Establishment Of Regeneration System And Cyclic Culture Of Somatic Embryo Of Camellia Vietnamensis T.C.Huang Ex Hu

Camellia vietnamensis T.C.Huang ex Hu is one of the excellent varieties of Camellia oleifera Abel.The seed oil contains tea polyphenols and other antioxidant substances with high content of unsaturated fatty acids,which has excellent nutritional value and taste.To obtain high yield and good quality of Camellia vietnamensis,it is necessary to improve varieties by using biotechnology breeding based on regeneration system.In this study,the young leaves of Camellia vietnamensis were used as explants to study the effects of different mediums,growth regulators,and light on callus induction,somatic embryo differentiation,adventitious bud induction,young leaf regeneration,root meristems,and plant regeneration.The regeneration system of Camellia vietnamensis was initially established,which laid the foundation for the establishment of genetic transformation system and molecular breeding of Camellia vietnamensis.To solve the problem of the low potential and probability of somatic embryos from embryogenic callus differentiation of Camelia vietnamensis in vitro,a cyclic culture system of somatic embryos→cotyledons→brittle embryogenic callus→somatic embryo was established by using the cotyledons from somatic embryos to dedifferentiate and redifferentiate.The cyclic culture system increased the probability of somatic embryogenesis and expanded the number of somatic embryos in a short time,providing high-quality materials for genetic transformation research.The main results of this study are as follows:1.Disinfection method of Camellia vietnamensis leaves:Firstly,it was quickly disinfected with 70%alcohol for 10s,followed by sterilizing with 0.1%HgCl2 for 8min,the explants had the lowest mortality and contamination rates.2.Method for eliminating browning of explants:The callus was pre-cultured for seven days on the MS basic culture medium without hormones under the conditions of 27±1℃,120001x,and 16h/d,and then transferred to the medium supplemented with hormone to induce callus through dark culture.3.The most suitable medium for white and crisp callus was MS+16mg/L Picloram+2uM/L CuSO4;the most suitable medium for green and dense callus was WPM+1mg/L NAA+1mg/L 6-BA,and the most suitable medium for nodular callus was WPM+0.5mg/L KT+0.5mg/L NAA.4.The factors affecting callus regeneration and differentiation were optimized by L8(27)orthogonal experimental design.After eight weeks of culture,four different types of callus were obtained.The suitable culture condition for white callus with brittle structure was MS+1mg/L 6-BA+0.1mg/LNAA+0.5mg/L ZT+1mg/L TDZ.5.The white callus with the brittle structure was induced into yellow embryogenic callus on WPM+2 mg/L NAA+2 mg/L ZT medium for six weeks,the induction rate was 38%.Somatic embryos were induced from the yellow embryogenic callus on MS+0.5 mg/L NAA+2 mg/L 2,4-D medium for eight weeks,the induction rate was 18.75%.Then somatic embryos germinated into green cotyledons on MS+1 mg/L 6-BA+0.5mg/L NAA medium for six weeks.The germinated cotyledons grew into buds after four weeks on MS+1 mg/L 6-BA medium.The buds were separated into young leaves and root tissue after four weeks on MS+1 mg/L IB A medium.6.The cotyledons of somatic embryos were chopped and transferred to MS+1 mg/L 6-BA medium for callus induction in the process of dedifferentiation,and then the cyclic culture of somatic embryos was realized by non-embryogenic callus elution method.A large number and strong activity of somatic embryos of Camellia vietnamensis are obtained in a short time,and the somatic embryogenesis frequency of Camellia vietnamensis in this way will be greatly improved.