Application and optimization of in vivo bud regeneration technique via callus in Chinese jujube and sour jujube

Abstract:【Objective】Asexual polyploidization is an effective way for creating new germplasm in jujube.The occurrence and purification of mixoploid is always the bottleneck of polyploid induction breeding in fruit tree and could be avoided by combing bud regeneration via callus with chromosome number doubling using colchicines, by which pure polyploid could be acquired directly. Bud regeneration is the basis and key point of field in vivo polyploid induction via callus. This study is to examine the universality of in vivo Bud Regeneration Technique via Callus(IVBRTC) we previously created in Chinese jujube and to make a optimization of the approach.【Methods】A total of 115 Chinese jujube(Ziziphus jujuba Mill.) and 4 sour jujube(Z. acidojujuba Cheng et Liu) genotypes were employed for IVBRTC application. The 119 genotypes covered a wide genetic diversity including the main cultivars(such as‘Huizao'‘Junzao'‘Zanhuangdazao'‘Jinsixiaozao'‘Linyilizao'‘Yuanlingzao'), newly released cultivars(‘Jingzao39'‘Lengbaiyu'‘Jingudazao'‘Liuyuexian', et al), famous local cultivars(‘Fengmiguan'‘Changxindianbaizao', et al), South Korea cultivars(‘Wudeng'‘Hongyan'‘Jincheng'‘Fuzao'and‘Yuechu') as well as sour jujube(‘Taigudasuanza'‘Yesuanzao', and so on). The protocols of IVBRTC are as follows: First, cutting the randomly selected strong branches with branch shears or hand saw; Second, treating the cross sections of brancheswith 1.5 m L inducer(4.0 mg·L^-1 TDZ+ 2.0 mg·L^-1 Ag NO3) for 24 h; At last, putting wet mud on the cross section and then covering the wet mud with white plastic bag to keep moisture; Fourth, replacing the wet mud about every 10 days; Fifth, removing the wet mud after the adventitious buds formed from callus reaching 2 cm long and making them growing naturally. The callus status on the cross section were observed 20 days after the branches being cut. The bud number per branch cross section of all tested genotypes were counted 50 days after. The bud regeneration ability is represented by average bud number per branch. The effects of genotype, callus status, branch diameter and location on in vivo bud/shoot regeneration were analyzed.【Results】80.67% of the genotypes regenerated buds via callus in vivo, of them the average bud number per branch cross section of‘Beijingsuanzao'‘Longhuzao'and‘Beijingmayazao'reached up to 10.33, 9.40 and 8.00, respectively. The results showed that there was no significant correlation between average bud number per cross section and callus ratio. The callus quantity influenced the bud regeneration ability obviously. With the increase of callus quantity, the average bud number per cross section increased and topped 2.86 when the callus grade was 5. Then bud regeneration ability slightly decreased on callus in grade 6 of which the width of the callus circle accounts for over 1/2 of the cut section's radius. The results showed that branch diameter somewhat influenced bud regeneration and the branches between 1.4 cm and 1.9 cm in diameter were easier to regenerate buds with average bud number per cut section reaching 2.16, which was significantly higher than those of branches with diameter of 0.9~(-1).4 cm and 1.9-2.4 cm. The upright branches in the up part of the canopy showed higher bud regeneration ability than those in the mid or lower part and branch angle showed no significant effects on bud regeneration. It is also noted that using wet mud to keep moisture did not affect the amount of callus occurrence, but could improve the quality of callus. The wet mud treatment could change the callus status from light yellow,dense with smooth surface into white, dense with granular structure that easy to regenerate bud.【Conclusion】The IVBRTC has a quite good universality in different genotypes of Chinese jujube and sour jujube;The most dominant factor affecting in vivo bud regeneration is genotype, followed by callus status, branch diameter and branch vertical location. The angle and horizontal position of branch showed no obvious effects on bud regeneration. Based on the above-mentioned, an optimized IVBRTC in jujube was put forward. The optimized IVBRTC protocols are as follows: In the mid or late of May, strong branches of 1.4~(-1).9 cm in diameter located in the upper part of the tree are selected and cut; Then using 1.5 mL inducer(4.0 mg·L^-1 TDZ+ 2.0 mg·L^-1 Ag NO3) to treat the cross section of branch for 24 h; After that putting wet mud on the cross section and covering the wet mud with white plastic bag to keep moisture, replacing the wet mud every 10 days; At last, removing the wet mud after the adventitious buds reaching 2 cm long and allowing them a natural growth.