Ajay Kohli

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.

Transgene integration, organization and interaction in plants

It has been appreciated for many years that the structure of a transgene locus can have a major influence on the level and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing number of experiments have been carried out with the primary aim of characterizing transgene loci and studying the fundamental links between locus structure and expression. Cereals have been at the forefront of this research because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between higher order locus organization, nuclear architecture, chromatin structure and transgene expression.

Transgene expression in rice engineered through particle bombardment : molecular factors controlling stable expression and transgene silencing

Abstract. Transgenic rice (Oryza sativa L.) lines were generated through particle-bombardment-mediated transformation. Hygromycin phosphotransferase (hpt) was used as a selectable marker gene on co-integrate plasmids containing either one or two unselected genes, the Bialaphos-resistance gene (bar) coding for phosphinothricin acetyltransferase and the β-glucuronidase gene (gusA), respectively. Transformants were analyzed to determine possible correlation between expression, integrated transgene copy number and/or complexity of integration patterns. We observed that an increase in transgene copy number did not always lead to a concomitant decrease in expression levels or to silencing through co-suppression. Transgenic lines with four to five copies of integrated transgenes expressed the protein product of both unselected genes stably and at levels comparable to transformants with one or two copies. In the majority of lines we analyzed, expression patterns of the two unselected genes were similar. In lines where transgene silencing was observed, this was independent of position effects. In specific cases, silenced transgenes could be reactivated by treatment with 5-azacytidine, suggesting methylation of cytosine residues. We report that methylation of cytosines may not spread to adjacent regions, hence other transgenes in the vicinity of the silenced transgene remain active. By comparing the structure of transgenic loci with expression patterns of introduced genes, we concluded that the integrity of integrated transgenes was a major factor in the onset of silencing. We observed that the presence of truncated sequences of transgenes capable of generating incomplete transcripts, resulting in aberrant RNA species, may be responsible for silencing.

Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-IΔD86) for nematode resistance in transgenic rice plants

Abstract We have used a genotype-independent transformation system involving particle gun bombardment of immature embryos to genetically engineer rice as part of a programme to develop resistance to nematodes. Efficient tissue culture, regeneration, DNA delivery and selection methodologies have been established for elite African varieties (‘ITA212’, ‘IDSA6’, ‘LAC23’, ‘WAB56-104’). Twenty-five transformed clones containing genes coding for an engineered cysteine proteinase inhibitor (oryzacystatin-IΔD86, OC-IΔD86), hygromycin resistance (aphIV) and β-glucuronidase (gusA) were recovered from the four varieties. Transformed plants were regenerated from all clones and analysed by PCR, Southern and western blot. Detectable levels of OC-IΔD86 (up to 0.2% total soluble protein) in plant roots were measured in 12 out of 25 transformed rice lines. This level of expression resulted in a significant 55% reduction in egg production by Meloidogyne incognita.

Development, field evaluation, and agronomic performance of transgenic herbicide resistant rice

The commerical cultivars ‘Gulfmont’, ‘IR72’ and ‘Koshihikari’ were genetically engineered using electric discharge particle bombardment to express the bar gene which confers resistance to the broad-range herbicide glufosinate. Southern and northern blot analyses of transgenics material revealed stable integration and expression of introduced transgenes in the lines evaluated. In a few plants, silencing of the uidA marker gene was detected at the transcriptional level. Field studies were conducted in 1993 and 1994 at the Rice Research Station near Crowley, LA. This report summarizes results from the first two years of field trial for transgenic Gulfmont and Koshihikari. Transgenic cultivar IR72 was tested in 1995 and preliminary results are similar to those reported for transgenic Gulfmont. All 11 independently derived transgenic lines produced fertile, normal looking seed at maturity. Significant differences were observed in the absence of the herbicide between parental cultivars and transgenic Gulfmont-and Koshihikari-derived lines for days to 50% heading (20% of transgenic lines), plant height (13%), and grain yield (7%). Foliar application of glufosinate had little or no effect on agronomic performance of all transgenic Gulfmont and IR72 lines, while herbicide applications affected grain, yield and plant height of some transgenic Koshhikari. Non-transgenic plants of all three cultivars at the 4-leaf stage were killed within 7 days after 1.12 or 2.24 kg/ha glufosinate applications. Significant differences among certain transgenic lines were observed for agronomic traits after herbicide applications. These results demonstrate that the bar gene was effective in conferring field-level resistance to glufosinate in rice. Variation among transgenic lines required traditional breeding selection procedures to identify superior agronomic types with high levels of herbicide resistance and showed the necessity to generate several independent transgenic lines of each cultivar.

Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Abstract. Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene effective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene effective against sap-sucking insect pests, specifically the brown plant hopper. Using three plasmids, we co-transformed 5- to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene β-glucuronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as confirmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also co-expressed all four genes, as confirmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-polymerase chain reaction (Xa21) analysis. The co-expression efficiency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integration frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integration of any particular gene(s). Genetic analysis confirmed Mendelian segregation of the transgenes in subsequent generations. We report, for the first time, generation and analysis of transgenic rice lines carrying genes effective against more than one taxa of pathogen or pest, substantiating that particle bombardment represents an effective way to introduce unlinked complex multiple traits into plants.

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

Abstract An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T0) and their progeny (T1). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed.

Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels

We transformed rice (Oryza sativa L.) simultaneously with five minimal cassettes, each containing a promoter, coding region and polyadenylation site but no vector backbone. We found that multi-transgene cotransformation was achieved with high efficiency using multiple cassettes, with all transgenic plants we generated containing at least two transgenes and 16% containing all five. About 75% of the plants had simple transgene integration patterns with a predominance of single-copy insertions. The expression levels for all transgenes, and the overall coexpression frequencies, were much higher than previously reported in whole plasmid transformants. Four of five lines analyzed for transgene expression stability in subsequent generations showed stable and high expression levels over generations. A simple model is proposed, which accounts for differences in the molecular make-up and the expression profile of transgenic plants generated using whole plasmid or minimal cassettes. We conclude that gene transfer using minimal cassettes is an efficient and rapid method for the production of transgenic plants containing and stably expressing several different transgenes. Our results facilitate effective manipulation of multi-gene pathways in plants in a single transformation step.

Transgenic rice as a system to study the stability of transgene expression: Multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

Abstract The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world.

The Quest to Understand the Basis and Mechanisms that Control Expression of Introduced Transgenes in Crop Plants

We discuss mechanisms and factors that influence levels and stability of expressed heterologous proteins in crop plants. We have seen substantial progress in this field over the past two decades in model experimental organisms such as Arabidopsis and tobacco. There is no question such studies have resulted in furthering our understanding of key processes in the plant cell and the elaboration of sophisticated models to explain underlying mechanisms that might influence the fate, levels and stability of expression of recombinant heterologous proteins in plants. However, very often, such information is not applicable outside these laboratory experimental models. In order to generate a knowledge basis that can be used to achieve high levels and stability of heterologous proteins in relevant crop plants it is imperative to perform such studies on the target crops. With this in mind, we discuss key elements of the process at the DNA, RNA and protein levels. We believe it is essential to discuss recombinant protein production in crops in a holistic manner in order to develop a comprehensive knowledge base that will in turn serve plant biotechnology applications well.