Agriculture Reference
In-Depth Information
Analytical grade chemicals were used for preparation of stock solution and cul-
tured media. Stock solution for MS/N
6
media preparation can be summarized as
follows:
• Macronutrient - 10X; Micronutrient - 20X; Iron stock - 20X; Vitamins - 20X
• Growth regulator is used at a concentration of 1 × 10
−3
M
All constituents were stored at 4 ºC for further use. Myo-inositol and sucrose were
added freshly at the time of media preparation. The pH of the medium was adjusted
at 5.8 with 1N HCl or 1N NaOH. The media were solidified with 0.8 % agar and
autoclaved for 15 min at 1.06 kg cm
−2
and 121 ºC, before pouring in 60 mm size
Petriplates.
Culture Conditions
The anthers on each media were subjected to an array of pre-treatment conditions,
like cold and heat shock for various duration under both dark and light incubation.
Light and dark incubation at 25 ºC temperature served as a control in all the ex-
periments. All the cultures were maintained continuously at 25 ± 2 ºC and 50-60 %
relative humidity under 16/8 h (light/dark) photoperiod with diffuse light (1,000-
2,000 lx) provided by cool daylight fluorescent tubes (Philips TL40 W). Anther
cultures were initially kept continuously in the dark, but after eight weeks, the calli
that had developed from these cultures were transferred to multiplication medium
and maintained in light.
Twentyfour cultures were raised for each treatment, and each experiment was
repeated at least three times. The cultures were observed periodically and the mor-
phological changes were recorded at weekly intervals.
2.1.2 Pollen/Microspore Culture
Anther culture is beset with a number of problems. One of the major problems of
anther culture is the concomitant callusing of anther wall along with the pollen, as
a result of which the final tissue derived may not be of purely gametophytic ori-
gin. Moreover, the plants arising from an anther would constitute a heterogenous
population. It has been observed in some species that anther cultures show asynchro-
nous pollen development, the older grains may suppress the androgenic potential of
younger grains by releasing toxic substances (Bhojwani and Razdan
1996
). Kameya
and Hinata (
1970
) reported for the first time callus formation in isolated pollen cul-
tures of
Brassica oleracea
and the hybrid
B. oleracea
X
B. alboglabra
following
which successful pollen-derived androgenic plants have been produced in many crop
plants. There are numerous advantages of microspore culture over anther culture for
haploid plant production. Unlike anther culture, isolated microspore culture allows
haploid plant regeneration directly from microspores, assuring pure gametophytic
origin and, thus, avoiding mixing of proliferating anther walls. A homogeneous prep-
