Tomato (Lycopersicon esculentum Mill.) is a highly favored solanaceous vegetable crop in the world. Development and maintenance of completely homozygous lines of tomato, thus, have prime importance for its quality and yield augmentation. Among different techniques, anther culture is an effective way to achieve doubled haploids in tomato though it is poorly developed yet as tomato is highly recalcitrant to androgenesis. To address this issue an anther culture experiment is set up for two commercial F1 cultivars of tomato- ‘Campari’ and ‘Temptation’. Normally, flower buds of larger size possess microspores at advanced development stages. To study the microspore development stage at particular bud length and its effect on haploid/dihaploid regeneration, the anthers were squashed with 1% acetocarmine and microspore development stages were studied under light microscope. Flower buds of below 5.11 mm length were found to contain microspore mother cells exclusively while flower buds of 5.11-6.13 mm were found to have microspores from pre-meiotic to mid microspore. Flower buds longer than 15.05 mm were found to contain pollens at late developmental stage.
Among the anthers from different lengths of flower buds, the ones showing higher percentage of callus formation were from flower buds of 4 mm (63%) in Campari and 7 mm (72%) in Temptation which were not significantly different (p<0.05, DMRT) with that of 5 mm in Campari and 6 mm in Temptation respectively. Regarding the effect of cold pretreatment, anthers pretreated at 48 hrs. were found to be more responsive to callogenesis for both cultivars (67±5.78% in Campari and 53±5.18% in Temptation). Among different media treatments, the MS media supplemented with 2 ppm IAA and 1 ppm 2ip was found best for callogenesis (71±3.79% in Campari and 31±6.23% in Temptation) and shoot regeneration (29±4.58% in Campari and 9±3.15% in Temptation) which was not significantly different, in case of Campari, with that supplemented with 2 ppm IAA and 3 ppm BA (p<0.05, DMRT) giving 65±5.43% callus induction and 26±3.71% shoot regeneration. For elongation of regenerated shoots, MS media supplemented with 0.5 mg/l GA was found to be effective (100% for both cultivars) but the elongated shoots were weak and lanky while those elongated at MS media supplemented with 0.25 mg/l each of BAP and GA were found to give normal growth to the regenerants (98±1.3% for Campari and 97±2.13% for Temptation. For rooting, half-strength MS (2.2% MS + 10% sugar) was found highly responsive for both cultivars (66±4.76% in Campari and 59±5.86% in Temptation) as compared to other treatments. The effect of callus age on ploidy level of its own and its regenerants were also studied. Calli of 1 and 14 day/s were found to be highly mixoploid of n+2n cells (53.33±6.67% at 1 and 14 day/s and 60.00±11.55% at 7 days respectively). At the age of 28 days 53.33±6.67% of the initial calli were found to be at diploid state which is comparatively higher for diploid state than at any other ages. Ploidy level of calli was found to increase up to 8n (13.33±6.67) by 112 days. With increase in calli age mixoploidy was also went on higher ploidy state. Too earlier as well as prolonged age of calli were found to regenerate few if not no normal diploid shoots (0% at 1days and 13.33% at 112 days). Beyond 42 days, the number of total regenerants were found to plunge down with simultaneous decline of diploid, transient increase in mixoploidy (20.00±11.55% at 56 days), and gradual increase in chimera.
Regarding the direct androgenesis of tomato anthers, Cysteine was found to give initial positive effects on the cultured anthers with their swollen characteristics. Among 3 media (N6, MS, and CP) former two were found effective while CP was not.
Among 37 total regenerants (22 from Campari and 15 from Temptation), ploidy analysis showed that 67.57% were diploid, 16.22% were tetraploid, 5.41% were mixoploid, 5.41% were chimera and 2.70% were found aneuploid. The chromosome number in diploids (2n=24), tetraploids (4n=48), mixoploid (n+2n) and (2n+4n), and aneuploid (n=14) were counted in 1% acetocarmine squash of root tips of the regenerants. The comparative stomatal study showed that there is highly positive (p<0.01) correlation between increased ploidy level with each of stomata length (0.581), stomata width (0.664) and chloroplast number (0.852) and highly negative (p<0.01) correlation with each of stomatal density (-0.843) and stomatal index (-0.722). Stomata characteristics and chloroplast number in its guard cell can be a simple and inexpensive tool to determine ploidy level.
For the determination of the homozygosity and confirm the gametic origin of the regenerants, SSR analysis was carried out. Three-step marker analysis method was devised. Different 17 SSR markers were used in the study. SSR polymorphism was determined by developing F2-SSR profile for both cultivars. Marker analysis showed only 8.11% (5.41% Campari + 2.70% Temptation) of total regenerants to be homozygous. Morphological study of the homozygous regenerants showed variation in leaf and fruit characteristics. C-2 and C-5 (regenerants of Campari) were found to have smaller leaf size and distinct morphology while T-2 (a regenerant of Temptation) was found to have nearly similar plant morphology to the respective anther donors. C-2 was found to bear the smallest fruits among all the regenerants with 20.36±2.46 g. fruit weight and 33.62±1.32 mm equatorial diameter.