crop. Besides being the cheapest source of fuel for the tea industry, Eucalyptus is one of the trees of choice for the pulp and paper industry. In addition, the leaves of some species are the source of certain essential oils.
Eucalyptus is generally raised through seed and hence the plants exhibit wide variation owing to heterozygosity. Vegetative propagation through cuttings is attempted on small scale largely because of the want of reliable methods for rooting of cuttings from mature trees. In India, to overcome these problems, a programme for the tissue culture of elite lines of Eucalyptus globulus and E. grandis were established.
The in vitro production of plantlets in Indian laboratories has been reported earlier in respect to E. grandis (Lakshmi Sita and Shobha Rani, 1985) andE. globulus (Mascarenhas et. al, 1982). The work reported here was largely on the modification and scaling-up of the technology for commercial production. Maximum attention was paid to achieve high rates of multiplication and survival in the field.
MATERIAL AND METHODS
A massive selection procedure was initiated to identify elite trees from an existing seedling population of over 7 million trees. After stringent selection procedures, 50 lines were selected with average girth
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.
The rooted plantlets are transferred to poly pots in the nursery without special acclimatization in the laboratory. The transplanted plantlets are kept under polythene tents and are ready for planting out in the field in 4-5 months time.
Micropropagated plants ofE. globulus and E. grandis were initially tested in pots and subsequently in the field. The tissue cultured plants have been planted side by side with seedlings of the same age for observation and comparison.
RESULTS AND DISCUSSION
The complete lab-to-land procedure for micropropagation of two species of eucalypts has been achieved at an industrial level for the first time in India. Several features of the technique standardized are noteworthy, particularly from the industrial view point. These are:
33
(1) The standardisation of a common medium for two species at the multiplication and rooting stages saves considerable cost and time.
(2) Good multiplication rates have been obtained in both species - 1:5 in E. globulus and 1:6 in E. grandis, during the first 6 to 7 subcultures after which multiplication starts.
(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.
Some of the significant results obtained are presented in table 1. The tissue cultured plants have established well and growth is very satisfactory. Data pertaining to height and girth measurements were monitored and analysed.
Table 1: Summary of significant results obtained E. globulus E. grandis
Initial Explant 40% 45% Establishment
Multiplibation rate 1:5 1:6
Rooting in vitro 85% 95%
Survival in the nursery 80% 95%
Period for Establishment in the nursery 5 months 4 months
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:
Table 2: Comparative performance of seedlings and tissue cultured plants
Year Elevation Rainfall Seedlin g plants Tissue Plants
Height Girth Height Girth
(ft) (inches) (cm) (cm) (cm) (cm)
1988 5174 94.00 10.00 29.40 10.00 31.30
1988 5636 123.94 5.71 17.31 6.48 20.94
1988 4540 62.69 10.46 38.53 12.12 37.29
1989 5048 239.14 4.59 13.20 6.30 17.68
1989 4944 164.82 3.36 13.08 3.13 13.24
1989 4291 127.97 4.80 23.00 4.95 23.00
1990 5014 95.00 1.48 4.60 1.67 5.96
1990 5636 129.80 1.32 3.44 1.54 4.88
The success in the establishment of in vitro propagated plantlets of different species of Eucalyptus and perusal of tables 2 and 3 indicates the potential of this method for improving the productivity of fuelwood plantations. It is expected that yield can be increased considerably lithe present seedling population is replaced by in vitro raised elite trees.
The primary result of this developmental work is amply demonstrated by its commercial scale application. Already 1.45 lakhs of the tissue culture raised plants have been planted and a further 50,000 are currently under production for planting out by June, 1992.
35
Table 3: Statistical analysis of girth measurements of some tissue cultured and seedling plants
Year Elevation Tiss ue cultured plants Seedlings plants
N Mean SD CV N Mean SD CV
(ft) (Girth in cm) (Girth in cm)
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:
Table 4: Demand and supply gap in fuel and industrial wood
Year Fuel wood Industrial wood for pulp and paper
Demand Supply Gap (In million tonnes)
Demand Supply Gap (In million tonnes)
1980 184 17 167 25 9 16
1985 202 20 182 30 10 20
2000 225 47 — — — —
Source: Anonymous 1976, 1981
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
Anonymous, 1976. National Commission on Agriculture: Abridged Report 1976. Ministry of Agriculture, Government of India, New Delhi.
Anonymous, 1981. Forest Resources of Tropical Asia, 1981. Food and Agriculture Organization (FAO), Rome.
Anonymous, 1988. National Forest Policy. Ministry of Environment and Forests, Government of India, New Delhi, December 7, 1988.
Lakshmi Sita G. and Shobha Rani B., 1985. In vitro propagation of E. grandis by tissue culture. Plant Cell Reports 4: 63-65.
Mascarenhas A.F., Hazara S., Potdar U., Kulkarni D.K. and Gupta P.K., 1982. Rapid clonal multiplication of mature forest trees through tissue cultures. In: A. Fujiwara (editor), Plant Tissue Culture. Japan Asso. Plant Tissue Cult., Tokyo, pp. 719-720.
Rajasekaran P., Mohan Kumar P., Haridas P. and Lai R.D., 1991. Large scale propagation of Eucalyptus for energy plantations. XW Annual Conference of the Plant Tissue Culture Association, New Delhi.
37
Factors Affecting Somatic Embryogenesis in Four-year-old Callus of a Fabaceous Tree -
Albizia richardiana King & Pram
Uttar Tomar* and Shrish C. Tissue Culture Pilot Plant
Tata Energy Research Institute New Delhi - 110 003
** Department of Botany
University of Delhi - 110 007
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.
An osmotic shock given to green calli with 1 M mannitol or sucrose for 45
minutes, enhanced the percentage of embryogenic. cultures. Of the two
sugars, sucrose proved more effective. But a longer period of osmotic shock
(90 minutes) in I M sucrose inhibited both, callus growth as well as somatic
embryogenesis. The plausible enhancement of somatic embryogenesis by
mechanisms involving ABA, BAP and osmotic shocks, have been discussed.
Key words: Albizia richardiana, abscisic acid, osmotic shock, somatic embryogenesis
38
INTRODUCTION
In recent years, considerable attention has been devoted to in vitro propagation of plants via somatic for obvious reasons. Inspite of immense economic importance, the induction of somatic embryogenesis has been reported so far only in three fabaceous trees, namely Acacia koa (Skolmen, 1986), Albizia lebbek (Gharyal and Maheshwari, 1981) and Albizia richardiana (Tomar and Gupta, 1988a). Of the three species, embryos have been observed only up to globular stage in A. koa and the cotyledonary stage in A. lebbek. But complete plantlets have developed only in A. richardiana from dicotyledonous somatic embryos (Tomar and Gupta 1988a). Even in this species, the frequency of well-organized somatic embryos was quite low. Therefore, it was considered worthwhile to enhance the frequency and normalize the growth of somatic embryos for any effective large scale utilization. With this aim, the present investigations were carried out to assess the effect of different combinations of BAP and ABA as well as the osmotic shock treatments given to green callus cultures of A. richardiana.
MATERIAL AND METHODS
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.
Callus cultures were grown under 16 hr photoperiod exposed to 650 p. w cm2 light produced by cool, white fluorescent tubes (Crompton, 40W). Temperature and relative humidity of the culture room were maintained at 25 ± 2°C and 55 ± 10%, respectively. Morphogenic responses were recorded considering the following criteria: (i) per cent cultures producing embryos, (ii) per cent cultures developing shoot buds, (iii) number of embryos per responding culture, (iv) number of shoot buds per responding culture, (v) callus growth reckoned as relative scores on the basis of visual observations, i.e. nil (-), little (÷), moderate (+÷), good (+++), or profuse (++++), and (vi) the degree of browning of calli represented in relative scores as light brown (+), brown (+÷), dark brown (+++), or intense dark brown (++++).
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
Regenerable long-term callus cultures have been established by selective subculturing of A. richardiana embryogenic calli (Tomar and Gupta, 1988a). They were maintained on MS basal medium by regularly subculturing at an interval of 40 days. The long term green embryogenic calli reared on MS medium, were transferred to the same medium but supplemented with ABA either alone or along with 1 pM BAP: If added individually, ABA (0.01, 0.1 and 1 pM) increased the percentage of embryogenic cultures. At its optimal level (1 pM), 29.5 per cent of the cultures differentiated embryos. On the other hand, caulogenesis was adversely affected at 0.1 p.M and higher concentrations. On ABA containing media, calli eventually turned brown and the degree of browning was directly proportional to the concentration of ABA used (Table 1).
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.
Table 1: Effects of ABA and BAP individually and in combination on embryo and shoot bud differentiation in green and globular callus masses of Albizia after 40 days of subculture. Basal medium: MS
ABAJ BAP
M)
Embryo- genic culture (%)
Average number of embryos per embry- ogenic culture
Organog- enic cultures (%)
Average number of shoots per organo- genic culture
Rela- tive deg- roe of row- ning
0/0 98b 2.8a 4.6a 1.Oa +
oil 167ab 37a 6.9a 2.Oa +
0.01/0 242ab 43a 2.2a 1.Oa ++
0.01/1 103ab 3.2a 7.6a 1.7a
0.1/0 216ab 4.Oa 0.Oa 0.0 +++
0.1/1 46b 1.8a 5.la 35a
hO 29.5a 2.Oa 0.Oa 0.0 +++
7.6k 2.8a 0.Oa 0.0 +4-
10/0 63b 2.Oa 0.Oa 0.0 ++++
10/1 167ab 2.8a 0.Oa 0.0 ++++
A minimum of 18 cultures were observed for each treatment. Experiment has been repeated twice. Mean within a column followed by
41
the same superscript are not significantly different as determined by Chi-squate test at 5% level.
The 45 minutes osmotic shock with 1 M mannitol and sucrose enhanced the percentage of cultures producing embryos by two- and three-folds, respectively. However, the average number of somatic embryos did not change significantly (Table 2). But longer pretreatment (90 minutes) in sucrose solution was inhibitory to both, callus growth as well as somatic embryogenesis.
Individual normal somatic embryos on transfer to Knoop's modified medium (Tomar and Gupta, 1988a) developed into complete plantlets within one week. A minimum of 20 cultures were recorded for each treatment and the experiment was repeated twice.
Table 2: Effect of I M mannitol and sucrose, as osmotic shock, on morphogenic response of A. richardiana green caffi subeultured on MS basal medium for 40 days
Duration of osmotic shock
Calli producing embryos (mean±S.E.)
Average no. of embryos per responding culture (mean±S.E.)
Relative callus growth
Control (0 mm.)
15.3 ± 2.0 2.4 ± 0.9 ++++
Mannitol (45 mm.)
32.5 ± 4.7 2.8 ± 0.7 ++÷
Sucrose (45 mm.)
49.5 ± 12.9 2.9 ± 0.5 ++++
Sucrose (90 mm.)
9.1 ± 6.4 2.3 ± 1.7 ++
42
DISCUSSION
Abscisic acid was found to be quite critical for induction of somatic embryogenesis (Quatrano, 1986). It enhances the response in monocots (Renger, 1986; Brown et al., 1989), dicots (Kochba et a!., 1978) as well as gymnosperms (von Arnold and Hakman, 1988). At non-inhibitory levels (0.01-1 it increased the incidence of embryogenesis and also helped maturation of Carum carvi embryos but it inhibited production of secondary embryos as well as precocious germination of embryos (Ammirato, 1973, 1974, 1977). Similarly, promotary effect of ABA on somatic embryo formation has also been observed in callus cultures of tree species, i.e. Citrus sinensis (Kochba et al., 1978), Picea abies (von Arnold and Hakman, 1988) and Picea glauca Engelmannii complex (Roberts, 1991). The present investigations also indicate that 1 j.tM ABA increases the percentage of embryogenic cultures ofA. richardiana and reduces the abnormal growth of embryos.
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.
A significant observation presently made is that both, ABA and BAP promote embryogenic response of calli if added to the MS medium individually but if supplied simultaneously, they act antagonistically. Several membrane-related physiological processes are already known to be antagonistically effected by ABA and kinetin (van Steveninck and van Steveninck, 1983). Kinetin negates the effect of ABA on stomata! closure (Ta! and Imber, 1971), root exudation (Collins and Kerngan, 1974) and
and Cl uptake in storage (van Steveninck, 1974). It is opined that in all these situations, the initial site of action in membranes are the specific proteins. On the other hand, Stiliwell and Hester (1984) have suggested that membrane permeability is increased by undissociated ABA and kinetin individually interacting with phosphatidylethanolamine in bilayers, but kinetin inhibits the ABA-PE permeability.
43
An osmotic shock, given as pretreatment by exposing the cells to 1
M sucrose or mannitol for 45 minutes enhanced the embryogemc response of Daucus carota cell suspension in which sucrose proved more effective (Wetherell, 1984). Similar treatment given to A. richardiana hypocotyl-raised callus cultures, improved the embryogenic response (present investigation). Sucrose pretreatment was more effective than that of mannitol for this taxon as well. However, an extended pretreatment (1 M sucrose for 90 minutes) inhibited the response. Wetherell (1994) considered that the increase in embryogenic response by osmotic shock was caused by physiological isolation ofembryogenic cells from the neighbouring non-embryogenic cells. According to him, plasmolysis resulted in disriiption of plasmodesmata and thus led to the isolation of embryogenic cells. Recently, Roberts (1991) has also reported the enhanced development of globular embryos in embryogenic cultures ofPiceaglauca by low levels of mannitol (2-6%). Higher levels of mannitol (13 and 20%) inhibited the precocious germination as well as promoted the accumulation of storage proteins during cotyledon maturation.
Besides plasmolysis, water stress under the influence of high osmoticum is known to increase the endogenous levels of ABA (Henson, 1984; Jones et al., 1987). Therefore, the other possibility is that the embryogenic response by osmotic shock is caused through an increase in the endogenous ABA level, which in turn, enhances the somatic embryogenesis.
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 of Plant 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.
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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.
Von Arnold S. and Hakman I., 1988. Regulation of somatic embryo development in Picea abies by abscisic acid (ABA). J. P1. Physiol., 132: 164-169.
Wetherell D.F., 1984. Enhanced adventive embryogenesis resulting from plasmolysis of cultured wild carrot cells. Plant Cell Tissue Organ Cult., 3: 221-227.
47
B. COMMERCIAL ASPECTS OF MICROPROPAGATION
Commercialization of Plant Tissue Culture Research in India
LV. Ramanuja Rao Khoday Biotek
7th Mile Kanakapura Road Bangalore - 560 062
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.
Key words: Commercial tissue culture, protocol development, micropropagation.
INTRODUCTION
Commercialization of tissue culture or the mass propagation of plants using in vitro techniques (micropropagation) started in the early sixties and has since become a multi-million dollar industry in several countries. While most of this activity is in the Western countries, several commercial tissue culture laboratories have been established in Asia in the recent past. According to 1989 figures, well over 700 miffion plants are being produced all over the world through plant tissue culture techniques.
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.
Nature of the Problem Why is it that this excellent research, has had so little impact when
it comes to actual application. For long we have tended to side-line the main causes of the problem which essentially is about why we in India have not benefitted even from our own research while those abroad have been able to do so. A common refrain is that research in universities and other research institutions is of the pure kind and that plant tissue culture as practised in a laboratory is not the same as that at the field level. While the above is not entirely incorrect, it is certainly not the reason for lack of commercialization. The problem lies more in difference in perceptions, focus of research and overall attitudes. There is also a divergence in objectives - whereas the commercial enterprises look at what is profitable and sells, research institutions tend to concentrate on achieving a research goal. There are other reasons as well which are discussed later.
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.
There are three major players in the process of generation and utilization of research in India. These are the universities and other research institutions, the industry and other users, and the government. A brief look at the depth and content of the relationships between these groups is both revealing and educative. The relationship between scientists and the government is a reasonably good, comfortable and a mutualistic one with scientists helping define the goals, areas and priorities for funding of the government and the latter funding the work of the scientists (based on the priorities suggested by the latter). Unfortunately the research tends to get short-circuited in this process and only the scientific aspects emphasized as against commercial ones, members of such committees/task forces set up by the government being eminent scientists. Such a system has little provision for input from the public and industry (users) which could facilitate the development of proper focus leading to generating of usable technology.
Universities Vs. National Laboratories Even among the government institutions, a difference needs to be
made between ten national laboratories and the universities. While the
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.
Internal Brain Drain Two kinds of people are needed for effective tissue culture and its
commercialization - researchers and managers. The new companies that are coming up, view the best researchers as the best bet for the success of the company. This has led to an exodus of some of our best scientists from the universities and national laboratories to industry. This has created a research vacuum, which in many instances will be filled up on considerations other than merit. The latter can only result in a deterioration of research capabilities in the field of tissue culture at a time when the demands on the system are on an increase. At the same time, scientists who have moved over and joined companies are unable to find time for research and largely turn into administrators. It is imperative that this new form of brain drain is contained as soon as possible. The measures will need to go much beyond job security which is the only attraction a government position at present holds for the scientist.
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.
Interaction with Industry Research parks (on the lines of science parks) with facilities that
could be utilized by industry on payment should be established. Collaborative tie-ups between industry and R&D institutions should be entered into. Participation by industry in research projects should be made mandatory. This will make the work more goal-oriented and will benefit both parties. Industry in particular can benefit by establishing strong contacts with R&D institutions since the former have extensive infrastructure for research. R&D institutions should be converted into extensions of industries and vice-versa - these should work hand in glove with no artificial distinction between the two. Industry could adopt science departments, fund positions (in situ scientists of industry) and provide research support. As it sees more benefit flowing from such research, funds from industry to research could only increase. Training of students in commercial labs would become possible through such a cooperation. R&D institutions could also greatly help industry in areas such as germplasm exploration, since the latter have little capacity at present in such areas. These could also help in training, producing virus-free plants, contamination-free cultures, etc.
Industry can also be helped by government. Government could provide soft loans for research and also contribute 50 per cent to the cost of industry-sponsored research projects.
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.
Factors relating to continued subculturability and predictability need to be worked out and defined, since the cost of initiating cultures is very high. The number of subcultures possible is very important. At the earliest possible opportunity, one must scale up and graduate to a larger container. Tubes should be used only at the initial stages. However, scientists have little incentive for this. Even if they go up to a certain level, they increasingly come up against hurdles for want of staff, facilities etc. A separate rooting step in vitro adds the biggest cost to a plant. Protocols should be designed such that rooting is either simultaneous or possible in vivo.
Cost rationalization also needs to be carried out. Our attitude right now is not a global, outgoing, competitive one. It is for this reason that we are still unable to produce a competitive plant with locally-generated technology. Even though our personnel costs are very low compared to that abroad (a technician abroad gets on an average over Rs. 25,000 a month which is equivalent to that of the CEOs of Indian tissue culture companies), they are still price competitive since their operations are very efficient. For example, upto 1500 inoculations are done per day per technician. For protocols to be taken up by industry, clear implementable schedules need to be designed along'with a simultaneous cost calculation. The latter should have a complete accOunting of all inputs, etc. and should take into account all hidden costs. Cost-benefit a4alysis, internal rate of return and allied economic analyses should be made an integral and mandatory part of research projects.
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:
1. Research strategies must, wherever, possible have a marketable end product.
2. There should be an even spread of research effort between social and industrial priorities.
3. Research strategy formation and execution should take industry into confidence.
4. Brain drain fromuUniversities/national laboratories to industry needs to be contained.
5. Methods should be devised to evaluate the progress of scientists engaged in applied research, since little can be published.
6. The amount retained by the parent institution from consultancies by scientists should be reduced to 10 per cent.
7. Enterpreneurship among scientists should be encouraged and science parks set up around universities/national laboratories. Academic 'enterpreneurship' while retaining their positions, must be encouraged.
8. Scientists who develop commercial techniques/technologies should be monetarily rewarded and receive royalties. Other incentives for industry-related research should also be devised.
9. Research parks (on the lines of science parks) with facilities that could be utilized by industry should be established.
10. Research linkages need to be developed among the universities, national laboratories and industry.
11. Collaborative tie ups with industry would be made mandatory in research projects.
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.
Key words: Biomass, field evaluation, micropropagation, pilot plant, tree tissue culture.
INTRODUCTION
Tissue culture for in vitro cloning is commercially exploited over the past two decades for ornamental and some horticultural species but the technique is still at a developing stage for the forest tree species.
59
The main reason which can be attributed to non-comniercialisation of tissue culture technique for tree species are:
1. Overall research on forest tree species had lacked behind as compared to agricultural and horticultural plants of high economic value.
2. Tree species have very long life spans with the result breeding is difficult
3. Most of the forests are on government land and thus are under the direct control of government
4. The returns from the forestry plantations are available after many years. Also, areas to be planted every year are massive and so is the demand of propagules. Therefore, the initial investment in terms of cost of propagules is a critical factor for raising forest plants through tissue culture.
Tissue culture techniques are being increasingly popular in industry and in the past five years, over a dozen companies became operative, each with production targets varying from one to ten miffion annually. These, companies have buy back arrangement with the overseas companies. They get the mother cultures and the protocol for multiplication from foreign company and the product (tissue culture produced plantlets) are sold back to them. The patent laws overseas being stringent restrict the sale of plants only to the company from where the stock cultures were received. Thus, essentially in India, we are exploiting the availability of cheap labour for multiplying the plants and both cultivar development and the field transfers are done overseas. The consumption of tissue culture propagated plants in our country is very low and presently only tissue culture propagated plants of cardamom, banana and some ornamentals are sold. The main problem perhaps is that we are still not conscious of the quality and because tissue culture propagated plants are more expensive, they are not acceptable to large masses.
The commercial companies, so far, set up for tissue culture in India are not interested in propagating trees species, inspite of the fact that biomass problem today in the developing countries is very grave. With the mounting population pressure and their ever increasing energy needs
60
coupled with limited land resources, the only alternative is to increase per unit biomass production.
Increasing Biomass Production It is important to improve our planting stock. Conventionally, this
can be achieved by marking the candidate plus trees, cloning them by conventional techniques of rooting of cuttings or subjecting the seed raised progeny to progeny testing to select the superior genotypes. The progeny of these elite trees is then used for raising seed orchards. Tissue culture will be of immense practical value as the species which can not be conventionally propagated through rooting of cuttings can be multiplied in vitro with the added advantage of fast multiplication under disease free conditions. The main probleifis associated with conventional techniques of vegetative propagation are:
1. Cuttings from all tree species do not root 2. Many tree species loose their ability to form root with age 3. The cuttings taken from the terminal shoot grow straight
while the cuttings taken from the side branches either give rise to crooked stems. The plantlets derived from them die at a young age or remember their physiology.
4. The functional cutting is of the size of 20-30 cms and could be rooted only in a particular season
Because of these inherent difficulties associated with cloning by conventional vegetative techniques there are very few examples of tree improvement programmes where cuttings were taken as the starting material.
Tissue Culture Pilot Scale Facilities for Mass Cloning of Tree Species
India has been a pioneering country in the field of tree tissue culture. The first report of hardwood species being propagated from mature tissue was from National Chemical Laboratory, Pune on Tectona grandis (Gupta et. aL, 1980) and the list is expanding fast (Mascarenhas et al., 1989; Dhawan, 1983, 1992).
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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.
The Department of Biotechnology, Government of India, has sponsored two Pilot Scale facilities for the mass cloning of forest tree species in 1989. These facilities are set up by National Chemical Laboratory, Pune and Tata Energy Research Institute, New Delhi. These facilities are fully operational and each has the capacity to produce over a million plants of tree species annually.
The main objectives of the project are:
1. To initiate Pilot Plant units for transfer of tissue culture technology from laboratory level to the field
2. To create institutional facilities for research and development 3. To serve as a training center for production.
The two pilot plants were designed independently. Most of the equipment is indigenous. The first batch of plants from TERI's pilot plant is due in July, 1992. Over 40,000 plants will be produced by October, 1992 and another 1,00,000 by March, 1993. Over 1,50,000 plants were produced and lifted by state forest departments of Bihar, Haryana, Madhya Pradesh, Rajasthan and Uttar Pradesh. From the research facilities at NCL, over 15,000 plants were already produced and field evaluation trials have been initiated. The plants from TERI's TCPP will be planted in the neighbouring states of Haryana, Uttar Pradesh and Rajasthan. The plants would be evaluated in collaboration with the concerned forest departments. At TERI, work is being undertaken on the following species:
Acacia nilotica, exotic acacias(A. bivenosa, A. moconochieana, A. scierosperma and A. victoriae), Anoegeissus pendula, bamboos (Bambusa tulda, B. vulgaris, Dendrocalamus longispathus, and D. strictus), Eucalyptus tereticornis, Leucaena hybrids and Poplars (Populus ciliata and P. deltoides).
62
At NC L's pilot plant, Eucalyptus tereticornis, Dendrocalamus strictus and Tectona grandis are being multiplied.
Problems Associated with Commercial Exploitation of Tree Tissue Culture
Tissue culture of hardwood species is still in the developing stage. The problems associated with tissue culture of mature trees were recently reviewed by Bonga (1987) and Ahuja (1993). The techniques are well developed and commercially exploited for most ornamentals and many fruit crops (Boxus and Druart, 1985), but it is still to be optimised for forest tree species. Even in the developing countries where tissue culture is routinely used for the multiplication of ornamental and horticultural species, the tissue culture of trees is largely restricted to the laboratory. Nowhere in the world large plantations of hardwoods has been raised by tissue culture. This is largely because the adult tissue of most economically important hardwood species are still proving to be recalcitrant to tissue culture techniques.
A survey of the existing literature (Dhawan, 1992) shows that protocols are largely developed from the seedling explants. Even for the species where protocols are developed starting from the adult tissues, many refinements are required before it can be applied for mass multiplication (Populus deltoides clone L-34, D-121, Prosopis juliflora and Acacia nilotica; unpublished work of TERI's laboratory). However, recently Populus deltoides was successfully multiplied by in vitro techniques by DrHC Chaturvedi of NBRI, Lucknow. When the aim is to produce few plants, it is not difficult to start with large number of explants. Such protocols, however, can not be applied for large-scale propagation. Limited multiplication could be achieved by rooting the in vitro formed shoots and putting the mother explant again on the fresh medium for bud break. The role of mother explant in bud break and shoot growth is still to be It could take two paths: either the mother explant is contributing a growth factor which stimulates the shoot growth or adult is sieving out certain nutrients of the medium which are toxic to the plant system under study. Interestingly, the seedling explants on a similar media multiply with no problem.
.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.
Another major problem especially in the developing countries, is that most forest land is under the Government Forest Departments. These departments have meager funds with large targets to meet; hence they are more concerned about the quantity of the planting material and not the quality.
For rooting, after repeated subculturing, two diverse responses are observed. In some species, in which the cultures are initiated from the adult tissues, repeated subculturing introduces juvenility. In 20-year-old material of Eucalyptus citriodora, the shoots could be rooted only after the third cycle of shoot multiplication. The rooting percentage increased from 35-40% in the fourth subculture to 45-50% in the 5th and subsequent passages (Gupta et. al., 1981). In some cultivars of apple also, rooting was improved by repeated subculturing (Zimmerman and Broome, 1981). On the other hand in some species, rooting frequency declined with subculturing, perhaps due to the increased level of endogenous cytokinins.
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
Fortunately many tree species can be coppiced and the coppiced shoots behave more like the juvenile tissue. For Eucalyptus tereticornis, we have seen that explants from the terminal branches show no bud break while those from the coppiced shoots showed good bud break (Sood, unpublished work). It is observed, especially in those species wherein multiplication involves a callusing phase, that with repeated subculturing loss of morphogenetic potential occurs. After a few subcultures shoot differentiation declines. Fortunately, this is less pronounced in the method involving axillary branching. As discussed earlier, for hardwood species axillary branching is the most desirable
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.
Field Evaluation of Tissue Culture Propagated Plants
As discussed earlier, tissue-culture techniques are good for two diverse applications:
(1) for increasing the quantity of the planting material and (2) to improve the quality of the planting material.
When the aim is to produce a large number of plants of any one species, the only factor for evaluation is to check whether these plants survive and grow similar to the seeding-raised plants under different agroclimatic regions. However, when the aim is to produce quality plants, the field evaluation becomes a crucial factor. The plants need to be evaluated for:
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
Bonga J.M., 1987. Clonal propagation of mature trees: Problems and possible solutions. In: J.M. Bonga and D.J. Durzan (editors), Cell and Tissue Culture in Forestry. Vol. 1: General Principles and Biotechnology. Martinus Nijhoff, Dordrecht, pp. 249-271.
Boxus P. and Druart P., 1985. Mass propagation of fruit trees. In: A. Schaefer-Menhur, (editor). In vitro Techniques:Propagation and Long Term Storage. Martinus NijhofffDr. W. Junk, Dordrecht, pp. 29-34.
Dhawan V., 1992. Tissue culture of hardwood species. In: J. Prakash and R.L.M. Pierik, Plant Biotechnology: Commercial Prospects and Problems. Oxford and IBH Publishers, Delhi. (In Press)
Gupta P.K., Nadgir A.L., Mascarenhas A.F. and Jagannathan V., 1980. Tissue culture of forest trees: Clonal multiplication of Tectona grandis L. (teak) by tissue culture. Plant Sci. Lett., 17:259-268.
Gupta P.K., Mascarenhas A.F. and Jagannathan V., 1981. Tissue Culture of forest trees-Clonal propagation of mature trees of Eucalyptus citriodora Hook, by tissue culture. Plant Sci. Lett., 20:195-201.
Zimmerman R.H. and Broome O.C., 1981. Phioroglucinol and in vitro rooting of apple cultivar cuttings. J. Am. Soc. Hortic. Sci., 106: 648-652.
67
C. CONVENTIONAL TECHNIQUES 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
The importance of tree improvement in silvicultural practices is well
recognized. The improvement programmes undertaken in Forest Research
Laboratory, Kanpur in respect of three widely cultivated tree species inUttar Pradesh viz., Acacia nilotica, Dalbergia sissoo and Prosopis are
summarised in this paper which include plus tree selection and progeny testing.
Key words Acacia nilotica, Dalbergia sissoo, plus trees, progeny testing, Prosopisjuliflora, provenance trial.
INTRODUCTION
Tree improvement may be applied under different circumstances and at varying degrees of intensity, ranging from the conservation of gene resources, species and provenance trials to seed orchard establishment, controlled crossings and progeny trials.
Provenance trials are considered to be an essential part of the genetic improvement work. A range wide provenance test usually
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.
For successful promotion of large scale afforestation projects, there is need for carefully planned and well directed research. Species and provenance trials provide some of the basic information on which policies concerning afforestation are made and there are obvious advantages if such trials can be initiated well in advance. This is true for tissue culture work as well. Tree improvement, in the sense of species - provenance selection and breeding can contribute directly to increased net economic yield through increasing, average growth rates.
Tree Improvement Programme at Forest Research Laboratory The main objectives of tree improvement are to select superior trees
(plus trees) which would have qualities of good form, rapid growth rate, straight stems, desired crown to trunk ratio, good seed producing and germinating ability and resistance to certain pests and diseases.
Three species of economic and social importance have been taken under this programme, viz. Acacia nilotica, Dalbergia sissoo and Prosopis juliflora.
Species-wise work undertaken are as under:-
(i) Acacia nilotica (Kikar) In recent years, much attention has been given toAcacia nilotica as
one of the important species for afforestation of arid and semi-arid areas in India. It grows quickly and is a nitrogen fixing species, thus improves the soil fertility.
In a nine provenance, FAO, IBPGR provenance trial at Kanpur, significant differences were observed among the provenances. Data recorded for a period of seven years showed that the provenance. (P6) - Banaskantha, Gujarat, (P') - Aloka - Maharashtra and (P8) - Adilabad, Andhra Pradesh proved best. These three provenances showed consistent
72
good performance. throughout the seven year period and hence could be marked as best provenances for the region.
Plus trees of A. nilotica have been selected from more than 20 provenances and progeny trials laid out in different locations.
(ii) Dalbergia sissoo (Sbishaxn)
Plus trees of shisham have been selected from various regions in the U.P. where both natural and plantation forests exist. They were selected with respect to the straightness of the stem, height and diameter growth. Data on the selected plus trees are given in table-i.
Progeny testing of the selected trees is essential to assess its genotypic worth and breeding value. By collecting open pollinated seeds from individual trees, raising seedling from them, trials can be established to compare the performance of the progeny from the different trees in respect of the characters we are interested in, such as height, growth, diameter, vigor, wood properties etc.
In a one parent progeny test conducted at Kanpur of shisham plants, the seeds of which were brought from straight stemmed plus trees of Mauranipur Range, Jhansi (U.P.). There was high co-relation between the parent - progeny for stem form. This was observed even in one year old trees. The unselected control plants used for this trial, showed both forking and crooked stem form. Genetics plays a major role in the development of stem form. Stem straightness is related to wood quality. Genetical data on this, is therefore of direct use to the silviculturist.
(ffl) Prosopiajuliflora (Jand) Plus trees ofProsopisjuliflora have been selected from Allen Forest.
Bovine block and Fisher forest, Etawah; Mathura; Kukrail, Kotwa etc. Progeny/provenance trials have been undertaken in degraded soil, so that selection is undertaken in such areas, since finally selected trees have to be planted in usar/degraded soils.
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Table 1: Plus trees of Dalbergia 8i8800
1.
Sl.No. Place Height Clear bole- Girth
(m) height (m) (m)
39.0 24.0 2.10 Mauranipur Range,Jhansi (UP)
2. " 37.0 19.0 1.80
3 " 360 250 180 4. " 33.0 18.0 1.60
5. Bindki Range, Fatehpur (UP) 33.0 16.0 1.30
6. " 38.0 10.0 1.40
7. 35.0 12.0 1.40
8. " 35.0 11.5 1.10
9. Fisher Forest Etawah (UP) 19.0 6.65 1.40
10. " 18.6 8.0 1.90
11. " 13.4 3.3 1.22
12 ." 13.0 1.75 1.27
13. Allen Forest Kanpur (UP) 34.5 17.5 1.69
14. " 25.5 13.5 1.10
15. " 25.0 8.5 1.42
16. 22.5 10.5 1.49
17. Brindavan Block Mathura (UP) 9.0 8.0 1.42
18 " 189 75 146
19. • 17.9 6.0 1.29
20. " 19.5 9.0 1.55
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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
R.M. DAYAL*, V.K. KOUL AND ANMOL Tata Energy Research Institute
101, Jor Bagh, New Delhi - 110 003
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.
Key words : Cloning, Tectona grandis, tissue culture, vegetative propagation, plus trees.
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.
* Of Indian Forest Service, presently on deputation to TERI. ** Of Indian Forest Service, presently Siviculturist & Director, State Forest Research
Institute, Chandrapur, Máharashtra.
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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.
Micropropagation has been extensively used for mass propagation of orchids and many ornamentals. The idea was that through tissue culture technique selection of desirable types is possible in moderate sized laboratory, equipped with greenhouse with low financial outlay (Deepesh et.al., 1985). In case of forest trees successful micropropagation techniques are available for Eucalyptus camaldulensis, E. citriodora, Leucaena leucocephala, Morus alba, Phoenix dactylifera, Prosopis cineraria, Santalum album and Tectona grandis (Khoshoo, 1989). However, nowhere large scale tissue culture raised plants of forest species have gone into field plantings. There are not many reports of comparisons of tissue culture plants against seedlings. Mascarenhas (1989) reported that the biomass increase in tissue culture raised plants of E. torelljana was 700% and 100% above the control at the end of 12 and 34 months, respectively. The most important factor which is not considered is that trees cannot be judged in the same way as annual plants during the first season. In case of trees, juvenile phase of growth does not show any correlation with the adult phase. A too early biomass assessment is probably the most serious error in such reports. It has been observed in Populus deltoides that initially some clones grow very fast but later on they do not perform well. Tectona grandis (teak) has a rotation period of 60 to 80 years in different areas. A clear comparison between tissue culture raised plants and seedlings will be possible only after 30 to 40 years. In the present exercise, a study has been made to assess the performance of tissue culture techniques by studying 't' value of their volume between tissue culture raised plants and seedlings of this species.
MATERIAL AND METHODS
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)
Si.
No.
Tree
No.
Tissue Cu! ture Plants Seedlings
Height Girth Height Girth
(m) (in) (m) (m)
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
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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,
Table 2: Volume calculations of the raised plants
Si. No. Tissue Culture Plants Seedlings
(m3) (m3)
1. 1.02 0.15
2. 0.16 0.18
3. 0.11 0.32
4. 1.23 0.17
5. 0.96 0.32
6. 0.30 0.88
7. 0.86 0.20
8. 0.85 0.60
9. 0.05 0.36
10. 0.65 0.88
11. 0.61 0.40
12. 1.14 0.14
13. 1.43 0.65
14. 0.94 0.14
15. 0.19 0.97
16. 0.30 0.35
17. 0.65 0.69
18. 0.13 0.08
19. 0.39 0.97
20. 0,47 1.11
21. 1.14 0.18
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.
RESULTS AND DISCUSSION
The analysis of mean volume of tissue culture raised plants and seedlings and their subsequent determination of 't value' indicated that tissue culture raised plants were statistically non significant at 5% level. This was arrived at by the following calculations.
Tissu e Culture Plants Seedlings
X (Mean) 0.65 0.47
Standard Deviation (S.D.) 0.42 0.32
Standard Error (S.E.) 0.09 0.07
Standard Error % 13.85 14.89
Xl-X2 t value for volume =
S.E.21 + S.E. 22
0.65 - 0.47
(0.09)2 + (0.07)2
= 1.58
Table value oft 0.05 at 20 d.f. = 2.086
X1 =Mean volume of tissue culture raised poles. X2 =Mean volume of seedling raised poles. S.E.1=Standard error for volume of tissue culture raised poles. S.E.2=Standard error for volume of seedling raised poles.
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.
In our country, about 1295.80 lakh ha area is under wastelands, so primarily any afforestation work undertaken aims to ameliorate these sites. The authors believe that tissue culture raised plants growing in sterile controlled environment are not likely to withstand adverse conditions of humidity, salinity, high temperature, high evapo.transpiration and other climatic vagaries of field. This results in high mortality of young seedlings. Further in tissue culture raised plants the genetic base is narrowed as the explants are collected from a small number of parent trees. This may be desirable in the ornamentals because
- market demand is for certain specific varieties with uniform color, form, leaves, etc. But in forestry, this may prove to be very risky. A classic example of this is poplar clone 1-214, from Italy, which was chosen for its excellent growth traits and freedom from pests and was once widely planted. Its large scale plantations as monoculture have generated new disease problems. Similarly,.elm clone "belgica" , planted on large scale in Netherlands, to be susceptible to Dutch elm disease (Wright 1976). When the planted trees have similar genotype, loss can occur resulting in many years of cumulative productivity loss, if the trees are not old enough to be salvaged. When catastrophe occurs in agriculture and crop is damaged or lost, adjustments can be made and farmer can start in the next season but in forestry the plants must survive for many years against the all natural disturbances that occur periodically. If genetic base in forestry is narrowed, the possibility for losses become greater.
In most of the cases, materials collected presently are only from phenotypically superior trees. Micropropagation is probably meaningful only where selected plus trees/plants with proven genetic superiority are used as source of the explant. Most scientists believe that propagules obtained from trees which are good in appearance will produce the same genotype. The selection of plus trees, however, is possible only after progeny testing. Further the tissue culture raised plants are more
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
Chaturvedi A.N., 1987. .Tissue culture of forest trees. J. Trop. Forestry, 3:239-241.
Deepesh N.D., 1985. Role of tissue culture in woody biomass productivity. Prospects, problems and strategies. Proc. Bio-Energy Society Second Convention and Symposium, 13-15 October, 1985. Hyderabad, pp. 94.
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
Sunil Khanna, Banwari La! and Alok Adholeya Microbiology and Molecular Genetic Unit
Tata Energy Research Institute 158 Jor Bagh, New Delhi 110003 India
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.
Key words: Acacia nilotica; biomass; Leucaena leucocephala; nodulation; Prosopisjuliflora; Rhizobium; YAM.
84
INTRODUCTION
The depletion of forest cover leading to scarcity of fuel wood, fodder and timber coupled with increasing areas being converted into wasteland is a major concern of developing countries. Leguminous trees and shrubs have been suggested as a renewable source of fuel and wood products and are known to have symbiotic association with Rhizobium and vesicular arbuscular mycorrhizal (VAM) fungi (NAS, 1977 ; 1979). Acacia species usually found in arid and semi-arid region are renowned for their fast growth rate even on marginal lands and are popular plants for use in land rehabilitation.
SomeAcacia species (Dreyfus and Dommergues, 1981a;b and Habish and Khairi, 1970) and Prosopis species (Basak and Goyal, 1975) require specific rhizobia which may not occur at all sites. Maximum growth of Acacia sp. in degraded soil can be achieved by inoculation with compatible and effective strains of rhizobia and VAM. However, information on the beneficial effect of such inoculation on plant growth in tree species is meagre. Hogberg and Kvarnstrom (1982) reported fixation of 110 kg N ha'1 yeaf1 equivalent by effective strain of Rhizobium with Leucaena leucocephala. Enhanced growth due to Rhizobium and mycorrhizal association have been reported by Roskoski et al. (1986) in L. seedlings. Similarly, with Acacia sp. beneficial effect of Rhizobium inoculation have also been reported by Cornet and Diem (1982) and Thapar et al. (1990). All these studies were done only for 30 to 90 days. Hence the information available pertains to seedlings only. Unfortunately, the renodulation potential of the introduced isolates and the consequent improvement in soil due to inoculation have not been studied adequately.
The objective of the present study was to evaluate the ability of different isolates of Rhizobium and VAM in biomass production of Acacia nilotica, Leucaena leucocephala and Prosopisjuliflora. The survivability and competitive ability of Rhizobium isolates of Acacia was also studied over a period of 12 months.
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.
Sampling and Nitrogenase Activity The plants were harvested 4,8 and 12 months after transplantation.
A pit ( 1 metre deep and 0.5 metre in radius) was dug around the plant and then filled with water. Roots at 1 metre were then cut and plants removed. All nodules with feeder roots were taken in 300 ml flask and 10%
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.
RESULTS AND DISCUSSION
Nitrogenase Activity and Nodule Occupancy Nitrogenase activity was assayed at 4, 8 and 12 months after
transplantation into field. Nitrogen fixation activity was much higher in Leucaena as compared to Prosopis indicating that enzyme activity in Leucaena is more than Prosopis. (Fig.1 and Fig.2). The nitrogenase activity in L. leucocephala plant increased during the first four months and then declined towards the eighth month (Fig.1). This coincided with the onset of winters and senescence of the nodules. The arrival of spring caused renodulation in both plants and thus an increase in nitrogenase activity. However, in P. juliflora the nitrogenase activity increased at 4 month and remained constant at 8 month except isolate P5 and decreased at 12 months (Fig.2). The extent of renodulation in Leucaena was much more than in Prosopis (as seen by the number and weight of nodules, data not shown). This observation prompted us to evaluate the extent of
87
a
0
Q)
(0
Q)
0 L
z
C.)
a) CO
a) an 0
z
Fig. 1 : Nitrogenase activity (p.mol
CjH4/h/plant) in root nodules of L.leucocephala at 4, 8 and 12 months after transplanting. Values are mean of 4 replicates.
100
Months Months
Fig. 2 : Nitrogenase activity CzH4fhIplant) in root nodules of P.juli/lora at 4, 8 and 12 months after transplanting. Values are mean of 4 replicates.
0 month 12 months
o
-c 0 z
75
50
25 I >
C.)
(Ii
a) 10 CO
a) an 05
z 0
0 (.0
0
0 (I) :3
Fig. 3: Percent nodule occupancy of introduced Rhizobium isolates in A.niolica at zero time (during transplanting to field) and 12 month old plants.
(- z 0
—
+ <
,.) < + ÷
Z 0 0 Z
Fig. 4 : Effect of VAM on nitrogenase activity in root nodules of L. leucocephala in 12 month old plants. Values are mean of 4 replicates.
renodulation by the introduced strains of Rhizobium in field trials with Acacia since this was initiated one year later than both Leucaena and Prosopis.
Immunological probes as well as antibiotic resistant pattern of the introduced isolates ofAcacja were used to determine the extent of survival of the-introduced isolates over a period of 12 months. Renodulation data with USDA 3325, AB3 and AD4 of Acacia showed that among these three strains AB3 was the most competitive for survival and renodulatioii in that ecosystem (Fig.3). In uninoculated control plants the nodulation at zero time(during transplanting from nursery to field) was very poor and renodulation was negligible after 12 months of transplanting. Renodulation in L. l.eucocephala was higher than in P. juliflora and A. nilotica. L. leucocephala is a surface rooter while P.juliflora andA. nilotica are deep rooters. The movement of Rhizobium isolates in the soil is very minimal. P. and A. nilotica being deep rooters, the renodulation of only the surface roots is possible thus decreasing the total nodule number. In L. leucocephala the extent of renodulation is higher, it being a surface rooter. Enhanced renodulation increased nitrogenase activity inL. leucocephalaat 12 months as compared toP.juliflora andA. nilotica.
Effect of VAM on Nitrogenase Activity In L. leucocephala the nitrogenase activity of Rhizobium isolate Al
was enhanced five fold with indigenous mycorrhizae (Fig.4). However, indigenous mycorrhizae did not enhance the nitrogenase activity of Rhizobium isolate A3. In A. nilotica the nitrogenase activity of only one Rhizobium isolate AD4 was increased with VAM isolate G. fasciculatum at 12 months but the activity of other two inoculum Rhizobium isolates AB3 and USDA 3325 did not increase with G. fasciculatum after 12 months of transplantation (Fig.5). In P. juliflora no positive effect of mycorrhiza on nodulation as well as nitrogenase activity was observed.
Effect of Rhizobium on Colonization P. juliflora was inoculated with VAM isolates G. calospora, G.
caledonius and indigenous mycorrhizae. Maximum colonization was observed in plants inoculated with G. calospora after 12 months of
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).
In gener,al with dry matter yield it was observed that microbial interaction was indeed beneficial to the plants at 12 months after transplantation. - In Leuceana dual inoculation due to Rhizobium and yAM enhanced total biomass production, while in Prosopis and Acacia inoculation with Rhizobium alone was the best. It could be that in deep rooters i.e. Prosopis and Acacia, mycorrhizae does not enhance/stimulate association of Rhizobium with the plants while in surface rooters i.e. Leucaena dual inoculation played an important role. Thus the root physiology may play an important role in determining the microbial benefits with tree legumes. Nodulation and nitrogenase activity were
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.
Initial observation with these three tree legumes suggest that microbial interaction viz Rhizobium and vesicular arbuscular mycorrhizae may play an important role in the enhancement of tree biomass in degraded or marginal soils.
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.
93
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 Microbiology, University of Agricultured Sciences, G.K.V.K. Campus, Bangalore, pp 107-108.
94
ANNEXURE I
List of Participants
Dr Abha Agnihotn
Tata Energy Research Institute
90, Jor Bagh,
New Delhi 110003
Dr HC Chaturvedi
National Botanical Research Institute
17, Rana Partap Marg,
Lucknow, UP
DR RM Dayat
Tata Energy Research Institute
101, Jor Bagh
New Delhi 110003
Dr Vibha Dhawan
Tata Energy Research Institute
90JorBagh New Delhi 110003
Dr SC Gupta
Department of Botany
University of Delhi
Delhi 110007
Dr V Jagannathan
19, Kale Park
15-A Someshwadi
Pune 411008
95
Dr VS Jaiswal
Banaras Hindu University
Varanasi 221 005
Mr Anil Kapahi
Tata Energy Research Institute
158, Jor Bagh
New Delhi 110003
Mr VK Kaul
Tata Energy Research Institute
101 Jor Bagh
New Delhi 110003
DrSunilKhanna
Microbiology and Molecular Genetic Unit
Tata Energy Research Institute
158, br Bagh
New Delhi 110003
DrG.LakshmiSita Department of Microbiology and
Cell Biology
Indian Institute of science
Banglore 560 012
DrBanwariLal
Tata Energy Research Institute
158, Jor Bagh
NewDethi 110003
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
Mr Ajay Panda Energy Sources
Block II CGO Complex Department of Botany Lodhi Road University of Delhi New Delhi 110003 New Delhi 110007
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
March 16, 1992
Venue: India International Centre (IIC), 40 Max Mueller Marg, New Delhi 110 003
&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
SESSION L TISSUE CULTURE TECHNIQUES
Chairman: Dr V. Jagannathan Ms Archana Nair
i) Experience with Forest Tree Tissue Culture
Dr G. Lakshmi Sita
ii) Micropropagation of Bamboos
Mr Sanjay Saxena
iii) Tissue Culture of Populus deltoides
Dr HC Chaturvedi
Lunch Break
99
SESSION I: CONTD.
Chairperson: Dr Deepak Pental
Ms Abha Agnihotri
iv) Tissue Culture of Grapes
Dr AK Singh
v) Role of Somatic Embryogeny in Mass Propagation of Guava (Psidium guajava L.)
Dr VS Jaiswal
Tea Break
Discussion
a) Rapporteurs presentation followed by Chairman's remark
b) Round Table Meeting on Problems Associated with Tree Tissue Culture
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)
SESSION COMMERCIALLSATION ASFWfS OF MICROPROPAGATION
Chairperson: Dr G Lakshmi Sita
Dr UK Tomar
i) Commercialisation of Plant Tissue Culture Research in India
Dr N Raxnanuja Rao
ii) Plant Regeneration of East Indian Walnut (Albizia lebbek) from Different Explants
Dr Asbis Taru Roy
iii) Large Scale Propagation of Eucalyptus grandis Mr P Mohan Kumar
iv) Tissue Culture of Albizia species
Dr UK Tomar
v) Tissue Culture Pilot Plant Facility for
Forest Tree Species
Dr Vibha Dhawan
Visit to Pilot Plant (Gual Pahari)
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
1) A Brief Account of Forest Tree Improvement in Uttar Pradesh
Dr Padmini Shivkuinar
ii) Comparison of Tissue Culture Plants Against Seeddlings in Tectona grandi8
Mr RM Dayal
iii) Role of Microorganisms in Tree Biomass Enhancement
Dr Sunil Khanna
Discussion
Panelists:
Mr ON Kaul
Dr HL Sharma Dr KK Joshi
Tea
102
IDRC-TIFNET
The IDRC's Tree Improvement and Farm Forestry Network (TIFNET) is an informal network for interchanging information, exchanging plant material and to serve as a think tank for the scientists involved in the IDRC sponsored projects relating to Tree Improvement, Farm Forestry and allied subjects. An important activity of the network is publication of periodical newsletters, research results and conference proceedings. The network projects-recently completed/on going (some in second or third phase) are listed below:
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
Printed at Nirmal Printers, T-304, Baijeet Nagar, New 008, Ph. : 5725388, 5728975, 5725105
