Mar 16, 2016
Micropropagation Technologyfor Multipurpose Trees : FromLaboratory to Farmers Fields
Research Bulletin
Central Research Institute forDryland AgricultureSantoshnagar, Hyderabad
B.Venkateswarluand
G.R.Korwar
In collaboration with
AP-NL Biotechnology ProgrammeBiotechnology 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: [email protected]
ForewordDr.M.V.Rao,Chairman, BPCInstitute of Public EnterprisesOsmania University CampusHyderabad – 500 007
Multi purpose trees (MPTs) like neem and teak are important components ofthe agroforestry systems in drylands. These trees provide supplementary income throughtimber, fuel wood, fodder, by-products development and also to improve soil fertilitythrough nutrient recycling. The Andhra Pradesh Netherlands Biotechnology Programme(APNLBP) initiated with an aim to apply different biotechnologies for improving thedryland farming systems in Nalgonda and Mahabubnagar districts of Andhra Pradesh,accorded high priority for identification of superior germplasm of locally importantMPTs and their mass propagation through micro propagation in order to make suchmaterial available to the farmers in these districts. Accordingly, a network project wassponsored to CRIDA and two collaborating NGOs i.e. SAIRD (Sri Aurobindo Instituteof Rural Development) and YFA (Youth For Action) on “Micro propagation technologydevelopment and participatory field evaluation of neem and teak”. The project madeexcellent progress and resulted in useful products, which are already widely adoptedby the farmers.
The scientists have identified plus trees of these species following a country wide surveyand successfully developed the protocols for micro propagation. More importantly, thetechnology was transferred to the field laboratories established by the NGOs in the targetdistricts of Nalgonda and Mahabubnagar. The technical personnel at the NGOs have beenwell trained by CRIDA, who also contributed their skills to further improve/refine theprotocol. The project served as a role model of Institute-NGO-Farmer linkage for developmentand transfer of this particular biotechnology. The project team also tried to introduce newinnovations like rural bio centers to reduce the cost of final products to the farmers. Thisproject is a good example of combining the basic and adaptive research to develop an usefulproduct for the farmers. I compliment Dr.B.Venkateswarlu and Dr.G.R.Korwar forcompiling the results and experiences from the project, in particular the data from theon-farm trials supported with good quality pictures. I hope this research bulletin will serveas an useful guide to all those researchers and developmental workers interested inapplication 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 (Y.S.Ramakrishna)Hyderabad Director, CRIDA
Acknowledgements
This bulletin summarises the experiences from a six year comprehensive projectentitled “Micropropagation technology development for neem and teak and farmerparticipatory field evaluation”. This project was entirely funded by Andhra PradeshNetherlands Biotechnology Programme (APNLBP). The project was implementedduring 1997-2004 by CRIDA as the lead institution and two collaborating NGOs inAndhra Pradesh i.e. Sri Aurobindo Institute of Rural Development (SAIRD) and YouthFor 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 assistanceand in particular Dr.M.V.Rao the Chairman, Biotechnology Programme Committee(BPC) and Dr.G.Pakki Reddy, Coordinator of the BTU for their guidance, counselingand 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 BTUalso facilitated the effective implementation of the project by timely release of funds,arranging various training programmes, field visits and reviews. We also wish to thankall external experts who reviewed the project and provided valuable suggestions.
The authors would like to place on record the excellent guidance, cooperationand 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 thetechnical programme. Dr.G.Subba Reddy, Head, Division of Crop Sciences also providedvaluable help for the success of the project at CRIDA. The contribution of both theNGOs and the staff of KVKs attached to them was immense in organizing the fieldtrials and farmers awareness programmes.
Large number of contractual staff worked in the project at all the 3 centres andput up their best to upscale the protocols successfully. The most important ones includecontractual 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 andMr.M.Eugine who provided effective assistance and also contributed their own ideasfor improvement of the protocols. Mr. M. Balakrishna (SAIRD) and Mr. Rajendra KumarReddy (YFA) helped in organising field trials. Mrs. M.A. Rekha prepared the manuscriptwho’s help is also gratefully acknowledged.
Authors
ContentsItem 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
Teak (Tectona grandis) . . . . 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
Micropropagation Technology for Multipurpose Trees
1
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
Research Bulletin
2
other constraints in wider use of this
technology are the lack of adequate data
on the field performance of micro-
propagated 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 Pradesh-
Netherlands 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
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 indicaA.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
3
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 andangiospermic 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 neemeco 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
Research Bulletin
4
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
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
Table 1: Range and means of azadirachtin content and related characters in neemseed samples collected from different locations (mean of 2 years)
Locations 100 seed wt. % Kernel % Oil % Azadirachtin(g) in seed in seed 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.
Micropropagation Technology for Multipurpose Trees
5
Table 2: Azadirachtin content of 30 neem trees (aged between 15-20 years) sampledin a grid of 15 sq.m area near Hyderabad (mean of 2 years)
Sl. No. Girth at Kernel 100 No. of Oil in Azadirachtinbreast height to seed seed wt. seeds seeds in kernels
(cm) ratio (%) (g) in 1 kg (%) (%)
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
Research Bulletin
6
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
Table 3: Azadirachtin and oil content (range and mean) in neem ecotype growing indifferent soil types in the arid, semi-arid and sub humid regions of India
Soil type No.of Oil content in Azadirachtin contentsamples seeds (%) 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
Inceptisols and
Entisols 21 16-28 20.5 0.16-0.62 0.39
Oxisols 12 15-26 22.5 0.25-0.69 0.41
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.
Germplasm Registration
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
Fig. 2. Variation in the azadirachtin content during1997-2004 in five selected trees of neem atHayathnagar Research Farm, CRIDA, Hyderabad
Aza
dira
chtin
(%
) in
ke
rne
ls
Micropropagation Technology for Multipurpose Trees
7
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.
Research Bulletin
8
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.
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.
Field transfer and progeny evaluation
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
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
Fig.3 : Flow chart of steps involved in micropropagation of neem
Micropropagation Technology for Multipurpose Trees
9
Plate 1: Stages in micropropagation of neem
Table 4: Summary of the micropropagation protocol for adult neem trees
Step Process/Method Result Remarks
Initiation of Explants inoculated in Shoot buds March to MayPrimary culture MS basal medium with produced from 90% optimum season for
1 mg/l BAP nodal axes explant collection
In vitro 0.5 – 1.0 cm long In 8 weeks, micro Maintenance of growthmultiplication micro shoots transferred shoots elongated room temperatureand elongation to the same medium upto 5 cm with a (25oC ± 1) and
with 0.2 mg/l BAP + multiplication ratio preventing excess0.5 mg/l kinetin of 1:6 moisture is critical
Rooting 4 to 5 cm long shoots 85% shoots rooted In rooting chambers,transferred to soilrite after 20-25 days humidity should bein rooting chamber after 100% but water loggingdipping into rooting should be avoided inhormone for 15 minutes the rooting medium
Hardening Rooted plantlets 85-90% plantlets Protection from fungaltransferred to soil and survived, but slow diseases andkept in mist chamber growth in polybags. preventing water(80-90% humidity) logging are criticalfor 30 days
Field transfer Two months old 100% plants survived T.C.plants attainhardened plants uniform growth, growtransferred to field just like seedlings
Research Bulletin
10
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 calluscells showing absence of vascular tissues, (c) cross section of the explant showing distinct vascularconnection between explant and the microshoot bud primordia
Plate 3: AFLP analysis of tissue cultured progeny of neem plus treefor confirmation of clonal fidelity (using Primer EAACxMCTC)
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
a b c
Micropropagation Technology for Multipurpose Trees
11
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
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
Fig.4. Growth of TC plants of clone CRI-8 at CRIDA complex after 42 months of planting
Fig.5: Azadirachtin content (A and B) in motherplants and tissue cultured progeny of twoplus trees of neem (MT : mother tree TCP:tissue cultured progeny)
Source: Venkateswarlu et al. (1999)
Research Bulletin
12
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
(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
Fig. 6: Performance of tissue cultured neem in the on-station trial at Hayathnagar ResearchFarm, Hyderabad (left) and on the farmers fields near Chakan, Maharashtra (right)
Micropropagation Technology for Multipurpose Trees
13
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 AP-
NL 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, micropro-
pagation 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
Fig.7 : Flow chart of steps involved inmicro propagation of teak
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
Research Bulletin
14
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
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.
Plate 4: Stages in micropropagation of teak
Micropropagation Technology for Multipurpose Trees
15
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 Cost of rootingcan be rooted in 1 kg* 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 stepmehtod
Fig. 8 : Single step rooting cum hardening of teak
Research Bulletin
16
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.
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).
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
Months after planting
Hei
ght (
m)
GB
H (
cm)
Fig.9: Growth pattern of TC teak plantation(cl. Teli) at KVK instructional farm, Gaddipalli
between 1998-2002
Fig.10: A view of the model plantation of TCteak (cl. Teli) at KVK instructional farm,
Gaddipalli, five years after planting
Micropropagation Technology for Multipurpose Trees
17
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 on-
farm 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
0Height (m) Girth (cm)
Fig.11: Height and girth of TC teak andstumps at HRF, 6 years after planting
Fig. 12: Six year old TC teak at HRF withfinger millet as intercrop
Research Bulletin
18
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 on-
farm 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.
Table 6. Annual increments in height and girth of tissue culture teakon farmers fields during the first 4 years
Age of the Mean annual increment Mean annual incrementplantation in height (m) in girth (cm)
Nalgonda* Mahabubnagar** Nalgonda* Mahabubnagar**
Year I 1.4 1.2 5.5 4.5Year II 1.5 1.3 5.6 5.2Year III 1.0 0.8 5.6 5.3Year IV 0.8 0.6 7.0 6.5
*Mean of 6 farmers **Mean of 5 farmers
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
Micropropagation Technology for Multipurpose Trees
19
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
Fig.13: Comparative performance of stump raised and tissue culture plants of teak (cl.Teli) on farmersfield (Mr. Ramkrishna Reddy) in Gaddipalli village, Nalgonda district, A.P. 2 years after planting
25
30
35
40TC Stumps
20
15
10
5
0Height (m) Girth (cm)
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
Fig 14: Comparative performance of tissue cultured teak (cl. Teli) and stumps on a clay loam soilin Ranga Reddy district (Rainfall-670 mm, age of the plantation - 6 years, average height (m) andgirth (cm), TC: 11.1 and 38.5, stumps: 6.25 and 24)
TissueculturedStumps
Research Bulletin
20
Fig.15: six months old plantation of tissue culture teak(cl. Teli) on farmers feild (Mrs. Anjamma at Gidwalvillage, Zaheerabad Mandal, Medak district, A.P. Plantingon contours and rainwater harvesting with half moonbasins resulted in 20% higher growth. Mean annualrainfall: 900 mm, soil type: Alfisol, average height: 2.7m, 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 18: Two year old plantation of tissue culture teak(cl. Teli) on farmers field (Mr.Kusu Nasaraiah atvillage kanchikacherla, Krishna district. Mean annualrainfall: 1100 mm, soil type: medium deep black soil,height: 8 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, Adilabaddist. Mean annual rainfall: 995 mm, soil type: red sandyloam, average height: 3.65 m and GBH: 13 cm)
Micropropagation Technology for Multipurpose Trees
21
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.
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.
3
2.5
2
1.5
1
0.5
0550 670 750 900 1250
3
2.5
2
1.5
1
0.5
0550 670 750 900 1250
MA
I in
girt
h (c
m)
Fig.19: Growth rate of teak in plantations as affected by rainfall (3-4 years)
Fig.20: Mean annual increment of teak indifferent soil types in rainfall zone of 670-900 mm(M=Medium; D=Deep)
MA
I (m
) in
hei
ght
1.2
1
0.8
0.6
0.4
0.2
0
Loam
y San
d
Sandy
loam
Sandy
clay
loam
Black s
oil (M
)
Black s
oil (D
)
Research Bulletin
22
Impact of soil amendments ongrowth 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 (Paulowniafortuneii)
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
Micropropagation Technology for Multipurpose Trees
23
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
chamber (Venkateswarlu et al., 2001). Plants
subjected to primary and secondary
hardening were successfully field transferred
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 withpublic/private sector
In addition to the on-farm and on-station
trials, collaborative trials with the following
organizations were initiated at different
periods during 1999-2002.
Name of the Species underorganization evaluation
Forest Research Centre, NeemMulugu, A.P. Teak
Maharashtra Forest TeakResearch Centre,Lohra, Chandrapur
A.P. Forest Development NeemCorporation, Nellore, A.P.
Sri Ramananda Tirtha TeakResearch Institute,Pochampally, Nalgonda, A.P.
EID Parry Research Centre, NeemCuddalore, Tamil Nadu
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.
Technology transfer to NGOs
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
Research Bulletin
24
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 vermi-
compost 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
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
25
CRIDA
18000
No
. of
pla
nts
16000
14000
12000
10000
8000
6000
4000
2000
1999 2000 2001 2002 2003 20040
SAIRD YFA
Fig.22: Production of tissue cultured plants (teak + neam) during 1999-2004 at differentcenters under the project (The project was discontinued at YFA after 2001)
Fig.21: Laboratory and hardening facilities for micropropagation establishedat the SAIRD and YFA under the project
Research Bulletin
26
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
Fig. 23: Components of the typical rural bio center for clonal propagation involving a shadehouse (a), TC orchard stumped (b), re-grown healthy coppice shoots (c) and nodal cuttingstransferred for rooting in root trainers (d)
b
dc
a
Micropropagation Technology for Multipurpose Trees
27
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
Research Bulletin
28
area covered in different states is given in
Table 7. Innovative methods of cost
reduction through village level bio centers
Table 7: Micropropagated neem and teak plants supplied by CRIDA between1998-2003 to farmers and entrepreneurs in 4 states
State No. of plants Farmers covered Area (ha) brought underblock 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
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 plantsfrom CRIDA complex, Hyderabad
Micropropagation Technology for Multipurpose Trees
29
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 on-
station 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) Micropropagationof Woody Plants. Kluwer AcademicPublishers, Netherlands, pp: 1-481.
Bonga, J.M. and Durzan, D.J. (1987)Cell and Tissue Culture in Forestry-Vol.1. General Principles andBiotechnology. Martinus NijhoffPublishers, Dordrecht.
Chandra, R. and Mishra, M. (2003)Comprehensive Micropropagationof Horticultural Crops. InternationalBook Distributing Co., Lucknow,U.P, India.
DBT (2000) Plant tissue culture: fromresearch to commercialization, adecade of support, Departmentof Biotechnology, Ministry ofScience and Technology, New Delhi,pp: 1-224.
Ermel, K., Pahlich, E. and Schumutterer,H. (1987) Comparison of theazadirachtin content of neem seedfrom ecotypes of Asian and Africanregion. In: Natural pesticides fromneem tree and other tropicalplants (Ed. H.Schumutterer, andK.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 WoodyPlants, (Eds. M.R.Ahuja) KluwerAcademic 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.
Research Bulletin
30
Rangaswamy, S. and Parmar, B.S. (1995)Azadirachtin A content of seeds ofneem ecotypes in relation to theagroecological regions of India,Pesticide Research Journal 7(2):140-148.
Schumutterer, H. (1995) The neem treeand other meliacious plants: Sourceof unique natural products forintegrated 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 micropropagated neem plants using AFLPmarkers for ascertaining clonalfidelity. In Vitro Cell. Dev. Biol.(Plant) 38(5): 519-524.
Sreenivasa Rao, M., Raman,G.V., Srimannarayana, G. andVenkateswarlu, B. (1999) Efficacyof botanicals against gram podborer. Pestology XXIII (1): 18-22.
Venkateswarlu, B., Katyal, J.C., Choudhuri,J. and Mukhopadhyay, K. (1998)
Micropropagation of plus neem(Azadirachta indica A. Juss) andevaluation of field transferred plants.Indian Forester. 124(7) : 537-543.
Venkateswarlu, B. and Mukhopadhyay,J. (1999) Azadirachtin content inthe seeds of micropropagated neemplants 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 ofPaulownia fortuneii through in vitroaxilliary shoot proliferation. IndianJournal of Experimental Biology39: 594-599.
Venkateswarlu, B., Mukhopadhyay, K.,Mukhopadhyay, J. and Katyal, J.C.(2002) Selection of plus trees ofneem with emphasis on azadirachtincontent and development ofmicropropagation protocol for masspropagation. In. Proceedings of theWorld Neem Conference, Vancouver,Canada (ed.H.M.Behl), NeemFoundation, Mumbai, India. pp:190-205.