RAPD fingerprinting of blackcurrant (Ribes nigrum L.) cultivars

Ribes nigrum germplasm was screened for random amplified polymorphic DNA (RAPD) markers. Fiftyfour markers were identified which generated individual fingerprints for each of 21 cultivars. Genetic variation within R. nigrum germplasm, as detected by RAPDs, demonstrated that the genetic basis for improvement of blackcurrant is narrower than would be expected by the analysis of parentage.     

Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.     

Efficient transformation and regeneration of carnation cultivars usingAgrobacterium

We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.     

Agrobacterium-mediated transformation of Javanica rice

Difficulties frequently encountered using direct DNA transfer methods for transformation of Javanica varieties of rice (Oryza sativa L.) have limited the application of biotechnology to these varieties. We now reportAgrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are, respectively, widely used or about to enter commercial cultivation in the southern USA. Vigorous, phenotypically normal, fertile plants expressing both the selectable marker and the gene of interest were obtained. Southern analysis showed that only one or two copies of the T-DNA insert were present. Sequence analysis of right border fragments of one line confirmed that insertion was into a coding region of rice nuclear DNA. This analysis also revealed the presence of relatively short regions of permuted T-DNA border sequences, similar to those found afterAgrobacterium-mediated transformation of dicots. Progeny analysis of lines bearing two copies showed co-segregation, indicating that they were located relatively closely on the same chromosome. The introduced genes were transmitted to the R₁ and R₂ generations in a Mendelian fashion, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.     

High-frequency regeneration and transformation ofRaphanus savus

We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s (MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing 0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin.     

Factors influencing Agrobacterium tumefaciens mediated transformation and expression of kanamycin resistance in pickling cucumber

Cucumber (Cucumis sativus L.) petiole and leaf segments of two pickling genotypes were transformed with Agrobacterium tumefaciens strain LBA 4404, an octopine Ti-plasmid deletion mutant that is avirulent (disarmed plasmid), but to which were added T-DNA inserts on binary plasmids (pBIN 19, ca. 10 kb, and pCGN 783, ca. 25 kb). Expression of neomycin phosphotransferase (NPT II) encoding resistance to the aminoglycoside kanamycin was used as a selectable marker. Factors which influenced the frequency of callus development on medium containing kanamycin (75 mg l^-1) were explant size, bacterial concentration and length of exposure, cocultivation period, and presence of acetosyringone. The optimal procedure involved exposing segments of petiole (4–6 mm) or leaf (0.5 cm²) segments to a bacterial suspension (10⁸ cells ml^-1) containing 20 M acetosyringone for 5 min, followed by a 48 h cocultivation period on a tobacco feeder layer. Explants were placed on MS medium containing 500 mg l^-1 carbenicillin, 75 mg l^-1 kanamycin, and NAA/BA (5.0/2.5 M) or 2,4-d/BA (5.0/5.0 M) and subcultured twice, each after a 2–3 week period, onto fresh media. The overall frequency of transformed callus was 20–50%; the frequency of plantlet regeneration from transformed callus was 8–15%. Twenty-one out of 23 individual plants recovered from two genotypes of pickling cucumber were NPT II positive (transformation frequency of 9%). Copy number of the NPT II gene insert (35S-NPT II-3 fragment, ca. 2.2 kb) in three transformed plants was estimated at ten per haploid genome, indicative of multiple insertions within the cucumber genome. Multimers of the gene (visible as 4.4 and 6.6 kb fragments in Southern analysis) were detected in one plant, suggestive of tandem duplications or repeats. Progeny from a cross between this transformed plant and a nontransformed control showed segregation for the NPT II gene in dot-blot assays; at least 24 plants out of 32 were kanamycin positive. Copy number in the progeny was variable, and ranged from none to ten.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA- napthaleneacetic acid - BA benzyladenine     

Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler

The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk. Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed by the GUS assay and PCR analysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant pretreatment medium and the co-culture medium improved the transformation efficiency by decreasing the escape frequency. More than 90% rooting frequency of transformed citrange shoots was achieved by two-step culture: first on media supplemented with auxins, and then on media without hormones. Inclusion of 20 mg l^–1 kanamycin in rooting medium efficiently discriminated transformed shoots from non-transgenic escaped shoots. Shoot grafting in vitro was used to regenerate transformed plants, due to the slow growth of most sweet orange shoots.     

Genetic transformation of green-colored cotton

Summary This study reports an Agrobacterium-mediated transformation of green-colored cotton (Gossypium hirsutum L.). A tissue culture procedure was optimized to induce callus formation from hypocotyl explants and subsequent differentiation into the embryogenic type. Callus formation could be induced by growing explants on Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Among the four genotypes studied, embryogenic calli and plant regeneration were observed only in var. G9803. Agrobacterium-mediated transformation of G9803 with the fiber-specific expansin gene GhExpl was achieved based on the establishment of these tissue culture methods. A total of 32 individual regenerants resistant to kanamycin were generated within 7 mo., with a transformation frequency of 17.8%. Transformation was confirmed by Southern blot analysis and RT-PCR. These results represent the first step towards genetic manipulation of the colors and fiber quality of green-colored cottons by biotechnology. These authors contributed equally to this work     

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.     

Rapid and efficientAgrobacterium-mediated transformation in rice

An improved protocol forAgrobacterium-mediated transformation of rice is described. The procedure permitted the regeneration of transgenic plants from callus in only two months.     

Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation

Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 g/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.     

Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissues for transformation

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.This project was funded by grants from Cotton Incorporated, Nisshinbo Industries, and a grant from the Texas Agricultural Experiment Station to RHS. Texas Agricultural Experiment Station Technical Article TA-25667.     

Regeneration of transgenic tamarillo plants

Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny.     

Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker

Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptII genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T₁ plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T₁ plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptII gene was confirmed in 13 T₁ plants. The transformation system enables the rapid transfer of agronomically important genes.     

An improved selection strategy and the use of acetosyringone in shoot induction medium increase almond transformation efficiency by 100-fold

An improved almond transformation system was developed with a 100× increase in efficiency (12.3%) as compared to the existing transformation method (0.1%) in spite of the lower regeneration ability of the explants used. Leaf transformation was performed with Agrobacterium EHA105/p35 SGUSINT. Main modifications introduced in the transformation protocol were the use of 150 μM acetosyringone (AS) during the 21-days induction period, and a different selection strategy. Transformed shoots were assayed using PCR, GUS analyses and Southern blotting. The improved methodology is being applied for transformation of in vitro propagated cultivars and opens the possibility of using almond as model for functional studies in Prunoideae.     

Plant transformation technology

Plant transformation has its roots in the research on Agrobacterium that was being undertaken in the early 1980s. The last two decades have seen significant developments in plant transformation technology, such that a large number of transgenic crop plants have now been released for commercial production. Advances in the technology have been due to development of a range of Agrobacterium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques for regenerating whole plants from plant cells or tissues in a large number of species. In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied. Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.     

A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.)

In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.     

Development of genotype-independent regeneration system for transformation of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> ssp. <Emphasis Type="Italic">indica</Emphasis>)

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L⁻¹ thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5–8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L⁻¹ carbenicillin or 250 mg L⁻¹ cefotaxime to kill the rice shoot apical meristem was 50 mg L⁻¹ and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T₀ transformant plantlets.     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction