Embryogenic callus induction and virus-free plantlet regeneration from seedless cultivars in citruses of different ploidy

Abstract: 【Objective】Citrus is the biggest fruit industry in China and in the world. China is the origin center of citrus, owning abundant and diverse citrus germplasm resources, and it is important to properly conserve and utilize the elite citrus germplasm. The embryogenic callus induced from the aborted ovules is genetically identical to the original explant and capable of regeneration, making it feasible for in vitro conservation of citrus germplasm. The embryogenic callus also provides in vitro materials for studies of important traits, such as somatic embryogenesis, fruit quality control and stress resistance.We have induced and preserved embryogenic callus germplasm of over 100 different genotypes. However, the embryogenic calluses are all induced from the diploid germplasm, while induction of embryogenic callus from polyploid germplasm has been rarely reported. The polyploid is characterized by giant organ, dwarf plant and stronger adaptability. The polyploid embryogenic callus would accelerate basic research and applications of citrus polyploidy, by providing in vitro materials with short growth cycle, stable status, and easily accessibility. In addition, the whole new plantlets regenerated from embryogenic callus are always virus-free, which are promising in resolving the problem of fruit yield and quality delineation in seedless cultivars caused by virus infection after many years propagation by grafting. In this study, the aborted ovules of diploid and tetraploid citrus germplasm were cultured to induce embryogenic callus, to provide in vitro materials for citrus ploidy research. Regeneration of virus- free plantlets from embryogenic callus achieves purification and rejuvenation of seedless cultivars like navel orange and Satsuma mandarin.【Methods】The aborted ovules from mature fruits were inoculated on three types of callus induction mediums (MES: MT + ME 0.5 g·L-1 + SAD 40 mg·L-1 ; MGS: MT + ME 0.5 g·L-1 + SAD 40 mg·L-1 + GA3 1 mg·L-1 ; MK: MT + KT 0.5 mg·L-1 ) under sterile conditions to induce and train embryogenic callus, and the ploidy of embryogenic callus was determined by flow cytometry. Embryogenic callus was induced to differentiate into embryoids and adventitious buds. When the regenerated buds grew to the size of 2-3 cm, they were grafted to the yellowing rootstock in test tube to form a plantlet. SSR (Simple Sequence Repeats) analysis was used to identify the genetic origin of the regenerated plantlets, and PCR was used to detect Citrus tristeza virus (CTV) and Citrus yellow vein clearing virus (CYVCV) in the plantlets.【Results】The embryogenic calluses were induced from 13 citrus accessions, and eight of them have been trained for subculture, including red tangerine diploid and tetraploid (2x, 4x), Nadorcott tangor (2x, 4x), Egan No. 1 Ponkan mandarin (2x, 4x), Sunburst mandarin (2x) and Zaohong navel orange. The ploidy of the embryogenic calluses was consistent with the source materials from which they were induced, as detected by flow cytometry. The embryogenic callus induction efficiencies were different among accessions. The induction rate of Zaohong navel orange was the highest (74.73% ), followed by Guoqing No. 1 Satsuma mandarin, Okitsu Satsuma mandarin, Oita 4 Satsuma mandarin and Egan No. 1 Ponkan mandarin, each with an induction rate over 37%. The induction rates of red tangerine 2x, Sunburst mandarin 2x, Lane Late navel orange and Nadorcott tangor 2x were lower than those of the other genotypes, with induction rates between 6.85% and 12.61%. Embryogenic calluses were induced from four pairs of ploidy materials, and the induction rate of diploid germplasm was 6.85%-37.75%. Among them, the callus induction rate of Egan No. 1 Ponkan mandarin 2x was the highest, while that of red tangerine 2x was the lowest. The callus induction rates of the tetraploids (0.56%-22.61%) were lower than those of the diploids. Among them, the callus induction rate of Egan No. 1 Ponkan mandarin 4x was also the highest, while that of Nadorcott tangor 4x was the lowest. As for the incidence of embryoids, they were 16.15%-58.28% for diploids and 1.13%-30.65% for tetraploids. In summary, the callus induction and embryoid incidence rates of Egan No. 1 Ponkan mandarin 2x and 4x were the highest among the four pairs of 2x and 4x materials, while those of Nadorcott tangor 4x were the lowest among the tetraploid germplasm. Notably, the callus induction and embryoid incidence rates of the tetraploids were lower than those of the corresponding diploids. Besides, the induction rate of embryogenic callus on different mediums was different. The embryogenic callus induction rates of Guoqing No. 1 Satsuma mandarin, Okitsu Satsuma mandarin, Oita 4 Satsuma mandarin, Nadorcott tangor 2x and Sunburst mandarin 2x on MGS medium were higher than on MK and MES. The callus induction rates of Egan No. 1 Ponkan mandarin 2x and red tangerine 2x on MK medium were thehighest, and significantly higher than on MES and MGS. A total of 23, 22, 20, 10 and 15 plantlets were regenerated from five seedless cultivars, including Zaohong navel orange, Lane Late navel orange, Guoqing No. 1 Satsuma mandarin, Oita 4 Satsuma mandarin and Okitsu Satsuma mandarin, respectively. SSR analysis showed that the regenerated plantlets of the chimeric Zaohong navel orange were indeed Robertson navel orange. According to PCR, CTV and CYVCV were undetectable in the regenerated plantlets of Guoqing No. 1 Satsuma mandarin, Lane Late navel orange and Zaohong navel orange, proving them to be virus-free.【Conclusion】In this study, the aborted ovules of mature citrus fruits were cultured in vitro, and the diploid and tetraploid embryogenic callus of three varieties were obtained by induction and training, which provides stable and easily accessible in vitro materials for the research of citrus polyploidy. The embryogenic callus induction rates differed among different citrus germplasms and induction mediums. The embryogenic callus induction rate and somatic embryogenesis rate of the diploids were higher than the corresponding tetraploid for each cultivar. The regenerated virus- free plantlets provide materials for purification and rejuvenation of five polyembryonic and seedless citrus cultivars. The molecular marker analysis proved that the regenerated plantlets of the grafting chimera Zaohong navel orange were indeed Robertson navel orange.