沙漠研究 24-1, 37-40 (2014)
Ἃ₍◊✲ 24-1, 37 - 40 (2014)
Journal of Arid Land Studies
Journal of Arid Land Studies－ DT11 Refereed Paper －㸫DT11 Refereed Paper㸫

Clonal Multiplication of Multipurpose Desert Tree Azadirachta indica - Neem
Ashok GEHLOT1), I.D. ARYA*1), Vinod KATARIA2), R.K. GUPTA1) and Sarita ARYA1)
Abstract: Azadirachta indica A. Juss (Neem) is an untapped natural resource as far as its genetic potential is concerned. It is an
important semi-arid tree, which provides fuel wood, shelter, shadow, foliage and a large canopy. It is planted in wind-breaks toprotect food crops from the desert winds. It is used in shelterbelt plantations to reduce wind velocity, and helps in ecologicalrestoration and regeneration of mined wastelands. The maximum industrial utilization of neem is for its seed oil, which contains
Azadirachtin. Neem trees show considerable variability in the azadirachtin content in seeds irrespective of the habitat. The seeds
lose viability within two weeks. Propagation by seed is also undesirable because of the highly heterozygous nature of the plant due tocross-pollination. In vitro regeneration of Azadirachta indica was achieved through axillary shoot proliferation in nodal segments
collected from mature trees. The season and types of explants showed direct influence on bud break. Nodal explants collectedduring April gave the highest shoot bud sprouting percentage (100%), number of off shoot regeneration (5.65 ± 0.11) and growth ofregenerated shoots (5.02 ± 0.27) as obtained on a medium containing 9.00 µM/l BAP + 0.25 µM/l NAA. These in vitro
regenerated shoots further multiplied on MS medium + 9.00 µM/l BAP + 0.25 µM/l NAA, which resulted in the highest number ofoff shoots regenerated (9.35 ± 0.55) and growth of regenerated shoots (7.13 ± 0.54). Regenerated shoots were successfully in vitro
rooted on MS medium + 4.92 µM/l IBA with 100% rooting percentage, 9.50 ± 1.16 root numbers and 5.13 ± 0.18 root length. The
in vitro rooted plantlets were successfully hardened and acclimatized in a poly house. These plants showed a good survival rate of
95% under field conditions.
Key Words: Azadirachta indica, Azadirachtin, In vitro culture, Micropropagation

1. Introduction
Neem (Azadirachta indica) is a versatile Indian tree ofgreat importance. Neem is an untapped natural resource as
far as its genetic potential is concerned. Azadirachta indica is
one of the most valuable arid zone trees belonging to thefamily Meliaceae. A native of dry forest areas of India andthe subcontinent, it is widely cultivated in the arid,
nutrient-deficient regions of India and Africa. Neem tree isknown to increase soil fertility due to its water holding capacity
coupled to its unique property of calcium mining, whichchanges the acidic soil into neutral. The tree is resistant tohigh temperatures and drought and has been employed forafforestation of dry localities, reforesting bare ravines andchecking soil erosion (Gill et al., 1996). Azadirachta indica
A. Juss is renowned for its insecticidal properties and Neem
seed extracts show great potential as environmentally
acceptable bioinsecticides for crop protection (Jacobson, 1988;
Schmutterer, 1990) due to the presence of Azadirachtincompound. Vegetative propagation of an adult Neem tree by
conventional methods is difficult (Kaushik, 2002). Therefore,
it is normally grown from seeds but the seeds are of recalcitrant
type; they lose viability within 2-3 weeks (Mohan et al., 1996).
* Corresponding Author: aryaid@gmail.com

Propagation by seeds is also undesirable because of the highly
heterozygous nature of the plant owing to cross-pollination
which brings enormous heterozygosity.
In the present investigation, the main objective was toestablish a procedure which can be used routinely to produce
complete micropropagated plantlets from nodal explants ofmature trees of Neem.

2. Materials and Methods
Plant Materials: four types of nodal explants i.e. mature,semi-mature, young and shoot tips were used for theestablishment of aseptic cultures. In vitro shoots proliferated
as a result of axillary bud break were used to regenerate plants.
2.1. Explants preparation and surface sterilization
Under aseptic condition, explants were treated with 0.1%(w/v) Bavestin (BASF India Ltd. Mumbai, India) and 0.1%(w/v) streptomycin for 8-10 min to reduce the chance of fungal
contamination. After treatment these explants were rinsed
with autoclaved water for 3-4 times and treated with 0.1%(w/v) HgCl2 and streptomycin for 6 min for surface
sterilization. After treatment, explants were again rinsed with
autoclaved water 3-4 times.
㸦Received, September 15th, 2013; Accepted, January 14th, 2014㸧

Jodhpur (Rajasthan) 342005 India
1) Forest Genetics and Tree Breeding Division, Arid Forest Research Institute2) Department of Botany, JNV University

38

2.2. Nutrient media and culture conditions
Murashige and Skoog’s (MS: Murashige and Skoog, 1962)
medium was supplemented with different concentrations of
BAP(6-Benzylamino
purine)andKinetin(6-Furfurylaminopurine) and in combination with NAA(Naphthalene acetic acid). 3% sucrose with additives (567.76µM/l ascorbic acid, 260.24 µM/l citric acid and 484.38 µM/l
adenine sulphate) was used in the medium for culture initiation.
Agar-agar (Hi-media, India) was added to media as a gelling
agent at the concentration of 0.6% (w/v). The pH of themedium was adjusted to 5.8 ± 0.2 with 1N NaOH and the
medium was autoclaved at 15 psi and 121°C for 15 min.
Culture initiation and multiplication were done aseptically in alaminar air flow hood. Cultures were incubated in tissue
culture racks in an aseptic growth room having a temperatureof 26 ± 2°C in 16 hours light and 8 hours dark photoperiod and1600 lux intensity light via white cool florescent tubes (Philips,
India).
2.3. Multiplication of micro-shoots
MS medium, supplemented with BAP + NAA andadditives (567.76 µM/l ascorbic acid, 260.24 µM/l citric acidand 484.38 µM/l adenine sulphate), was used for multiplicationof microshoots.
2.4. In vitro rooting of micro-shoots
In vitro regenerated micro-shoots were harvested fromclumps and transferred to a medium for rooting. Individual
micro-shoots were transferred to MS medium enriched with
IBA + additive (86.85 µM/l Proline and 48.96 µM/l
Tryptophan) with sucrose (3%). The in vitro rooted
micro-shoots were removed from culture vessels and washedwith autoclave RO water to remove adhered nutrient agar.
These were carefully transferred to jam bottles containing
autoclaved vermiculite moistened with ½ MS salts. The
bottles were kept for 2 weeks for in vitro hardening under
growth room conditions. After 2 weeks, jam bottles
containing regenerated plants were kept in a poly houseinitially near the pad section (RH 80 - 90% and temperature 28± 2°C) in order to harden the plantlets.
2.5. Hardening and acclimatization of regenerated plantlets
After one week of transfer of in vitro raised plantlets to thepoly house, the caps of jam bottles were gradually opened overa period of 2 weeks and finally removed. Bottles containing
plantlets were then shifted from the pad section towards the fan
section to provide growing conditions of low humidity (50 60%) and high temperature (30 ± 2°C). After 4 weeks,
acclimatized plantlets were transferred to poly bags containing
a mixture of soil + sand + FYM (2:1:0.5). Such plantlets wereTable 1. Effect of cytokinin (BAP) and auxin (NAA 0.25 µM/l) on
shoot bud proliferation of Azadirachta indica.

25.00 (29.73)c
40.00 (38.95)c

Number ofoffshoots
regenerated
1.40±0.24d
2.50±0.19cd

Growth ofregenerated
shoots (cm)0.82±0.14c
2.05±0.25b

2.25
4.5
9
18
M ean

55.00 (47.89)c
80.00 (66.59)b
100.00 (88.72)a
80.00 (69.75)b
63.33 (56.94)

3.09±0.49c
4.63±0.34ab
5.65±0.11a
4.44±0.48b
4.20±0.21

2.81±0.28b
3.99±0.01a
5.02±0.27a
3.98±0.47a
3.67±0.19

F- value

15.09*

14.39*

16.18*BAP (µM/l)

S hoot bud
S prouting %0
0.5

kept in the poly house for 3 - 4 weeks and then shifted to an
agro-shade house and later to the open environment.
2.6. Experimental design and statistical analyses
Data were analysed using a statistical package for social
sciences (SPSS 8.0). Duncan Multiple Range Test (DMRT) P= 0.05 was used to compare the means from main effects. A
minimum of 4 replicates with 5 samples (one explant) weretaken per treatment and with repetition of experiments thrice.
Degree of variation was shown by mean and standard error.
Data given in percentages were subjected to arcsine ξ)transformation (Snedecor and Cochran, 1967) before statisticalanalysis.

3. Results and Discussion
In the present investigation, axenic cultures of Azadirachta
indica were established from nodal segments derived frommature trees. The explants harvested during April were foundmost suitable for culture establishment. One way analysis ofvariance (ANOVA) showed that BAP + NAA had a highly
significant effect on shoot bud sprouting percentage (F5 = 15.09,
p<0.05), number of off shoots regenerated (F5 = 14.39, p<0.05)
and growth of regenerated shoots (F5 = 16.18, p<0.05). In
Table 1 it is shown that the different concentration of BAP in
combination with NAA had a significant effect on shoot bud
sprouting percentage, number of off shoots regenerated andgrowth of regenerated shoots. The highest shoot bud
sprouting percentage (100%), number of off shoot regenerated(5.65 ± 0.11) and growth of regenerated shoots (5.02 ± 0.27)
were obtained with the treatment of 9.00 µM/l BAP + 0.25
µM/l NAA. These in vitro regenerated micro shoots were
further amplified on BAP + NAA. Table 2 shows that BAP+ NAA had a highly significant effect on number of off
shoots regenerated (F5 = 35.30, p<0.05) and growth ofregenerated shoots (F5 = 24.68, p<0.05).

39

Table 2. Effect of cytokinin (BAP) and auxin (NAA) on shoot
Table 2. Effectof cytokinin(BAP) indica.
and auxin (NAA) on shootmultiplicationof Azadirachta
multiplication of Azadirachta indica.

BAP
NAA
BAP
NAA
BAP
NAA
(µM
(µM
/l)
(µM/l)/l)(µM/l)(µM /l)(µM /l)
00
00
0
0
0.5
0.25
0.5
0.25
0.5
0.25
2.25
0.25
2.25
0.25
2.25
0.25
4.5
0.25
4.5
0.25
4.5
0.25
99
0.25
0.25
9
0.25
18
0.25
18
0.25
18 M ean 0.25
M ean
M ean
FF- valuevalue
F- value

Numberof
Number of
Numberoffshoots
offshootsof
offshoots
regenerated
regenerated
regenerated
1.73±0.19d
1.73±0.19d
1.73±0.19d
3.10±0.41c
3.10±0.41c
3.10±0.41c
3.69±0.43bc
3.69±0.43bc
3.69±0.43bc
4.95±0.28b
4.95±0.28b
4.95±0.28b
9.35±0.55a
9.35±0.55a
9.35±0.55a
4.83±0.51b
4.83±0.51b
4.83±0.51b
5.12±0.31
5.12±0.31
5.12±0.31
35.30*
35.30*
35.30*

GrowthofGrowth of
Growthofregenerated
regenerated
regenerated
shoots(cm)
shoots (cm)shoots (cm)1.06±0.15d
1.06±0.15d
1.06±0.15d
2.36±0.35c
2.36±0.35c
2.36±0.35c
3.14±0.33bc
3.14±0.33bc
3.14±0.33bc
4.18±0.20b
4.18±0.20b
4.18±0.20b
7.13±0.54a
7.13±0.54a
7.13±0.54a
4.11±0.48b
4.11±0.48b
4.11±0.48b
4.09±0.26
4.09±0.26
4.09±0.26
24.68*
24.68*
24.68*

Table 3. Effect of IBA on in vitro rooting of Azadirachta indica.
Table 3. Effect of IBA on in vitro rooting of Azadirachta indica.
IBA
NumberM
IBA
Number ofof
Mean
ean rootroot
Rooting
Rooting %%IBA
Number
of
M
ean
root
(µM
/l)
Roots
length(cm)(µM /l)Roots
length (cm)Rooting %(µM /l)Roots
length (cm)00
35.00
(36.06)d2.14±0.14c
1.04±0.17d
35.00 (36.06)d2.14±0.14c
1.04±0.17d
035.00 (36.06)d2.14±0.14c
1.04±0.17d
0.49
45.00
2.67±0.29bc
2.12±0.43cd
0.49
45.00 (41.83)cd(41.83)cd
2.67±0.29bc
2.12±0.43cd
0.49
45.00 (41.83)cd
2.67±0.29bc
2.12±0.43cd
2.46
70.00
(60.26)bc
3.21±0.24bc
2.65±0.43bc
2.46
70.00 (60.26)bc
3.21±0.24bc
2.65±0.43bc
2.46
70.00 (69.76)ab(60.26)bc
3.21±0.24bc
2.65±0.43bc
3.94
85.00
5.00±0.48b
3.44±0.41b
3.94
85.00 (69.76)ab
5.00±0.48b
3.44±0.41b
3.94
85.00 (69.76)ab
5.00±0.48b
3.44±0.41b
4.92
100.00(88.72)a9.50±1.16a
5.13±0.18a
4.92
100.00 (88.72)a
9.50±1.16a
5.13±0.18a
4.92
100.00(57.39)bv(88.72)a9.50±1.16a
5.13±0.18a
9.84
70.00
3.09±0.39bc
9.84
70.00 (57.39)bv 4.79±0.48b
4.79±0.48b
3.09±0.39bc
9.84
70.00
(57.39)bv
4.79±0.48b
3.09±0.39bc
M
67.50
5.26±0.43
3.31±0.20
Mean
ean
67.50 (59.00)(59.00)5.26±0.43
3.31±0.20
M ean
67.50 (59.00)
5.26±0.43
3.31±0.20
F7.42*
13.16*
13.60*
F- value
value
7.42*
13.16*
13.60*
F- value
7.42*
13.16*
13.60*The different concentrations of BAP in combination withThe different concentrations of BAP in combination with
NAA had a significant effect on number of off shoots
NAA had a significant effect on number of off shoots
regenerated and growth of regenerated shoots. Highest
regenerated and growth of regenerated shoots. Highest
number of off shoot regeneration (9.35 ± 0.55) and growth ofnumber of off shoot regeneration (9.35 ± 0.55) and growth ofregenerated shoots (7.13 ± 0.54) were obtained on medium
regenerated shoots (7.13 ± 0.54) were obtained on medium
treated with 9.00 µM/l BAP + 0.25 µM/l NAA. For rooting
treated with 9.00 µM/l BAP + 0.25 µM/l NAA. For rootingof in vitro regenerated shoots, IBA was found to be the mostof in vitro regenerated shoots, IBA was found to be the mostsuitable for root induction. The individual in vitro shoots
suitable for root induction. The individual in vitro shoots
regenerated were tested for their ability to form roots. The
regenerated were tested for their ability to form roots. The
rooting treatment consisted of MS medium supplemented withrooting treatment consisted of MS medium supplemented with
IBA. One way analysis of variance (ANOVA) showed that
IBA. One way analysis of variance (ANOVA) showed that
the IBA treatment resulted in highly significant affect on
the IBA treatment resulted in highly significant affect on
rooting % (F5 = 7.42, P<0.05), root number (F5 = 13.16,
rooting % (F = 7.42, P<0.05), root number (F5 = 13.16,
P<0.05), and 5mean root length (F5 = 13.60, P<0.05).
The
P<0.05), and mean root length (F5 = 13.60, P<0.05). The
results showed that different concentrations of IBA
results showed that different concentrations of IBA
significantly affect the plant regeneration parameters,
significantly affect the plant regeneration parameters,rooting %, root per shoots, and mean root length. The
rooting %, root per shoots, and mean root length. The
optimal rooting of in vitro shoots was obtained on the medium
optimal rooting of in vitro shoots was obtained on the medium
with 4.92 µM/l IBA resulting in 100% rooting percentage, 9.50
with 4.92 µM/l IBA resulting in 100% rooting percentage, 9.50
± 1.16 root number and 5.13 ± 0.18 root length (Table 3).
± 1.16 root number and 5.13 ± 0.18 root length (Table 3).
Plantlets with well developed shoot and root systems werePlantlets with well developed shoot and root systems weretransferred to soil after hardening and acclimatization.
transferred to soil after hardening and acclimatization.
Establishment of aseptic cultures of Neem was not possible
Establishment of aseptic cultures of Neem was not possible

round the year since significant seasonal fluctuation affectedround the year since significant seasonal fluctuation affected
the explant viability in culture.
the explant viability in culture.
Season of collection of explants is one of the important
Season of collection of explants is one of the importantfactors in the establishment and growth of in vitro cultures
factors in the establishment and growth of in vitro cultures(Siril and Dhar, 1997).
Explants collected during(Siril and Dhar, 1997).
Explants collected during
March-April responded best in culture as during this period ofMarch-April responded best in culture as during this period of
the year, not only maximum bud-break leading to regeneration
the year, not only maximum bud-break leading to regenerationof shoots was obtained, but the infection percentage was alsoof shoots was obtained, but the infection percentage was also
low. To be more specific, mid-April was the best time forlow. To be more specific, mid-April was the best time for
explant collection for obtaining maximum bud-break which is
explant collection for obtaining maximum bud-break which isabout 80% with minimum infection, i.e., 20%. Similar
about 80% with minimum infection, i.e., 20%. Similar
results were obtained by Chaturvedi et al. (2004) in Neem.
results were obtained by Chaturvedi et al. (2004) in Neem.
BAP was found to be most effective cytokinin in inducing
BAP was found to be most effective cytokinin in inducing
multiple shoot formation. Similar findings have been
multiple shoot formation. Similar findings have beenreported by Arya et al. (1995), Chaturvedi et al. (2004) andreported by Arya et al. (1995), Chaturvedi et al. (2004) andQuraishi et al. (2004). IBA has also been reported suitable for
Quraishi et al. (2004). IBA has also been reported suitable forrooting in neem by several workers (Eeswara et al., 1998; Salvi
rooting in neem by several workers (Eeswara et al., 1998; Salvi
et al., 2001; Quraishi et al., 2004). The present work exhibits
et al., 2001; Quraishi et al., 2004). The present work exhibits
superiority over in vitro shoot multiplication rate and in vitro
superiority over in vitro shoot multiplication rate and in vitro
rooting without callus formation which was a bottle neck in all
rooting without callus formation which was a bottle neck in all
earlier work reported.
earlier work reported.

4. Conclusion
4. Conclusion
A reproducible protocol is developed for the regeneration
A reproducible protocol is developed for the regenerationof complete plants from nodal segments containing axillaryof complete plants from nodal segments containing axillarybuds. The Neem plant growth and development wasbuds. The Neem plant growth and development was
remarkably affected by growth regulator type andremarkably affected by growth regulator type andconcentration as described in the protocol.
concentration as described in the protocol.
Acknowledgements
Acknowledgements
Authors are grateful to the State Forest Department,
Authors are grateful to the State Forest Department,
Gujarat of India for financial support of the work.
Gujarat of India for financial support of the work.

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