<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) using root explants

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of niger [<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.] using seedling explants

A protocol was developed for Agrobacterium-mediated genetic transformation of niger [ Guizotia abyssinica (L.f.) Cass.] using hypocotyl and cotyledon explants. Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance. Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness. Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime. After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin. After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome.     

胡杨转基因体系的建立

胡杨（Populus euphratica）是唯一能在沙漠里生长的高大乔木树种,建立其转基因体系可为胡杨抗逆分子机制与应用技术研究提供基本方法。通过研究农杆菌介导的胡杨转GUS基因的技术体系得出以下结论：（1）胡杨再生植株体系,叶片在附加1.0μmol·L^-1 6-BA和5.0μmol·L^-1NAA的1/2MS培养基上不定芽诱导率较高;（2）转基因体系,胡杨叶片在含100μmol·L^-1乙酰丁香酮的OD600值为0.4-0.6的根癌农杆菌菌液中浸染15分钟,共培养2天,GUS基因转化效率较高;（3）转基因植株抗生素筛选,转GUS基因胡杨叶片用300mg·L^-1头孢霉素抑制农杆菌生长,在含9mg·L^-1G418的培养基上诱导不定芽以获得转基因的抗性植株。     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of Indian mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2: a time-phased screening strategy

An efficient and reproducible protocol for the production of transgenic plants was developed for Morus indica cv. K2 by Agrobacterium tumefaciens-mediated transformation. The hypocotyls, cotyledon, leaf and leaf callus explants precultured for 5 days on regeneration medium were co-cultivated with a bacterial suspension at 10(9) cells/ml for 3 days in the dark. Infectivity of A. tumefaciens strain LBA4404 was more than that of strains GV2260 and A281, and among the various plasmids tried, pBI121 and pBI101:Act1 transformed nearly 100% of the explants followed closely by p35SGUSINT. About 90-100% of the explants tested positive in the beta-glucuronidase (GUS) histochemical assay performed after 3 days of co-cultivation. This high level of transient expression, however, decreased to 20-25% after 15 days. Gus activity was most stable in the callus explants, which emerged as the explant of choice for transformation. The transformed explants were selected on 50-75 mg/l kanamycin for 1 month, and 25-50% of the explants developed adventitious buds. On the basis of kanamycin-resistant shoots produced from the total number of explants inoculated, the transformation efficiency was 44%. After 1 month, 40% of these shoots displayed high gus activity as assessed by the GUS fluorometric assay. On a selection-free root induction medium, 80% of the shoots developed roots and 90% of the potted plantlets acclimatized to the growth room conditions. The 3-month-old regenerates showed gus and nptII(neomycin phosphotransferase II) gene activity as assayed by the GUS fluorometric assay and nptII enzyme assay, followed by PCR polymerase chain reaction (54.5%) analysis after 6-months. Transgene integration into the nuclear genome of 1-year-old regenerates was confirmed in 10 of the 18 transformants tested by Southern analysis. The transformation efficiency as defined by the number of transgenic plants produced from the total number of explants co-cultivated was 6%.     

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.)

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.     

农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。     

胡杨转基因体系的建立

胡杨(Populus euphratica)是唯一能在沙漠里生长的高大乔木树种, 建立其转基因体系可为胡杨抗逆分子机制与应用技术研究提供基本方法。通过研究农杆菌介导的胡杨转GUS基因的技术体系得出以下结论: (1) 胡杨再生植株体系, 叶片在附加1.0 mmol.L^-1 6-BA和5.0 mmol.L^-1 NAA的1/2MS培养基上不定芽诱导率较高; (2) 转基因体系, 胡杨叶片在含100 mmol.L^-1 乙酰丁香酮的OD600值为0.4-0.6的根癌农杆菌菌液中浸染15分钟, 共培养2天, GUS基因转化效率较高; (3) 转基因植株抗生素筛选, 转GUS基因胡杨叶片用300 mg.L^-1头孢霉素抑制农杆菌生长, 在含9 mg.L^-1 G418的培养基上诱导不定芽以获得转基因的抗性植株。     

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.     

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus × P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.     

In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Study on the conditions of Cell Transformation in Astragalus sinicus

The conditions of genetic transformation of cells in Astragalus sinicus were studied. The experimental results showed that Agrobacterium tumefaciens strain C58 (pKIW 105), when incubated in medium of low pH and low phosphate concentration in presence of acetosyringone could be induced and activated. When the activated bacteria were used to infect A. sinicus, the GUS gene transient expression in the hypocotyl protoplasts of A. sinicus was immediately and remarkably enhanced. This indicated that the vir gene of A. tumefaciens was activated under the above-mentioned incubation conditions which facilitated T-DNA transfer. In PEG-mediated DNA direct transfer, transient expression of GUS gene was promoted by higher pH and higher Ca2+ concentration of fusion medium. In the same experimental condition, expression of GUS gene under the control of MAS-CaMV 35S chimeric promoter was more effective than that under the control of CaMV 35S promoter, and intensity of GUS gene expression was positively correlated with the amount of foreign plasmid DNA in the range of 10--100 μg. Adventitious shoots were induced from cotyledon and hypocotyls explants treated with Agrobacterium turnefaciens strain PGV 2260 (pBI 121) and were subcultured on MS medium containing 50 mg/L kanamycin to select transformants, and then the transformed shoots were rooted. Stable expression of the foreign genes in the transformed plants was confirmed by assay of neomycin phosphotransferase Ⅱ (NPT Ⅱ ) and β-glucuronidase (GUS) activity.     

Additional virulence genes and sonication enhance <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated loblolly pine transformation

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> mediated genetic transformation of selected elite clone(s) of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

Procedure for the Agrobacterium tumefaciens mediated T-DNA delivery into the elite clone(s) of Eucalyptus tereticornis using leaf explants from microshoots has been developed. Amongst two strains of A. tumefaciens namely, EHA105 and LBA4404 (harbouring pBI121 plasmid), strain EHA105 was found to be more efficient. Pre-culturing of tissue (2 days) on medium supplemented with 100 μM acetosyringone, before bacterial infection significantly increased transient expression of reporter gene (GUS). Co-cultivation period of 2 days and a bacterial density of 0.8 OD₆₀₀ resulted in higher transient GUS expression. Method of injury to tissue, presence of acetosyringone in co-cultivation medium and photoperiod during co-cultivation also influenced the expression of transient GUS activity. Amongst the three clones tested, maximum transient GUS activity was recorded in clone ‘CE2’ followed by clone ‘T1’. Regeneration of transformed shoots was achieved on modified Murashige and Skoog medium (potassium nitrate was replaced with 990 mg/l potassium sulphate and ammonium nitrate with 392 mg/l ammonium sulphate, and mesoinositol concentration was increased to 200 mg/l). Stable transformation was confirmed on the basis of GUS activity and PCR amplification of DNA fragments specific to uidA and nptII genes. The absence of bacteria in the stable transformed tissues was confirmed by PCR amplification of fragment specific to 16S rRNA of bacteria.     

Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens

A reproducible protocol has been established for the transformation of Ginkgo biloba by Agrobacterium tumefaciens . Embryos were co-cultivated with Agrobacterium tumefaciens GV3101 (pGV2260) carrying the binary vector pTHW136, which contained the gus reporter gene and the nptII selectable gene, encoding the enzymes β -glucuronidase (GUS) and neomycin phophotransferase II, respectively. Transient GUS activity has been used to screen the effects of different factors on the transfer of DNA into embryos (age of embryos, infection method, composition of co-cultivation medium). Then, experimental conditions have been defined to obtain transgenic kanamycin-resistant G. biloba calluses expressing GUS activity. The highest rate of transformation (45%) was reached using 1.5-month-old embryos co-cultivated on a medium lacking mineral elements. The integration of gus and nptII genes in calluses was confirmed by polymerase chain reaction analysis and Southern blot analysis.     

玉米核糖体失活蛋白基因z108在烟草原生质体中的转化及其表达

利用与根癌农杆菌共培养的方法将玉米核糖体失活蛋白基因z108及其融合基因GUS导入烟草叶肉原生质体细胞。试验结果表明,烟草叶片在含有1.0%纤维素酶和0.5%离析酶的裂解液中,以0.5M甘露醇、5000.0mg/LCaCl2为渗透压调节剂,25℃保温12-14h,可获得纯化的原生质体细胞;纯化的叶肉原生质体细胞与根癌农杆菌菌株pCam1301/91-108共培养30min,经25mg/L潮霉素筛选,转化率可达17.84%。转化体细胞经X-Gluc染色、PCR和RT-PCR检测证明玉米核糖体失活蛋白基因z108已整合到烟草原生质体细胞的核基因组中并获得表达。