Mutation of DEP1 facilitates the improvement of plant architecture in Xian rice (Oryza sativa)

Plant architecture is a complex trait and has a profound impact on crop performance and productivity. We applied the CRISPR/Cas9 system to mutate the DEP1 gene, which has been reported to function as regulator of plant architecture, in elite Xian cultivar ‘Huhan1509’. Sequencing analysis of T₀ transformed plants showed that the CRISPR/Cas9 system was highly efficient in mutagenesis of targeted DEP1 gene, with 30% of the homozygous mutations and 70% of the heterozygous mutations. T₂ homozygous mutants without T-DNA were further examined for the agronomic traits. The DEP1 mutants exhibited an altered plant architecture along with a shorter plant height and grain size and increased spikelets and grain density. Furthermore, phenotypes of raising primary branches and stem diameters were observed in the DEP1 mutants. Our results demonstrate that favourable alleles of the DEP1 gene, developed by CRISPR/Cas9 system, could be used to improve plant architecture in Xian rice.     

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T₁ lines, derived from 80 T₀ plants of 9 rice varieties. On average, about 33% T₁ lines had either morphological or agronomic variant plants. Most of the variations in T₁ plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T₃ or T₄ generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

采用RT-PCR法亚克隆了PDI基因保守区内450 bp的靶标序列作为干扰区段, 构建了含有内含子hpRNA (ihpRNA)的双元表达载体pTCK303-RiOsPDI, 经农杆菌介导转化日本晴, 获得转基因植株; 通过在T₀代对其潮霉素(Hyg)抗性基因的PCR鉴定, 确定携带有干扰片段的T-DNA区已整合到水稻基因组中, 且在转基因T₁代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明, PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低, 尤其是其籽粒中表达量较微, 几乎能引起靶基因80%左右沉默。对转基因T₂代植株的高温结实特性和籽粒理化品质的检测结果, PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低, 耐热性显著下降, 但其在常温处理下的结实率与对照之间无显著差异。此外, PDI基因沉默后, 稻米的透明度下降、垩白度增加, 但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。     

RFLP facilitated analysis of tiller and leaf angles in rice (Oryza sativa L.)

Plant type is an important composite trait associated with the yield potential in rice and other cereal crops. Several characters associated with the plant type of modern rice cultivars including tiller angle, leaf and flag leaf angle, were investigated using a complete linkage map with 115 well distributed RFLP markers and progeny testing of 2418 F₂ derived F₄ lines from a cross between O. sativa ssp. japonica cv. ‘Lemont’ and spp. indica cv. ‘Teqing’. One major gene (Ta) and 11 QTLs were largely responsible for the tremendous variation of the three plant type characters in the Lemont/Teqing F₂ population. The major gene, Ta, located between RZ228 and RG667 on chromosome 9, accounted for 47.5% of the phenotypic variation in tiller angle and had large pleiotropic effects on both leaf and flag leaf angles. This gene plus four QTLs accounted for 69.1% of the genotypic variation in tiller angle. Eight additional QTLs for leaf and flag leaf angles were also identified, which collectively explained 52.0 and 66.4% of the genotypic variation of these traits. Ta and three QTLs ( QFla2, QFla5 and QFla7) apparently affected the related plant type characters differently, suggesting their possible differential expression in different developmental stages of rice plants or possibly clustering of different genes affecting these traits. Plant type, and consequently grain yield of rice, may be improved by deliberately manipulating these QTLs in a marker-assisted selection program. This revised version was published online in July 2006 with corrections to the Cover Date.     

根癌农杆菌介导转Bt基因苎麻的获得及其抗虫鉴定

采用本研究室建立的根癌农杆菌介导的高效遗传转化体系,转化苎麻优良品种芦竹青,获得了转Bt基因苎麻候选植株。通过PCR和Southern杂交等分子检测,证明Bt基因已经整合到部分候选植株基因组中。选取PCR和Southern杂交均为阳性的部分株系(T₀)种植在大田,对这些植株进行室内抗虫鉴定并考察其主要农艺性状和品质性状,次年对T₁代植株进行PCR和Southern杂交检测。结果表明, 与对照相比,T₀代转Bt基因苎麻植株的抗虫性均强于对照,部分株系的抗虫性显著强于对照植株;且基本保持了亲本的优良性状;T₁代植株中也含有Bt基因,表明Bt基因能稳定遗传,且T₁代植株在大田的抗虫性明显强于非转基因植株。     

Somaclonal variation for some morphological traits, herb yield, essential oil content and essential oil composition in an Indian cultivar of rose-scented geranium

Somaclonal variation for plant height, plant spread, leaf shape, leaf size, leaf form, herb yield, essential oil content and 10 important constituents of the essential oil was studied in an Indian cultivar, ‘Bourbon’ of rose‐scented geranium (Pelargonium graveolens). Significantly larger variance was observed among in vitro‐regenerated plants of the SC1 generation (first generation following an in vitro phase) than among parental plants raised from stem cuttings for herb yield, plant height, leaf size, essential oil content, and for the contents of cis‐rose oxide, trans‐rose oxide, isomenthone and 10‐epi‐γ‐eudesmol in the essential oil. Plants of the SC₁ generation showing values beyond the higher or lower limit (depending on the trait) of the range observed among parental plants for different traits were developed into clones by their multiplication through stem cuttings, and evaluated again in the SC₂ generation (second generation following the in vitro phase). Somaclones selected for plant spread, and for the contents of linalool and trans‐rose oxide in their essential oils did not breed true in the SC₂ generation. However, for the remaining traits studied, 13–100% of the selected clones retained their selected traits in the SC₂ generation, suggesting that the changes in these somaclones were due to genetic causes.     

Allelic relationship between spontaneous and induced mutant genes for stem fasciation in chickpea

Stem fasciation is a morphological abnormality observed in plants where the stem is widened and leaves and flowers or pods are clustered at the apex. Several spontaneous mutants and one induced mutant for stem fasciation are found in chickpea (Cicer arietinum L.). This study was aimed at determining allelic relationship between spontaneous and induced mutant genes controlling stem fasciation and effects of stem fasciation on grain yield. Two spontaneous (ICC 2042 and ICC 5645) and one induced (JGM 2) stem fasciation mutants were crossed in all combinations, excluding reciprocals. The F₁ and F₂ plants from a cross between the two spontaneous mutants had fasciated stem. This indicated the presence of a common gene (designated fas1) for stem fasciation in the two spontaneous mutants. The F₁s of the crosses of the induced mutant JGM 2 with both spontaneous mutants had normal plants and segregated in a ratio of 9 normal : 7 fasciated plants in F₂. Thus, the gene for stem fasciation in the induced mutant JGM 2 (designated fas2) is not allelic to the common gene for stem fasciation in spontaneous mutants. The two genes in dominant condition produced normal non‐fasciated stem. The fasciated and the non‐fasciated F₂ plants did not differ significantly for number of pods per plant, number of seeds per plant, grain yield per plant and seed size, suggesting that it is possible to exploit the fasciated trait in chickpea breeding without compromising on yield.     

Rapid and high-precision marker assisted backcrossing to introgress the <Emphasis Type="Italic">SUB1</Emphasis> QTL into BR11, the rainfed lowland rice mega variety of Bangladesh

Flooding is one of the major hazards of rice production for the rainfed lowland rice ecosystem, and tolerant cultivars are urgently needed to help protect farmers from submergence damage. A quick and efficient strategy was implemented to introgress SUB1, a major QTL for submergence tolerance, into a rainfed lowland mega variety BR11 of Bangladesh by only two backcrosses and one selfing generation. In marker-assisted backcrossing (MABC), one tightly-linked simple sequence repeat (SSR) and two gene-based markers, four flanking SSR and 116 background SSR markers were used for foreground, recombinant and background selection, respectively, in backcrosses between a SUB1 donor IR40931-33-1-3-2 and BR11. BR11-Sub1, identified in a BC₂F₂ plant, possessed BR11 type SSR alleles on all fragments analyzed except the SUB1 QTL. The introgression size in BR11-Sub1 was 800 Kb indicating approximately 99.8% identity to BR11. BR11-Sub1 along with other introgression lines showed submergence tolerance similar to the tolerant parent. Yield, yield-component parameters and grain physico-chemical properties showed successful recovery of the BR11 traits in BR11-Sub1, with yield potential ranging from 5.2 to 5.6 t/ha, not significantly different from the recurrent parent mega variety BR11. Producing a large number (~1000) of backcross F₁ plants was considered essential to achieve recombination on both sides of the gene, limiting linkage drag with only two backcrosses. A large number of background markers ensured proper recovery of the recurrent parent genome in the BC₂F₂ generation. The study demonstrates a rapid and highly precise strategy to introgress a major QTL by BC₂F₂ generation into a modern rice variety using an unadapted donor. The variety can replace BR11 on more than 2 million of ha in Bangladesh and provide major increases in rice production.     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

Molecular dissection of the primary sink size and its related traits in rice

The genetic mechanism underlying the relationship between three traits of the primary sink size ‐ spikelet number per panicle (SNP), panicle number per plant (PN), and 1000‐grain weight (GWT), and their 10 component traits in rice was dissected in 292 F₁₃ recombinant inbred lines using a complete linkage map. A total of 43 genomic regions on 12 rice chromosomes were found to contain quantitative trait loci (QTLs) affecting the sink size traits, which revealed several important aspects of the genetic basis of sink capacity in rice. First, QTLs for SNP, PN and GWT were largely independent. Secondly, most QTLs affecting SNP and GWT showed close characteristics in both genomic locations and directions of effects to QTLs for their components, suggesting that pleiotropy, rather than linkage, was the primary genetic basis of the correlated panicle and grain traits. Thirdly, some QTLs affecting component traits did not contribute to SNP or GWT. In these cases, two or more QTLs with opposite effects on their component traits were detected, which could be due to either linkage or pleiotropy. Fourthly, some QTLs had large effects on panicle number (QPn4), panicle branching and length (QPbn3a, QPbn3b and QPb14), grain length and volume (QG13, QG15 and QGv2), and grain shape (QGs1 and QGs7), which were consistently detected in the related rice mapping populations and in different environments, providing good candidates and useful information for marker‐aided improvement of sink size and yield potential of rice.     

Semi-dominant genes confer additive tolerance to metribuzin in narrow-leafed lupin (<Emphasis Type="Italic">Lupinus angustifolius</Emphasis> L.) mutants

Narrow-leafed lupin (Lupinus angustifolius L.) is a grain legume well-adapted to sandy acid soils in a Mediterranean climate. Improved metribuzin tolerance in lupin cultivars is considered essential to protect crops from herbicide damage in Australia. This paper reports on the inheritance of metribuzin tolerance in two induced mutants Tanjil-AZ-33 and Tanjil-AZ-55 over the susceptible wild type cv. Tanjil. Both mutants were highly tolerant to 800 g/ha metribuzin with no foliage damage, but cv. Tanjil died and reciprocal F₁ hybrids had intermediate tolerance with foliage damage. The F₂ populations of both crosses, Tanjil-AZ-33 × Tanjil and Tanjil-AZ-55 × Tanjil, had a segregation ratio of 1:2:1 for highly tolerant: damaged:dead plants. Progeny tests (F₃) of selected F₂ single plants confirmed that highly tolerant F₂ plants were homozygous and damaged F₂ plants were heterozygous. Clearly a single semi-dominant gene conferred metribuzin tolerance in both mutants. An allelism test revealed that the two mutants had two non-allelic tolerance genes with F₂ plants segregating in a 15:1 ratio for survival and death at 800 g/ha metribuzin. The tolerance gene in Tanjil-AZ-33 was designated as Mt3 and the gene in Tanjil-AZ-55 as Mt5. At 4,000 g/ha metribuzin, 1/16 of F₂ plants from the cross between the two mutants had no herbicide damage, suggesting the additive effects of the two tolerance genes, whilst the rest were damaged or dead. Combining these two tolerance genes, Mt3 and Mt5, increased tolerance further by approximately five-fold.     

Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer

Transgenic Korean rice plants containing the cry1Ab gene were developed for resistance against yellow stem borer (Scirpophaga incertulas, YSB). More than 100 independent transgenic lines from three Korean varieties (P-I, P-II and P-III) were generated. The amount of Cry1Ab in transgenic T₀ plants was as high as 2.88% of total soluble proteins. These levels were sufficient to cause 100% mortality of YSB larvae. The majority of T₁ transgenic lines originated from the varieties P-I and P-II followed a Mendelian fashion of segregation. Deviation from the expected segregation ratio was observed in a small number of the transgenic lines of P-I and P-II origins. However, this deviation was primarily observed in the P-III originated lines. Segregation analysis of the T₁ generation indicated that 1–3 copies of the cry1Ab gene were integrated into the genome of the majority of the transgenic lines originating from varieties P-I and P-II. Stunted and semi-fertile mutants were observed in some transgenic lines. These aberrations were either independent or closely linked to the introduced cry1Ab gene loci in different transgenic lines. Reduction in GUS expression levels and loss of toxicity against YSB larvae were found in some transgenic lines. The transgenic T₃ and T₄ lines causing 100% mortality of third instar YSB larvae in the lab were completely protected in the field. Analysis of important yield components on nine selected transgenic lines indicated that stem length, panicle length, grain number per panicle, and seed setting rates were reduced in transgenic plants compared to those in non-transgenic parental rice lines. Number of panicles per cluster, however, was significantly higher in transgenic plants. The numerical value of the average yield was in general greater in the controls than in all the transgenic lines, indicating some ‘yield drag’. Since some selected lines were highly resistant to the YSB with good yielding potential, they offer effective potential for use in insect resistance management programs.     

Fine mapping of the quantitative trait locus <Emphasis Type="Italic">qFLL9</Emphasis> controlling flag leaf length in rice

The morphological traits of leaves, such as size and shape, are major determinants of plant architecture and strongly affect high yield performance. To understand the molecular mechanism governing flag leaf length, we analyzed quantitative trait loci (QTLs) affecting flag leaf length by employing 176 F₂ individuals derived from a cross between two japonica rice cultivars: Shennong265 (SN265) and Lijiangxintuanheigu (LTH). We identified qFLL3, qFLL6 and qFLL9 from this F₂ population. Flag leaf length was increased by SN265 alleles at qFLL3 and qFLL6, but by LTH allele at qFLL9. In order to eliminate the influence of qFLL3 and qFLL6, one single residual heterozygous plant for qFLL9 region, RH-qFLL9, was selected based on the genotypes of 114 simple sequence repeat (SSR) markers and used as the parent of a segregating population. Using this segregating population of 889 plants, this region was narrowed down to an interval between RM24423 and RM24434. According to the rice annotation project database, there are 17 predicted genes in the 198-kb target region.     

Quantification of critical factors affecting fitness of the sugary1 mutant in maize

Fitness of sugary1 (su1) is affected by some critical traits that depend on the genotypes and environments, while their effects have not been quantified with convincing statistical methods. The objective of this work was to identify and quantify the critical factors of su1 fitness with different genotypes and environments. We used two pairs of field corn inbreds that differentially affected su1 viability to develop F₁, F₂, BC₁ and BC₂. After selfing, Su1 and su1 kernels were evaluated under controlled environmental conditions and in field trials. Multiple regressions showed that dry weight of juvenile plant was affected by early vigour (plant size, colour and health) and emergence in cold conditions; ear weight by plant appearance, number of plants and chlorophyll content; number of ears by plant appearance, number of plants, chlorophyll content and female flowering; and kernel weight by ear weight, number of plants, row number and ear length. The main critical factors for su1 fitness were early vigour and emergence under cold conditions at initial stages, while several adult traits were related with final fitness.     

Comparative Morphological Studies of Microspore Derived and F2 Plants Obtained from an Interspecific Rice Hybrid Oryza sativa Linn. ×O. rufipogon Griff.)*

Anther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F₂ plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F₂ segregation. (3) It was possible to recover indica type recombinants among the anther-derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice.     

Genetic analysis of methionine content in indica-japonica hybrid rice (Oryza sativa L.) at different grain developmental stages

Genetics of methionine content in indica-japonica hybrid rice (Oryza sativa L.) was studied in 35 F₁ hybrids derived from crossing 7 male-sterile indica rice cultivars with 5 restorer japonica rice cultivars. Two genetic models and their corresponding statistical methods for quantitative traits of triploid endosperm in cereal crops were used for the analysis. One is the unconditional model, which refers to the analysis of cumulative measurements along the developmental stages, while another is the conditional model, which relates to analysis throughout the developmental stages. Results showed that methionine content of indica-japonica hybrid rice was controlled by expression of triploid endosperm nuclear genes, cytoplasm genes, diploid maternal plant nuclear genes, and their genotype-environment interaction effects. Dominant effects were important at the first three developmental stages, while additive effects were important at the next two stages of grain development under both unconditional and conditional analyses. With regard to the components of heritability, maternal and cytoplasm general heritabilities and their environmental interaction heritabilities were important. It was suggested that enhancing methionine content could be more efficient when selection is based on maternal plants in early breeding generations.     

Insect-mediated seed-set evaluation of 21 soybean lines segregating for male sterility at 10 different loci

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T₂ lines obtained from T₁ PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T₁ PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.     

Multiplex PCR genotyping for five bacterial blight resistance genes applied to marker‐assisted selection in rice (Oryza sativa)

Bacterial blight (BB) is the most economically damaging disease of rice in Asia and other parts of the world. In this study, a multiplex PCR genotyping method was developed to simultaneously identify genotypes of five BB resistance genes, Xa4, xa5, Xa7, xa13 and Xa21. The resistance R alleles were amplified using five functional markers (FMs) to generate amplicons of 217, 103, 179, 381 and 595 bp in IRBB66. Amplicons of 198, 107, 87, 391 and 467 bp corresponded to susceptible alleles in Taiwanese japonica rice cultivars. In backcross breeding programmes, the multiplex PCR assay was integrated into selection from a population using BB resistance donor IRBB66 crossed to rice cultivar ‘Tainung82’. Two plants with homozygosity for Xa4, xa5, Xa7, xa13 and Xa21 were selected from 1100 BC₂F₂ plants. In addition, the five BB resistance genes were also accurately identified in F₂ populations. This multiplex PCR method provides a rapid and efficient method for detecting various BB resistance genes, which will assist in pyramiding genes to improve durability of BB resistance in Taiwanese elite rice cultivars.     

Individual and combined effects of plant height reducing genes in periwinkle

Inheritance of an induced bushy mutant (EMS 24-5) of periwinkle (Catharanthus roseus) and its genetic relationship with an earlier reported dwarf mutant (NEU 6-15) was studied by crossing it with its parental variety, Nirmal and the dwarf mutant. The bushy mutant was found to be under the control of a monogenic recessive gene (by), which was inherited independently of the recessive gene (dw ₁ ) involved in the dwarf mutant. The double mutant (bydw ₁ ) recombinant, which was recovered from the F₂ generation of the cross (NEU 6-15) × (EMS 24-5), its parental mutants and variety, Nirmal were used for determining the individual and combined effects of plant height reducing genes dw ₁ and by. Individually, the recessive genes by and dw _1, reduced plant height by 27–30, 64–68%, respectively, while together they reduced plant height by 73–77%. The double mutant recombinant was found to be 63–68 and 25–29% shorter than its parental mutants (EMS 24-5) and (NEU 6-15), respectively. The effects of the recessive gene by on plant height were discernable only after the age of 4 months while those of dw ₁ and combined effects of dw ₁ and by became apparent one to 2 weeks after germination. The double mutant recombinant was found to have higher content of total alkaloids in the roots than parental mutants as well as the variety, Nirmal.