A study of plant regeneration from the leaflets of microspores-derived homozygous plants generated from anther culture of 'Gala' apple

Abstract：【Objective】Apple is highly heterozygous due to its self-incompatibility, but the microsporesderived plantlets from the in vitro culture of‘Gala'apple anther are highly homozygous and can be a better material for genetic transformation. This study aimed to establish a stable and highly efficient in vitro regeneration system from leaves of the homozygous microspores-derived‘Gala'plantlets, so as to provide a good system for genetic transformation. These plantlets were regenerated on MS medium containing phytohormone (6-BA and IBA) , and the method was optimized by optimizing the combinationof plant growth regulators, the way of leaf wounding and dark culture【.Methods】The genotype of microspores-derived homozygous plantlets from anther culture of‘Gala'apple differed among plantlets.The leaves of microspores-derived plantlets with cutting wounds were used as the explants and cultured on MS medium with different hormone combinations and concentrations (M1: MS+1.0 mg· L-1 IBA+4 mg· L-16-BA; M2: MS+1.0 mg· L-1 IBA+5 mg· L-16-BA; M3: MS+1.0 mg· L-1 IBA+6 mg· L-16-BA;M4:MS+0.5 mg· L-1 IBA+4 mg· L-16-BA; M5: MS+0.5 mg· L-1IBA+5 mg· L-16-BA; M6: MS+0.5 mg· L-1 IBA+6 mg· L-16-BA) , and were incubated at 25 ℃/17℃ (14/10 h) for 14 days in dark. The leaves of 40 d, 80 d and 120 d were used as the explants for regeneration culture to explore the effect of leaf age on regeneration. Both the entire leaves with cutting wounds and leaflet slices were used as the explants for regeneration culture to find the best explant form. And the explants were cultured under light or in darkness for 14 days. The progresses of regeneration were observed, and the callus rate and regeneration rate were calculated. The regenerated buds were subcultured on MS medium containing 0.1 mg · L-1 IBA+1 mg· L-16-BA, and 1/2 MS medium+3 mg· L-1 IBA was used as the rooting medium for the regenerated plantlets.【Results】The anther culture obtained homozygous plants, which had been analyzed by flow cytometry (BD Accuri C6) and proved to be haploid (plantlets of DH2-3 and DH2-41) , diploids (plantlets of DH2-10, DH2-20, DH2-24 and DH2-35) , triploid (plantlet of DH2-40) or tetraploid (plantlet of DH2-15) . Shoot regeneration frequency was different among plantlets. The regeneration rate of the diploid plantlet DH2-10 was the highest, followed by tetraploids DH2-15 and haploid plantlets, and the tritraploid DH2-41 had the lowest regeneration frequency. The regeneration frequency of the haploid plantlets DH2-10 and DH2-20 was 31.1% and 5.0%, respectively. That of the diploid plantlets DH2-10, DH2-20, DH2-24, DH2-35 was 100%, 78.89%, 60% and 33.33%, respectively. The regeneration frequency of the tetraploid plantlets DH2-15 was 93.33%. The results showed that plant genotype greatly affected plant regeneration. DH2-10 was a good material for highly efficient plant regeneration. The period of regeneration of the diploid DH2-10 was only 23-35 d, shorter than the other plantlets, and its average bud number per explant was 7.27. Furthermore, hormones had impact on plant regeneration. MS medium containing 6-BA and IBA could be used for callus differentiation and for adventitious bud regeneration. When the concentration of 6-BA was 5 mg · L-1, the induction of callus from leaf explants was good. The best hormone combination for different genotypes varied. The optimal auxin concentration suitable for the haploid plantlets was higher than those for the diploid plantlets and tetraploid plantlets. The regeneration of the tetraploid DH2-15 required a higher level of cytokinin than that of the diploid plantlets and haploid plantlets. In regeneration process, the explant of whole leaf with wounds had a higher regeneration rate than that of small leaf slices. A higher regeneration rate was obtained using the 40 day old leaves as the explants. Culture under dark increased the regeneration rate significantly.The regenerated plants were subcultured and rooted plants were obtained after root induction【.Conclusion】A high-frequency in vitro regeneration system for homozygous apple plantlets was established.The diploid plantlets of DH2-10 had a regeneration rate of 100% after 14 d dark culture in the optimized regeneration medium of MS+0.5 mg· L-1 IBA+5 mg· L-16-BA. The tetraploid homozygous plantlets DH2-15, had a regeneration rate of 93.33% after 14 d dark culture in the optimized regeneration medium of MS+0.5 mg· L-1 IBA+6 mg· L-16-BA. The regeneration system could be used for the rapid propagation and genetic transformation.