Studying the effect of genotype and culture medium, biochar and 2,4-D pretreatment on androgenesis efficiency through tomato anther culture (Lycopersicum esculentum L.)

Introduction: Tomato (Lycopersicum esculentum L.) is one of the most economically and nutritionally important vegetable crops cultivated worldwide and is a major target of plant breeding programs. The development of new cultivars and pure lines through conventional breeding requires several generations, extensive labor, and considerable time. Anther culture is an effective technique for producing haploid and doubled haploid plants and allows breeders to obtain completely homozygous lines within a single generation. The success of androgenesis through anther culture depends on several factors, including genotype, culture medium composition, plant growth regulators, and supplementary compounds added to the medium. Therefore, this study was conducted to investigate the effects of genotype, culture medium, 2,4-dichlorophenoxyacetic acid (2,4-D) pretreatment, and biochar application on androgenesis and callus induction in tomato anther culture.
Materials and Methods: The responses of tomato anthers to the interaction between genotype and different culture media, various concentrations of 2,4-D pretreatment, different exposure times to 500 µM 2,4-D, and different concentrations and types of biochar incorporated into the culture medium were evaluated. Callus induction percentage and the percentage of calli larger than 5 mm were recorded as the main indicators of androgenic response. Experimental data were analyzed using Minitab 14 and SPSS 16 software, and mean comparisons were performed using Duncan’s multiple range test at the 5% probability level. Graphs were prepared using Microsoft Excel.
Results: The interaction between genotype and culture medium significantly affected both callus induction percentage and the percentage of calli larger than 5 mm (P < 0.001). The highest callus induction was observed in the cultivar Isabella on N6 medium (88.88%), whereas the cultivar Ramtin showed the highest response on B5 medium (86.66%). Significant differences among different concentrations of 2,4-D were detected for both traits (P < 0.01). Pretreatment with 500 µM 2,4-D for one hour resulted in the highest callus induction rate (100%). Different exposure durations to 500 µM 2,4-D also significantly influenced callus induction (P < 0.001) and the percentage of calli larger than 5 mm (P < 0.05). The highest percentage of large calli (40%) was obtained after 0.5 h of pretreatment. A significant interaction between biochar type and concentration was observed for both traits (P < 0.001). Double-filtered biochar at a dilution of 1:1000 produced the highest callus induction (95.55%) and the highest percentage of calli larger than 5 mm (35.55%), whereas no callus formation occurred in the treatment containing single-filtered biochar at the same concentration. Chromosome counting confirmed the origin of the regenerated tissues. Root-tip cells contained 24 chromosomes (2n = 2x = 24), while cells derived from anther culture calli contained 12 chromosomes (n = x = 12), confirming the haploid nature of the induced calli.
Conclusion: The results demonstrated that the androgenic response of tomato anthers is highly dependent on genotype, culture medium, 2,4-D pretreatment, and biochar application, with each genotype exhibiting its optimum response under a specific culture medium. Appropriate 2,4-D pretreatment and biochar supplementation significantly enhanced both the frequency and quality of callus induction. Cytological analysis confirmed the successful production of haploid tissues through anther culture, highlighting the effectiveness of this technique for haploid production in tomato. These findings provide valuable information for optimizing androgenesis protocols, improving haploid and doubled haploid plant regeneration, and accelerating tomato breeding programs through the rapid development of completely homozygous lines.