Transgenic Rice: Characterization of Protoplastderived Plants and their Seed Progeny

Thirty eight green and 2 albino plants were regenerated from400 kanamycin-resistant colonies derived from protoplasts isolatedfrom cell suspensions of Oryza sativa variety Taipei 309 andelectroporated with pCaMVNEO carrying the neomycin phosphotransferaseII (nptII) gene. Twenty of the green transgenic R_o plants weretransferred to the glasshouse, where 3 flowered after 7 months.Of 15 plants analysed by DNA hybridization, all carried thenptll gene, but only 2 of 11 plants assayed for NPTII activityexpressed the nptll gene. One transgenic R_o plant produced 59seeds following self-pollination. The seeds, when germinatedon medium containing kanamycin sulphate, gave 16 green transgenicR, plants. Five transgenic R₁ plants flowered and set seed,7 flowered but failed to produce seeds, while 4 did not producepanicles. Transgenic R_o and R₁ plants were shorter, requiredlonger to flower, and had reduced pollen viability comparedto non-transformed R₁ protoplast-derived plants. The nptII genewas present in all 16 transgenic R₁ plants, but NPTII activitywas detected in only 8 of these plants. Key words: Oryza sativa variety Taipei 309, rice, protoplasts, direct DNA uptake, kanamycin-resistant tissues, transgenic plants, DNA hybridization, neomycin phosphotransferase II (NPTII), gene expression and inheritance     

Oryzacystatin Exogenously Introduced into Protoplasts and Regeneration of Transgenic Rice

Oryzacystatin (OC) is a proteinaceous cysteine proteinase inhibitor involved in the biodefense of rice seeds. To create transgenic rice plants with increased OC activity, we introduced an OC expressing vector into rice protoplasts and obtained transformed calli. The expression vector contained a bacterial inaA DNA fragment in the 3′-noncoding region as a tag to distinguish the introduced DNA from the intrinsic OC gene. The OC vector and a selection marker gene conferring hygromycin resistance were used together to transfect into rice protoplasts. A number of hygromycin-resistant calli were obtained and studied by polymerase chain reaction and genomic Southern blotting to find if the exogenous OC gene had been integrated. The calli were studied by northern blotting as well to examine mRNA expression. The results showed that integration and expression of the introduced OC gene occurred in 51% and 27%, respectively, of 156 subcultures from 15 hygromycin-resistant calli. As a final step, transgenic rice plants were regenerated from the calli expressing OC. Leaves and seeds from the plants had higher OC activities than those from nontransgenic plants.     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene. Received: 10 October 1997 / Revision received: 18 March 1998 / Accepted: 29 November 1998     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Development of transgenic rice pure lines by particle bombardment‐mediated transformation and genetic analysis‐based selection

Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection.     

Efficient Production of Transgenic Cassava Using Negative and Positive Selection

In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.     

Characterization of transgenic rice plants that express rgp1, the gene for a small GTP-binding protein from rice

 The rgp1 gene, which encodes a small GTP-binding protein from rice, was introduced into rice protoplasts by electroporation. Transformed protoplasts were cultured on liquid protoplast-culture medium for 1 month, and then cells that had proliferated were transferred to a selection medium that contained 50 mg/l hygromycin B. Among 50 colonies that were selected and transferred to regeneration medium, 3 colonies generated shoots. However, two of the three shoots failed to form roots and ceased growing. A single regenerated shoot that formed roots was planted in soil and transferred to a greenhouse. Southern hybridization showed that the regenerated plant harbored a single copy of the introduced gene. The transformant (T₀) plant was shorter than the controls, it developed three times as many tillers as controls, it developed three times as many tillers as control plants but it produced mostly sterile seeds. In a test of hygromycin resistances, viable seeds segregated into resistant and sensitive seedings at a ratio of approximately 1 : 3. The progeny (T₁) plants were short with many tillers, and some produced seeds normally. The T₂ seedlings grew more rapidly than control seedlings for the first 28 days after germination, but control plants subsequently outgrew the T₂ plants. Northern blotting analysis revealed that the rgp1 gene in T₂ plants was expressed consitutively throughout all developmental stages. The results suggest that the observed phenotypic changes were due to expression of the exogenous rgp1 gene. Received: 21 September 1997/Accepted: 31 March 1998     

农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和 Ｇ Ｕ Ｓ 基因的双元载体将杀虫结晶蛋白基因ｃｒｙ Ｉ Ａ（ ｂ） 和ｃｒｙ Ｉ Ａ（ｃ） 导入到籼、粳稻幼穗愈伤组织中,然后经过在含有不同浓度潮霉素的培养基上进行数次筛选,获得一批 Ｂｔ 转基因株。经 Ｐ Ｃ Ｒ、 Ｓｏｕｔｈｅｒｎ 杂交及 Ｗｅｓｔｅｒｎ 印迹分析证实此二基因已整合进水稻中,饲虫试验结果表明,转基因株具有１００ ％ 杀虫率。     

Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system.

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.     

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

INTRODUCTIONArachiS hypogaea L., Peanut or groundnut, isan importal commercial crop worldwide. It provides an excellellt source of protein and other nutrients. Its production and quality can be severelyimpacted under stressful growing conditions such ascdriate factors, pests and diseajses. Genetic engineering provides a prospective way to reduce certainproblems by transferring individual genes for pestresistance or other traits into elite germplasm of acultiVated species. Thansgenic pea…     

Analysis of a large number of independent transgenic rice plants produced by the biolistic method

Summary Over 500 independent transgenic rice plants have been obtained by the biolistic method with an average transformation frequency of 9.7% for japonica variety Taipei 309. A tight selection procedure using 50 mg/l of hygromycin B successfully prevented the growth of nontransformed tissues. Analysis of the T0 transgenic rice plants revealed that more than 97% of the transgenic plants were morphologically normal and more than 80% were at least partially fertile. The hyg^r trait was inherited as a dominant trait in a Mendelian manner in 8 out of 11 transgenic events assayed. Thirty-seven out of fifty transgenic plants were estimated to contain no more than five copies of the transgenes. In six out of seven transformation events, unlinked, co-transformed genes co-segregated in the T1 generation. The hyg^r trait has been stably inherited to the T4 generation. No chimerical transgenic plant has been found in an intensive search. Novel phenomena observed in transgenic rice plants are also reported.     

Agrobacterium tumefaciens — mediated Transformation of Rice Using Coleoptile and Mature Seed-derived Callus

Morphologically normal, fertile transgenic rice plants (Oryza sativa L cv Taipei 309) were obtained using Agrobacterium tumefaciens strain LBA4404 harbouring the plasmid pTOK233. Two transgenic systems were developed. The first involved callus derived from mature seeds (scutellum) and, the second, used callus derived from 4-d-old coleoptiles. This is the first time that a coleoptile-based system has been used for producing transgenic rice plants. In the development of coleoptile based system, we have evaluated the effect of the length of callus induction period of the coleoptiles on transformation efficiency. The proportion of GUS positive plants was 23% in coleoptile experiment while in mature seed experiments it was 21%. Southern analyses were done to confirm the presence of the transgene. It was found that one to three copies of the transgene integrated in the transgenic plants.     

Over Expression of Rice chitinase Gene in Transgenic Peanut (Arachis hypogaea L.) Improves Resistance Against Leaf Spot

A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T₀) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T₀) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.     

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.