Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

A transgenic perspective on plant functional genomics

Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype. To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops. This revised version was published online in August 2006 with corrections to the Cover Date.     

Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

Identification of Stigma Specific Expression Fragment in the Promoter of a Soybean Chitinase Class I Gene

Molecular Biology - The expression level of heterologous genes in transgenic plants serves as an important indicator of gene efficiency. The small number of currently known effective promoters,...     

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.     

Recombinant Protein Expression in <Emphasis Type="Italic">Leishmania tarentolae</Emphasis>

A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals, and plants. Recently Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has been suggested as candidate for heterologous genes expression. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein; the oligosaccharide structures are similar to those of mammals with N-linked galactose, and fucose residues. To date several heterologous proteins have been expressed in L. tarentolae including both cytoplasmic enzymes and membrane receptors. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of those tools coupled with a better understanding of the biology of Leishmania species will lead to value and power in commercial and research labs alike.     

Investigation of cytokinin-deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1

The plant hormone cytokinin plays a major role in regulating plant growth and development. Here we generated cytokinin-reduction Arabidopsis plants by overexpressing a heterologous cytokinin oxidase gene DSCKX1 from Dendrobium orchid. These transgenic plants exhibited reduced biomass, rapid root growth, decreased ability to form roots in vitro, and reduced response to cytokinin in growing calli and roots. Furthermore, the expression of KNAT1, STM, and CycD3 genes was significantly reduced in the transgenic plants, suggesting that cytokinin may function to control the cell cycles and shoot/root development via regulation of these genes.     

Molecular genetic analysis of collection of transgenic tobacco plants with buckwheat serine proteases inhibitor gene during long-term subculture

In this paper, the results of long-term screening of independently derived transgenic tobacco plants carrying the synthetic BWI-1a gene of serine proteases inhibitor from buckwheat are presented. For several years periodic spot checks of persistence and expression of the heterologous protective genes in vegetatively cloned collections and seeds of transgenic plants were conducted. The persistence of expression of the target gene after ten years of passage of plants in aseptic culture without selective pressure in their seed progeny for at least three generations and derived callus was shown. Extracts of tissues of all the variants of transgenic plants inhibited the growth of phytopathogenic bacteria and the germination of spores of fungi. The degree of the suppression of a pathogen in this case was hardly reduced. In the second seed generation, the number of defective seeds increased and there was a sharp decline of germination ability of the seeds even in nonselective conditions.     

植物基因工程表达载体的改进和优化策略

外源基因在转基因植物中表达效率低一直是令相关研究困扰的一个问题。转基因植物的生物安全性最近在全球范围内开始引起世人的担优。本简要介绍了近年来在植物表达载体构建方面所采用的一些新策略,这些策略有助于增强外源基因的表达水平,提高生物工程体的安全性。     

Sugar transport across the plasma membranes of higher plants

The fluxes of carbohydrates across the plasma membranes of higher-plant cells are catalysed mainly by monosaccharide and disaccharide-H⁺ symporters. cDNAs encoding these different transporters have been cloned recently and the functions and properties of the encoded proteins have been studied extensively in heterologous expression systems. Several of the proteins have been identified biochemically in these expression systems and their location in plants has been shown immunohistochemically or with transgenic plants which were transformed with reporter genes, expressed under the control of the promoters of individual transporter genes. In this paper we summarize the current knowledge on the molecular biology and biochemistry of higher-plant sugar transport proteins.     

Constitutive expression of Arabidopsis NPR1 confers enhanced resistance to the early instars of Spodoptera litura in transgenic tobacco

In Arabidopsis , NPR1 ( AtNPR1 ) regulates salicylic acid (SA)-mediated activation of PR genes at the onset of systemic acquired resistance. AtNPR1 also modulates SA-induced suppression of jasmonic acid-responsive gene expression, and npr1 mutants manifest enhanced herbivore resistance. We have raised stable transgenic tobacco lines, expressing AtNPR1 constitutively, which showed elevated expression of PR1 and PR2 genes upon SA treatment. Herbivore bioassays with a generalist polyphagous pest, Spodoptera litura , revealed that the transgenic lines exhibited enhanced resistance compared to the wild-type plants, particularly with respect to younger larval populations. Insect-mediated injury induced several protease inhibitors (PIs), more significantly a 40-kDa serine PI in all the tobacco lines, but the induction was higher in the transgenic plants. We show in this communication that heterologous expression of AtNPR1 provides enhanced resistance to early larval populations of the herbivore, Spodoptera in transgenic tobacco plants.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

Improvement of freezing tolerance in tobacco plants expressing a cold-responsive and chloroplast-targeting protein WCOR15 of wheat

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.