TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

A new method to identify and improve the purity of hybrid rice with herbicide resistant gene

There is a close relationship between the hy—brid rice production and seed purity.Two-linehybrid rice with higher heterosis is producedthrough the hybridization between a photo-thermo sensitive genetic male sterile(GMS) rice line and a paternal variety.But the fertili-

RICE TRANSFORMATION WITH A PHYTOALEXIN GENE AND BIOASSAY OF THE TRANSGENIC PLANTS

Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 (100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae ) and bacterial blight of rice ( Xanthomonas oryzae ). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L.

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O.sativa and O.meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety （Digu） by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 （pZH01-tp309） and three different expression constructs, pCB-Pi-d25.3kb （pCB5.3kb）, pCB-Pi-d26.3kb （pCB6.3kb） and pZH01-Pi-d22.72kb （pZH01-2.72kb） of Pi-d2, driven by Pi-d2 gene’s own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

Genetic Analysis of Stripe Disease Resistance in Rice Restorer Line C224 Using Major Gene plus Polygene Mixed Effect Model

The inheritance of stripe disease resistance in a rice restorer line C224 was analyzed using the mixed effect model of major gene plus polygene for quantitative traits.In addition,the resistance was investigated in seven crosses of C224 with maintainer lines.The results showed that the stripe resistance of C224 was controlled by two major genes with additive-dominance-epistasis effects plus polygenes with additive-dominance effects （E-1 model）.These two genes had additive effects of-12.47% and-24.75%,respectively,showing negative dominance effects.There were significant epistasis and interaction effects between the two major genes.The heritability of the two major genes was 92.12%,while that of polygenes was 2.74%,indicating that the stripe resistance had dominant major gene effect.Of the seven crosses,five displayed high or medium resistance to the stripe disease.

Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice(Oryza sativa L.)caused by the fungus Magnaporthe oryzae(M.oryzae),and neck blast is the most destructive phase of this illness.The underlying molecular mechanisms of rice blast resistance are not well known.Thus,we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining,Shandong Province,China in 2017.Results showed that 92(61.3%)and 58(38.7%)rice varieties were resistant and susceptible to M.oryzae,respectively.Among the 150 rice varieties screened for the presence of 13 major blast resistance(R)genes against M.oryzae by using functional markers,147 contained one to eight R genes.The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers.The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance.Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties,and were meaningful for rice disease resistance breeding.

Development of Hybrid Rice Variety FY7206 with Blast Resistance Gene Pid3 and Cold Tolerance Gene Ctb1

Hybrid rice Fanyou 7206（FY7206）, derived from the cross between a sterile line Fanyuan A and a restorer line Fuhui 7206, was bred by the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. FY7206 was characterized by moderate blast resistance, cold tolerance, as well as wide adaptability, and high yields. The blast resistance results indicated that the frequencies of blast races in race B, race C and the total resistance frequency for FY7206 were 95.5%, 100.0% and 97.2%, respectively. The disease resistance results showed that the leaf blast grade for FY7206 was level 1 and panicle blast was level 5. The indoor spray results indicated that FY7206 was resistant to 11 isolates of Magnorpathe oryzae. The blast resistance of FY7206 might be derived from the high expression of blast resistance gene Pid3. The results for simulated cold resistance in an artificial climate chamber indicated that the cold tolerance for FY7206 was moderate at the booting and flowering stages. The cold tolerance results also indicated that FY7206 could be tolerant to temperatures as low as 10 °C at the seedling stage. The q RT-PCR results showed that the expression of cold tolerance gene Ctb1 in FY7206 was relatively high. These results suggested that FY7206 is a hybrid indica rice variety with good comprehensive characteristics, including blast resistance and cold tolerance.

Influence of different inoculation methods on the evaluation of the resistance to rice sheath blight(Rhizoctonia solani Kuhn)

To establish a standard system for geneticstudies on sheath blight resistance, a field testwas conducted at the experimental farm ofYangzhou University to compare several pro-cedures for inoculating rice plants with R.solani Kuhn (RH9). The varieties used wereJasmine 85, Teqing (resistant or moderatelyresistant), and Lemont (susceptible). They

Rice planthopper resistance of interspecific protop last fusin line"pf9279" between O.sative and O.officinalis

An interspecific hybrid line pf9279 was obtained by proto-plast fusion between 02428(japonica, with a wide com-patibility gene) and CNW240 (O. officinalis, fromMalaysia) at CNRRI in 1992. Possible introgression of

Relationship between changes of endogenous ABA level and resistance in hybrid rice under temperature stress

We studied the relationship between endogenous ABA and resistance to temperature stress in seedlings of hybrid rice. The hybrid rice Combinations Shanyou 63, Shanyou 287, Weiyou 63, and Weiyou 287 were treated under 2℃ and 42℃ respectively for 48 h. Endogenous ABA concentrations were obtained by gas chromatography with electron capture detector (GC-ECD) and a nickel labelled external standard. Leakage of electrolytes were determined. At optimal temperature (27℃), there was difference between the level of endogenous ABA and the leakage of electrolytes in hybrid rice combinations, which attributed to genetic difference. ABA level in Weiyou system was higher than that in Shanyou, and there were distinctions between Shanyou system itself (see table). At 2℃, the ABA content of rice seedling was increased. ABA level of Weiyou system maintained higher than Shanyou system at 27℃. The leakage quantities of

Protein changes in rice seedlings during the enhancement of chilling resistance by different stress pretreatment

The changes of proteins in the rice (Oryzasativa L.) Tesanai 2 seedling under salt (NaCl,4 g/L), heat shock (42℃, 3h ), and cold(14℃, 3d ) pretreatments were compared toexplore the mechanism of the cross adaptationto different environmental stresses. The chill-ing resistance of rice seedling after 1℃, 150pmol·msPFD(photo flux density) for 2d was enhanced distinctly by salt, heat shock,

Effects of yield and resistances on rice by application of Si-fertilizer

According to the analysis of 654 soil samples collected from 45 counties in Zhejiang Provence, 73.7% of paddy field showed Si deficient. During 1991 and 1993, in Lishi, Longquang, Qingtian and Tonglu Counties, field experiments were conducted to test the effects of basal or additional Si-fertilizer on rice. Si-fertilizer was produced by Nanjing Inorganic Chemical Manufacture, containing 50% water solu

Inheritance study on the stable herbicide resistance of transgenic rice

The transgene technology showed a potentiali-ty in crop improvement such as disease and in-sect resistance,anti-adversity,and grain qual-ity.The inheritance of bar gene for herbicideBASTA resistance in stable transformed ricelines was studied for an understanding of theforeign gene inheritance pattern. BASTA resistant transgenic rice plantletswere regenerated from the embryos of rice va-rieties JY119 bombarded with bar gene.A sta-ble BASTA resistant TRline was consecutiv-

Effect of exogenous salicylic acid on resistance of rice seedlings to blast

Salicylic acid(SA)is an endogenous regulatorymolecule for plant flowering and thermogene-sis,and has been reported as a signal moleculeof plant disease resistance in the plant-pathogen interaction.Exogenous SA is capableof inducing disease resistance in a wide range ofplants.This study was to evaluate the poten-tial for SA to induce resistance of rice seedlings

Verification test on an innovated method for the studies on inheritance of resistance to rice sheath blight

We have recently developed a systematic method for the study on the inheritance of resistance to sheath blight. The key of the system is an innovated method of inoculation and investigation along with the employment of the permanent population. This paper reported the procedure of the system and the result of its verification.

Mechanism of rice variety resistance to the yellow stem borer,Scirpophaga incertulas (Walker)

Yellow stem borer(YSB),Scir-pophaga incertulas(Walker)has be-come the main rice insect pest onceagain along the Changjiang Riversince 1990s.Unfortunately,no ricevariety with resistance to YSB hasbeen reported so far.Japonica Zhen-dao 2 with moderate resistance toYSB was found.In 10 rice vari-eties with different resistance levels,YSB individuals showed distinct de-velopmental rates,and the asyn-chronous development even occurredin the same rice variety.In this

New technique for identifying varieties resistance to rice blast

After 8 yrs lab experiments and field tests, an advanced technique for identifying varieties resistance to rice blast was developed by a research group in Plant Protection Institute, Zhejiang Academy of Agricultural Sciences. With this technique, the inoculum was prepared on a maize-rice-straw-agar media which was suitable for sporulation of most rice blast pathogen isolates. To avoid possible virulence variation during the vegetative growth, the conidia were harvested and preserved for later different sets of resistance evaluation tests. A simple and precise new method—smear inoculation was used for infection. The experi-

Analyses of gene effects and inheritance of resistance to bacterial blight in rice

We analyzed the resistant inheritance of ajaponica variety,Jia23,to two bacterial blightpathogen strains,KS-6-6 and Zhe 173,rep-resenting respectively the two predominantpathogenic types(Ⅱ and Ⅳ)in the rice crop-ping area along the Yangtze River Valley.Jia23 was crossed with susceptible vari-eties,Ewan8 and 7416.Fplants were back-