Establishment And Application Of Rapid Propagation And Regeneration System In Camellia Vietnamensis In Hainan

Camellia oleifera is a general term for plants with high oil content in Camellia plants of Theaceae family,and is an important woody oil tree species native to China.Camellia seed oil is a nutritious and healthy pure natural high-grade plant edible oil recommended by the International Food and Agriculture Organization.Camellia oleifera,olive oil,oil palm and coconut are also known as the world's four largest woody edible oil plants.Camellia oleifera is a unique plant resource in China,an irreplaceable strategic resource for the development of China's woody grain and oil industry,and an important native oil tree species in Hainan Province.The lack of improved varieties and seedlings which adapted to the local climate and soil conditions are the two major bottlenecks that restrict the promotion of the cultivation of Camellia oleifera in Hainan and the further development of the Camellia oleifera industry.Excavating,conserving and utilizing excellent germplasm resources;breeding high-quality,high-yielding new varieties of high-yield oil-containing Camellia oleifera with high oil content,high quality,and strong adaptability,as well as accelerating the breeding and promotion of new varieties of high-quality varieties,is to accelerate the development of Hainan's camellia industry and maintain an effective strategy for sustained and healthy development.The main plant variety in Hainan is Camellia vietnamensis TC Huang ex Hu.In this study,immature embryos and anthers of Camellia vietnamensis were used as explants to establish the regeneration system in vitro through two pathways:meristem nodulation,somatic embryoid.And top buds,and stem segments containing axillary buds were used as explants to establish the rapid propagation system in vitro through organogenesis.This re-system has laid a technical foundation for the future preservation of germplasm resources,improved breeding of strong seedlings,shortening the generation cycle of hybrid breeding,and the use of genetic engineering for functional gene research and directed molecular breeding of Camellia oleifera.The main findings are as follows:1. When the immature embryos of Camellia vietnamensis was used as explants,the immature embryo at 240-300 days after full flowering stage cultured at the modified MS+Kt 2.0 mg/L+NAA 0.2 mg/L+Zt 5.0 mg/L medium,the initial induction rate of the immature embryo was more than 90%and the meristematic nodules are primary.The suitable medium for the proliferation of meristematic nodule tissues was the modified MS supplemented with?1.0?2.0?mg/L Kt,0.2 mg/L NAA,and the proliferation coefficient was approximately 4.6.The immature meristematic nodule embryo cultured at the 1/2 modified MS containing 2.0 mg/L 6-BA,0.2 mg/L NAA,0.2 mg/L IAA,5.0 mg/L Zt mainly differentiate into adventitious buds,with the average adventitious bud number was 11.3.Adventitious shoots rooted by sugar-free exposed cultivation,and plant transplant survival rate was 91.6%.The immature meristematic nodule embryo cultured at the 1/2 modified MS containing 2.0mg/L6-BA,0.2 mg/L NAA,Zt 5.0 mg/L mainly differentiate into somatic embryoid body with the average somatic embryoid body number was 6.5.Somatic embryos with dicotyledonate were effectively germinated and developed into plantlets on modified MS supplemented with 0.5 mg/L 6-BA,2.0 mg/LIAA,0.5 mg/L GA₃.The germination rate was 72.1%,the germ and the radicle were effectively extended to form a somatic embryo regeneration plant and plant transplant survival rate was94.7%.2. When the anthers were used as explants,the anthers which pollen development in the mononuclear marginal stage were suitable explants for inducing anther embryogenic callus;the primary medium suitable for camellia anther embryogenic callus induction was Modified MS+2,4-D 2.0 mg/L+Zt 2.0 mg/L;embryogenic callus in proliferation and redifferentiation medium 1/2 modified MS+6-BA 2.0 mg/L+Zt 1.0 mg/L+NAA 0.5 mg/L can effectively proliferate and differentiate into somatic embryos by dark culture;somatic embryos can induce somatic embryo maturation and somatic embryo germination to form regenerated plants on 1/2 modified MS+6-BA 0.5mg/L+NAA 0.1 mg/L medium,but the induction rate was low,and the medium ratio still needed to be further optimized.3. When the axillary bud and top bud were used as explants,the optimal disinfection method was a combination of 2%sodium hypochlorite solution for 15minutes and 0.1%Hg Cl₂solution for 12 minutes.The pollution rate can be reduced to9.7%.The stem segments with 1/3 leaves retained were inoculated in the first generation induction medium modified MS+GA₃0.5 mg/L+Zt1.0 mg/L,the bud formation rate,shoot formation rate and multi-bud rate of top buds were 68.9%,40.3%and 41.9%,the axillary bud formation rate,shoot formation rate and multi-bud rate were 82.2%,40.6%and 38.7%;the best propagation medium was modified MS+Zt 2.0 mg/L+IAA 0.3 mg/L,the coefficients reached 5.6 and 6.8;the best medium for strong buds was improved MS+IAA 0.5mg/L+GA₃0.5 mg/L+CH 200 mg/L,and the fixed bud stem thickness increased effectively by 85%,and the bud length increased effectively by 79%.4. Tissue cultured buds were used as grafting roots for grafting,and soaking in 6-BA for 2?3min before grafting could significantly improve the survival rate of grafting.Using conventional semi-lignified branches scion as a control,tissue culture seedlings with sterile single buds as scion for bud seedling root grafting,the growth in the early stage was relatively slow,but showed an advantage in the later stage.There was no significant difference in the length of new shoots and newly added leaves of grafted seedlings of tissue culture buds at 92 d and 252 d from the control,but the shoot thickness of new shoots was significantly increased by 171%and 41.8%compared to the conventional semi-lignified branches scion.