A regeneration system for zygotic embryo callus induction of Ziziphus jujuba ‘Xiangfenyuanzao'

Abstract: 【Objective】Chinese jujube is known for severe embryo abortion especially at globular or heart-shape stage;and the artificial zygotic embryo culture starting from pre-globular stage is confronted withlow seedling rate. So far,thereis no data about the seedling regeneration from immature zygotic embryocallus of jujube. This study attempts to generate the offspring developed from embryos by optimizing theculture conditions including the composition of growth regulators and the factors for callus regeneration. Itis expected to improve the techniques of zygotic embryo culture and to provide new data for enhancing thetheoretic basis for efficient development of new jujube varieties.【Methods】One genotype(Ziziphus jujube‘Xiangfenyuanzao') with moderate embryo abortion were used in this study The naturally pollinated em-bryos sampling at 30 d(at globular stage) and 40 d(at heart-shaped stage) were used as explants for callus regeneration and seedling development. First,the invisible embryos at the stage of 30 and 40 days pri-marily underwent embryo culture with nurse endosperm to maintain the embryonic growth and then thethree-step culture process. Second,after 30 days of embryo culture with nurse endosperm the visible zy-gotic embryos were removed under ultra-clean conditions and transferred into BA medium for callus in-ducing. Third,the callus from the second successive culture was transferred to the medium with TDZ fordifferentiation,at that time the callus turned yellowish green in color and densely tumor-like in shape.The budding point appeared at 30 days. When the adventitious buds grew to at least 2 cm the callus wasdivided for proliferation. The embryonic seedlings were treated for rooting and later transferred to pots todevelop into independent seedlings. Specifically,the chalazal end of ovule without epidermis was insert-ed downward into nine MS medium cultures(the L9(34) orthogonal design) containing the correspondinghormone composition,sucrose at 7%(for 30 d-old embryos) and 5%(for 40 d-old embryos) and LH at0.5 g·L-1to test the effects of growth regulators(IBA,ZT,GA3,NAA) in various compositions and con-centrations on facilitating the embryonic growth and on generating the visible zygotic embryos after em-bryo culture with nurse endosperm of the jujube variety. The zygotic embryos obtained after the embryoculture with nurse endosperm were inoculated into the MS medium of different hormone composition,along with 4% sucrose,LH at 0.5 g·L-1,and 6-BA at 0.3-2.0 mg·L-1. The purpose of the design is tocompare the effect of the growth regulator sets on callus induction. After this,the MS medium containing3% sucrose,TDZ at 0.4-1.0 mg·L-1,NAA at 0-0.4 mg·L-1and IAA at 0-0.5 mg·L-1was used for succes-sive transfer culture and the differentiation of callus derived from zygotic embryos. The gradient of plantgrowth regulators was set up to examine the effect of hormone composition on callus differentiation. Afterthe proliferation of adventitious buds,the lignified vigorous individuals were inoculated into the mediumwith 1/2MS + IBA1.0 mg·L-1+ sucrose 2% for rotting. The culture bottles,each containing 3-5 individu-als,were incubated at 23-29 ℃ with light intensity of 2 000-3 000 lx in photoperiod of 14 h/10 h(light/dark). After 20 days the seedlings with root were first acclimatized and then transferred into the soils ingreenhouse. The growth condition were relative humidity of 85%-100%,temperature 26-36 ℃ and lightof 4 000-6 000 lx intensity. After 3-5 days the aeration was conducted and the light was intensifiedto 6 000-30 000 lx according to the growing conditions. The greenhouse shed was removed in 20-30 days and the seedlings were transferred to field in 35 days for growing into independent seedlings.【Re-sults】For 30 and 40 days old zygotic embryos of jujube variety of‘Xiangfenyuanzao',the optimal medi-ums turned out to be MS + IBA 0.2 mg·L^-1+ ZT 1.0 mg·L^-1+ GA35.0 mg·L^-1+ NAA 0.2 mg·L^-1+sucrose7%+LH 0.5 g·L^-1,and IBA 0.5 mg·L^-1+ ZT 0.5 mg·L^-1+ GA35.0 mg·L^-1+ NAA0.3 mg·L^-1+sucrose5%+LH 0.5 g·L^-1,the embryonic growth rate of these mediums were 54.17% and 55.25%. The demandfor growth regulators varied depending on the embryo age at inoculation. The embryonic growth is mainlyaffected by NAA for 30-day-old embryos but IBA for 40-day-old ones. The optimal medium for callus in-duction was MS + BA 1.5 mg·L^-1+ sucrose 4%,in which the callus exhibited satisfactory growth and qual-ity in favor of bud differentiation,with an induction rate of 70.59%. It is noted that TDZ effectively im-proved the callus growth. The optimal medium for callus differentiation was MS+TDZ 0.8 mg·L^-1+ IAA0.5 mg·L^-1+ sucrose 3%,which resulted in good adventitious buds in green color in favor of the followingsuccessive culture,with the differentiation rate of 58.90%. The optimal medium for rooting was 1/2MS +IBA 1.0 mg·L^-1+ sucrose 2%,resulting in rooting rate of more than 80%. After acclimatization the seed-ling were transferred into the pot containing vermiculite : grass peat : pot soil(1:1:1 in volume),and thesurvival rate was more than 85%.【Conclusion】The successful culture of 30- and 40-day-old zygotic em-bryos of jujube variety‘Xiangfenyuanzao'significantly depends on different composition and concentra-tion of growth regulators. The embryonic growth rete positively relates to embryo age at inoculation. Theoptimal condition is workable in the whole process covering the endosperm nursing culture,callus induc-tion for adventitious buds,rooting,seedling acclimatization and proliferation in field. The final survivalrate was more than 85%. Although jujube regeneration from organogenic callus is featured by longer peri-od and more complicated process,it renders more surviving seedlings derived from zygotic embryos. Thetechnique is effective in rescuing the zygotic embryos of jujube trees,especially those with favorable phe-notypes but suffering from massive embryo abortion in nature,through generating the offspring individu-als carrying the parental genotypes. The artificial culture of zygotic embryos turns out useful in copingwith the embryo abortion in early developmental stage of jujube. Therefore,jujube seedlings derived fromcallus effectively supplement the plantlets directly from zygotic embryo culture and are useful in rescuingthe embryos prone to severe abortion in early development stage as well as in selecting the desirable geno-types. In addition,the callus from zygotic embryo culture results in the intermediate embryos carrying theparental genes. The technique reduces the loss of embryonic offspring and improves the breeding efficien-cy,thus creates an innovative way for developing the optimal breeds of jujube trees.