Agriculture Reference
In-Depth Information
growth inhibiting properties against a wide range of pests and thus, has been well
accepted as an eco-friendly, biodegradable biopesticide. Today, due to the remark-
able properties shown by azadirachtin and other related triterpenoids, the tree has
attained universal significance. Almost each and every part of this tree, particularly
the leaves, bark and seeds, has manifold applications. Besides being a popular av-
enue tree, with a large crown, the wood of this tree is used as timber for house build-
ing, furniture and other domestic and agricultural tools.
Androgenesis is a very useful technique for resolving the problem of self-incom-
patibility, heterozygosity and long gestation period in this tree species. However,
limited attempt has been made for the overall improvement of this valuable tree
through in vitro haploid production. Gautam et al. (
1993
) made the initial attempts
to produce haploids of neem for which they cultured anthers at the uninucleate stage
of microspores and observed formation of multicellular pollen. However, all the
plants regenerated from anther callus were diploids. Chaturvedi et al. (
2003
), for the
first time, generated androgenic haploids of neem by anther culture, at early-to-late
uninucleate stage of pollen. Callusing from anthers was induced on MS basal me-
dium (with 9 % sucrose) supplemented with 2, 4-D (1 μM), NAA (1 μM), and BAP
(5 μM). The calli multiplied best on MS medium (with 3 % sucrose) supplemented
with 2, 4-D (1 μM), and Kinetin (10 μM). MS supplemented with BAP (5 μM) was
optimum for regeneration from younger calli (75 % cultures differentiated shoots),
but older calli showed the best regeneration at 7.5 μM BAP. As per histological
investigations, in four-week-old cultures the anther-wall cells had started dividing,
while the microspores appeared unchanged. However, in eight-week-old cultures, it
was observed that the entire anther locules were filled with microcalli. Calli main-
tained on MS medium containing 2, 4-D exhibited good regeneration potentiality,
but those calli that were maintained on MS medium containing 2, 4-D (1 µM) and
Kinetin (10 µM), retained the regeneration potential for a longer period. Elongation
of shoots was achieved at a lower concentration of BAP at 0.5 µM. These shoots
were multiplied by forced axillary branching and were rooted through in vitro on ¼
strength of MS medium supplemented with IBA (0.5 µM). The plants were subse-
quently established in soil. Of the plants that regenerated from anther callus, 60 %
were haploids (2n = x = 12), 20 % were diploids (2n = 2x = 24) and 20 % were aneu-
ploids (2n = 2x−2 = 22). Srivastava and Chaturvedi (
2011
) reported a new improved
method of haploid production from androgenic cultures of
Azadirachta indica
A.
Juss. In this investigation, the best callus induction response was obtained in the
induction medium with 12 % sucrose concentration on MS + 2,4-D (1 µM) + NAA
(1 µM) + BAP (5 µM). Maximum shoot regeneration frequency obtained was
98.5 % with an average of 8.5 shoot-buds/ explant on MS + BAP (2.2 μM) + NAA
(0.05 μM), as against 75 % with cultures forming an average of 4.5 shoot-buds/
explants on MS + BAP (5 μM) in the earlier report of anther culture of neem by
Chaturvedi et al. (
2003
). Cytological analysis of the calli and regenerants confirmed
their haploid status with the chromosome number as 2n = x = 12.
To date, there is only one single report on in vitro ovary culture of neem by Sriv-
astava et al. (
2009
). However, the regenerated plants were diploid in nature. Un-
