micropropagation technology for multipurpose trees, crida

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,


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.


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


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


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


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)


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


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


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


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)


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


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


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


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


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.

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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.

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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.

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Micropropagation of plus neem(Azadirachta indica A. Juss) andevaluation of field transferred plants.Indian Forester. 124(7) : 537-543.

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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.

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