Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Co-transformation of indica rice protoplasts with gusA and neo genes

Protoplasts of the indica rice (Oryza sativa L.) variety, IR54, were transiently transformed with the gusA gene and stably transformed with both the neo and gusA genes. We show that PEG-mediated co-transformation of protoplasts with two genes on separate plasmids coupled with selection on kanamycin is an effective way of transferring foreign gene(s) into the indica rice genome. The efficiency of co-transformation was generally 20–30%, i.e. the frequency of kanamycin-resistant calli having both the neo and gusA active genes. Southern blot analysis using a probe for gusA indicated integration of several copies of the gene, often as head to tail tandem repeats.Abbreviations GUS ß-glucuronidase - PEG polyethlene glycol - PCV packed cell volume     

Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection

Perennial ryegrass is one of the most widely cultivated grasses in temperate regions. However, it is recalcitrant for in vitro manipulation. In this study, various parameters affecting Agrobacterium tumefaciens-mediated infection were tested to optimize transformation efficiency in perennial ryegrass. The effects of heat shock and maltose concentration during Agrobacterium infection were evaluated along with variations in callus induction medium, bacterial infection media and callus age. Our results suggest that Agrobacterium infection at 42 °C for 3 min and co-cultivation of Agrobacterium-infected callus on a high maltose medium (6 %) significantly enhances the transformation efficiency in perennial ryegrass. The most optimal conditions proved to be use of four-month-old embryogenic callus induced on a modified N6 medium, infected with Agrobacterium grown on a modified Murashige and Skoog (MSM) medium, and a 42 °C heat shock treatment followed by the co-cultivation of the Agrobacterium and the callus on medium containing 6 % maltose (instead of 3 %). Using this optimized protocol, we were able to increase the transformation efficiencies for regenerated plants from approximately 1 % to over 20 %. Significant improvement in rice stable transformation efficiency was also observed when the optimized conditions were applied to this important cereal, indicating the method described here may apply to other monocots as well.     

Mapping QTLs that control the performance of rice tissue culture and evaluation of derived near-isogenic lines

Quantitative trait loci (QTLs) that control the performance of tissue culture in rice were detected by using 116 RFLP markers and 183 BC₁F₃ lines derived from two varieties, Koshihikari and Kasalath. With time, the seed callus of Koshihikari tends to turn brown and stop growing, while that of Kasalath remains yellowish-white and proliferates continuously. The performance of tissue culture in the induction of calli from seed, the subculture of induced calli, and shoot regeneration were evaluated by five indices: induced-callus weight, induced-callus color, subcultured-callus volume, subcultured-callus color, and regeneration rate. Through callus induction and subculture, eight putative QTLs (P < 0.001) were located on chromosomes 1, 4, and 9. Among these QTLs, five Kasalath alleles and three Koshihikari alleles improved tissue culture performance. No QTL for regeneration was found. Among all the QTLs, qSv1 explained the largest phenotypic variance, 33%, in subcultured-callus volume. In induced-callus color, two detected QTLs accounted for 36.4% of the total phenotypic variance; this was the highest score among the five indices used to evaluate the performance of tissue culture. Three near-isogenic lines for QTLs, located in two regions on chromosome 1, were developed to evaluate their tissue culture performance. The Kasalath alleles in qSv1 and qSc1-1 improved callus color through callus induction and subculture, and increased the subcultured-callus volume and the fresh weight of regenerated calli, including shoots, roots, and differentiated structures. In qSc1-2, the Kasalath allele improved callus color through induction and subculture. These results verified the presence of QTLs for the volume and color of subcultured callus on chromosome 1, qSv1, qSc1-1, and qSc1-2.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T₀ and T₁ plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T₁ progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.     

The Promoter of a Metallothionein-Like Gene from the Tropical Tree Casuarina Glauca is Active in Both Annual Dicotyledonous and Monocotyledonous Plants

A chimeric gene consisting of the -glucuronidase (gusA) reporter gene under the control of the metallothionein-like promoter cgMT1 from the tropical tree Casuarina glauca was introduced into Nicotiana tabacum via Agrobacterium tumefaciens and into Oryza sativa by particle bombardment. The strongest histochemical staining for GUS activity was observed in the root system of the transgenic plants, and especially in lateral roots. In contrast, a relatively low level of reporter gene expression was seen in the aerial tissues and GUS staining was located mainly in the plant vascular system. The average ratio of GUS activity between root and leaf was found to be 13:1 in tobacco and 1.5:1 in rice. The pattern of cgMT1 promoter activity in floral organs was found to be different in tobacco and rice. High levels of gusA gene expression were detected in the ovules, pollen grains and tapetum, whereas in rice PcgMT1 directs expression to the vascular system of the floral organs. These results suggest that PcgMT1 is potentially useful in molecular breeding to express genes of interest whose products are preferentially needed in roots.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.     

A procedure for regenerating Japonica and Indica varieties of rice from protoplasts

Fertile rice plants have been regenerated from protoplasts of two japonica rice varieties (Radon and Baldo) using a protocol initially developed for plant regeneration from protoplasts of an indica rice. Embryogenic calli were developed from immature embryos of Radon and Baldo rice on a callus induction medium, and then used to establish cell suspensions. Protoplasts were isolated from the cell suspensions, and cultured on a Millipore filter placed on a Kao/agarose medium that contained cell clusters from suspensions of IR52 or IR45. The protoplasts grew vigorously on Kao medium and developed into embryogenic calli within two to three weeks. Somatic embryo development occurred during a subsequent transfer of the calli to an LS medium for two to three weeks. The calli were then transferred to MS or N6 plant regeneration medium, and within one to three weeks, plants regenerated from 21 to 32% of the Radon calli, and 33 to 35% of the Baldo calli. Based upon these results and the previous success in regenerating an indica variety from protoplasts, this procedure has great promise for regenerating a range of rice varieties, and probably for regeneration of other monocotyledonous plants from protoplasts     

Induction of a polyubiquitin gene promoter by dehydration stresses in transformed rice cells

The expression of the maize polyubiquitin gene promoter UBI1 in rice cells has been used to study the involvement of ubiquitin in cell protection responses to dehydration caused by osmotic, saline or freezing stress. The effect of these stresses on UBI1 activity was investigated by the use of stably transformed rice calli (UBI1:GUS), as well as by transient expression experiments performed with cell lines with high or low tolerance to each type of stress. The theoretical analysis of the UBI1 promoter shows several putative stress-regulated boxes that could account for the stress-related UBI1 induction pattern described in this work. We suggest that the study of the differential UBI1 promoter-driven expression in rice cell lines with different level of tolerance to stress might be useful to elucidate complex signal transduction pathways in response to dehydration stresses in monocots.     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

Asymmetric protoplast fusion aimed at intraspecific transfer of cytoplasmic male sterility (CMS) in Lolium perenne L.

Summary Techniques have been developed for the production of cybrids in Lolium perenne (perennial ryegrass). Gamma-irradiated protoplasts of a cytoplasmically male-sterile breeding line of perennial ryegrass (B200) were fused with iodoacetamide-treated protoplasts of a fertile breeding line (Jon 401). After fusion 25 putative cybrid calli were characterized to determine mitochondrion type and composition of the nuclear genome. Analysis of phosphoglucoisomerase isozyme profiles and determination of the ploidy level by flow cytometry indicated that all of the calli tested essentially contained the nuclear DNA of the fertile line. However, the presence of parts of the nuclear DNA from the sterile line could not be excluded. Southern blotting of total DNA isolated from the parental lines and putative cybrids combined with hybridizations using the mitochondrial probes cox1 and atp6 revealed that the mitochondria of the calli originated from the fertile line (5 calli), the sterile line (5 calli) or from both parental lines (15 calli). The hybridization patterns of the mtDNA from the cybrid calli showed extensive quantitative and qualitative variation, suggesting that fusion-induced inter- or intramolecular mitochondrial recombination had taken place.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Tissue-specific expression of the rolC promoter of the Ri plasmid in transgenic rice plants

Summary Transgenic rice plants were obtained from protoplasts treated with two plasmids by electroporation. Primary transformants were selected on the basis of resistance to hygromycin, conferred by one of the co-transferred plasmids. Out of 26 hygromycin-resistant plants 2 showed the reporter gene activity due to another plasmid possessing a chimeric gene consisting of the promoter (about 900 by upstream non-coding region) of the ORF12 gene (roIC of the Ri plasmid and the coding region for -glucuronidase (GUS). Using a colorimetric reaction, the GUS enzyme was found to be localized in vascular tissues, demonstrating the similar expression of the roIC gene promoter in monocots and dicots (Sugaya et al. 1989; Schmülling et al. 1989).