Engineering Liguleless,Super-sweet And Waxy Compound Maize Varieties Via CRISPR-Cas9

Yield and nutritional quality improvement are the core mission of the maize breeding.The gain of yield obtained by increasing planting density is significant,so breeding the tight type maize varieties is an important way to increase the yield of maize.In addition,the market demand for fresh corn continue increase,so breeding the fresh sweet-waxy maize varieties is an important objectives of maize breeding.However,the traditional maize breeding has a long breeding cycle,low efficiency and the linkage drag,which seriously restricts the breeding process.Fortunately,the CRISPR-Cas9 gene editing technology can mutate the target gene and solve the defects of traditional breeding.The main research results of this experiment are as follows:1.Based on CRISPR-Cas9 gene editing technology,we constructe the CPB-lgl vector.A total of 113 T0 transgenic plants were obtained by Agrobacterium-mediated stable genetic transformation of maize.PCR-RE technology was used to detecte the ZmLgl gene target of 113 T0 plants.The mutation efficiency was 91.2%(103/113).The 81 individuals were biallelic mutations accounting for 78.64%of the total.19 plants were detected as homozygous mutations accounting for 16.81%of the tota.4 plants were detected as chimeric mutations accounting for 3.54%of the total.1 plant was detected as heterozygous.Phenotypic showed that 54.9%of the mutant plants showed a tight plant type.The mutant plants had no auricle and ligulelees structure,the leaf angle was about 12 degrees,and the mazie showed a tight plant type.2.The DTM lines were assessed by phenotypic statistics and amplicon deep sequencing systems.The DTM line was selected to induce mutations in the inbred lines.B73,Mo17,Dan 340,Huangzao 4,X178,Y478 and other backbone inbred lines were selected for DTM-induced mutation.The phenotypic statistics showed that DTM can induce mutations in different genetic backgrounds maize.The efficiency ranges from 4.35%to 43.59%,and the DTM line developed by the experimental phenotype can mutate different inbred lines.3.Based on the above experimental data,DTM-induced F1 were used to measure the photosynthesis efficiency and other parameters.It is showed that the pHotosynthetic efficiency and other parameters of the mutant material which was densely implanted were significantly increased compared with the control group.Through high-density planting,we found that the yield per plant is significantly increased by densely planted tight type maize compared to normal plant type maize,so the population yield is high.4.Based on CRISPR-Cas9 gene editing technology,we designed a directional mutation vector that can mutate ZmSh2 and ZmWxl genes,and obtained 22 To transgenic plants by Agrobacterium-mediated stable genetic transformation.55 F1 and T1 plants were obtained by T0 hybridization or self-crossing,and the ZmSh2 gene and ZmWx1 gene target of each plant were detected by Sanger sequencing.It was found that 27 plants of the ZmSh2 gene target were detected as biallelic mutations,17 plants were detected as homozygous mutations,and 3 plants were detected as heterozygous.Thirty plants of the Zm Wx1 gene target were detected as biallelic mutations,20 plants were detected as homozygous mutations,and two plants were detected as heterozygous.5.Through the above genotype data,the wx172,wx179,wx155,and wx170 containing no transgenic components were screened for waxy maize line which more than 95%of the amylopectin content in the grain starch.At the same time,the sw893,sw144 and sw891 no transgenic components were screened for sweet maize,and the soluble sugar content in the grains was 7.3 8%-10.28%.The genotype of sweet maize line P1(sh2,sh2;wx1,wx1)was crossed with the the genotype of waxy maize line P2(Sh2,Sh2;wx1,wx1)and obtain the genotype F1(Sh2,sh2;wx1,wx1),the obtained F1 hybrids are open pollinated.According to Mendel's law of inheritance,the genotypes of the seeds in the freshly harvested F1 are(Sh2,Sh2;wx1,wx1):(Sh2,sh2;wx1,wx1):(sh2,sh2;wx1,wx1)=1:2:1,showing a waxy corn material with a sweetness ratio of 1:3.This study was based on CRISPR-Cas9 technology to obtain compactly plant type maize and sweet waxy corn material.It also provided important theoretical and practical basis for CRISPR-Cas9-mediated genetic improvement of other traits in maize.