Main tillers were harvested from field grown plants soon after the emergence of flag-leaf ligule and stored at 7±1°C with their base dipped in plain water for 3-5 days. Before isolating anthers, the spikes enclosed in leaf sheath were surface sterilized with 70% ethyl alcohol. Anthers from only such spikes were selected for culturing which on microscopic examination revealed mid to late uninucleate stage of microspore development.

For all the experiments, media modified with various supplements were used for anther culture. For culture, 90 mm diameter glass petri dishes were used. In each dish, approximately 100 anthers were cultured. To avoid variations within ear head, anthers from each spike were evenly distributed among culture media tested in the respective experiments. All the cultured dishes sealed with Parafilm were incubated at 28±1°C in absence of light for 7 days and then transferred under dim light conditions for 16 hr photoperiod at 22±2°C.
Effect of culture media and genotypes for anther culture (Exp. 1): Experiment was conducted with 16 genotypes of durum wheat (2n=4x=28) (Table 1). Seeds were supplied by the Wheat Improvement Research Programme, JNKVV, Jabalpur, India. Cultivars were selected for their high yielding ability, crop duration, drought tolerance and disease resistance properties due to which they are often included in the wheat breeding programs of Central India. Anthers from sixteen durum wheat genotypes were cultured on two modified N₆ (Chu 1978) media, (i) N₆ NB supplemented with 1.0 mg/l NAA +1.0 mg/l BA and (ii) N₆ DK with 2.0 mg/l 2,4-D + 0.5 mg/l kinetin. Both the media were added with 326 mg/l 1, glutamine + 100 mg/l m- inositol + 90 g/1 sucrose + 7.5 g/l agar.

The experiment was conducted in completely' randomized design in three replications. The statistical significance of treatment difference was evaluated by the t-test (Steel and Torrie 1980). For each experiment, percentages were transformed to arcsin √P.

The calli attaining 2-3 mm diameter size were transferred for plant regeneration to MS (Murashige and Skoog 1962) medium containing 0.4 mg/l IAA, 0.4 mg/l BA, 20 g/l sucrose and 7.5 g/l agar. The cultures were kept under light (fluorescent 3600 lx intensity) and dark cycle of 16/8 h at 22±2°C. In case, where no root formation took place, shoots were transferred to MS medium containing 0.5 mg/l IBA, 20g/l sucrose and 7.5 g/l agar.

All observations were based on initial culture media, irrespective of regeneration medium or rooting medium.
Effect of sugars and gelling agents on anther culture (Exp.2): Anthers from two high responding genotypes for in vitro androgenesis, A 9-30-1 and HI 8381 were cultured on three N₆ DK media (as described in Exp. 1) and as carbon source and gelling agent supplemented with (i) sucrose 90 g/l + agar 7.5 g/l, (ii) maltose 90 g/l + agar 7.5 g/l, and (iii) maltose 60 g/l + agarose 5 g/l. For plant regeneration, similar methodology as described in the Exp. 1 was used.

The experimental design used was split plot design, approximately. 480 anthers were plated for each genotype and medium combination. Comparisons were made by Fisher's protected LSD test after transforming the data (Steel and Torrie 1980).
Long tern maintenance of androgenic callus cultures (Exp. 3): Opaque and nodular embryogenic calli, generated after 45 days culture of anther of HI 8381 during previous experiments were sub-cultured on three different MS maintenance media supplemented with (i) 1.0 mg/l 2,4-D (ii) 2.0 mg/l 2,4-D and (iii) 2.0 mg/l 2,4-D + 5.0 mg/l silver nitrate. All the media were supplemented with 30 g/l sucrose and 8 g/l agar. Sub-culturing in fresh media was done at every 1-month interval. The culture conditions for callus maintenance were 22±2°C under 16/8h light/dark cycle of 2000 lux illumination.

The experiment was conducted in completely randomizes design and the data were analyzed by Duncan's new multiple range test (Steel and Torrie 1980).