Creation Of Engineered Female Sterile Restorer Line And Application In Mechanized Hybrid Rice Seed Production

The large-scale application of hybrid rice has made great contribution to the food security in China,but the further development of hybrid rice is seriously restricted by the complicated seed production procedures,high labor costs and expensive seeds.Realizing the mechanized seed production of hybrid rice,especially in the way of mixed-planting and mixed-harvesting,is the development direction of hybrid rice,and of great significance to the development of hybrid rice in China and even the world.Using female sterile lines of rice is one of the feasible ways to solve the mechanized seed production of hybrid rice in the way of mixed planting and harvesting.The female sterile line used as the male parent,and crossed with the male sterile line,and all the harvested seeds are hybrid seeds.while,the reproduction of female sterile line is a difficult problem.In this paper,three expression cassettes(a rice female fertility gene expression cassette;a pollen-lethal gene expression cassette and a red fluorescence protein gene expression cassette)were introduced into female sterile rice to solve its reproductive problems,and a method for mechanized hybrid rice seed production using female sterile rice was established,which was applied to mechanized hybrid rice seed production in the way of mixed-planting and mixed-harvesting.The main results are as follows:1.A vector pFS4 with three expression cassettes(DsRed2+ZmAA1+PTB1)was constructed and transformed into Taibei 309 and Minghui 86 to obtain stable transgenic positive plants.The female fertility gene PTB1 expression cassette digested from vector PHB,the ZmAA1 gene expression cassette was synthesized,and the red fluorescence protein gene DsRed2 expression cassette digested from vector pLJ02 were cloned into the vector pC1300,generating plant expression vector pFS4.Plasmid pFS4 was transformed in the japonica rice Taibei 309 and indica rice Minghui 86.12 positive transgenic plants from TP309 and 17 positive transgenic plants from MH86 were obtained using red fluorescence protein as screening marker.Southern hybridization also showed that PTB1 sequences were inserted into the genome of transformed plants.2.A engineered self-reproducible female sterile restorer line Mfs4B was obtained.Using the T0 generation positive plants of the indica restorer line MH86 transferred from pFS4 as the female parent,and the indica restorer line ptb1 mutant as the male parent,the engineering female sterile restorer line Mfs4B with stable phenotype,contained homozygous ptbl and three expression cassettes was obtained in the F5 generation through hybridization,multi-generation back-crossing and self-crossing.The expression level of PTB1 gene in engineered female sterile restorer line Mfs4B was significantly increased.The ratio of fertile pollen to sterile pollen was basically maintained at 1:1.Its agronomic traits such as plant height,panicle number per plant and panicle length were stable.3.Five engineered female sterile restorer lines were rapidly created by gene editing.Five excellent hybrid rice parents,Yazhan,Huazhan,Huanghuazhan,R278 and Zhong8B,which were widely used in China,were introduced into pFS4 plasmid.1 positive transformation plant from Yazhan,1 from Huazhan,1 from Huanghuazha,7 from R278 and 7 from Zhong8B were successfully obtained.These transformed plants were hybridized with the corresponding female restorer sterile lines developed by gene editing in our laboratory,and the engineered female sterile restorer lines of these 5 parents were obtained.4.Editing intron region of PTB1 gene using CRISPR/Cas9 and co-transformation with three expression cassettes.The target was designed in the intron region of PTB1 gene,gene editing vector/PTB1-CAS9 was constructed,and transformed into Huazhan.12 positive transformed plants were obtained,among which one plant had a large fragment deletion between the target regions,with ratio of 8.3%.The three expression cassettes were amplified from plasmid pFS4 and cloned into vector PTB1-CAS9,then transformed into Huazhan and Yazhan.5.Non-fluorescent female sterile restorer lines without transgenic components can be efficiently separated by color sorter.The ratio of fluorescent seeds and non-fluorescent seeds produced by self-crossing of engineered female sterile restorer lines was basically stable at about 1:1.6 kg self-crossing seeds were randomly collected and sorted by a color sorter with 2 kg per time for three times.After sorting through the color sorter,fluorescent seeds were manually sorted by reinspection.1000 non-fluorescent seeds were randomly selected for PCR amplification of exogenous genes,and no transgenic components were detected.6.Male sterile line 301A and female sterile line fs4 were tested,and excellent hybrid F1 were produced.Male sterile lines 301A and 2182S were crossed with female sterile restorer line fs4.The F1 301 A/fs4 was superior to the control in panicle number,flag leaf length,panicle length,and other agronomic traits.At the same time,mechanized hybrid rice seed production was carried out,the flowering stage of female sterile restorer line fs4 in accordance with male sterile line 301A,and the F1 yield of 301 A/fs4 was satisfied,which was suitable for mechanized hybrid seed production by mixed-planting and mixed-harvesting.This study obtained a engineered female sterile restorer line Mfs4B,Self-pollination of Mfs4B generates the fluorescent seeds with transgene and non-fluorescent seeds without transgene in a 1:1 ratio.By means of fluorescence-assisted sorting,homogeneous female sterile rice seeds without transgene were sorted out from other seeds and used for mechanized hybrid rice seed production;heterozygous seeds carrying the transgene can then be used in the reproduction of female sterile restorer rice.This technology solved the reproductive problems of female-sterile rice,and proved especially valuable in systems using a mixed-planting and mixed-harvesting approach.The male sterile line 301A and female sterile restorer line fs4 were selected suitable for mechanized F1 hybrid rice seed production.Meanwhile,with the help of gene editing technology,the engineering female sterile restorer line was created quickly,and the breeding period of the engineering female sterile restorer line was significantly shortened.