Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Summary Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from 55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expression of gfp, demonstrating efficiency and repeatability of the selection system. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the University of Wisconsin or the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Transgenic oat plants via visual selection of cells expressing green fluorescent protein

 New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines. Received: 11 May 1999 / Revision received: 31 August 1999 / Accepted: 2 September 1999     

The usefulness of the <Emphasis Type="Italic">gfp</Emphasis> reporter gene for monitoring <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of potato dihaploid and tetraploid genotypes

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. ‘Baltica’, respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.     

High transformation frequencies obtained from a commercial wheat (Triticum aestivum L. cv. ‘Combi’) by microbombardment of immature embryos followed by GFP screening combined with PPT selection

Improved gene transfer techniques are necessary to obtain adequatenumbers of stable transgenic wheat plants needed for practical purposes.Considering that wheat transformation is genotype-dependent, we used cv. Combiin all experiments, which had been selected from agronomically important Germanspring wheat cultivars because of its high transformation ability. In mostwheatgene transfer attempts, immature embryos or embryogenic scutellar calli weremicrobombarded. We compared both methods under optimised conditions, usingbar, uidA, andgfp as markers in co-transformation attempts. Integrationof the genes mentioned above was proven by Southern blotting, expression levelswere measured by assays on phosphinothricin acetyltransferase and-glucuronidase activities, and by monitoring for green fluorescentproteinin most developmental stages. Following bombardment of scutellar calli, anaverage transformation frequency of 0.13% was attained. Using immature embryos,mean transformation frequency (1.06%) was 8-fold higher. In addition, embryotechniques were over 2 weeks faster than scutellar callus procedures.Introducing gfp as a vital marker led to an improvement ofembryo-based techniques. In a first screening, transientgfp-expressing embryos were transferred tophosphinothricincontaining callus medium. Only gfp-expressing calli whichdeveloped on it were cultured further on phosphinothricin containingregeneration medium. Shoots obtained from gfp-expressingcalli were rooted on phosphinothricin-free medium, and cultured exvitro. Average transformation frequency (4.93%) was 38-fold higherthan with scutellar callus techniques. Differences between the transformationstrategies used were of high statistical significance. Combining greenfluorescent protein screening with phosphinothricin selection in embryo-basedtechniques offers a promising system to obtain high wheat transformationfrequencies.     

An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)

An efficient transformation and regeneration system was established for the production of transgenic spinach (Spinacia oleracea L.) plants. Cotyledon explants were infected with Agrobacterium tumefaciens strain LBA4404 carrying the selectable marker gene, neomycin phosphotransferase II (nptII), and the reporter gene smgfp, encoding soluble-modified green-fluorescent protein, driven by the cauliflower mosaic virus 35S promoter. The infected explants were cultured on Murashige and Skoog medium, containing 1 mg/l benzyladenine and 0.4 mg/l naphthaleneacetic acid. Shoots were regenerated on selection medium containing 50 mg/l kanamycin. Regenerated kanamycin-resistant shoots were rooted on medium containing 1 mg/l indolebutyric acid and subsequently grown in soil in the greenhouse. Southern blot analysis indicated that the smgfp gene had been integrated into the spinach genome. Northern and Western blots showed that the smgfp gene was expressed in progeny plants. Received: 31 March 1998 / Revision received: 27 September 1998 / Accepted: 10 Ocotber 1998     

Green fluorescent protein as a visual marker for wheat transformation

 Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed. We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could be targeted for optimization. Received: 29 March 2000 / Accepted: 29 March 2000     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of GNA transgenic sugarcane

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.     

A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants

The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor^TM Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 g and 2.11 g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 g mGFP5-ER per mg extractable protein.     

Sporisorium scitamineum colonisation of sugarcane genotypes susceptible and resistant to smut revealed by GFP‐tagged strains

Sporisorium scitamineum is the causal agent of sugarcane smut disease. The fungus establishes a biotrophic interaction with sugarcane tissues, and unlike smut fungi of other monocot hosts, the primary meristem of sugarcane plants develops a whip‐like structure instead of a tumour‐like galls emerging from floral structures (tassels and ears). We examined (GFP)‐tagged S. scitamineum infecting tissues of three sugarcane genotypes with distinct responses to smut (susceptible, intermediate resistant and resistant). Mating compatible haploid cells gfp‐expressing were obtained by Agrobacterium tumefaciens‐mediated transformation (ATMT) using the integrative vector pFAT‐gfp. Regardless of the inoculation method (drop inoculation and hypodermal syringe inoculation), all genotypes were colonised by the fungus. GFP‐tagged strains of opposite mating reaction were able to: (a) grow in vitro as fluorescent yeast‐like cells; (b) generate infectious dikaryon; (c) penetrate sugarcane tissues; (d) colonise tissues by growing a filamentous network; and (e) form the characteristic highly branched hyphae within host cells. Fungal colonisation 160 DAI revealed an association of the fungus with vascular vessels disrupting their organisation in all three genotypes analysed. However, the resistant plants did not develop whips spanning the experiment time. The first whips emerged 76 DAI from plants of the susceptible genotype whereas for intermediate resistant plants whips were detected at 137 DAI. These whips were dissected and fluorescent sporogenesis and teliospore maturation were analysed. In vitro germination of recovered teliospores revealed after meiosis the formation of a three‐celled hyphal filament, where the fourth cell was likely maintained in the teliospore coat. These cells showed independent segregation of the gfp marker, as a result of gfp insertions in different chromosomes of each compatible haploid strain. This work presents the complete fungal life cycle of GFP‐marked S. scitamineum to study developmental stages in planta.     

Transformation of floral organs with GFP in Medicago truncatula

 A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108–1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants. Received: 25 May 1999 / Revision received: 2 August 1999 / Accepted: 2 August 1999     

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but β‐glucuronidase (GUS)‐stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5–plant interaction. PAL5 could be isolated from the root surface (10⁸ CFU g^?1) and from surface‐disinfected root and stem tissues (10⁴ CFU g^?1) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria–plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.     

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low <Emphasis Type="Italic">Agrobacterium</Emphasis> density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD₆₀₀) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD₆₀₀ being more effective for the transformation of Crimson Seedless and 0.2 OD₆₀₀ for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.     

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.     

Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean

 Short-season adapted soybean [Glycine max (L.) Merrill] genotypes (maturity group 0 and 00) were susceptible to Agrobacterium tumefaciens in tumor-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase and β-glucuronidase (gus) transgenes. The transgenes were transmitted to the progeny and stable gus expression was detected in the T₇ generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27–92%, but transformation events were usually restricted to non-regenerable callus. Received: 8 January 1998 / Revision received: 30 June 1999 / Accepted: 12 July 1999     

Overexpression of SoACLA-1 Gene Confers Drought Tolerance Improvement in Sugarcane

Drought is one of the most severe stresses which limit sugarcane production in China. ATP citrate lyase (ACL) is a major enzyme responsible for the production of acetyl-CoA in cytoplasm and plays an important role in plant metabolism and stress response. In this study, sugarcane ACL gene SoACLA-1 was cloned. The plant overexpression vector of SoACLA-1 was built and transformed into sugarcane calli by Agrobacterium-mediated transformation, and PCR analysis confirmed that SoACLA-1 gene had been stably present in the T0, T1, and T2 generations of the transgenic sugarcane. In order to evaluate the drought resistance of the transgenic lines and verify the function of SoACLA-1 gene in the transgenic sugarcane, T1 generation of the SoACLA-1 transgenic sugarcane lines was used as the material to investigate the physiological and biochemical characteristics at 0 day, 3 days, 6 days, and 9 days after water stress and rewatering for 3 days. Comprehensive evaluation of four indicators (chlorophyll, malondialdehyde, proline, soluble sugar) related to drought resistance was done with membership fuzzy function method. The results showed that the drought resistance of five transgenic sugarcane lines from strong to weak, in turn, was RT2?>?RT4?>?RT3?>?RT1?>?WT, and the recovery ability after drought, in turn, was RT1?>?RT2?>?RT4?>?RT3?>?WT. The T2 generation of the SoACLA-1 transgenic sugarcane lines was used to analyze the physiological and biochemical changes and the expression of drought-related genes under water stress. The results showed that the transgenic sugarcane lines were more tolerant to drought as compared with the wild-type plants. Our findings indicated that SoACLA-1 gene plays an important role as a positive factor in response to water stress, and overexpression of SoACLA-1 can enhance drought tolerance in transgenic sugarcane plants.

     

A protocol for consistent, large-scale production of fertile transgenic rice plants

A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates, when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure, transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0 plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies. Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998     

In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase

Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 μg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 μg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.