Agrobacterium-mediated transformation of the wetland monocot Typha latifolia L. (Broadleaf cattail)

An Agrobacterium-mediated model transformation system was standardized for the wetland monocot Typha latifolia L. to achieve the long-term objective of introducing candidate genes for phytoremediation. Two binary plasmid vectors, pCAMBIA1301/EHA105 and pTOK233/LBA4404, both containing the gus (beta-glucuronidase) and hptII (hygromycin phosphotransferase II) genes, were used for transformation. Fifty-day-old 5 mg/l picloram-derived calli were cocultivated and selected on medium containing 20 mg/l or 40 mg/l hygromycin. Resistant calli were regenerated on medium supplemented with 5 mg/l 6-benzylaminopurine, with or without 20 mg/l or 40 mg/l hygromycin and with or without charcoal (10 g/l). Transient GUS activity in explants ranged between 28% and 36%. Hygromycin-resistant calli, selected after 3 months, showed stable GUS expression. A total of 46 plants were regenerated and established in the greenhouse; 13 showed stable GUS expression. Cocultivation of dark culture-derived calli, directly selected on regeneration medium containing 20 mg/l hygromycin and rooted on medium with 20 mg/l hygromycin was the best protocol. The addition of charcoal did not have any effect on regeneration. PCR and Southern analyses of transgenic calli and transgenic plants confirmed the presence of the introduced genes. In conclusion, T. latifolia could be genetically transformed by Agrobacterium tumefaciens.     

Agrobacterium-mediated transformation of Javanica rice cv. Rojolele

The Agrobacterium-mediated transformation system was extended to a famous Javanica rice variety, Rojolele, that is cultivated in Indonesia now. Efficient callus induction from immature and mature seeds of Rojolele did not succeed by any previous method for any rice cultivar. In this study, the callus from mature seeds of Rojolele exhibited a compact and nodular appearance on C medium after the carbon source and medium pH was modified. Scutellum-derived calli from mature seeds were co-cultivated with Agrobacterium tumefaciens strains EHA101 or LBA4404 that carried plasmid pAFT14, which contained the genes for beta-glucuronidase (gus) and hygromycin resistance (hpt). Finally, the transformation efficiency of Rojolele variety using A. tumefaciens strain EHA101 (pAFT14) was improved to about 23%, similar to that of the Japonica rice variety Nipponbare. The seed fertility of transgenic Rojolele was more than 90%. The copy number of the transgene varied from one to three copies in the T(0) transgenic lines. Both the gus and the hpt genes were inherited and expressed in the progeny.     

简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101／pHQ9,EHA101／pHQ10,EHA101／pHQT3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤／g愈伤外植体,抗性愈伤的分化频率平均高达85％。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3：1。该系统的建立将有助于应用T—DNA标签法和基因打靶法进行水稻功能基因组的研究。     

High Efficiency of Genetic Transformation of Rice Using Agrobacterium Mediated Procedure

Four japonica varieties and two indica varieties were used for the genetic transformation of rice （Oryza sativa L.） by using Agrobacterium tumefaciens (Smith et Townsend) Conn EHA101 harboring binary vector containing GUS gene and selectable marker gene of NPTⅡ and HPT. Calli derived from mature and immature embryos of rice were infected and cocultured with Agrobacterium at logarithmic phase. The highest transformation frequency was 55.1% (indica) and 85.2% (japonica) respectively according to the estimation of hygromycin resistant calli produced. The ratio of transgenic plants regenerated from the calli of indica and japonica varieties was 37.8% and 69.0% respectively. The putative transformed plants were confirmed by GUS assay, PCR analysis and Southern blotting. The segregation of foreign genes in T1 progeny corresponded to the Mendelian ratio. This transformation procedure of rice will provide an efficient model for the transformation of monocots.     

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.     

Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.     

高赖氨酸蛋白基因导入水稻及可育转基因植株的获得

构建了一个植物高效表达质粒,使来源于四棱豆（Psophocarpus tetragonolobus(L.)DC)的高赖氨酸蛋白基因（lys）受控于单子叶植物ubiqutin强启动子下表达。用基因枪法将其导入水稻(Oryza sativa L.)幼胚诱导的愈伤组织,经潮霉素抗性筛选,得到可育的再生植株。经PCR和Southem blotting检测,表明该基因已整合到水稻的基因组织。GUS组织化学染色表明转基因水稻植株的叶、茎和根中均有gus基因的表达。测定112株转基因水稻叶片中赖氨酸叶量,大部分植株有不同程度的提高,最高幅度为16．04％。     

Genetic transformation of two species of orchid by biolistic bombardment

We report here the transformation of two species of orchid, Dendrobium phalaenopsis and D. nobile,by biolistic bombardment. Calli or protocorm-like bodies (PLBs) were used as target explants. Gold particles (1.0 microm) coated with plasmid DNA (pCAMBIA1301) encoding an intron-containing beta-glucuronidase gene (gus-int) and a hygromycin phosphotransferase (hpt) gene were introduced into the PLBs or calli using the Bio-Rad PDS-1000/He Biolistic Particle Delivery System. Calli and PLBs were then chopped up and pre-cultured in 1/2-strength MS medium supplemented with 0.4 M mannitol for a 1-h osmoticum treatment before bombardment. Immediately after bombardment, the calli and PLBs were transferred to 1/2-strength MS medium without mannitol for recovery. Putatively transformed plantlets were obtained by selection and regeneration on medium supplemented with 30 mg/l hygromycin. The highest efficiency of transformation was obtained when selection was conducted at 2 days post-bombardment. For D. phalaenopsis and D. nobile, respectively, about 12% and 2% of the bombarded calli or PLBs produced independent transgenic plants. Integration and expression of the transgenes were confirmed by Southern hybridization and Northern hybridization. No nontransformed plants were regenerated, indicating a tight selection scheme. However, separate incorporation of the gus gene and the hpt gene was observed, and in one transgenic line the gus gene was integrated into the genome of the transgenic plant, but not expressed.     

Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus

Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar ‘Gravesac’, 72% of the excised root tips initiated hairy root cultures on growth regulator-free media. According to the nature of the strains used in co-inoculation, co-transformation frequencies of the hairy root clones ranged from 4 to 16%. Co-transformed roots showed resistance to kanamycin and hygromycin but responses varied from clone to clone. Fluorometric GUS expression and GCMV coat protein production showed a large variability among hairy root clones co-transformed by pKHVG2+. Though the presence of gus, nptII and GCMV coat protein genes was checked by polymerase chain reaction and Southern blotting, it was difficult to establish a clear relationship between expression of the different transgenes. The regeneration of plants was not achieved, but the possibility to graft in vitro transgenic roots to non transformed shoot systems could permit rapid testing of the resistance induced by nepovirus coat protein in roots of cultivars that are recalcitrant to A. tumefaciens-mediated transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Festuca arundinacea</Emphasis> (Schreb.) and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> (Lam.)

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.     

转基因培育抗除草剂水稻

以ｐＡＨＣ２０（含Ｂａｒ基因）和ｐＷＲＧ１５１５（含ＧＵＳ基因和潮霉素抗性基因）以及含Ｂａｒ基因和雪莲凝集素（ＧＮＡ）基因的ｐＣＡＭＢＩＡ３３００ ＲＧ为供体ＤＮＡ,选用水稻品系８７２０３、上农香糯及鄂宜１０５的成熟胚诱导出的愈伤组织及微不定芽为受体材料,分别采用基因枪和根癌农杆菌（ＬＢＡ４４０４,含ｐＡＬ４４０４）导入法进行基因转化;经抗性筛选、ＧＵＳ检测和ＰＣＲ分析。结果表明,外源基因已通过基     

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。     

Agrobacterium-mediated transformation of Bangladeshi Indica rices

Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233. Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation. Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis. Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number. Mendelian segregation of transgenes occurred in T1 seed progeny.     

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.     

Agrobacterium tumefaciens-mediated transformation of Robinia pseudoacacia

Robinia pseudoacacia (black locust) plants were regenerated after co-cultivation of stem and leaf segments with Agrobacterium tumefaciens strain GV3101 (pMP90) that harbored a binary vector that included genes for β-glucuronidase (GUS) and hygromycin phosphotransferase. Successful transformation was confirmed by the ability of stem and leaf segments to produce calli in the presence of hygromycin, by histochemical and fluorometric assays of GUS activity in plant tissues, and by Southern blotting analysis. In this transformation system, about 2 months were required for regeneration of transgenic plants from stem and leaf segments. The frequency of transformation from stem segments was approximately 24%, and the morphology of regenerated plants resembled that of the original parental strain. Received: 2 September 1999 / Revision received: 30 November 1999 / Accepted: 4 December 1999     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

pib基因启动子及其诱导启动性初探

将pib基因上游5.7 kb区段取代pCAMBIA1301中gus基因上游的35S启动子构建了pib拟启动区-GUS+ 35S-hpt 基因表达载体pNAR604。经农杆菌介导转化水稻成熟胚愈伤,获得了转基因抗潮霉素愈伤和36株转基因水稻植株。 转基因抗性愈伤和转基因植株根的组织化学GUS活性检测表明,光照培养下的抗性愈伤和转基因植株根不能使X-gluc显色,而暗处理24 h后的抗性愈伤和定植后转基因植株的根能使X-gluc显色。转基因植株GUS荧光定量分析结果表明,GUS表达具有器官特异性,黑暗处理前根的GUS活性最高、茎次之,分别是是叶片的7倍和3倍,叶片中仅有痕量本底。24 h黑暗处理后根、茎、叶中GUS活性都有增加,且叶片中的增加比例最大,其活性仅次于根。5 mmol/L水杨酸和0.3 mol/L NaCl叶面喷施转基因植株24 h后叶片中GUS活性分别为处理前的2.7和3.6倍。初步确定pib拟启动区是一个诱导型启动子。黑暗、水杨酸和NaCl能诱导该启动子启动活性。