Agriculture Reference
In-Depth Information
Fig. 8.1 a
Neem flower buds of 2 mm size bearing correct stage of microspores (6X)
b
An uni-
nucleate microspore of neem stained with acetocarmine (2300X)
Plant Material
For anther culture, 2 mm size flower buds (Fig.
8.1a
) were collected early in the
morning during the flowering season (April-May). The stage of microspore de-
velopment was determined by acetocarmine squash preparations. Anthers contain-
ing early-to-late uninucleate stage of microspores (Fig.
8.1b
) were cultured in the
laboratory.
Establishment of Aseptic Cultures
The flower buds, collected from a field, were rinsed with sterile distilled water
(SDW) several times, followed by surface sterilization in a glass vial with 0.1 %
mercuric chloride solution (HgCl
2
) for 7 min. Finally, the buds were washed three to
four times with SDW. The buds were dissected out with the aid of a binocular micro-
scope using pre-sterilized Petriplates, forceps and fine needles. Damaged anthers, if
any, were discarded. Twenty anthers, from two buds, were cultured in 55 × 15 mm
pre-sterilized, disposable Petriplates containing 10 ml of MS (Murashige and Skoog
1962
) medium. The Petriplates were sealed with parafilm and subjected to specific
treatment conditions.
Culture Media Preparation
A number of media compositions containing different concentrations of auxins and
cytokinins were used for inducing in vitro androgenesis in neem. MS media, devoid
of any growth regulator, did not induce any morphogenic response in anther cul-
tures. For successful induction of callus from anthers, a combination of auxins and
a cytokinin was found to be necessary in most of the cultures.
