Effect of immature zygotic embryo development stage on in vitro culture for 'Renong 1' mango

Abstract: 【Objective】Mango(Mangifera indica L.) is one of the most important fruit crops in tropical and sub-tropical regions of the world. However, most commercial mango cultivars are highly susceptible to pathogens and some are highly disease-resistant but are not tasteful. Cross breeding is not very efficient because of low seed setting rate in mango. Somatic embryogenesis is a key way for genetic improvement, and embryo rescue is an important way to solve the early embryo abortion in fruit trees. Immature embryo or nucellus is the most commonly adopted explant in somatic embryo induction of mango. It had been verified that the embryo development phase affected the induction of somatic embryo or embryo rescue in plants. However, the relation between the embryo development phase and the induction of embryogenic cultures(EMs) or embryo rescue in mango is not very clear up to now. Immature embryos at different developmental stages were used to survey the effects of the developmental stages of emboyos on the efficiency of induction and survival rate of the embryos.【Methods】The embryos of'Renong 1'mango from 10 days to 60 days after pollination(DAP) were used to determine the the embryo development phase according to microscopic and cytological observation. Embryos from 25 DAP to 60 DAP were cultured on PM0-PM7 medium containing different concentrations of auxin analog(i.e., 2,4-D) and cytokinin analogs(i.e., KT and ZT), and the effect of the embryo development phase and plant growth regulators on the induction of Ems, somatic embryo and zygotic embryo germination were compared.【Results】Stereoscopic and cytological observations showed that globular embryos were found on 25 DAP, heart stage on 30 DAP, torpedo embryos on 35 DAP, and embryos were at the cotyledon-stage from 40 DAP.The immature embryos collected at seven developmental stages(globular embryo, heart embryo, torpedo embryo, early cotyledon embryo, cotyledon embryo 1, cotyledon embryo 2, cotyledon embryo 3) were cultured on eight embryogenic culture initiation media PM0-PM7. The immature embryos became bulgy and white in color after 7 days of incubation on induction media PM0-PM7. A fraction of the embryos gradually developed a brown color, demonstrating symptoms of necrosis. After 3 weeks of culture, A fraction of the surviving immature embryos developed into two kinds of Ems by dedifferentiation. The embryogenic calli or the embryo-like structures were pale-yellow and transparent. A fraction of the surviving immature embryos developed into plantlets with white cotyledons with crinkled margins and elongated radical. These immature embryos of seven developmental stages presented significant differences in the rate of Ems induction after 4 weeks of dedifferentiation culture on media PM0-PM7. The induction rates(including the embryogenic calluses or the embryo-like structures) of Ems on media PM0-PM7 increased gradually along with embryo development and then decreased gradually. The average rate of the early cotyledon-stage embryos was 36.5%, higher than the other stage embryo. Media PM0-PM7 were characterized by higher auxin concentrations (0-5.0 mg·L-12, 4-D), which were 0.67-5 times greater than the those of cytokinin concentrations (KT and ZT). The average rate of Ems induction from early cotyledon-stage embryo increased gradually along with the enhancement of the concentration of 2,4-D and then decreased gradually. The highest rate(54.4%-62.2%) of Ems induction were observed on medium PM3 and PM4 supplemented with 3.0 mg·L-12,4-D and 1.0 mg·L-1 cytokinin (KT or ZT), with three-fold difference in the auxin/cytokinin rate. In addition, the germination of the immature embryos collected at seven developmental stages increased gradually along with embryo development and then decreased gradually on media PM1, PM3 and PM4, the average rate of torpedo-stage embryo was 47.2%, higher than those of the embryos collected at other stages.【Conclusion】The rate of induction of embryogenic cultures or embryo rescue weas significantly influenced by the embryo development phase. The optimum stage for the somatic embryo induction and embryo rescue of'Renong 1'mango was early cotyledon-stage(40 DAP) and torpedo-stage embryo(35 DAP) respectively in Zhanjiang, Guangdong province.