Research Bulletin
Micropropagation Technology for Multipurpose Trees : From Laboratory to Farmers Fields
B.Venkateswarlu and G.R.Korwar
Central Research Institute for Dryland Agriculture Santoshnagar, Hyderabad
In collaboration with
AP-NL Biotechnology Programme Biotechnology Unit, Institute of Public Enterprise, Hyderabad
Citation:
Venkateswarlu, B. and Korwar, G.R. 2005, Micropropagation Technology for Multipurpose Trees: From Laboratory to Farmers Fields, Research Bulletin, Central Research Institute for Dryland Agricutlure, Hyderabad, India, pp. 1-30.
January, 2005 300 copies
Other Contributors: Dr. G.Pratibha (CRIDA) Dr. M.Vanaja (CRIDA) Dr. Kunal Mukhopadhyay (CRIDA) Dr. Jayjayanthi Mukhopadhyay (CRIDA) Mr. Abdul Rasul (CRIDA) Dr. G.Satyanarayana (SAIRD) Mr. E.Venkata Ramanaiah (YFA) Mr. M.Balakrishna (SAIRD) Mrs. M.Neeraja (SAIRD)
Published by Director, Central Research Institute for Dryland Agriculture, Santoshnagar, Saidabad P.O, Hyderabad, Telefax: 040-24535336, www.dryland.ap.nic.in
Printed at : Heritage Print Services Pvt. Ltd., Hyd. : 2760 2453, 2760 8604; Email: heritageprint@yahoo.com
Foreword Dr.M.V.Rao, Chairman, BPC Institute of Public Enterprises Osmania University Campus Hyderabad – 500 007
Multi purpose trees (MPTs) like neem and teak are important components of the agroforestry systems in drylands. These trees provide supplementary income through timber, fuel wood, fodder, by-products development and also to improve soil fertility through nutrient recycling. The Andhra Pradesh Netherlands Biotechnology Programme (APNLBP) initiated with an aim to apply different biotechnologies for improving the dryland farming systems in Nalgonda and Mahabubnagar districts of Andhra Pradesh, accorded high priority for identification of superior germplasm of locally important MPTs and their mass propagation through micro propagation in order to make such material available to the farmers in these districts. Accordingly, a network project was sponsored to CRIDA and two collaborating NGOs i.e. SAIRD (Sri Aurobindo Institute of Rural Development) and YFA (Youth For Action) on “Micro propagation technology development and participatory field evaluation of neem and teak”. The project made excellent progress and resulted in useful products, which are already widely adopted by the farmers. The scientists have identified plus trees of these species following a country wide survey and successfully developed the protocols for micro propagation. More importantly, the technology was transferred to the field laboratories established by the NGOs in the target districts of Nalgonda and Mahabubnagar. The technical personnel at the NGOs have been well trained by CRIDA, who also contributed their skills to further improve/refine the protocol. The project served as a role model of Institute-NGO-Farmer linkage for development and transfer of this particular biotechnology. The project team also tried to introduce new innovations like rural bio centers to reduce the cost of final products to the farmers. This project is a good example of combining the basic and adaptive research to develop an useful product for the farmers. I compliment Dr.B.Venkateswarlu and Dr.G.R.Korwar for compiling the results and experiences from the project, in particular the data from the on-farm trials supported with good quality pictures. I hope this research bulletin will serve as an useful guide to all those researchers and developmental workers interested in application of biotechnology through participatory methods.
(M.V.Rao)
Preface Neem and teak are two important Multi Purpose Trees (MPTs), which are quite popular with farmers in most parts of the country due to their proven economic benefits. These species are planted on farm boundaries, block plantations and as components of agri-silvi culture and agri-pasture systems. Identification of plus trees and production of adequate planting material through mass propagation are pre requisites for supporting any successful plantation effort. With a view to augment the planting material supply, CRIDA under took a project on development of micro propagation protocols for these two important MPTs through a project sponsored by Andhra Pradesh – Netherlands Biotechnology Programme (APNLBP). The unique feature of this project was the involvement of Non Governmental Organisations (NGOs) and farmers as participants in the technology development, upscaling and field evaluation. The protocols for micro propagation of neem and teak were developed successfully at CRIDA, pilot tested and transferred to SAIRD and YFA in A.P. These protocols were adapted by these organizations quite successfully as a result of which the production of planting material could be taken up simultaneously at all the 3 centres. More than one lakh planting material has been produced during the last 5 years which is utilized both for on-farm research and pilot scale commercial plantations. The farmer participatory research enabled CRIDA to generate extensive field data on both the species, which will be valuable to make recommendations in future. Data so far indicated that micro propagated plants show higher uniformity and equal or marginally superior growth performance over that of planting material produced through traditional methods. The performance however varied with soil depth and rainfall. A long-term evaluation is required under different agro-ecological conditions to draw valid conclusions. I compliment Dr.B.Venkateswarlu, PI of the project and the two NGO partners for their collaborative effort in not only developing the technology but also its upscaling through the production centers established at the KVKs. I hope this pilot effort will grow into a larger and self-sustainable model in future and contribute towards greater adoption of agroforestry systems in the country.
January, 2005 Hyderabad
(Y.S.Ramakrishna) Director, CRIDA
Acknowledgements This bulletin summarises the experiences from a six year comprehensive project entitled “Micropropagation technology development for neem and teak and farmer participatory field evaluation”. This project was entirely funded by Andhra Pradesh Netherlands Biotechnology Programme (APNLBP). The project was implemented during 1997-2004 by CRIDA as the lead institution and two collaborating NGOs in Andhra Pradesh i.e. Sri Aurobindo Institute of Rural Development (SAIRD) and Youth For Action (YFA). The programme was coordinated by the Biotechnology Unit (BTU) of Institute of Public Enterprises (IPE), Hyderabad. We wish to acknowledge the APNLBP and the BTU for the financial assistance and in particular Dr.M.V.Rao the Chairman, Biotechnology Programme Committee (BPC) and Dr.G.Pakki Reddy, Coordinator of the BTU for their guidance, counseling and encouragement, which resulted in the successful implementation of the project. Dr.M.L.N.Rao, Dr. (Mrs.) Janaki Krishna and Mr.Anji Raju subject experts from BTU also facilitated the effective implementation of the project by timely release of funds, arranging various training programmes, field visits and reviews. We also wish to thank all external experts who reviewed the project and provided valuable suggestions. The authors would like to place on record the excellent guidance, cooperation and facilities provided by Dr.Y.S.Ramakrishna, Director, CRIDA and former Directors, Dr.H.P.Singh and Dr.J.C.Katyal which helped in effective implementation of the technical programme. Dr.G.Subba Reddy, Head, Division of Crop Sciences also provided valuable help for the success of the project at CRIDA. The contribution of both the NGOs and the staff of KVKs attached to them was immense in organizing the field trials and farmers awareness programmes. Large number of contractual staff worked in the project at all the 3 centres and put up their best to upscale the protocols successfully. The most important ones include contractual research staff like Dr.Kunal Mukhopadhyay, Smt. Jayjayanthi Mukhopadhyay, Dr.E.Srinivasan, Mr.V.Moses Kumar, Mr.B.V.Mashesh Kumar, Mr.V.Srinivas, Mrs.V.Aparna, Mrs. A. Annapurna and technical staff like Mr.Abdul Rasul, Mr.J.S.Mani Babu and Mr.M.Eugine who provided effective assistance and also contributed their own ideas for improvement of the protocols. Mr. M. Balakrishna (SAIRD) and Mr. Rajendra Kumar Reddy (YFA) helped in organising field trials. Mrs. M.A. Rekha prepared the manuscript who’s help is also gratefully acknowledged. Authors
Contents Item
Page #
Introduction
....
1
Neem (Azadirachta indica A.Juss)
....
2
Selection of plus trees
....
3
Germplasm registration
....
6
Micropropagation
....
7
Field transfer and progeny evaluation
....
8
Performance in on-station and on-farm trials
....
11
....
12
The protocol
....
13
Field performance
....
15
On-farm trials
....
18
Paulownia (Paulownia fortuneii)
....
22
Collaborative Trials with Public/Private Sector
....
23
Technology Transfer to NGOs
....
23
Establishment of Bio Centers
....
24
Linkages and Participatory Research
....
26
Adoption and Impact
....
27
Conclusions
....
29
References
....
29
Teak (Tectona grandis)
Micropropagation Technology for Multipurpose Trees
Introduction Micropropagation is the application of tissue culture technology for mass propagation of any economically important plant species. It offers an alternative to vegetative propagation and is mainly aimed at enhancing the rate of multiplication. Micropropagation can be done through i) shoot bud proliferation ii) adventitious shoot production iii) meristem culture iv) in vitro tuberization and v) somatic embryogenesis (Bonga and Durjan, 1987). The choice of technology depends on the species of interest, the availability of competing technologies and the cost advantage. Large number of protocols for micropropagation of horticultural, ornamental, forest and medicinal plant species have been developed over the last two decades, some of which have been successfully applied for routine nursery production and supply of planting material to farmers (Ahuja,1993; DBT, 2000; Chandra and Mishra, 2003). The most spectacular has been the application of micropropagation for multiplication of high quality ornamentals by export-oriented units (EOUs) and for quality banana saplings production for domestic markets. In addition to these examples, micropropagation technology has also been widely used in India for commercial
production of papaya, cardamom, vanilla, sugarcane, teak, bamboo, Populus and Anogeissus. Considering the potential of this technology, the Department of Biotechnology (DBT) established two pilot projects for mass multiplication of multipurpose trees at NCL, Pune and TERI, New Delhi and hardening facilities at selected universities (DBT, 2000). These centers have spurred the commercialization of micropropagation technology for various species in horticulture and forestry in the last two decades. Currently, more than 50 units in the private sector and large number of universities and research institutes are producing and supplying lakhs of economically important plants to farmers. With growing emphasis on crop diversification towards horticulture, afforestation to increase the green cover and planting of multipurpose trees for value added products like bio diesel and medicinal plants, micropropagation is likely to play more important role in future for production of quality planting material in the country. The chief advantages of micropropagation are the possibility of producing large number of “true to type� plants in a limited space through out the year. However, it is skill demanding and energy intensive compared to conventional propagation methods. The 1
Research Bulletin
other constraints in wider use of this technology are the lack of adequate data on the field performance of micropropagated plants, non-availability of economic viability information and the high cost of final product. Promotional efforts of the Government through subsidies to some extent helped in generating demand for species like banana, but use of micropropagated material of many other species still remains quite small compared to the actual potential in the country. This bulletin summarizes the work carried out by CRIDA and the collaborating institutions on i) development of micropropagation technology for elite clones of neem and teak, ii) achieving cost reduction by improvement of the protocols and iii) participatory evaluation of the tissue cultured material on farmers fields across Andhra Pradesh and neighbouring states under a financial grant from Andhra PradeshNetherlands Biotechnology Programme (APNLBP) during 1997-2004. Extensive work was carried out on selection of elite germplasm before initiating the work on micropropagation. Not only the technology was developed and scaled up under this project, but also was transferred to two Non Governmental Organizations (NGOs) in A.P. i.e. Sri Aurobindo Institute of Rural 2
Development (SAIRD) in Nalgonda and Youth For Action (YFA) in Mahabubnagar districts by setting up laboratories, hardening facilities and training the manpower. This resulted in significant adaptation/refinement of the technology to local conditions. It also led to the development of an innovative mechanism of combining micro and macropropagation technologies through linking the district level laboratory at the NGO and the village level biocenters.
Neem (Azadirachta indica A.Juss) Though neem has been known as a multi purpose tree with immense application in agriculture and health care for long time, the interest on this species revived in the last two decades owing to the discovery of large number of limonoids in the seeds which exhibited significant insect repellent, anti feedant and growth retarding properties (Randhawa and Parmar, 1993, Schumutterer, 1995). This led to the development of large neem based bio pesticide industry and increased use of neem seed extracts in on-farm pest management by farmers across the country. All these developments resulted in a growing interest on neem plantations in social forestry and as commercial block plantation on private lands to produce quality seed. Therefore,
Micropropagation Technology for Multipurpose Trees
the need arose for identifying superior germplasm for undertaking such plantations. Selection of plus trees
Though the potential of neem is realized by all, there are several issues to be considered before embarking on promotion of large scale plantations in the country. Extensive variability is found across the country for economic traits like seed yield, oil per cent and azadirachtin content. This was found to be the main reason for the highly variable bio efficacy results when seed extracts are used. Therefore in the project, it was hypothesized that if plus trees of neem with high azadirachtin in the kernels are selected and seeds/clonal planting material from such trees are used for planting trees on field boundaries, the seed harvested from these plantations would have higher bio efficacy when used as extracts. Studies carried out at CRIDA did confirm this hypothesis in case of lepidopteren pests (Sreenivasa Rao et al., 1999). Therefore, to begin with, efforts were made by the project team for selection of plus trees by considering the following characters: 1. Length of the clean bole (more than 2 m) 2. Seed yield 3. Kernel to seed ratio 4. Per cent oil in the seed 5. Azadirachtin content in the kernels
6. Resistance/tolerance against diseases and angiospermic parasites Accordingly, a countrywide survey was carried out for 2 years mostly in arid and semi-arid regions and seeds from more than 400 eco types were analyzed. The location map of areas surveyed against the agro-eco sub region background is depicted in Fig.1.
Fig 1. Locations representing the neem eco type survey against agro-eco regions background
Variability for economic traits
High variability was found for important traits like seed yield, seed-kernel ratio, oil per cent and azadirachtin content (Venkateswarlu et al., 2002). It varied from as low as 0.1 to as high as 1.0% (Table 1). This was also reflected in other parameters like oil per cent, 100 seed weight and kernel to seed ratio. No clear 3
Research Bulletin Table 1: Range and means of azadirachtin content and related characters in neem seed samples collected from different locations (mean of 2 years) Locations
100 seed wt. (g)
% Kernel in seed
% Oil in seed
% Azadirachtin in kernel
Range
Mean
Range
Mean
Range
Mean
Range
Mean
Indore (7)
15-21
18
42-55
52
14-21
18
0.25-0.60
0.39
Akola (11)
14-26
20
29-54
46
18-24
21
0.19-0.43
0.26
Dantiwada(15)
15-24
21
44-64
52
19-31
23
0.17-0.80
0.35
Varanasi (5)
17-24
20
36-52
47
16-26
20
0.14-0.25
0.19
Solapur (9)
12-16
14
22-58
44
11-26
20
0.12-0.65
0.32
Rajkot (3)
15-20
17
43-52
47
18-20
19
0.22-0.42
0.34
Kovilpatti (5)
18-23
20
51-58
54
16-24
18
0.08-0.42
0.20
Anantapur (9)
14-30
20
49-56
53
19-25
23
0.19-0.33
0.20
Phulbani (4)
15-20
17
53-55
54
27-34
31
0.21-0.69
0.42
Bijapur(8)
17-21
19
42-51
46
17-20
19
0.31-0.65
0.43
Hisar (3)
16-20
18
42-52
47
21-26
23
0.23-0.27
0.25
Rajahmundry (3)
17-18
18
42-46
44
25-27
26
0.26-0.36
0.31
Hyderabad (30)
11-23
15
43-62
51
14-29
22
0.21-0.95
0.55
Bellary(5)
47-52
50
17-23
20
21-26
23
0.19-0.34
0.27
Gulbarga(4)
31-42
36
19-21
20
15-19
17
0.20-0.45
0.32
Bangalore(3)
19-50
39
18-20
19
8-23
18
0.28-0.47
0.40
Dantiwada(5)
30-41
36
12-25
18
12-18
16
0.26-0.44
0.34
*Figures in parentheses represents number of samples analysed.
relationship was found between seed size, yield, oil per cent etc. with azadirachtin content. To over come the effect of soil type and rainfall, grid sampling was done within 15 sq.m area at Hyderabad. Five fold variation was observed among individual trees even with in this grid indicating both genotype and environment are involved in influencing the aza content (Table 2). Soil type or rainfall of the sampling location also did not clearly explain the variability in azadirachtin 4
content (Table 3). Some reports (Rangaswamy and Parmar, 1994; Ermel et al., 1987) tried to link the aza content in neem eco types to rainfall and soil type, but no such relationship was found in the present survey. Samples with low and high aza content could be found in all soil types/rainfall zones. Inter annual variation for aza content
However, high inter annual variation was found for azadirachtin content in the same
Micropropagation Technology for Multipurpose Trees Table 2: Azadirachtin content of 30 neem trees (aged between 15-20 years) sampled in a grid of 15 sq.m area near Hyderabad (mean of 2 years) Sl. No.
Girth at Kernel breast height to seed (cm) ratio (%)
100 seed wt. (g)
No. of seeds in 1 kg
Oil in seeds (%)
Azadirachtin in kernels (%)
1
54
58.07
10.89
9183
21.88
0.585
2
57
59.60
13.37
7479
23.03
0.800
3
66
52.93
20.73
4824
25.57
0.627
4
67
49.68
18.82
5826
19.16
0.326
5
67
58.89
10.12
9881
26.56
0.572
6
68
50.86
15.51
6447
22.23
0.544
7
73
46.00
10.78
9276
18.95
0.313
8
75
51.02
18.23
5685
17.82
0.516
9
77
55.48
12.06
8291
25.63
0.287
10
80
52.03
13.32
7507
19.89
0.735
11
82
44.57
12.09
8271
21.31
0.718
12
84
34.22
15.79
6333
15.18
0.498
13
85
44.27
22.91
4369
19.89
0.705
14
85
51.21
16.35
4621
15.08
0.419
15
92
53.88
12.56
7962
19.15
0.522
16
93
45.75
17.38
5754
16.76
0.786
17
94
53.18
14.56
6868
27.75
0.393
18
101
47.33
16.09
6215
18.03
0.335
19
105
49.63
14.81
6752
19.60
0.533
20
106
53.23
13.64
7331
22.17
0.959
21
108
49.68
17.12
5841
19.18
0.453
22
111
48.67
15.64
4562
18.96
0.432
23
115
49.46
14.07
7281
18.34
0.309
24
120
45.93
17.33
5776
18.74
0.854
25
122
56.96
17.40
5747
24.21
0.458
26
127
44.01
11.85
8439
17.38
0.611
27
129
55.61
14.97
6680
21.22
0.913
28
153
47.23
18.56
5275
15.72
0.231
29
176
36.35
15.30
6536
13.98
0.456
30
192
43.52
16.51
6057
17.15
0.432
5
Research Bulletin Table 3: Azadirachtin and oil content (range and mean) in neem ecotype growing in different soil types in the arid, semi-arid and sub humid regions of India Soil type
No.of samples
Oil content in seeds (%)
Azadirachtin content in kernels (%)
Range
Mean
Range
Mean
Aridisols
15
12-32
23
0.11-0.75
0.35
Alfisols
40
14-29
22
0.21-0.95
0.46
Vertisols
76
11-31
21
0.15-0.82
0.40
Entisols
21
16-28
20.5
0.16-0.62
0.39
Oxisols
12
15-26
22.5
0.25-0.69
0.41
trees. Sampling identified trees at the same location continuously for 5 years clearly revealed high variability year to year but the relative ranking of these trees remained constant more or less throughout the period (Fig.2) indicating that both genetic and environmental factors are important in influencing the azadirachtin content (Venkateswarlu et al., 2002). While selecting a high azadirachtin containing plus tree and using its seed for plantation does not guarantee same azadirachtin content in
the progeny due to the effect of season and location, using a plus tree is still useful as the high aza tree generally maintained its rank with respect to other trees at the same location. But a plus tree selected at a given location did not produce same aza content when planted at other locations. Therefore from the project results, it was evident that plus trees of neem may be selected from the location/agro eco subregion where the plantations are to be undertaken and the seed/planting material from such trees may be utilized for plantations.
Azadirachtin (%) in kernels
Inceptisols and
Germplasm Registration
Fig. 2. Variation in the azadirachtin content during 1997-2004 in five selected trees of neem at Hayathnagar Research Farm, CRIDA, Hyderabad
6
After an extensive study of 400 ecotypes, five plus trees were selected based on the traits described above. The total seed yield and azadirachtin yield per tree were considered in selection of these trees rather than aza per cent. It was ensured that the plus trees are relatively free from foliar diseases and infestation by angiospermic parasites. One
Micropropagation Technology for Multipurpose Trees
of the plus tree (CRIDA-8) with an estimated age of 25 years was registered with the NBPGR, New Delhi under registration No.INGR No.03038 dated 20th September, 2001. It exhibited consistently high yield (air dried fruit yield of more than 50 kg/year), oil (25% in seeds) and azadirachtin (>0.75% in kernels) contents. Vegetative Propagation
In view of the high variability in seed raised progeny and conflicting reports on the pollination mechanism in neem, it was thought to rely on clonal propagation to produce “true to type� material. Accordingly, macropropagation was tried using soft wood cuttings with different hormone combinations. The cuttings took more than 100 days to root and in the meanwhile high humidity in the poly tunnels led to fungal infection of leaves and significant mortality of the cuttings. Therefore, this method was not considered viable for mass propagation. Other techniques like air layering were standardized at National Research Centre for Agroforestry (NRCAF), Jhansi (Gupta,V.P., Personnel Communication), but these techniques are more useful for research and breeding rather than mass propagation. Micropropagation
Alternatively, an attempt was made to standardize micropropagation protocol for
two of the five plus trees selected (Venkateswarlu et al., 1998). The protocol which was refined over a period of 5 years from 1998 to 2004 consisted of the following key steps. Juvenile shoots from plus trees are selected during March to May and used as primary explants for initiating the culture. After bud break, the cultures are transferred to MS medium containing 0.2 mg/l BAP and 0.2 mg/l kinetin for in vitro shoot elongation. The multiplication is done by repeated sub culture of nodal explants from elongated shoots. In each culture bottle, two explants could be accommodated which produced two micro shoots of six nodal length in eight weeks. In other words, a multiplication ratio of 1:6 was achieved in two months. A flow chart of steps involved in the protocol is depicted in Fig.3. However, there are some critical steps in success of the protocol (Table 4). These includes i) the establishment of sterile primary explant, ii) size of the transferable node for multiplication and iii) maintenance of optimum moisture in the culture vessels. Excess humidity in culture vessels always resulted in more callus development and delay in the shoot elongation. Although, 3 shoots could be accommodated in each bottle, optimum elongation/shoot proliferation occurred only with two shoots. 7
Research Bulletin
Selection of mother plant Nodal explant from juvenile shoots 2 weeks Axilliary bud induction 6 weeks In vitro multiplication (1:6) 25 days Rooting in soil rite (85%) 2 weeks Primary hardening in poly tunnels/ mist chamber (90%) 8 weeks Secondary hardening in shade house (85%) Field planting
8
Studies on clonal fidelity
Both anatomical studies of the in vitro originated shootlets originating from nodal explants and molecular analysis of the leaf DNA from progeny from different batches were carried out to prove the “true to type� nature of the TC plants (Singh et al., 2002). Pictures from the callus sections of secondary cultures of neem obtained after microtomy clearly established that lateral shoots originated directly from the explants and not from the callus (Plate 2). Characterisation of progeny from 5-6 batches through AFLP technique using standard primers (EACG x MCTA) also confirmed the clonal fidelity (Plate 3). The identical banding pattern of DNA from all leaf samples of different batches of TC progeny can be seen in plate 3 (EAAC x MCTC). Other samples like Thai neem and tomato used for comparison showed dissimilar banding patterns.
Fig.3 : Flow chart of steps involved in micro propagation of neem
Field transfer and progeny evaluation
During hardening also, neem plants are highly susceptible for excess humidity and therefore sufficient care has to be taken to regulate watering/misting in green house/ mist chamber. Fungicide treatment of freshly rooted shoots during transfer to soil is also critical.
Three months old hardened neem plants were transferred to the field at CRIDA Institute Complex at Santoshnagar and also at the Hayathnagar Research Farm. The plantlets were transplanted in 45 cm x 45cm pits filled with soil + FYM and watered weekly during the first summer. There after, they were grown under rainfed
Micropropagation Technology for Multipurpose Trees Table 4: Summary of the micropropagation protocol for adult neem trees Step
Process/Method
Result
Remarks
Initiation of Primary culture
Explants inoculated in MS basal medium with 1 mg/l BAP
Shoot buds produced from 90% nodal axes
March to May optimum season for explant collection
In vitro multiplication and elongation
0.5 – 1.0 cm long micro shoots transferred to the same medium with 0.2 mg/l BAP + 0.5 mg/l kinetin
In 8 weeks, micro shoots elongated upto 5 cm with a multiplication ratio of 1:6
Maintenance of growth room temperature (25 oC Âą 1) and preventing excess moisture is critical
Rooting
4 to 5 cm long shoots transferred to soilrite in rooting chamber after dipping into rooting hormone for 15 minutes
85% shoots rooted after 20-25 days
In rooting chambers, humidity should be 100% but water logging should be avoided in the rooting medium
Hardening
Rooted plantlets transferred to soil and kept in mist chamber (80-90% humidity) for 30 days
85-90% plantlets survived, but slow growth in polybags.
Protection from fungal diseases and preventing water logging are critical
Field transfer
Two months old hardened plants transferred to field
100% plants survived
T.C.plants attain uniform growth, grow just like seedlings
Plate 1: Stages in micropropagation of neem
9
Research Bulletin
a
b
c
Plate 2: Histological studies on origin of microshoots from the neem explants (a) profuse callus cells originating through rupture of the epidermis of the explant, (b) close view of callus cells showing absence of vascular tissues, (c) cross section of the explant showing distinct vascular connection between explant and the microshoot bud primordia
Lane
Sample
1
14yr mother plant (plus)
2
3 yr TC progeny of 1
3
6m TC progeny of 1
4
TC progeny of 1
5
TC progeny of 1
6
TC progeny of 1
7
3 yr TC progeny of 1
8
2m TC progeny of 1
9
22 yr plus tree
10
20 yr plus tree: high aza
11
15 yr normal tree
12
Poor tree: low aza
13
Same as 1
14
Same as 2
15
Thai neem
16
Tomato
Plate 3: AFLP analysis of tissue cultured progeny of neem plus tree for confirmation of clonal fidelity (using Primer EAACxMCTC)
10
Micropropagation Technology for Multipurpose Trees
Fig.4. Growth of TC plants of clone CRI-8 at CRIDA complex after 42 months of planting
conditions. These isolated plants attained a height of 5m and GBH of 25 cm 24 months after planting (Fig.4) and were normal in phenotype. The first flowering was noted after 30 months. Seeds collected from the progeny of tissue cultured plants were analysed for azadirachtin content and oil per cent. The
Fig.5: Azadirachtin content (A and B) in mother plants and tissue cultured progeny of two plus trees of neem (MT : mother tree TCP: tissue cultured progeny) Source: Venkateswarlu et al. (1999)
oil % in the progeny seed was 24 as against 25% in the mother plant. The seeds produced comparable azadirachtin to that of the mother tree (Fig.5). The seeds from TC progeny produced 56% kernels as against 56% in the mother tree. Performance in on-station and on-farm trials
The micropropagated neem plants were evaluated on-station at Hayathnagar Research Farm (HRF) of CRIDA and on farmers fields at different locations in AP and Maharashtra. At HRF, the plants were compared with seed raised progeny from same plus tree on two soil types under rainfed conditions (mean annual rainfall 670 mm). On a sandy loam soil (phase I), the TC plants attained an average height of 657 cm and girth of 40.2 cm in 5 years and 4 months (Fig.6), while seed raised 11
Research Bulletin
Fig. 6: Performance of tissue cultured neem in the on-station trial at Hayathnagar Research Farm, Hyderabad (left) and on the farmers fields near Chakan, Maharashtra (right)
plants attained average height of 680 cm and girth of 46.5 cm. The seed raised and TC progeny didn’t exhibit significant differences either in rate of increment of height or girth. Plants from both the treatments flowered after 4 years. Because of the differences in the soil depth within the experimental block, there was some heterogeneity in the growth of plants and canopy development and plants from same replication did not flower in one season. At the second site on a loamy sand soil, after 4 years and 4 months, TC plants showed marginal superiority in terms of height (385.3 cm) over seedlings (360 cm) but no differences were noted in girth (19 and 20 cm, respectively). The performance of plants on the farmers fields was quite variable. Locations with higher soil depth supported significantly higher growth and girth increments. For example at Chakan, near Pune, two year old plants attained a height of 2.85 m 12
(Fig.6) as against 2.40 m with seedlings, while at Mahboobnagar on a rocky out crop, TC plants took 4 years to attain the comparable height and girth. At Chakan, the TC plants showed higher uniformity in growth, 15% more height and girth by 4 years while at Mahabubnagar both were on par.
Teak (Tectona grandis) Teak (Tectona grandis) is the most important timber tree in India. Over the years, many clonal plantations were raised all over the country both by the forest departments and private sector. Vegetative propagation techniques have been used by the forest department and private nurseries on a limited scale with elite clones. Dr.A.F.Mascerenhas and his group standardised the micropropagation protocol for teak at NCL, Pune (Mascerenhas et al., 1993), which was latter successfully upscaled to pilot stage
Micropropagation Technology for Multipurpose Trees
and formed part of the Micropropagation Technology Park of DBT. Limited data was also generated on the field performance of micropropagated plants at different locations in comparison to different clones produced through vegetative propagation or raised through stumps. During 90s, enormous interest was generated among farmers and plantation companies in A.P. on prospects of planting teak around farm boundaries and as block plantations to generate supplementary income. To meet this demand and also generate scientific data on the micropropagation technology itself and field performance, teak was included as the second species in the APNL project. Under the project, clones from south India which are suitable for raising plantations in A.P. were selected in consultation with the state forest departments. Accordingly, micropropagation work was initiated on two teak clones i.e. Teli from north Kerala and Nallamalai from Andhra Pradesh. The Protocol
In case of teak also, the protocol was based on culturing nodal explants from juvenile shoots of mature plus trees. The new shoots growing at the nodes after pruning served as best explants. The initial problems with browning of explants and contamination
were overcome by using anti oxidants and antibiotics in the media. Secondary cultures were raised in normal media without these chemicals. As in case of neem, the protocol involved 4 stages i.e establishment of the primary explants, in vitro multiplication, rooting and hardening. Single nodal explants from primary cultures were used for
Nodal explant from mother plant 3 weeks Axilliary bud induction 8 weeks In vitro multiplication (1:6) 3 weeks Ex vitro rooting in soil rite (85%-90%) 10 days Primary hardening in soil in mist chamber (95%) 20 days Secondary hardening in shade house (98%) Field planting Fig.7 : Flow chart of steps involved in micro propagation of teak
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Research Bulletin
Plate 4: Stages in micropropagation of teak
secondary multiplication. In each culture vessel, 4 explants could be accommodated for multiplication stage. The multiplication ratio achieved was 1:6 in 6 weeks. Though initially in vitro rooting was tried, subsequently a highly reproducible and cost effective ex vitro rooting technique was standardized with excellent results. A flow chart of the protocol is given in Fig.7. Cost reduction
Efforts were made to improve the protocol for teak to reduce the cost of production 14
and also make it more amenable for scale up. The multiplication hormone was changed and concentration reduced, which brought down the cost by 15% of the total media cost involved in multiplication stage. Similarly, the soil rite used in the initial stages for ex vitro rooting was replaced with cocopeat. The cost of rooting medium was brought down from Rs.0.35 to Rs.0.15/ plant (Table 5). At SAIRD, Gaddipalli (collaborating NGO) excellent results were achieved by using vermicompost in the rooting medium in place of cocopeat.
Micropropagation Technology for Multipurpose Trees Table 5: Reduction in cost of rooting medium due to substitution of soil rite with cocopeat Rooting medium
Cost Rs/kg
No of plants that can be rooted in 1 kg*
Cost of rooting Rs/plant
Coco peat
6.00
40
0.15
Soil rite
28.00
80
0.35
* based on repeat use
Single step rooting
Even the ex vitro rooting was further improved subsequently by direct rooting of the shootlets in poly bags. This improvement from two step process of rooting in plastic trays containing soil rite followed by transferring to poly bags (for hardening) was changed to a single step method wherein shoots were directly transferred into the poly bags filled
with sterile soil rite in the planting hole and soil in the remaining part of the bag (Fig.8). This improvement saved two man days per each cycle. Field performance
The ex vitro rooted TC plantlets of teak showed 95% survival during hardening and 100% survival after field transfer. During
Two Step method
Single step mehtod
Fig. 8 : Single step rooting cum hardening of teak
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Research Bulletin
the project period, extensive data was generated on the growth and survival of tissue cultured teak of clone Teli both from on-station and on-farm trials. During 1997-2003, nearly one lakh plants were produced at pilot labs of CRIDA and SAIRD and planted on more than 100 farmers fields in 4 states (see impact section). Two main experimental plantations were established at Hayathnagar Research Farm (HRF) of CRIDA, Hyderabad and KVK instructional farm at Gaddipalli in Nalgonda district, A.P. Both the experimental plantations and farmers fields were followed with regular data collection and analysis during 1999-2004.
In the Hayathnagar Research Farm trial also, a steady growth of TC teak was observed upto 5 years. However, from year five onwards, the increment in height slowed
GBH (cm)
Height (m)
The height and GBH increments were influenced by a number of factors like soil type and rainfall. The increment pattern of height and GBH of TC teak (Teli) at Gaddipalli (on-station) is presented in
Fig.9. This is a typical shallow Alfisol of Nalgonda district with 10 cm of soil depth. The plantation was given protective irrigation during summer for 2 years and vermicompost @ 5kg/tree from 4th year onwards. So far, a steady and uniform annual increments in the height and GBH was noted in the plantation with a mean annual increment (MAI) of 1.5-1.8 m in height and 6-8 cm in girth during the five years after plantation. These increments are not very high, but considering the limitation of soil depth, this growth rate can be described as average to good. The plantation has remained fairly uniform till 5 years with an uniformity index of 0.75 (Fig. 10).
Months after planting
Fig.9: Growth pattern of TC teak plantation (cl. Teli) at KVK instructional farm, Gaddipalli between 1998-2002
16
Fig.10: A view of the model plantation of TC teak (cl. Teli) at KVK instructional farm, Gaddipalli, five years after planting
Micropropagation Technology for Multipurpose Trees
down where as the girth increment has accelerated. Up to first three years, stumps showed superiority in terms of height and girth increments. By sixth year, both the treatments remained on par (Fig.11). However, in case of stumps, some individual plants proved superior to TC plants. This was perhaps due to the food reserves in these stumps which benefited them in the initial boosting of the growth. However, TC plants showed higher uniformity as compared to stumps. The TC plants used in this trial were from the initial batches where the rooting was done in vitro and there was 10% field mortality. The protocol was subsequently improved with ex vitro rooting and most of the onfarm trials were carried out with TC plants produced through ex vitro rooting.
Irrigation and inter crop sub treatments were introduced in the trial from 4th year onwards. Data so far indicated no significant impact of irrigation on the height or girth increment in teak. Intercrops like greengram, groundnut and fingermillet were grown successfully. Compared to sole crop, the yield of intercrop was lower in all the treatments. The yield of intercrops also showed a gradual decline with increasing age of the teak from 4th to 6th year after planting. The impact of other treatments was not significant. The results showed that intercrop can be successfully grown in widely spaced (3x3m) teak plantation up to 6 years but a 25-40% yield reduction was recorded compared to the sole crop.
25
20
15
10
5
0 Height (m)
Girth (cm)
Fig.11: Height and girth of TC teak and stumps at HRF, 6 years after planting
Fig. 12: Six year old TC teak at HRF with finger millet as intercrop
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Research Bulletin
On-Farm Trials
Extensive on-farm trials were carried out in 6 districts of A.P. and few locations in Maharashtra, Karnataka and Tamil Nadu. Since Mahabubnagar and Nalgonda are the two target districts for the project, data was compiled from the initial on-farm trials on 6 farmers fields. Data presented in Table 6 indicate that in Nalgonda district, the mean annual increment in height and girth of TC teak plants was marginally superior to the Mahabubnagar district but the differences were not significant. Individual plantations in both the districts performed well with good management. However, soil depth has a distinct influence on the growth rate. To assess the comparative performance of TC vs stumps on farmers field, a large onfarm trial was taken up on the field of progressive farmer Mr. Ramkrishna Reddy of Gaddipalli village during kharif 2002. On a 16 ha field, tissue culture and stump derived plants were raised on 8 ha each.
Initial growth data showed a marginal superiority of TC plants over stumps. After 6 months of planting, the height of TC plants ranged from 90-200 cm while stumps attained height of 40-170 cm. The girth of TC ranged from 5.45-8.5 cm while that of stumps from 4.55-7.25 cm. The uniformity index of TC plants was 0.86 as against 0.45 for stumps (Fig. 13). However, the growth differences disappeared by 2 years, but TC plants still maintained high uniformity over stumps. In Rajendranagar mandal of Rangareddy district, a distinct difference was observed between stumps and tissue cultured plants in a clay loam soil both in terms of height and girth (Fig. 14). In an Alfisol, near Zaheerabad Mandal of Medak district, tissue cultured teak attained an average height of 2.7m and girth of 9 cm after 6 months which was significantly superior to stumps. There was also high degree of uniformity in the TC plants (Fig.15). Data on the field performance of TC teak in
Table 6. Annual increments in height and girth of tissue culture teak on farmers fields during the first 4 years Age of the plantation Year Year Year Year
I II III IV
Mean annual increment in height (m) Nalgonda*
Mahabubnagar**
Nalgonda*
Mahabubnagar**
1.4 1.5 1.0 0.8
1.2 1.3 0.8 0.6
5.5 5.6 5.6 7.0
4.5 5.2 5.3 6.5
*Mean of 6 farmers **Mean of 5 farmers
18
Mean annual increment in girth (cm)
Micropropagation Technology for Multipurpose Trees Tissue cultured
Stumps
Fig.13: Comparative performance of stump raised and tissue culture plants of teak (cl.Teli) on farmers field (Mr. Ramkrishna Reddy) in Gaddipalli village, Nalgonda district, A.P. 2 years after planting
Adilabad, Medak and Rangareddy districts are given in Fig.16 to 18. In a plantation in Gidwal village of Medak district, water harvesting through half moon terraces resulted in 20% higher height gain over control. Impact of rainfall and soil properties on growth rate
Since soil depth and rainfall play important role in influencing the growth rate, data
from farmers fields was analyzed to understand the impact of the above two parameters. Since plantations in different rainfall zones/soil depths were not of same age, the MAI data over a period of 3 to 4 years was used to study the impact of rainfall and soil parameters. In addition to the plantations in the target districts, data from other locations in A.P. and states like Maharashtra and West Bengal where the planting material was supplied were 40 TC
Stumps
35 30 25 20 15 10 5 0 Height (m)
Girth (cm)
Fig 14: Comparative performance of tissue cultured teak (cl. Teli) and stumps on a clay loam soil in Ranga Reddy district (Rainfall-670 mm, age of the plantation - 6 years, average height (m) and girth (cm), TC: 11.1 and 38.5, stumps: 6.25 and 24)
19
Research Bulletin Fig.15: six months old plantation of tissue culture teak (cl. Teli) on farmers feild (Mrs. Anjamma at Gidwal village, Zaheerabad Mandal, Medak district, A.P. Planting on contours and rainwater harvesting with half moon basins resulted in 20% higher growth. Mean annual rainfall: 900 mm, soil type: Alfisol, average height: 2.7 m, GBH: 9 cm)
Fig. 16: Six year old plantation of tissue culture teak (cl. Teli) on farmers field ( Mr. Mohanlal) at Uppal village, R.R. district AP. Mean annual rainfall: 700 mm, soil type: loamy sand, average height: 11.5 m, GBH: 40 cm)
Fig. 17: Seven months old plantation of tissue culture teak (cl. Teli) showing high uniformity on farmers field (Mr. Satyam Reddy at village Kaluva in Nirmal mandal, Adilabad dist. Mean annual rainfall: 995 mm, soil type: red sandy loam, average height: 3.65 m and GBH: 13 cm)
Fig 18: Two year old plantation of tissue culture teak (cl. Teli) on farmers field (Mr.Kusu Nasaraiah at village kanchikacherla, Krishna district. Mean annual rainfall: 1100 mm, soil type: medium deep black soil, height: 8 m, GBH: 40 cm)
20
Micropropagation Technology for Multipurpose Trees 3
2.5
2.5
MAI in girth (cm)
3
2 1.5 1
2 1.5 1 0.5
0.5
0
0 550
670
750
900
550
1250
670
750
900
1250
Fig.19: Growth rate of teak in plantations as affected by rainfall (3-4 years)
1.2
MAI (m) in height
1 0.8 0.6 0.4 0.2
so il (D )
Bl ac k
so il (M ) Bl ac k
lo am cl ay
Sa nd y
Sa nd y
lo am
0
Sa nd
Similarly, efforts were made to understand the impact of effective soil depth on growth rate. During the first 2 years, there was no significant impact of soil depth on height. However, from year 3 onwards, soils with more than 1m effective depth supported significantly higher girth increments (Fig. 20) than shallow soils while the impact on height increment was not marked. It is likely that soil depth will have a profound influence on girth increments, as the trees grow further. A
number of soil chemical properties like organic carbon, pH, EC, available N and P at the experimental locations were correlated with the growth rate but upto 5 years of growth, but no definite relationship could be established up to 5 years. However, in future, important information on the growth of teak may come out from these trials which will help in identifying suitable soil type and management practices for optimum growth of TC teak.
Lo am y
also included in the analysis. Data on the effect of rainfall on growth increment is presented in Fig. 19. Plantations raised in areas with high rainfall showed higher height and girth increment although the trend is not linear. Between 600 – 900 mm, the differences were not significant, which may be because of the interactive influence of the soil type and rainfall.
Fig.20: Mean annual increment of teak in different soil types in rainfall zone of 670-900 mm (M=Medium; D=Deep)
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Research Bulletin
Impact of soil amendments on growth rate
Teak is generally grown in deep soils of more than 0.75 m for effective growth increment. However, farmers continue to grow teak in marginal lands also. In order to improve the performance of teak in such degraded soils, fly ash was incorporated in the planting pit @ 30 kg/pit at the time of transplanting in an on-station trial at CRIDA. The growth of tissue culture teak (cl. Teli) was monitored at periodical intervals. The average height and collar girth in the degraded soil at Hyathnagar
Research Farm 9 months after planting were 2.25 m and 16 cm, respectively. No significant differences were noted between control and fly ash amended plots upto 2 years after planting.
Paulownia (Paulownia fortuneii) Paulownia popularly known as empress tree is native of eastern Asia. It has revolutionized the agroforestry in China and is widely grown in temperate areas of Tiwan, China and Australia. More recently Paulownia has been introduced into tropical
Plate 5: Steps in the micropropagation of Paulownia
22
Micropropagation Technology for Multipurpose Trees
and sub tropical areas of the world including India. In view of the demand generated by farmers and plantation companies during late 90s who were importing planting material from Australia at high costs, CRIDA took up the work on micropropagation protocol development for this species. Primary explants were collected from some of the actively growing plantations in Karimnagar district of A.P. and nodal explants are cultured on MS medium containing 1 mg/l of BAP. Buds collected during May to June showed maximum response. Half strength MS medium gave better response. The secondary multiplication was done with half strength MS containing 0.5 mg/l of BAP. The multiplied shoots were successfully rooted ex vitro in soil rite and hardened in the mist
organizations were initiated at different periods during 1999-2002. Name of the organization
Species under evaluation
Forest Research Centre, Mulugu, A.P.
Neem Teak
Maharashtra Forest Research Centre, Lohra, Chandrapur
Teak
A.P. Forest Development Corporation, Nellore, A.P.
Neem
Sri Ramananda Tirtha Research Institute, Pochampally, Nalgonda, A.P.
Teak
EID Parry Research Centre, Cuddalore, Tamil Nadu
Neem
Though detailed data was not available from these trials, information supplied by the collaborators from time to time indicated that the plant material provided by CRIDA showed good survival rate, uniformity and the field performance has been satisfactory.
chamber (Venkateswarlu et al., 2001). Plants subjected to primary and secondary
Technology transfer to NGOs
hardening were successfully field transferred
The unique feature of the AP-NL project was the development and scale up of the technology at CRIDA and its transfer subsequently to NGOs for field level implementation. Accordingly, the project provided adequate grants for setting up of the production units including laboratory and green houses at SAIRD, Nalgonda and YFA, Mahabubnagar. The technical staff recruited at the NGOs were given hands on training at CRIDA for a period of 3 months. The stock cultures of the mother
with 95% survival rate. The technology was transferred to M/s.EPC Irrigation, Nasik through an agreement entered in June, 2000 and the firm has been successfully producing and marketing Paulownia with the technology provided by CRIDA.
Collaborative trials with public/private sector In addition to the on-farm and on-station trials, collaborative trials with the following
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Research Bulletin
plants were maintained at CRIDA and supplied to the satellite centers from time to time till they could establish the cultures in sufficient numbers. During the first one year, the laboratory and greenhouse infrastructure were established and from year two onwards the production started. The design parameters of the greenhouse were changed to suit to the local conditions. Educated youth from the villages were trained at the production centers particularly on hardening. The laboratory and greenhouse infrastructure at the SAIRD and YFA are depicted in Fig. 21. CRIDA coordinated the technology transfer including providing details on improvements made in the protocol from time to time to both the NGOs but flexibility was given to make local adaptation and refinements, particularly in rooting and hardening stages. For example, by using vermicompost instead of soil rite and coco peat, the center at SAIRD significantly reduced the cost and achieved comparable results. Minor modifications were also made in the poly tunnels and humidity control systems. The successful transfer of the technology to the NGOs was evident from the fact that more than 1.5 lakh tissue cultured plants could be produced during 6 years (1999 to 2004) in the pilot laboratories 24
at CRIDA and SAIRD (Fig.22). Initially the plants were given free of cost to the farmers in the target villages as a part of awareness generation activities. During second phase, however, the plants were marketed within and outside the target districts on cost basis by SAIRD while at CRIDA these were supplied both free for collaborative trials and on cost basis for individual farmers for block/ boundary plantations.
Establishment of Bio Centers Despite the successful transfer of micropropagation technologies to the production centers run by KVKs, the cost of micropropagated plants remained high. To reduce the product cost to the farmer an innovative mechanism of combining the micro and macropropagation methods through a farmer level bio center was tried in the project. This concept is based on using the plantations established through micropropagated plants as base material for under taking macropropagation at the farmers level. This was successfully tried with teak by SAIRD in Nalgonda district. A farmer who has a two year old TC plantation of an elite clone is provided a “bio center� facility involving a shade net, root trainers, plastic trays and chemicals. He is also trained on rooting technique. Alternate rows of the TC plantations
Micropropagation Technology for Multipurpose Trees
Fig.21: Laboratory and hardening facilities for micropropagation established at the SAIRD and YFA under the project 18000 16000
CRIDA
SAIRD
YFA
No. of plants
14000 12000 10000 8000 6000 4000 2000 0
1999
2000
2001
2002
2003
2004
Fig.22: Production of tissue cultured plants (teak + neam) during 1999-2004 at different centers under the project (The project was discontinued at YFA after 2001)
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Research Bulletin
are stumped to the ground and the stumps are provided with water and optimum nutrition. Within 3-4 months, profused coppice shoots grow from the ground level of the stumps which are then used as nodal cuttings for vegetative propagation. The detached coppice shoots from the stumps are cut into single nodal segments and kept for rooting in the root trainers. Within 3 weeks, these stumps develop excellent root system. From each stump, nearly 60-75 plants can be produced per season. By this technology, clonal planting material could be produced with one third
of the cost of the final product as compared to micropropagation. Three such bio centers were initiated during the second phase of the project. These centers can also produce other planting material in demand at the village level. The detailed steps involved in the bio center are depicted in Fig.23.
Participatory Research: A new model of Institute – NGO – Farmer linkage The technology development and evaluation of products was done in a
a
b
c
d
Fig. 23: Components of the typical rural bio center for clonal propagation involving a shade house (a), TC orchard stumped (b), re-grown healthy coppice shoots (c) and nodal cuttings transferred for rooting in root trainers (d)
26
Micropropagation Technology for Multipurpose Trees
participatory manner involving the stakeholders. Farmers were involved from the beginning of the project in choosing the tree species as relevant to their conditions. The technology was developed by the research institute (CRIDA) and transferred to an NGO (SAIRD) who also were actively involved in training the farmers and organizing demonstrations. This process enabled in an effective linkage between the research institutes and farmers through an NGO. Decentralization of the technology application at the NGO level also resulted in generation of new ideas and suitable refinement of the protocol to the local conditions. Overall, an effective Institution – NGO – Farmer linkage emerged, which worked quite well during the project period. In view of the constraints in organizing large number of on-farm trials across the state, CRIDA experimented with a unique model of farmer participatory research during second phase where in the planting material for 1 acre plantation are supplied by the institute free of cost and the participating farmers provide all inputs for plantation and maintenance. The farmer also provides information on site characteristics (rainfall, soil type, soil test report) etc. and collaborates in collection of data on height and girth at 3 monthly intervals. At the end of one year, the technician from CRIDA visits the site, verifies the observations of
the farmer and records the annual growth measurements and a video/still picture of the plantation. Progressive farmers who were interested in participating in these trials were given one day training at CRIDA on planting and data collection. This arrangement worked successfully and during 2001-2004, more than 50 such on-farm trials were organized in A.P. and neighbouring states, which generated useful data across locations with varying rainfall and soil types.
Adoption and Impact While the technology of production of planting material including the identification of mother plants was done successfully, the real impact of the technology can be measured only after harvesting the timber from these plantations and the proceeds realized by the farmers. So far, TC plantations have by and large demonstrated greater uniformity and at some sites marginally superior height increment over stumps and seedlings. However, the demonstrations and on-farm trials laid out so far generated good interest among the farmers, particularly on TC teak. There have been large number of requests from farmers all over the country during the participation of the institute scientists and technicians at Kisan melas and trade fairs. The total number of plants supplied by the institute, the farmers and 27
Research Bulletin Table 7: Micropropagated neem and teak plants supplied by CRIDA between 1998-2003 to farmers and entrepreneurs in 4 states State
No. of plants
Farmers covered
Area (ha) brought under block or border plantation
Andhra Pradesh
65,000
120
105
Maharashtra
14,500
18
26
Tamil Nadu
8,000
20
12
Karnataka
6,000
15
18
area covered in different states is given in Table 7. Innovative methods of cost reduction through village level bio centers
could reduce the cost of plants to the farmers and result in better diffusion of the technology.
Fig. 24 : Farmers training, sale and transport of micropropagated plants from CRIDA complex, Hyderabad
28
Micropropagation Technology for Multipurpose Trees
Conclusions It was possible to demonstrate through the project that neem and teak can be mass propagated through micropropagation and the technology can be successfully upscaled to produce more than one lakh plants/year at the district level production units run by NGOs/KVKs. The cost of plants can be further reduced if vegetative propagation is combined with micropropagation by setting up of village level bio centers. The model of Institute – NGO – Farmer linkage worked successfully in the project for production and participatory evaluation of micropropagated planting material. The project demonstrated that micropropagated plants successfully establish and grow under field conditions. Field evaluation of micropropagated plant material both onstation and on-farm showed variable results. In most trials, these plants showed equal performance upto the first six years with greater uniformity. The field performance depended largely on rainfall and effective soil depth. Long term evaluation of such planting material is required before drawing valid conclusions.
References Ahuja, M.R. (1993) Micropropagation of Woody Plants. Kluwer Academic Publishers, Netherlands, pp: 1-481.
Bonga, J.M. and Durzan, D.J. (1987) Cell and Tissue Culture in ForestryVol.1. General Principles and Biotechnology. Martinus Nijhoff Publishers, Dordrecht. Chandra, R. and Mishra, M. (2003) Comprehensive Micropropagation of Horticultural Crops. International Book Distributing Co., Lucknow, U.P, India. DBT (2000) Plant tissue culture: from research to commercialization, a decade of support, Department of Biotechnology, Ministry of Science and Technology, New Delhi, pp: 1-224. Ermel, K., Pahlich, E. and Schumutterer, H. (1987) Comparison of the azadirachtin content of neem seed from ecotypes of Asian and African region. In: Natural pesticides from neem tree and other tropical plants (Ed. H.Schumutterer, and K.R.S.Ascher), Eschborn, Germany. pp: 83-90. Mascarenhas, A.F., Kendurkar, S.V. and Khuspe, S.S. (1993) Micro-propagation of teak In: Micro-propagation of Woody Plants, (Eds. M.R.Ahuja) Kluwer Academic Publishers, Netherlands, pp:247-262. Randhawa N.S. and Parmar B.S. (1993) Neem: Research and development, Society for pesticide science, IARI, New Delhi, India, pp.283. 29
Research Bulletin
Rangaswamy, S. and Parmar, B.S. (1995) Azadirachtin A content of seeds of neem ecotypes in relation to the agroecological regions of India, Pesticide Research Journal 7(2): 140-148. Schumutterer, H. (1995) The neem tree and other meliacious plants: Source of unique natural products for integrated pest management, medicine industry and other purpose, VCH press, Weinheim, Germany, pp: 696. Singh, A., Negi, M.S., Moses, V. K., Venkateswarlu, B., Srivastava, P.S. and Lakshmikumaran, M. (2002) Molecular analysis of micro propagated neem plants using AFLP markers for ascertaining clonal fidelity. In Vitro Cell. Dev. Biol. (Plant) 38(5): 519-524. Sreenivasa Rao, M., Raman, G.V., Srimannarayana, G. and Venkateswarlu, B. (1999) Efficacy of botanicals against gram pod borer. Pestology XXIII (1): 18-22. Venkateswarlu, B., Katyal, J.C., Choudhuri, J. and Mukhopadhyay, K. (1998)
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Micropropagation of plus neem (Azadirachta indica A. Juss) and evaluation of field transferred plants. Indian Forester. 124(7) : 537-543. Venkateswarlu, B. and Mukhopadhyay, J. (1999) Azadirachtin content in the seeds of micropropagated neem plants in relation to its mother tree. Current Science 7(5): 626-627. Venkateswarlu, B., Mukhopadhyay, J., Sreenivasan, E. and Moses Kumar, V. (2001) Micro-propagation of Paulownia fortuneii through in vitro axilliary shoot proliferation. Indian Journal of Experimental Biology 39: 594-599. Venkateswarlu, B., Mukhopadhyay, K., Mukhopadhyay, J. and Katyal, J.C. (2002) Selection of plus trees of neem with emphasis on azadirachtin content and development of micropropagation protocol for mass propagation. In. Proceedings of the World Neem Conference, Vancouver, Canada (ed.H.M.Behl), Neem Foundation, Mumbai, India. pp: 190-205.