Culture In Vitro By Unpollinated Ovaries And Ovules Of "Sanhua" Camellia Oleifera

Camellia oleifera Abel.is an important tree species and a major economic tree species unique to the south of China.The cultivation and utilization of Camellia oleifera has a long history and a wide planting area.However,low yield and low efficiency have been restricting the healthy development of Camellia oleifera industry,and the traditional clonal breeding and cross breeding methods both have long breeding cycle and unstable genetic traits.By inducing haploid plants using plant tissue culture technology,homozygous double haploid plants can be quickly screened out,and excellent varieties with stable characters can be obtained to improve breeding efficiency,which can accelerate the breeding process of oil tea good varieties and establish an efficient breeding system for oil tea.In this study,the unpollinated ovary and ovule of"Sanhua" Camellia oleifera were used as explants to explore the effects of various factors,such as genotype,exogenous hormones,pretreatment and dark culture,on callus induction.Through morphological cytological analysis and ploidy identification of the callus,somatic embryogenic callus generated from the division and proliferation of outer ungume cells was obtained.A stable and efficient technique system for embryogenic callus induction of unpollinated ovule of Camellia oleifera was established to provide scientific basis for somatic embryogenesis and haploid breeding system of Camellia oleifera.The main research results are as follows:1.The optimal culture system for callus induction of unpollinated ovary of Camellia ’huashuo’ was established,and the disinfection method,the optimal medium formula and culture conditions were defined.Using unpollinated ovary as material,the effects of disinfection method,hormone type,ovary cutting method,developmental stage and preconditioning on ovary callus induction were studied.The results showed that the ovary disinfected with 75%ethanol for 60 s and 0.1%mercuric solution for 5 min had the lowest Browning degree after 7 days of culture.NAA and TDZ were the best hormone combinations to induce ovary callus,and the induction rate was 63.89%.The highest induction rate was 64.58%when the ovary was cut longitudinally.The highest callus induction rate of unpollinated buds collected on the day of flowering was 66.67%.The best pretreatment method was to pretreat ovary at high temperature(35℃)for 1 day,and the induction rate reached 70.83%.In the orthogonal treatment of hormone and sucrose combination,the effect of TDZ on induction rate was higher than that of NAA,sucrose had the least effect.The optimal medium formula for callus induction of unpollinated ovary of Camellia oleifera’huashuo’ was MS+60 g/L sucrose+7 g/L AGAR+0.3 mg/L NAA+2.0 mg/L TDZ,and the callus induction rate of ovary could reach 72.34%after 30 days of culture.2.The optimum culture system for callus induction of unpollinated ovule of Cameltea ’huajin’ was established,and the optimum medium formula and culture conditions were determined.Firstly,the embryogenic callus induction rate of unpollinated ovule of "Sanhua" varieties in different medium and hormone types was compared to screen the best genotype and the most suitable hormone combination.The results showed that in MS medium combined with NAA+TDZ,the highest callus induction rate of ’huashuo’ was 91.94%in "Sanhua",and the highest callus induction rate of ’uajin’ was 30.17%in "Sanhua".Since this study focused on embryogenic callus induction,the subsequent treatment group took the unpollinated ovule of ’huajin’ variety as the material to study the effects of sucrose concentration,dark culture,pretreatment,anti-browning agent and other factors on embryogenic callus induction.The results showed that the highest embryogenic callus induction rate was 38.85%when sucrose concentration was 40 g/L.The embryogenic callus induction rate of unpollinated buds pretreated at high temperature(30℃)for 2 days reached 45.76%.Dark culture and anti-browning agent were not effective in inducing ovule callus.In the orthogonal treatment of hormone and sucrose,the effect of TDZ on callus induction rate was greater than that of NAA,and the effect of sucrose was the least.The optimum culture-medium formula for callus induction from unpollinated ovule of ’Huajin’ was MS+40 g/L sucrose+7 g/L AGAR±1.0 mg/L NAA+2.0 mg/L TDZ.After 30 days,the highest callus induction rate of ovule was 92.27%and the highest embryogenic callus induction rate was 48.60%.3.Through morphological and cytological analysis and ploidy identification,the developmental process of embryogenic callus was investigated,and the embryogenic callus originating from outer integument cells was obtained.The embryogenic callus obtained in this study were compact in texture,light green in color,with dense spherical prominences on the surface.The sections showed that the cells were small in size,tightly packed,with large nuclei that were easily stained and strong in division.Embryogenic callus development process:the initial embryogenic cell division activity of internal haploid cells,due to the restriction of space in the later period,the division stops.The outer integuments initially produced non-embryonic callus,and then became embryonic and formed meriform nodules.The meriform nodules moved towards the epidermis and gradually separated from the maternal callus,and the protruding part formed proembryo,which may develop into embryoid structure in the later stage.The ploidy of embryogenic callus was identified as hexaploid cells.Combined with the development process of callus,it was speculated that callus cell clusters originated from the division and proliferation of outer integment cells after dedifferentiation.