| Camellia oleifera has low efficiency in conventional breeding due to the characteristics of complex ploidy and self-incompatibility,while haploid culture techniques can be used to quickly obtain stable homozygous haploid breeding intermediate material,save breeding time and workload,improve the breeding speed and efficiency.Many studies on haploid induction of Camellia oleifera by anther culture in vitro were reported in the early stage,but they were all stagnated in obtaining callus and failed to cultivate the haploid plants of the Camellia oleifera.In this study,six genotypes of Camellia oleifera microspore cells in the anthers at the mononuclear edge stage,’ Huashuo ’ natural pollinated immature embryos,and irradiated pollen pollination-induced parthenogenesis embryos were used as the test materials.The haploid of Camellia oleifera was induced by anther culture and parthenogenesis,and the regeneration system of young embryos was studied,aiming to create a pure germplasm of Camellia oleifera.The main results are as follows:1.Anther culture regenerates callus of different ploidy.The anthers of ’Huashuo ’,’ Huajin ’,’ Huaxin ’,Diping 47,Dabaohuangzhenzhu,and NR-7,were cultured in vitro using 22 media,and the induced healing ploidy identification of wounded tissue,it was found that 58 anther-induced callus ploidy of the hexaploid ’Huashuo’ was different,including 25 tetraploid,9 hexaploid,15 octoploid,8 decaploid,and mixed ploidy(three/hexaploid)1 copy;there are 10 tetraploid,1 pentaploid,37 hexaploid,8 octoploid,and 2 decaploid callus induced by 58 anthers of hexaploid’Huajin’;there are 32 tetraploid,15 hexaploid,1 octoploid and decaploid callus induced by 49 anthers of hexaploidy ’ Huaxin ’;there are 1 triploid and 12 tetraploid callus induced by 13 anthers of tetraploid Diping 47;all 111 anther-induced callus of tetraploid Dabaohuangzhenzhu were tetraploid;all 61 anther-induced callus of diploid NR-7 were diploid.2.Different genotypes of Camellia oleifera have different anther callus induction media.The best medium for ’ Huashuo ’ anther callus culture was MS(deNH4+)+30 g/L sucrose+7 g/L agar+1 mg/L 2,4-D+0.2 mg/L 6-BA,the induction rate is 98.33%;’ Huajin ’ anther callus induction the best medium is MS(de-NH4+)+30 g/L sucrose+7 g/L agar+5 μM 2,4-D+7 μM KT+800 mg/L glutamine+200 mg/L serine,and the induction rate is up to 98.50%;the best medium for callus induction of anthers of ’Huaxin ’ is MS(de-NH4+)+30 g/L sucrose+7 g/L agar+5 μM 2,4-D+5 μM KT+800 mg/L glutamine+200 mg/L serine,the induction rate is 97.40%;the best medium formula for the induction of callus of Diping47 anther is B5+30 g/L sucrose+7 g/L agar+1.2 mg/L 2,4-D+0.4 mg/L 6-BA,the induction rate is 96.13%;the best medium for callus induction of Dabaohuangzhenzhu anthers is MS(de-NH4+)+30 g/L sucrose+7 g/L agar+5 μM 2,4-D+9 μM KT+800 mg/L glutamine+200 mg/L serine,the induction rate is 87.80%;the best medium for NR-7 anther callus induction is MS(de-NH4+)+30 g/L sucrose+7 g/L agar+0.6 mg/L 2,4-D+0.05 mg/L NAA,the induction rate reached 69.73%.3.Establishment of in vitro regeneration culture system for young embryos of Camellia oleifera and identification of ploidy of regenerated plants.The immature embryos from July to August after the natural pollination of the hexaploidy ’Huashuo ’ were used as explants,and the culture was induced in vitro with 16 different media,the results showed that:different media have different effects on inducing immature embryo regeneration,among them,WPM+30 g/L sucrose+8 g/L agar+1.5 mg/L NAA+2.0 mg/L 6-BA medium has the best effect,and the seedling rate of immature embryos is up to more than 34.86%.The ploidy identification of 146 seedlings regenerated from the hexaploidy ’ Huashuo ’ natural pollinated immature embryos showed that 2 seedlings were octoploid,1 seedling was nineploid,and the remaining 143 seedlings were identical to the female parent ploidy.4.Radiation pollen pollination induces parthenogenesis to cultivate Camellia oleifera haploid.Different radiation doses(0Gy,150Gy,250Gy,350Gy,450Gy,550Gy)are applied to pollen of ’Huashuo ’,’Huajin’ and ’ Huaxin’ with 60-Co,and ’ Huashuo’(hexaploid),Diping47(tetraploid),NR-2(diploid)pollination to induce parthenogenesis by radiation pollen.The results showed that:the pollen after radiation treatment still had high vigor and germination rate,which was not significantly different from the normal pollen germination rate;observe the pollen behavior after pollination,and the pollen tube after irradiation can germinate normally,grow into the ovule,and obtain fruit.The embryo development of fruits pollinated by radiation pollen is quite different from that of normal pollinated fruits,the number of normal embryos in the fruits with normal pollination of ’Huashuo’,Diping47,and NR-2 is divided into 3-10,1-3,2-6,respectively,while there are only 0-3(’ Huashuo ’),0-2(Diping47),0-3(NR2)normal embryos in the fruit after radiation pollination,and all the remaining embryos were aborted.While there are only 0-3 normal embryos in the fruits after radiation pollination,and the rest all embryos were aborted.Based on the established immature embryo regeneration culture system,embryo rescue of immature embryos after radiation pollination was carried out,and 12 regenerated plants,24 embryoids,and 36 callus were obtained,ploidy identification confirmed that 1’ Huashuo ’ haploid plant,2’ Huashuo ’ haploid embryoids,and 1 NR-2 haploid callus were obtained. |
PDF Full Text Request