Advances in Physiological-biochemical and Genetic Mechanisms of Seed Aging in Rice(Oryza sativa L.)

Reasons causing or accelerating seed aging are mainly damage of mem- branes, DNA and proteins, decline of protein synthesis capacity and excessive ac- cumulation of reactive oxygen species. With the application of natural aging or artifi- cial aging methods, it was reported that quantitative trait loci （QTLs） of seed stora- bility in rice were widely distributed on the chromosomes except the 10th chromo- some. In this paper, we reviewed the progresses in the research on physiological- biochemical and genetic mechanisms of seed aging, and analyzed the existing problems and developing prospect in molecular breeding of rice with improved seed storability, in order to provide reference for the basic research and genetic improve- ment of rice seed storabUity.

Irrigation regimes modulate non-structural carbohydrate remobilization and improve grain filling in rice(Oryza sativa L.)by regulating starch metabolism

Recently developed ‘super’ rice cultivars with greater yield potentials often suffer from the problem of poor grain filling, especially in inferior spikelets. Here, we studied the activities of enzymes related to starch metabolism in rice stems and grains, and the microstructures related to carbohydrate accumulation and transportation to investigate the effects of different water regimes on grain filling. Two ‘super’ rice cultivars were grown under two irrigation regimes of well-watered(WW) and alternate wetting and moderate soil drying(AWMD). Compared with the WW treatment,the activities of ADP glucose pyrophosphorylase(AGPase), starch synthase(StSase) and starch branching enzyme(SBE), and the accumulation of non-structural carbohydrates(NSCs) in the stems before heading were significantly improved, and more starch granules were stored in the stems in the AWMD treatment. After heading, the activities of α-amylase, β-amylase, sucrose phosphate synthase(SPS) and sucrose synthase in the synthetic direction(SSs)were increased in the stems to promote the remobilization of NSCs for grain filling under AWMD. During grain filling, the enzymatic activities of sucrose synthase in the cleavage direction(SSc), AGPase, StSase and SBE in the inferior spikelets were increased, which promoted grain filling, especially for the inferior spikelets under AWMD.However, there were no significant differences in vascular microstructures. The grain yield and grain weight could be improved by 13.1 and 7.5%, respectively, by optimizing of the irrigation regime. We concluded that the low activities of key enzymes in carbon metabolism is the key limitation for the poor grain filling, as opposed to the vascular microstructures, and AWMD can increase the amount of NSC accumulation in the stems before heading, improve the utilization rate of NSCs after heading, and increase the grain filling, especially in the inferior spikelets, by altering the activities of key enzymes in carbon metabolism.

The auxin transporter OsAUX1 regulates tillering in rice(Oryza sativa)

Tillering is an important agronomic trait of rice(Oryza sativa)that affects the number of effective panicles,thereby affecting yields.The phytohormone auxin plays a key role in tillering.Here we identified the high tillering and semi-dwarf 1(htsd1)mutant with auxin-deficiency root characteristics,such as shortened lateral roots,reduced lateral root density,and enlarged root angles.htsd1 showed reduced sensitivity to auxin,but the external application of indole-3-acetic acid(IAA)inhibited its tillering.We identified the mutated gene in htsd1 as AUXIN1(OsAUX1,LOC_Os01g63770),which encodes an auxin influx transporter.The promoter sequence of OsAUX1 contains many SQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL)binding sites,and we demonstrated that SPL7 binds to the OsAUX1 promoter.TEOSINTE BRANCHED1(OsTB1),a key gene that negatively regulates tillering,was significantly downregulated in htsd1.Tillering was enhanced in the OsTB1 knockout mutant,and the external application of IAA inhibited tiller elongation in this mutant.Overexpressing OsTB1 restored the multi-tiller phenotype of htsd1.These results suggest that SPL7 directly binds to the OsAUX1 promoter and regulates tillering in rice by altering OsTB1 expression to modulate auxin signaling.

Genotypic Difference in the Responses of Seedling Growth and Cd Toxicity in Rice(Oryza sativa L.)

The experiment was carried out to study the genotypic difference in the responses of seed germination, growth and physiological characters of rice seedlings to Cd toxicity. The result showed that the germination was slightly stimulated under low Cd concentration （0.01-1.5 mM Cd）, while severely depressed under higher Cd concentration （2.0 mM）. Rice seedlings exposed to 0.01 mM Cd showed slight increases in plant height, root volume, biomass and chlorophyll concentration. These parameters were significantly reduced when Cd level in the medium was increased to 0.5 mM, and meanwhile corresponding increase in superoxide dismutase （SOD） and peroxidase （POD） activities, and MDA （malondialdehyde） content was observed. However, SOD and POD activities declined when plants were exposed to 1 mM Cd when compared with those under 0.5 mM Cd. Cadmium addition lowered Fe, Cu and Mn concentrations in roots and shoots. There was significant genotypic difference in the response of these parameters to Cd stress. Under Cd stress, Xiushui 110 had the least inhibition of growth and increase in MDA content, higher shoot Cd concentration, and greatest increase in POD and SOD activities, indicating its higher tolerance to Cd toxicity, while Bing 9914 had the greatest reduction of growth, and Zn, Cu, Fe, and Mn contents, but greatest increase in MDA content, and least increase in activities of antioxidative enzymes, indicating its sensitivity to Cd toxicity.

Mapping of three QTLs for seed setting and analysis on the candidate gene for qSS-1 in rice(Oryza sativa L.)

The lower seed setting is one of the major hindrances which face grain yield in rice. One of the main reasons to cause low spikelet fertility （seed setting） is male sterility or pollen abortion. Notably, pollen abortion has been frequently observed in advanced progenies of rice. In the present study, 149 BC2F6 individuals with significant segregation in spikelet fertility were generated from the cross between N040212 （indica） and Nipponbare （japonica） and used for primary gene mapping. Three QTLs, qSS-1, qSS-7 and qSS-9 at chromosomes 1, 7 and 9, respectively, were found to be associated with seed setting. The recombinant analysis and the physical mapping information from publicly available resources exhibited that the qSS-1, qSS-7 and qSS-9 loci were mapped to an interval of 188, 701 and 3741 kb, respectively. The seed setting responsible for QTL qSS-1 was further fine mapped to 93.5 kb by using BC2F7 population of 1 849 individuals. There are 16 possible putative genes in this 93.5 kb region. Pollen vitality tests and artificial pollination indicated that the male gamete has abnormal pollen while the female gamete was normal. These data showed that low seed setting rate relative to qSS-1 may be caused by abnormal pollen grains. These results will be useful for cloning, functional analysis of the target gene governing spikelet fertility （seed setting） and understanding the genetic bases of pollen sterility.

Physiological and genome-wide gene expression analyses of cold-induced leaf rolling at the seedling stage in rice(Oryza sativa L.)

Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanisms underlying these two traits remains unknown. In the present study, a cold-tolerant rice cultivar, Lijiangxintuanheigu, and a cold-sensitive cultivar, Sanhuangzhan-2, were subjected to low-temperature treatments and physiolog-ical and genome-wide gene expression analyses were conducted. Leaf rolling occurred at temperatures lower than 11℃, whereas discoloration appeared at moderately low temperatures such as 13℃. Chlorophyll contents in both cultivars were significantly decreased at 13℃, but not altered at 11℃. In contrast, the relative water content and relative electrolyte leakage of both cultivars decreased significantly at 11℃, but did not change at 13℃. Expression of genes associated with calcium signaling and abscisic acid (ABA) degradation was significantly altered at 11℃ in comparison with 25℃ and 13℃. Numerous genes in the DREB, MYB, bZIP, NAC, Zinc finger, bHLH, and WRKY gene families were differentially expressed. Many aquaporin genes and the key genes in trehalose and starch synthesis were down regulated at 11℃ in comparison with 25℃ and 13℃. These results suggest that the two chilling injury symptoms are temperature-specific and are controlled by different mechanisms. Cold-induced leaf rolling is associated with calcium and ABA signaling pathways and is regulated by multiple transcriptional regulators. The suppression of aquaporin genes and reduced accumulation of soluble sugars under cold stress results in a reduction in cellular water potential and consequently leaf rolling.

Natural Homologous Triploidization and DNA Methylation in SARII-628,a Twin-seedling Line of Rice (Oryza sativa)

A total of five pairs of diploidotriploid twin-seedlings （a diploid seedling and a triploid seedling emerged from a grain） were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ＇MSAP （methylation-sensitive AFLP）＇ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on Mogeneration after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.

Analysis of QTL for Seed Dormancy and Their Response to Dry Heat Treatment in Rice (Oryza sativa L.)

Quantitative trait loci (QTL) controlling seed dormancy in rice were identified usingrecombinant inbred lines (RILs) population derived from the cross between a japonicavariety Kinmaze and an indica variety DV85. Seeds of two parental cultivars and each RILwere harvested in 35d after heading. The germination percentage of these seeds at 30℃for 7 days were measured as the degree of seed dormancy. QTL analysis was performed withWindows QTL Cartographer 1.13a program by composite interval mapping. A total of four QTLfor seed dormancy were detected on chromosome 2 (two regions), 5 and 11, respectively.Phenotypic variation explained by each QTL ranged from 8.37 to 17.40%. Responses of suchloci to a dormancy-breaking treatment with dry heat were further detected. The resultsshowed that two alleles of qDOR-2-1 and qDOR-5 from DV85 as well as the allele of qDOR-11 from Kinmaze increased the seed dormancy, which seemed to be easily broken by dry heattreatment. Such loci of seed dormancy may be applied to rice genetic improvement. Theallele of qDOR-2-2 from DV85 increased the seed dormancy, which could not be broken bydry heat treatment.

Mapping QTLs for Salt Tolerance at Seeding Stage of Landrace Rice (Oryza sativa L.)

[Objectives] The identification of salt tolerant genetic loci in rice can provide a research basis for the molecular mechanism of salt tolerance and gene resources for improving salt tolerant cultivars. [Methods] Recombinant inbred lines(RILs) derived from Zhaxima, an indica landrace variety from Yunnan Province and Nanjing 46, an elite japonica variety with superior grain quality from Jiangsu Province were used. The salt tolerance at seeding stage in the RIL population was investigated as the phenotypic value. [Results] Combined with the linkage map, a total of 4 QTLs were detected: qSST-1, qSST-3, qSST-5 and qSST-11, located in rice chromosomes 1, 3, 5 and 11, respectively. All positive alleles were from the parent Nanjing 46. Three QTLs among them were not included in chromosome intervals the same as cloned rice salt tolerance genes, and thus were described as new candidate gene loci associated with seeding-stage salt tolerance. [Conclusions] This study provides important information for further exploration and utilization of new salt tolerant QTLs in rice. It is of great significance for improving the utilization of saline land in China and ensuring the stable rice production.

Influence of Epistasis and QTL×Environment Interaction on Heading Date of Rice(Oryza sativa L.)

QTLs for heading date of rice （Oryza sativa L.） with additive, epistatic, and QTL × environment （QE） interaction effects were studied using a mixed-model-based composite interval mapping （MCIM） method and a double haploid （DH） population derived from IR64/Azucena in two crop seasons. Fourteen QTLs conferring heading date in rice, which were distributed on ten chromosomes except for chromosomes 5 and 9, were detected. Among these QTLs, eight had single-locus effects, five pairs had double-locus interaction effects, and two single-loci and one pair of double-loci showed QTL × environment interaction effects. All predicted values of QTL effects varied from 1.179 days to 2.549 days, with corresponding contribution ratios of 1.04%-4.84%. On the basis of the effects of the QTLs, the total genetic effects on rice heading date for the two parents and the two superior lines were predicted, and the putative reasons for discrepancies between predicted values and observed values, and the genetic potentiality in the DH population for improvement of heading date were discussed. These results are in agreement with previous results for heading date in rice, and the results provide further information, which indicate that both epistasis and QE interaction are important genetic basis for determining heading date in rice.

Comparative Studies on the Changes of Microtubule Distribution and Reorganization During the Meiotic Stages of Development in Normal (IR36) and a Temperature/photoperiod Sensitive Male Sterile Line (Peiai 64S) of Rice ( Oryza sativa )

Changes in the pattern of organization of microtubules in the meiotic stages of development of pollen (i.e. from pre-meiotic interphase to more or less metaphase I) of a normal (IR36) and a temperature/photoperiod sensitive male sterile line (Peiai 64S) of rice were studied using immunofluorescence confocal microscopy. In IR36, from pre-meiotic interphase to metaphase I, the pattern of microtubule distribution in the meiocytes underwent a series of changes. Some new organizational patterns of microtubules (that have not been described before) were observed during microsporogenesis, including the existence of a broad band of perinuclear microtubules at the diakinesis stage of development. The pattern of microtubule distribution in the meiocytes of the male sterile line, Peiai 64S, was quite different front that seen in IR36. In Peiai 64S, the microtubules showed abnormal patterns of distribution from pre-meiotic interphase to metaphase I. For example the broad band of perinuclear microtubules seen at diakinesis in IR36 was much disorganized and loosened in Peiai 64S. The spindles formed were also very abnormal and different from the normal spindle. The appearance of abnormal microtubule distribution in the early stages of microsporogenesis may contribute to the malformation and ultimate abortion of pollen in Peiai 64S.

Calcineurin B-like interacting protein kinase OsCIPK23 functions in pollination and drought stress responses in rice (Oryza sativa L.)

Drought is very harmful to grain yield due to its adverse effect on reproduction, especially on pollination process in rice. However, the molecular basis of such an effect still remains largely unknown. Here, we report the role of a member of CBL （Calcineurin B-Like） Interacting Protein Kinase （CIPK） family, OsCIPK23, in pollination and stress responses in rice. Molecular analyses revealed that it is mainly expressed in pistil and anther but up-regulated by pollination, as well as by treatments of various abiotic stresses and phytohormones. RNA interference-mediated suppression of OsCIPK23 expression significantly reduced seed set and conferred a hypersensitive response to drought stress, indicating its possible roles in pollination and drought stress. In consistent, overexpression of OsCIPK23 induced the expression of several drought tolerance related genes. Taken together, these results indicate that OsCIPK23 is a multistress induced gene and likely mediates a signaling pathway commonly shared by both pollination and drought stress responses in rice.

Bulked Segregant Analysis to Detect QTL Related to Heat Tolerance in Rice(Oryza sativa L.) Using SSR Markers

The study was undertaken to assess the genetic effect of quantitative trait loci （QTLs） conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats （SSR）. The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis （SMA）. The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.

Studies on the Mechanism of Single Basal Application of Controlled-Release Fertilizers for Increasing Yield of Rice (Oryza sativa L.)

This paper was to explore the mechanism of single basal application of controlled-release fertilizers for increasing yield of rice （Oryza sativa L.）. Pot trials and cylinder trials were carried out from 2002 to 2005 to study the influences of single basal application of 3 controlled-release fertilizers on the changes of soil available N, root development, senescence and lodging resistance at late growth stages. Results showed that at 30 days after fertilization, single basal application of controlled-release fertilizers coated with vegetal-substance （CRF1） and polymer materials （CRF3） increased soil available N to 12.0 and 147.9%, respectively, in comparison to split fertilization of rice-specific fertilizer （RSF1）. Treatments of the two CRFs obviously benefited the development of root system, resulting in greater rice root weights with extensive distribution and higher root activity. In addition, the two CRF treatments, in comparison to RSF1, enhanced chlorophyll consents of the flag leaves to 9.5 and 15.5%, and soluble protein up to 89.7 and 108.0% respectively. Application of the two CRFs also made the base of rice stems strong and large, declined the proportion of shoot and root, increased root depth index. Though relatively low K rate, single basal application of the CRF3 coated with NH4MgPO4 could also promote the development of root system, enhance root activity and some physiological functions of flag leaves. Based on these results, it was concluded that major mechanisms for increasing rice yield by single basal application of the CRFs should be attributed to grater soil available N supply, superior development of root systems, better nutrient absorption capacity, slower senescence and enhancement of lodging resistance at late stages.

Biomass-Based Rice (Oryza sativa L.) Aboveground Architectural Parameter Models

To quantify the relationships between rice plant architecture parameters and the corresponding organ biomass, and to research on functional structural plant models of rice plant, this paper presented a biomass-based model of aboveground architectural parameters of rice （Oryza sativa L.） in the young seedling stage, designed to explain effects of cultivars and environmental conditions on rice aboveground morphogenesis at the individual leaf level. Various model variables, including biomass of blade and blade length, were parameterized for rice based on data derived from an outdoor experiment with rice cv. Liangyou 108, 86You 8, Nanjing 43, and Yangdao 6. The organ dimensions of rice aboveground were modelled taking corresponding organ biomass as an independent variable. Various variables in rice showed marked consistency in observation and simulation, suggesting possibilities for a general rice architectural model in the young seedling stage. Our descriptive model was suitable for our objective. However, they can set the stage for connection to physiological model via biomass and development of functional structural rice models （FSRM）, and start with the localized production and partitioning of assimilates as affected by abiotic growth factors. The finding of biomass-based rice architectural parameter models also can be used in morphological models of blade, sheath, and tiller of the other stages in rice life.

Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination

Phospholipase D （PLD） plays a critical role in plant growth and development, as well as in hormone and stress responses. PLD encoding genes constitute a large gene family that are present in higher plants. There are 12 members of the PLD family in Arabidopsis thaliana and several of them have been functionally characterized; however, the members of the PLD family in Oryza sativa remain to be fully described. Through genome-wide analysis, 17 PLD members found in different chromosomes have been identified in rice. Protein domain structural analysis reveals a novel subfamily, besides the C2-PLDs and PXPH-PLDs, that is present in rice - the SP-PLD. SP-PLD harbors a signal peptide instead of the C2 or PXPH domains at the N-terminus. Expression pattern analysis indicates that most PLD-encoding genes are differentially expressed in various tissues, or are induced by hormones or stress conditions, suggesting the involvement of PLD in multiple developmental processes. Transgenic studies have shown that the suppressed expression office PLDβ1 results in reduced sensitivity to exogenous ABA during seed germination. Further analysis of the expression of ABA signaling-related genes has revealed that PLDβ1 stimulates ABA signaling by activating SAPK, thus repressing GAmyb exoression and inhibiting seed germination.

Genetic Analysis and Primary Mapping of pms4, a Photoperiod-Sensitive Genic Male Sterility Gene in Rice (Oryza sativa)

To understand the genetic characteristics of a new photoperiod-sensitive genic male sterile line Mian 9S, some reciprocal crosses were made between Mian 9S and six indica rice materials, Yangdao 6, Luhui 602, Shuihui 527, Mianhui 725, Fuhui 838 and Yixiang 1B. Genetic analysis results suggested that the photoperiod-sensitive genic male sterility （PGMS） of Mian 9S was controlled by a single recessive nuclear gene. Thus, the F2 population derived from the cross of Yangdao 6/Mian 9S was used to map the PGMS gene in Mian 9S. By using SSR markers, the PGMS gene of Mian 9S was mapped on one side of the markers, RM6659 and RM1305, on rice chromosome 4, with the genetic distances of 3.0 cM and 3.5 cM, respectively. The gene was a novel PGMS gene and designated tentatively as pms4. In addition, the application of the pms4 gene was discussed.

Enclosed stigma contributes to higher spikelet fertility for rice(Oryza sativa L.) subjected to heat stress

With global warming, rice plants may be subjected to heat stress more regularly during the heatsensitive flowering stage, causing spikelet sterility and grain yield loss.Stigma exsertion is considered to increase pollen reception and promote female reproductive success.The aim of this study was to investigate the role of stigma exsertion on spikelet fertility at high temperatures.Five rice cultivars(Liangyoupeijiu, Shanyou 63, Huanghuazhan, Nagina 22, and IR64) with differing degrees of stigma exsertion were cultivated and exposed to high temperature at anthesis.Heat-tolerant cultivars did not always show a high percentage of spikelets with exserted stigmas, and vice versa.Irrespective of the presence of more pollen grains on exserted stigmas, spikelets with exserted stigmas did not show greater spikelet fertility than spikelets with fewer exserted stigmas or hidden stigmas under heat stress.GA3 application augmented the percentage of spikelets with exserted stigmas;however, it did not increase spikelet fertility under heat stress.Spikelet fertility of whole panicles was negatively correlated with the percentage of spikelets with exserted stigmas, but positively with that with hidden stigmas.Viability of the hidden stigmas was less reduced than that of exserted stigmas under heat stress, suggesting that hidden stigmas have an advantage in maintaining viability.Heat stress delayed anther dehiscence and reduced the viabilities of both exserted stigmas and pollens, thereby causing low spikelet fertility.Together, these results suggest that high spikelet fertility does not depend on stigma exsertion and that enclosed stigma generally contributes to higher spikelet fertility and heat tolerance under high-temperature conditions during flowering in rice.

Dissection of two quantitative trait loci for grain weight linked in repulsion on the long arm of chromosome 1 of rice(Oryza sativa L.)

Grain weight is a key determinant of grain yield in rice. Three sets of rice populations with overlapping segregating regions in isogenic backgrounds were established in the generations of BC2 F5, BC2 F6 and BC2 F7, derived from Zhenshan 97 and Milyang 46, and used for dissection of quantitative trait loci(QTL) for grain weight. Two QTL linked in repulsion phase on the long arm of chromosome 1 were separated. One was located between simple sequence repeat(SSR) markers RM11437 and RM11615, having a smaller additive effect with the enhancing allele from the maintainer line Zhenshan 97 and a partially dominant effect for increasing grain weight. The other was located between SSR markers RM11615 and RM11800, having a larger additive effect with the enhancing allele from the restorer line Milyang 46 and a partially dominant effect for increasing grain weight. When the two QTL segregated simultaneously, a residual additive effect with the enhancing allele from Milyang 46 and an over-dominance effect for increasing grain weight were detected. This suggests that dominant QTL linked in repulsion phase might play an important role in heterosis in rice. Our study also indicates that the use of populations with overlapping segregating regions in isogenic backgrounds is helpful for the dissection of minor linked QTL.

Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization

The progress of research on transferring elite genes from non-AA genome wild rice into Oryza sativa through interspecific hybridization are in three respects, that is, breeding monosomic alien addition lines （MAALs）, constructing introgression lines （ILs） and analyzing the heredity of the characters and mapping the related genes. There are serious reproductive barriers, mainly incrossability and hybrid sterility, in the interspecific hybridization of O. sativa with non-AA genome wild rice. These are the ＇bottleneck＇ for transferring elite genes from wild rice to O. sativa. Combining traditional crossing method with biotechnique is a reliable way to overcome the reproductive barriers and to improve the utilizing efficiency of non-AA genome wild rice.