An improvedAgrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars

We report here a high-efficiency transformation protocol for recalcitrant indica rice cultivars IR64 and IR72 with the selectable marker genehph and thegusA reporter gene. Factors that favor high-efficiency transformation were found to be use of 2-month-old mature seed-derived embryogenic calli, maltose as a source of carbon, a higher concentration of 2,4-dichlorophenoxyacetic acid, and both phytagel and agar as gelling agents. The putative transgenic (T₀) plants were analyzed for integration of the transgene through polymerase chain reaction and Southern blotting analyses. Various factors thought to be responsible for increased transformation efficiency are discussed.     

Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.)

Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.     

Effect of Callus Induction Media on Morphology of Embryogenic Calli in Rice Genotypes

Effects of four culture media on callus induction, regeneration and number of plants per unit culture were studied with mature seeds from five indica rice genotypes as explants. Based on the morphology, the calli were classified into four types as I to IV. Type I and type II are most suited to initiate suspension cultures or as target material for transformation. Number of plants regenerated per unit culture, formation of easily dissociating cell clusters and frequency of type I and type II calli were highest on NBKNB medium. Thus NBKNB medium is suitable for in vitro culture of even the hitherto recalcitrant indica genotypes.     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.     

Reasons for lower transformation efficiency in indica rice using Agrobacterium tumefaciens-mediated transformation: lessons from transformation assays and genome-wide expression profiling

Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In this study, transient and stable transformation assays using different japonica and indica cultivars showed that the lower transformation efficiency in indica rice was mainly due to the low efficiency in T-DNA integration into the plant genome. Analyses of the global gene expression patterns across the transformation process in different varieties revealed major differences in the expression of genes responding to Agrobacterium within the first 6 h after infection and more differentially expressed genes were observed in the indica cultivar Zhenshan 97 (ZS), with a number of genes repressed early during infection. Microarray analysis revealed an important effect of plant defense response on Agrobacterium-mediated transformation. It has been shown that some genes which may be necessary for the transformation process were down-regulated in the indica cultivar ZS. This dataset provided a versatile resource for plant genomic research to understand the regulatory network of transformation process, and showed great promise for improving indica rice transformation using genetic manipulation of the rice genome.     

Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes

Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or -glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.Communicated by W. Harwood     

Evaluation of parameters affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of citrus

An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars. It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 μM acetosyringone were required for improvement of transformation efficiency. Co-cultivation duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency as did OD₆₀₀ value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for maximum transgenic production are required for each individual cultivar.     

A tissue culture system for different germplasms of indica rice

Agrobacterium-mediated transformation of indica rice has been manipulated in only a limited number of cultivars because the majority of indica varieties are recalcitrant to in vitro response. Establishment of a highly efficient and widely used tissue culture system for indica rice will accelerate the application of transformation technology in breeding programs and the study of the functions of indica-specific genes. By manipulating plant growth regulators, organic components and salts within the culture media, we established two media for callus induction and subculture, respectively, in tissue culture of indica rice. The modified media could guarantee the production and proliferation of a great number of embryogenic calli with high regeneration capacity from mature seeds representing different indica rice germplasms. The calli obtained from this system should be ideal material for Agrobacterium-mediated transformation. The results suggest that this optimized tissue culture system will be widely applicable for the tissue culture of indica varieties. Electronic Supplementary Material Supplementary material is available for this article at The first two authors contributed equally to this work.     

Regeneration of transgenic plants from two indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars using shoot apex explants

We have established a reproducible procedure for transformation of shoot apices and regeneration of transgenic plants for two indica rice cultivars, white ponni (WP) and Pusa Basmathi 1 (PB 1). Four-day-old shoot apex explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA 101 harbouring a binary plasmid pRIT1. The vector contained an improved hygromycin phosphotransferase (hpt) gene for hygromycin resistance driven by actin 1 promoter and the reporter gene beta-glucuronidase intron (INT-GUS) controlled by CaMV 35S promoter. Rice shoots were induced on media containing 0.1 mg/l napthalene acetic acid (NAA), 1.0 mg/l kinetin (kn), 1.0 mg/l N(6)-benzyleaminopurin (BAP), 300 mg/l casaminoacid, 500 mg/l proline, 50 mg/l hygromycin and 500 mg/l cefotaxime. Transgenic plants were raised in pots and seeds were collected. Histochemical and polymerase chain reaction (PCR) analyses of field established transgenic rice plants and their offsprings confirmed the presence of GUS gene. Integration of T-DNA into the genome of putative transgenics was further confirmed by southern analysis. The transformation efficiency of WP was found to be ranging from 5.6 to 6.2% whereas in the case of PB1, it was from 7 to 8%. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both hpt and GUS gene.     

Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens

The efficiency of transformation was improved by treating immature embryos with heat and centrifugation before infection with Agrobacterium tumefaciens in rice and maize. Because the effects were detected both in the levels of transgene expression after co-cultivation and in the number of independent transgenic plants obtained per embryo, conditions were first optimized based on the transgene expression, and then transformants were produced. The optimal conditions varied considerably depending on species and genotypes, but reasonably good parameters were identified for Japonica rice, Indica rice or maize. As a general tendency, the effect of centrifugation was greater than that of heat in Japonica rice, whereas that of heat was greater than that of centrifugation in Indica rice and maize A188, and the combination of the treatments was the most effective in all of the genotypes tested. The frequency of transformation was improved several fold in rice and maize. In addition, transformation of certain genotypes of maize, which were not transformable before, and transformation of maize with a less efficient vector, which could not transform maize before, became possible by these pre-treatments. In the highest case, 18 independent transgenic plants were obtained from a single immature embryo of Japonica rice. Although nothing is known about the mechanism, these pre-treatments seemed to render cells of rice and maize more competent for transformation mediated by A. tumefaciens.     

Effect of sorbitol concentration on regeneration of embryogenic calli in upland rice varieties (Oryza sativa L.)

This study describes the impact of sorbitol on plantlets regeneration frequency (PRF) of four rice cultivars (japonica, Oryza sativa L.) both of which mature and immature embryo-derived calli were investigated. The variance analysis results showed that PRF of the three elite upland rice cultivars, Handao297 (HD297), Handao502 (HD502), Handao65 (HD65) and one lowland rice cultivar Zhongzuo93 (ZZ93) were significantly increased with addition of appropriate amount of sorbitol in culture media. Supplementing appropriate sorbitol in the media of a continous culture from induction and maintenance to regeneration for mature embryo-derived calli could improve PRF dramatically, originally from 27.6% up to a maximum of 71.8%. Especially to low regenerative capacity (LRC) cultivar HD65, the PRF was increased over 7-fold (from 9.7% to 74.0%). The optimum concentrations of sorbitol for calli induction, subculture and differentiation were 5, 20 and 40 g/l, respectively. Adding sorbitol, only in maintenance media at concentration of 20 g/l, also enhanced the PRF greatly in all the cultivars from 27.6% to 43.3%. Similar results were observed when incorporating with maltose in regenerating media both in immature and mature embryo-derived calli. The optimal concentration was 25 g/l sorbitol + 20 g/l maltose and 20 g/l sorbitol + 25 g/l maltose, respectively. HD297 appeared to be the most responsive genotype compared to other cultivars in PRF, 99.2% in immature embryo-derived calli and 76.8% in mature embryo-derived calli. The results and relevant conclusions might be valuable to establish an efficient plant regeneration system from somatic embryogenesis culture in upland rice.     

Efficient transformation of Korean rice cultivars (Oryza sativa L) mediated byAgrobacterium tumefaciens

The purpose of this study was to improve transformation efficiency for three Korean rice cultivars, Ilpum, Dasan, and Namyang. Using two different media with or without light, efficiencies of callus induction, regeneration, and transformation of the Korean cultivars were compared to Japanese cultivar, Nipponbare, as a control. Immature cv. Nipponbare seeds produced 35.5% and 16.1% regeneration efficiency on CIM and N6D media, respectively. Among the Korean cultivars, only cv. Ilpum induced on CIM in the dark was actively regenerated with efficiency of 8.2%. With LBA4404 (pTOK233), no difference for the efficiency of transformation was found between mature and immature seeds of cv. Ilpum. This result reveals that mature seeds can be substituted for this study with no difference. The anther-derived calli of cv. Namyang inoculated with either LBA4404 (pTOK233) or EHA101 (pSMABuba) showed regeneration efficiencies of 14.5% and 20.9%, respectively, even though efficiency of transformation did not differ with these two vectors. We suggest that the anther-derived calli are better-materials for transformation experiment due to their genotype-independent regeneration. In the assay of GUS, all of the calli that survived on the second selection medium were strongly stained. PCR-Southern blot analyses confirmed that T-DNA was stably transformed into all tissues selected. Cvs. Nipponbare and Namyang transformed by LBA4404 (pTOK233) showed positive color in the NPTII ELISA.     

提高农杆菌转化水稻频率的研究

以16种重要的籼稻和粳稻栽培品种为材料,研究了影响农杆菌转化水稻频率的有关因素,结果表明,CC培养基是绝大多数水稻全国组织的最适诱导与继代培养基;添加2．5－5mg/L ABA可以有效地改善水稻愈伤组织的质量,籼稻愈伤组织所需的筛选剂浓度低于粳稻愈伤组织所需的浓度,根癌农杆菌EHA105菌株对水稻的转化效果优于LBA4404和AGL1菌株的效果,头孢霉素对农杆菌的抑制效果优于羧苄青霉素的效果,共培养后进行适当的干燥处理既可增强脱菌效果,又可提高转化频率,应用我们所优化的农杆菌转化技术体系,获得了10个品种的水稻转基因植株。     

Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene

Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1 B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1 B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology.No additional selective pressure was needed to enrich the precise replacement events. This work demonstrates the feasibility of replacing any genes with elite alleles within one generation, greatly expanding our ability to improve agriculturally important traits.     

Agrobacterium-mediated transformation of élite indica and japonica rice cultivars

A rapid, efficient, routine system has been established forAgrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1. Calli induced from embryos of mature rice seeds were cocultivated withA. tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system. Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively. T1 seeds were harvested within 7–8 mo of initiation of mature embryo cultures. Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers. Mendelian inheritance of the transgenes was confirmed in T1 progeny.     

An improved technique for culture of rice panicles

An improved technique for long-term culture of rice caryopses is necessary for physiological and genetic studies. Panicles of three rice (Oryza sativa) cultivars `Lemont', `Gummo-byeu' and `Hwasung-byeu' were cultured in liquid media with combinations of light versus dark, panicle position, nitrogen level (5-40 mM), and sucrose level (29–351 mM). Grain growth was increased when panicles were positioned horizontally, partially submerged in the media, owing to greater media contact and apoplastic uptake as observed by fluorescent dyes. The optimal media assimilate supply included 175 mM sucrose and 5 mM nitrogen. Grain fill occurred for up to four weeks; grain dry weight reached 80% of that on intact plants, with 50% germination. This technique should allow for future physiological studies with rice or other panicle-bearing species.     

药用野生稻TAC克隆转化籼稻的体系初探

采用已构建的药用野生稻TAC文库,比较研究4个籼稻品种在不同培养基的诱导率及愈伤组织对潮霉素抗性。通过2种不同诱导方法产生的愈伤组织,将携带药用野生稻大片段DNA的TAC克隆转化到籼稻品种中的结果表明,除了‘粤香占’以外,其它3个品种在心诱导培养基中添加微量B,后,其愈伤诱导率最高。不同品种的潮霉素筛选适合浓度存在差异,其中‘华粳籼74’的适合筛选浓度为50mg．L-1,‘粤香占’为40mg．L-1。用预培养5d的愈伤组织进行遗传转化,在潮霉素筛选之前先以头孢拉定抑菌处理的,容易获得转基因植株。4个品种中以‘粤香占’的抗性愈伤组织筛选率和分化率最高,分别为22．58％和14．86％。分子检测11株转基因水稻的结果表明,其中8株含有抗性标记基因。据此认为,药用野生稻TAC文库在水稻创新育种中可能有一定的利用前景。     

Subspecies-specific intron length polymorphism markers reveal clear genetic differentiation in common wild rice （Oryza rufipogon L.） in relation to the domestication of cultivated rice （O. sativa L.）

It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice （O. sativa）. However, how the two subspecies of O. sativa （indica and japonica） were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism （SSILP） markers by comparison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups： one group （Group I） comprises all the O. rufipogon accesions from tropical （South and Southeast） Asia as well as the standard indica rice; the other group （Group II） comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domestication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. ruffpogon might occur after its speciation from other AA-genome species.     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.