Optimization Of Embryogenic Cell Mass Induction System And Somatic Embryogenesis In Immature Embryos Of Pinus Massoniana

Masson pine(Pinus massoniana Lamb.)is an important afforestation pioneer species in the southern subtropical region of China.It has strong adaptability,fast-growing and high-yield,which has the functions of material,lipid-producing and pine pollen utilization,and the fiber is excellent,and the comprehensive utilization value of the whole tree is high.In the past 40 years of genetic improvement of Masson pine,a number of seed orchards have been established,but the seed yield is low,resulting in insufficient supply of improved varieties,which greatly limits the productivity of masson pine forests.Somatic embryogen-esis technology provides an effective way to achieve rapid propagation of Masson pine,However,the embryonic cell mass induction rate is low,and the embryoid body matures and differentiates difficult,which limits the scale-up effect of somatic embryo development.In this study,Immature female gametophytes of masson pine were used as experimental materials,and LP was the basic medium.The goal is to optimize the induction system of the embryonic cell mass of masson pine and establish a highly efficient somatic embryogen esis system of masson pine.The main results are as follows:1.The occurrence and type of embryogenic cell mass:Most embryogenic cultures are produced by the micropyle of the female gametophyte.The status of embryogenic cell mass induced by different treatments and different families is different.according to the change of the micropyle.,color,texture,is divided into 5 categories;(1)Spit out filaments or clusters from the micropyle,white and translucent;(2)The crack of the micropore is white granular and translucent in texture;(3)Browning of cultures at the micropyle;(4)The micropyle cracked,but the crack was smooth and there was no white granular protuberance;(5)There was no change at the micropyle,but the female gametophyte was enlarged;The first two types of culture tissues have polar structure,while the last three types of culture tissues have no polar structure.2.The optimum hormone combination for induction of embryogenic cell mass of masson pine is 1.Omg·L-1 2,4-D+0.5mg·L-16-BA+1.Omg·L-1NAA+1.Omg·L-1KT,the induction rate is up to 40.00%;Excessive or low concentration of agar medium is not conducive to the induction of embryogenic cell mass,and when the concentration is 8.5g·L-1,the induction rate of embryogenic cell mass is the highest,the induction rate is up to 43.33%;the immature embryos of Masson pine were cultured at 23℃,which was more conducive to the formation of embryogenic cell mass,the highest induction rate,up to 43.33%;There were significant differences in induction and status of mesodermal cell clusters among different maternal tree genotypes.It was also found that the fuller the immature seeds,the higher the induction rate of embryonic cell mass.3.There is a significant difference in the proliferation ratio of the inner and outer layers of the embryonic cell mass.The outer multiplication ratio of the embryonic cell mass is larger than that of the inner layer and has more polar structure.Therefore,it is maintained when the embryogenic cells are transferred to fresh medium.The same position is necessary because the change in direction has an effect on the growth of the embryonic cell mass.4.The mature differentiation of masson pine cells is sensitive to the concentration of ABA.When the concentration of ABA is in the range of 10-20mg·L-1,it is beneficial to the formation of somatic embryos of masson pine.5.Histological and cytological observations on the somatic embryo development of Masson pine showed that the embryogenic cell mass was composed of a circular dense cytoplasmic meristem cell and a long strip of embryogenic suspensor cells with large vacuoles.Different types of embryonic cell clusters induced by different treatments and different genotypes have different state and structure;only the embryonic cell mass of specific structure can proliferate for a long time,and mature into somatic embryos;The induction phase and the proliferative phase are mainly in the pro-embryonic stage(PEMs).After the three stages of PEMⅠ,PEMⅡ and PEMⅢ,the embryonic cell masses enter the early somatic embryo stage when the embryonic head of the polar structure appears bullet-shaped.Embryogenic suspensor cells gradually degenerate,and the embryonic head is full,and then the cotyledonary embryos around the embryonic head elongate and grow into green plantlets.