Research On Camellia Oleifera Radiation Pollination Combined With Embryo Rescue To Breed Haploids

Camellia oleifera Abel is an important woody oil tree species in China,but its breeding work has been plagued by long cycle time and low efficiency..Haploid breeding is reliable and fast to obtain pure lines and significantly shorten the breeding years.The group obtained one common Camellia oleifera haploid germplasm by radiation pollination combined with embryo rescue technology in the early stage,but the embryo rescue efficiency and haploid induction rate were extremely low.Therefore,in the present study,the embryo rescue period,early germination culture,somatic embryo succession culture and seedling proliferation culture were systematically investigated by using the excellent germplasm Wangmu 25 as the test material,in orderto establish an efficient embryo rescue technology system for Camellia oleifera;The pollination of the state-examined Camellia oleifera variety ’Huashuo’ and the excellent germplasm NR-2,NR-5,DB(1)and Longan tea by radiation pollination,the study of the effect of radiation pollination on the fruit set rate and fruit development of Camellia oleifera,At the same time,a number of new haploid germplasm were produced by embryo rescue of radiation pollinated young embryos.The main results are as follows:,the main results are as follows:1.An efficient technical system for Camellia oleifera embryo rescue was established.The optimal period for Camellia oleifera embryo rescue was determined to be 250 d after flowering,and the most suitable medium for inducing early germination of young embryos was selected as WPM+2.0 mg/L 6-BA+30 g/L sucrose+7 g/L agar,and the seedling success rate reached 35%;At the same time,the embryonic guardian tissue produced in the early germination of young embryos was used to establish a technical system for the regeneration of Camellia oleifera somatic embryos:the best medium for inducing embryonic guardian tissue was WPM+1.0 mg/L 6-BA+0.5 mg/L NAA+30 g/L sucrose+7 g/L agar,with an induction rate of 60.00%;the best medium for inducing spherical embryos to differentiate into heart-shaped embryos was WPM+1.0 mg/L 6-BA+0.2 mg/L NAA+30 g/L sucrose+7 g/L agar,and the induction rate was 38.89%.The best medium for inducing the differentiation of heartshaped embryos and torpedo-shaped embryos to form cotyledons was WPM+2.0 mg/L 6-BA+30 g/L sucrose+7 g/L agar,with induction rates of 22.22%and 27.78%,respectively;the best medium for inducing cotyledons into seedlings was MS+1.5 mg/L 6-BA+1.0 mg/L NAA+1.0 mg/L riboflavin+30 g/The optimum medium for seedling proliferation culture was determined as MS+2.0 mg/L 6-BA+1.0 mg/L IBA+30 g/L sucrose+7 g/L agar with a proliferation coefficient of 3.60.219 seedlings obtained from embryo rescue were identified for ploidy,and one plant was measured to be tetraploid while the rest were hexaploid.2.The effect of radiation pollination on the fruit set rate and fruit development of Camellia oleifera was investigated.The pollen of‘Huajin’,‘Huaxin’‘Huashuo’ and DB(1)were treated with different radiation doses(150 Gy,250 Gy,350 Gy,450 Gy,and 550 Gy)and found that pollen viability and germination rate were significantly reduced after radiation treatment;pollen treated with radiation was used for ’Huashuo’,’Huajin’,’Huaxin’,Hainan Camellia oleifera-2,DY7,U2,NR-2,NR-5,Longan tea,LPXG-4,DB(1),Wangmu25,GTDB(02)were pollinated with radiation pollen,and it was found that pollination with radiation pollen significantly reduced the fruit set rate,with most pollination combinations having a fruit set rate of less than 20%,and ’Huajin’×’Huaxin’ 450 Gy and 13 other combinations had a fruiting rate of 0.Only five combinations,including ’Huaxin’ × ’Huajin’ 150 Gy,had a fruiting rate higher than 20%,of which Longan tea × ’Huashuo In addition,radiation pollination also had a greater impact on fruit development,and compared with normally pollinated fruits,radiation-pollinated fruits were smaller and more deformed,and the seed embryos were highly abortive.3.7 copies of new haploid germplasm were successfully obtained by embryo rescue.Embryo rescue was performed on young embryos of radiation-pollinated fruits,and 88 seedlings and 84 healing tissues were obtained.One haploid plant of’Huashuo’,two haploid plants of NR-2,two haploid plants of NR-5,one haploid plant of DB(1)and one haploid plant of Longan tea were obtained after ploidy identification;also two haploid healing tissues of’Huaxin’ were obtained.2 copies of haploid guardian tissues,and 1 copy of U-2 mixed ploid(triploid/hexaploid)guardian tissues.In addition,198 haploid plants and 813 embryonic healing tissues of ’Huashuo’ were obtained by multiplication and expansion;56 rooted plants were obtained by micrografting,and 12 plants were transplanted and became viable.