PIG-mediated cassava transformation using positive and negative selection

 In order to develop new selection systems for production of transgenic cassava (Manihot esculenta Crantz), two different selection regimes were assessed for their efficiency on regeneration of transgenic cassava plants: positive selection using mannose and negative selection using hygromycin. Explants from somatic cotyledons and embryogenic suspensions were used as target tissues in the transformation experiments and bombarded using the particle inflow gun. Different culture and selection strategies were assessed to optimise the selection protocols. For the first time transgenic plants could be obtained using positive, and in the case of embryogenic suspensions, hygromycin-based negative selection. The stably transformed nature of the regenerated cassava plant lines and the expression of the transgenes were verified with PCR, RT-PCR, Southern and northern analyses. A rooting test for transgenic plants on a medium supplemented with mannose was developed to further improve the efficacy of the positive selection system. Our results demonstrate that it is possible to obtain transgenic cassava plants using non-antibiotic positive selection. Received: 21 February 2000 / Revision received: 2 May 2000 / Accepted: 5 May 2000     

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     

Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint

 Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein, osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is a viable alternative to plant breeding and clonal selection for improvement of this crop. Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999     

Plant regeneration from transformed embryogenic callus of an elite indica rice via Agrobacterium

The present study describes a simple and efficient protocol for plant regeneration from scutellar-derived embryogenic calli of an elite basmati indica rice (Oryza sativa L., cv Pusa Basmati 1) transformed with Agrobacterium. A supervirulent plasmid pTOK233 as well as a non-supervirulent plasmid pJB90GI containing -glucuronidase (gus) and hygromycin phosphotransferase (hpt) chimeric genes were used to assess transformation and regeneration efficiency. The effects of some factors like the bacterial density and inclusion of sorbitol in the medium on the co-culture and transformation have been evaluated; the procedure for selection and regeneration from transformed calli was found to be critical. Furthermore, co-culture and selection on regeneration medium was found to be better than callus medium and led to minimal media manipulations. Regeneration medium supplemented with 3% maltose was found to be better for regeneration as compared to 3% sucrose. The transformed calli were subjected to three cycles of regeneration, thus converting a higher number of transformation events into regenerants. The selected calli as well as leaf sections and roots of the transformants were GUS positive. The stable integration of the transgene was confirmed by polymerase chain reaction and Southern blot analysis of the transformants. Interestingly, the presence of three additional vir genes in supervirulent plasmid pTOK233 was not required for transformation as transformation was successful with non-supervirulent plasmid pJB90GI, although the transformation and regeneration frequency was higher with the former. This effective protocol for regeneration from transformed calli resulted in a relatively high transformation frequency.     

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures

A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.     

Regeneration of cassava plants via shoot organogenesis

A novel regeneration system based on direct shoot organogenesis is described for cassava. Plants could be regenerated at high frequency by inducing shoot primordia on explants derived from cotyledons of cassava somatic embryos. After a passage on elongation medium, the regenerated shoots were easily rooted in hormone-free medium and could be successfully transplanted to soil. Using the shoot-organogenesis-based regeneration method, up to eight transplantable plantlets per explant could be regenerated. The system was optimised first for one cassava cultivar, and then its transferability to three other cultivars was demonstrated. This method widens the scope of in vitro regeneration modes of cassava, and is also compatible with Agrobacterium-mediated transformation. To develop an efficient system for production of somatic embryos for regeneration experiments, conditions for inducing primary and cycling somatic embryos were also studied, and highly efficient plant regeneration via germination of somatic embryos was achieved using maltose instead of sucrose in the culture medium, and combining paclobutrazol with 2,4-dichlorophenoxyacetic acid in the embryo induction medium. Received: 25 January 1997 / Revision received: 10 February 1997 / Accepted: 20 February 1997     

High-efficiency,microprojectile-mediated cotransformation of sugarcane,using visible or selectable markers

Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×10³ cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.     

Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T₁ and T₂ progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.Abbreviations 2,4-D: 2,4-Diclorophenoxyacetic acid - IAA: Indole acetic acid - ICRISAT: International Crops Research Institute for the Semi-Arid Tropics - IZEs Immature zygotic embryos Communicated by H. Lörz     

Factors influencing <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn

Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic nodules of seed origin were used as the target tissue for Agrobacterium tumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated and incubated at 22°C. Incorporation of higher level of CuSO₄ in the regeneration medium had significantly positive effect on the recovery of transformed plants. PCR analysis of T ₀ and T ₁ generation plants with nptII-specific primers revealed the amplification of nptII gene. Southern blot analysis of six regenerated plants confirmed selectable marker gene integration in three plants. This is a first report on Agrobacterium-mediated genetic transformation of finger millet and will pave the way for further studies in this and other millet crops.     

Use of fused <Emphasis Type="Italic">gfp</Emphasis> and <Emphasis Type="Italic">gus</Emphasis> reporters for the recovery of transformed <Emphasis Type="Italic">Medicago truncatula</Emphasis> somatic embryos without selective pressure

We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP) and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported, with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the previously selected embryos.     

Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.     

Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.     

Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica)

 An improved broccoli transformation system was developed by optimising several factors that affect the rate of effective Agrobacterium-mediated transformation. Leaf explants of cultivar Shogun were co-cultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II (NOS-NPTII-NOS) gene for kanamycin resistance and a β-glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated including combinations of arginine, mannopine, acetosyringone and the use of feeder cell layers. The new protocol includes the use of 200 μm acetosyringone in LB medium for bacterial growth, the use of a Brassica campestris feeder cell layer, 10 mm mannopine and 50 μm acetosyringone in the co-cultivation medium and 1 mm arginine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary experiments transforming broccoli with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene from pTOM13. Received: 2 July 1998 / Revision received: 9 February 2000 / Accepted: 17 February 2000     

Efficient regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated stable transformation of perennial ryegrass

We utilized gene transfer technology for genetic perennial ryegrass improvement, efficient regeneration, and Agrobacterium-mediated transformation of phosphinothricin acetyltransferase gene (bar). Four growth regulator combinations were compared and intact seeds of six turf-type cultivars as mature embryo sources were tested to optimize the regeneration conditions. Callus formation and regeneration were observed in all seeds. The highest callus formation frequency was observed in the seeds cultured on MS medium supplemented with 9 mg/l 2,4-D, without benzyladenine. Cv. TopGun revealed the highest callus induction and regeneration frequencies of 96 and 48.9%, respectively. By using an optimized regeneration system, embryogenic calli were transformed by an Agrobacterium strain LBA4404 containing the plasmid pCAMBIA3301. After the selection of the potentially transgenic calli with phosphinothricin, a herbicide, 22 transgenic resistant plants were regenerated. With PCR, Southern-blot hybridizations, and GUS expression techniques, we confirmed that some regenerants were transgenic. Two of the tested transgenic plants showed herbicide resistance. Our results indicated that embryogenic calli from mature seeds can be directly used for perennial ryegrass efficient regeneration and transformation and this protocol is applicable for genetic engineering of herbicide-resistant plants. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 590–596. The text was submitted by the authors in English.     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.     

Transformation of different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> infection of in vitro cultured ovules

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.     

Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars

Perennial ryegrass (Lolium perenne L.) is the most important grass species in areas with a temperate climate. Biolistic transfer of a ubiquitin promoter driven nptII expression cassette into mature or immature tissue derived calli of perennial ryegrass followed by paromomycin selection, resulted in the rapid and efficient production of fertile transgenic ryegrass plants. Transformation efficiencies after paromomycin selection in combination with the nptII selectable marker compared favourably with hygromycin selection in combination with the hph selectable marker. In total 83 independent nptII expressing plants were produced. Transformation frequency was highly affected by genotype, explant, selection regime and the duration of the callus induction period. The optimised transformation protocol for mature embryo derived calli of turf-type or forage-type cultivars resulted in an average transformation efficiency of 5.2% or 6.6% respectively. This converts into 1.7 or 2.2 independent transgenic plants per bombardment. Immature inflorescence- and immature embryo-derived calli were also successfully used as target for the gene transfer, resulting in transformation efficiencies of up to 3.7% or 11.42% respectively. Transgenic plants were transferred to soil 12 or 9 weeks after excision of mature and immature embryos or inflorescences respectively. Transgene integration and expression were confirmed by PCR and ELISA or western blot analysis. Southern blot analysis confirmed the independent nature of the transgenic lines. The majority of lines showed the integration of two to six transgene copies, while 21% of the analysed lines had a single copy insert. A short tissue culture period in comparison to recently published reports seems to be beneficial for the production of normal and fertile transgenic ryegrass plants. Consequently we report for the first time molecular evidence for sexual transgene transmission in fertile transgenic perennial ryegrass.     

Transformation of barrel medic (Medicago truncatula Gaertn.) by (Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12

Summary Fertile and stable transgenic plants of the model legume Medicago truncatula Gaertn. were obtained through transformation of leaf tissue with the disarmed Agrobacterium tumefaciens strain LBA4404 and in vitro regeneration via somatic embryogenesis. An optimised transformation/regeneration protocol has been established for two genotypes of the cultivar Jemalong, including a previously described highly embryogenic line (Nolan et al. 1989, Plant Cell Rep. 8: 278–281). Using this protocol, transgenic plantlets were obtained within 4–10 months following cocultivation with Agrobacterium. We have introduced into M. truncatula a chimeric fusion between the early nodulin MtENOD12 promoter and the gus (-glucuronidase) reporter gene, and shown that symbiosis-specific gene expression can be elicited in the roots of such transgenic plants following the addition of purified Rhizobium nodulation factors.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine tetracetic acid - GUS gb-glucuronidase - IBA indolebutyric acid - MES 2-(N-morpholino) ethane sulfonic acid - OD optical density - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

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