Breeding of a Water-saving Drought-resistant Two-line Hybrid Rice Variety Wanliangyou 1008

Drought is one of the important factors limiting rice production,and the application of new water-saving rice varieties is urgently needed in rice production.Wanliangyou 1008 bred by Institute of Rice Research,Anhui Academy of Agricultural Sciences is a new two line hybrid rice variety with some good traits,including high drought resistance,high yield and good disease resistance.Wanlingyou 1008 was obtained by crossing drought-resistant high-quality restorer line DJ1008 and disease-resistant two-line sterile line Wan 25S.In this paper,the breeding process,characteristics and seed production technology of the variety were described in detail.

Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea(Pisum sativum L.)

Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.

Development of plant cytosine base editors with the Cas12a system

Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors(CBEs). The main elements of Cas12aCBEs were engineered and their efficiency was evaluated in stably transformed rice cells. An optimized ttCas12a-hyA3Bctd editor, consisting of a LbCas12a variant carrying catalytic inactive D832A and temperature-tolerance D156R double mutations, a truncated human APOBEC3B deaminase, a human RAD51 single-stranded DNA-binding domain, and double copies of UGI, outperformed other Cas12aCBEs in base editing efficiency. In T0transgenic rice plants, ttCas12a-hyA3Bctd edited an average of42.01% and a maximum of 68.75% of lines at six genomic targets. A-to-G conversions were generated in rice by an adenine base editor with a similar architecture to the optimized CBE. Our results provide preliminary evidence for the feasibility of robust and efficient plant Cas12a base editing systems, which could be useful for precise crop breeding.

Study on the Variation of the Distant Crossing Rice by Ion Beam Implantation

In this paper, the following contents including the original receptor EI213 and other two control materials, RAPD polymorphism, photosynthetic efficiency, and the number of vascular bundles of the first internodes below the peduncle have been studied for the eight F7 transgenic lines obtained from ion beam implantation. The results showed that there was a significant variation in genomes of maize-rice line, compared with the receptor EI213, after the total exogenous maize DNA was introduced into EI213. The number of the vascular bundles of maize-rice progeny’s lines was obviously much larger than those of the original receptor EI213 and other two controls GER-3 and MH63, and along with the photosynthetic efficiency of maize-rice progeny’s lines being gone up. Moreover, the parenchyma cells around the vascular bundles of the transgenic lines became much larger in number and in size than those around the controls. All these indicated that the maize-rice progeny’s lines are really different from and superio

Prime editing-mediated precise knockin of protein tag sequences in the rice genome

Dear Editor,Accurately labeling proteins in living plant cells has long been a challenge and can be addressed by targeted insertion of tag sequences in a given locus.Recent optimized plant prime editors(PEs)enable efficient programmable installation of small insertions or deletions,including insertions of short sequences(Li et al.,2022a,2022b;Jiang et al.,2022;Xu et al.,2022;Zong et al.,2022;Zou et al.,2022).To investigate whether prime editing can be used to tag endogenous proteins in rice,we made use of the enpPE2 system described in our previous report(Li et al.,2022b).

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.

Developing a CRISPR/FrCas9 system for core promoter editing in rice

Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths.However,targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s.In this study,we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing.Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM.In addition,FrCas9 demonstrated activity against all 16 possible NNTA PAMs,achieving an efficiency of up to 35.3%in calli and generating homozygous or biallelic mutations in 31.3%of the T_(0)transgenic plants.A proof-ofconcept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content.Multiplex mutations and deletions were produced by bidirectional editing,mediated by FrCas9,using a single palindromic TATA sequence as a PAM.Moreover,we developed FrCas9-derived base editors capable of programmable conversion between AT and GC pairs in plants.This study highlights a versatile FrCas9 toolset for plant core promoter editing,offering great potential for the fine-tuning of gene expression and creating of new germplasms.

Development of an efficient plant dual cytosine and adenine editor

An enhanced CDA-like(eCDAL)was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors(CT-CBE).Then,a novel plant dual-base editor version1(pDuBE1)was developed by integrating TadA-8e into eCDAL.The editing efficiency of pDuBE1 could reach to 87.6%,with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7%in stably transformed plant cells.Our results showed that pDuBE1 could mediate robust dual editing in plant genome,providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.

Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants

Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in plants.The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope of application.Recently,five SpCas9 variants,SpCas9-NRRH,SpCas9-NRCH,SpCas9-NRTH,SpG,and SPRY,were developed to recognize non-canonical PAMs in human cells.In this study,these variants were engineered for plant genome editing,and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice(Oryza sativa)by stable transformation.Moreover,both cytosine base editors using a rat APOBEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 variants.Our results demonstrated that the developed SpCas9 variantsbased base editors readily generated conversions between C.G and T.A in the target sites with noncanonical PAMs in transgenic rice lines.Collectively,the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.