Agriculture Reference
In-Depth Information
marized below. The following methods were used to induce in vitro gynogenesis in
tea plants (Hazarika and Chaturvedi; unpublished data).
Plant Material
For ovary slice culture in tea, unopened and unpollinated mature flower buds
(6-10 mm) size were collected early in the morning. Some of the buds were fixed
in FAA (5:5:90 v/v/v Formaldehyde: Acetic acid: 70 % Ethanol) for 48 h and then
stored in 70 % alcohol. Later, the appropriate developmental stage of the embryo
sac was determined by histological analysis.
Establishment of Aseptic Cultures
The mature unpollinated tea flower buds were surface sterilized with 0.1 % HgCl
2
for seven minutes, followed by rinsing with sterile distilled water at least thrice.
Carefully dissected transverse sections of ovaries were cultured on Murashige
and Skoog's (MS) media supplemented with varying concentrations of auxins
and cytokinins. Six ovary slices containing unpollinated ovules were cultured in
60 × 15 mm pre-sterilized disposable Petriplates containing 10 ml MS medium.
The sealed Petriplates were subjected to various regimes of temperature and light
treatments.
Culture Media Preparation
Murashige and Skoog (
1962
) basal medium was used throughout the studies to raise
ovary slice cultures. MS basal medium was supplemented with a range of growth
regulators, viz., 2, 4-Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene acetic
acid (NAA), N
6
- Furfuryladenine (Kinetin), 6-Benzylaminopurine (BAP) and Thid-
iazuron (TDZ) either individually or in combinations. The media contained 3 %
sucrose and was gelled with 0.8 % Agar.
Culture Conditions
The ovary slice cultures from each media were subjected to an array of pre-treat-
ments, like cold (4 °C) and heat shock (33 °C), for various durations under both
light and dark incubation. The light and dark incubation at 25 °C served as a control
in all the experiments. After the application of treatments, the cultures were main-
tained continuously at 25 ± 2 °C condition with 50-60 % relative humidity under
16/8 h (light/dark) photoperiod irradiance (1000-2000 lx) provided by cool daylight
fluorescent tubes. The cultures were observed periodically and the morphological
changes were recorded at weekly intervals.
