Aluminum borate whisker-mediated production of transgenic tobacco plants

An aluminum borate whiskers-mediated transformation system for calluses of tobacco (Nicotiana tabacum, cv. SR-1) has been developed. A total of 50 small pieces of calluses were vigorously agitated in a liquid medium containing aluminum borate whiskers, pBI221 plasmid carrying the -glucuronidase (GUS) gene, and pBI222 plasmid carrying the hygromycin phosphotransferase (HPT) gene. After treatment, calluses were cultured to select for hygromycin resistance, and three resistant calluses were obtained. Adventitious shoots were produced from each hygromycin-resistant callus and were transferred to rooting medium. A total of three plantlets obtained from each hygromycin-resistant callus were acclimatized and established in soil. Polymerase chain reaction analysis revealed that all the plantlets were cotransformed with both the GUS and HPT genes. Detached leaves of transgenic individuals showed clear hygromycin resistance when cultured in liquid medium. Histochemical assay for GUS revealed that one of these transgenic plants expressed the GUS gene, indicating coexpression of foreign genes.     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts

Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 g/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9–6.8 × 10^–4 (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.     

Genetic transformation of sweet potato by particle bombardment

Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.Abbreviations BA 6-benzylaminopurine - CaMV cauliflower mosaic virus - 2,4-D 2, 4-dichlorophenoxyacetic acid - GUS ß glucuronidase - NAA naphthaleneaceticacid - nos nopaline synthase gene - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962) - MS-CP MS cell proliferation medium     

Transformation system of rice suspension-cultured microcolonies by Electroporation

For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and root apex.     

Expression of a foreign gene in electroporated pollen grains of tobacco

Summary The incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding -glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.     

Stable transformation of the food yam Dioscorea alata L. by particle bombardment

Summary A biolistic particle gun was used to deliver genetic material into intact yam cells. Cultured suspension cells of D. alata were bombarded with microprojectiles coated with pBI221.2 DNA and histochemical assays were carried out to show transient GUS expression in bombarded cells. Stably transformed D. alata cells were recovered from cultured cells after bombardment with microprojectiles coated with pRT99gus harbouring both the nptII and uidA genes. Bombarded cells were selected on a medium containing geneticin (G418). Two months after bombardment, calli resistant to G418 were assayed for GUS expression. There was a 100% correlation between resistance to G418 and GUS expression. From these calli, four cell lines were established and GUS activity in each line was determined fluorometrically. The use of a specific GUS inhibitor showed that the GUS activity was due to the introduced uidA gene rather than to any intrinsic GUS-like activity originating from the plant. Incorporation of the introduced DNA into the plant genomic DNA was confirmed by Southern analysis.Abbreviations GUS -glucuronidase - MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--D-glucuronide - PVP Polyvinylpyrrolidone - SDS Sodium dodecyl sulphate - TAE Tris-acetate-EDTA buffer - X-Gluc 5-Bromo-4-chloro-3-indolyl--D-glucuronide     

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.     

A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.)

Summary A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.) was developed using epicotyl segments. The segments were infected with Agrobacterium harboring the binary vector pBI121 or pBI101-O12-p1. Both vectors contained the neomycin phosphotransferase II (NPTII) and the -glucuronidase (GUS) genes. In the plasmid pBI101-O12-p1, the GUS gene was directed to the promoter region of ORF12 (rolC) of the Ri plasmid. On a selection medium containing 100 or 200 g/ml kanamycin, adventitious shoots were formed from 21.7–44.6% of the segments. Histochemical GUS assay showed that 55.4–87.7% of the shoots expressed the GUS gene. The stable integration of this gene was also confirmed by polymerase chain reaction (PCR) analysis and by Southern blot analysis. When the pBI101-O12-p1 plasmid was used, the GUS activity was found to be located in phloem cells of leaf, stem and root. More than 100 transformed plants were obtained using this method within 2–3 months.     

Transient and stable transformation of wheat with DNA preparations delivered by a biolistic method

An optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PDS 1000/He to deliver foreign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo- derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

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     

Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble)

The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).     

花粉管通道法介导的铁皮石斛转基因技术

该研究以含有GFP和GUS基因的质粒和农杆菌为载体,采用花粉管通道法对铁皮石斛进行转基因技术研究。结果表明:(1)铁皮石斛种子萌发和原球茎生长对卡那霉素的最低致死浓度分别为90和150 mg·L~(-1)。进一步研究证实,在筛选转化种子和原球茎时,可分别向培养基中添加100和150 mg·L~(-1)的卡那霉素进行选择培养。(2)以携带GFP和GUS基因的质粒(pSuper1300和pBI121)和农杆菌为载体,用无菌去离子水重悬质粒pSuper1300和pBI121至浓度为100 ng·μL~(-1),用2%蔗糖+1/2MS+0.1%silwet-77+0.1%AS或5%蔗糖+0.1%silwet-77+0.1 mmol·L~(-1)AS重悬携带质粒pSuper1300和pBI121的农杆菌至菌液浓度为OD_(600)=0.7~0.8;在授粉后0.5~2.5 h内使用柱头滴加法导入携带外源基因的质粒或农杆菌溶液,收集成熟的转化种子,经选择培养及PCR检测发现,几乎所有处理的转化材料均能检测出外源GFP和GUS基因片段。另外,与农杆菌相比,以质粒为载体进行转化,可获得更高的结实率。该研究结果为铁皮石斛的基因工程育种提供了参考。     

Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’

Efficient callus formation was achieved from cotyledon, stem, and leaf expiants of the domestic safflower cultivar Centennial on MS salts medium containing 1 mg/L BAP and 1 mg/L NAA. Shoot buds were regenerated from 26% of leaf-derived calli on callus induction medium, although attempts to root regenerated shoots were not successful. Centennial expiants inoculated with Agrobacterium tumefaciens containing NPT II and GUS genes produced kanamycin-resistant calli from which buds were regenerated. Transformation and stable integration of transgenes was confirmed by GUS assay and DNA hybridization in kanamycin-resistant calli, and GUS assay in regenerated shoots.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - GUS -glucuronidase - IAA indole-3-acetic acid - NPT II neomycin phosphotransferase II     

植物激素和抗生素调节的转基因火炬松愈伤组织的生长和分化

包含质粒载体pBI121的农杆菌菌株LBA4404被用于转化火炬松的成熟合子胚。质粒载体pBI121含有胭脂碱合成酶基因的启动子驱动的新霉素磷酸转移酶基因（npt Ⅱ)和花椰菜花叶病毒的35S启动子驱动的GUS基因。器官发生的转基因愈伤组织和转基因的再生植株已经获得,并经GUS组织化学染色、聚合酶链式反应和Southern杂交分析证实。植物激素（BA/IBA）和抗生素对转基因愈伤组织的生长和分化的影响被研究。500mg/L羧苄青霉素和2mg/LBA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加54.2%,分化增加45.7%。500mg/L Claforan和2mg/L BA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加40.8%,分化增加38.7%。高浓度的植物激素和抗生素降低了转基因愈伤组织的生长和分化。实验结果有助于建立一个高效的农杆菌介导的火炬松遗传转化系统,也有助于未来针叶树的遗传转化研究。     

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

Preliminary Study on Transformation of Wheat Apical Point

Apical points of young seedlings of wheat (Triticum aestivum) cultivar "Jing 411” and somatic calli of cultivar "FK8” were transformed with plasmids pBI121 and (or) pBIAH-A+ by using microprojectile bombardment. Histochemical assay of GUS activity showed positive reaction on some of the transformation processed apical points and calli. This demonstrated that foreign genes were introduced into the apical meristematic cells as well as the callus cells. The plantlets of cv. "Jing 411” survived after apical point transformation with pBIAH-A+ were transplanted into the field and the progenies were screened with kanamycin. 4 % of the screened seeds germinated into green seedlings with kanamycin resistance. Dot hybridization of total DNA from kanamycin resistant plants showed the existence of foreign DNA in some of the detected plants.     

Transgenic coffee fruits from <Emphasis Type="Italic">Coffea arabica</Emphasis> genetically modified by bombardment

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T₁ progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.