Mat (Multi-Auto-Transformation) vector system. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants

Summary We have developed a new transformation method called MATVS (Multi-Auto-Transformation Vector System). The oncogenes (ipt or rol genes) of Agrobacterium are used as selectable markers to regenerate transgenic cells and to select marker-free transgenic plants in the MATVS. The chimeric ipt gene or the rol genes are combined withthe site-specific recombination R/RS system to remove the oncogenes from the transgenic cells after transformation. We report here the application of MATVS to transformation of tobacco, aspen, rice and snapdragon. (I) The GST-MAT vector pMAT8 has the native ipt gene and the R gene with a chemical inducible promoter (GST-II-27). Use of the GST-MAT vector generated marker-free transgenic tobacco plants cotaining a single copy transgene at high frequency. (2) Use of the GST-MAT vector pRBI11 containing the rbcS 3B-ipt gene produced transgenic marker-free hybrid aspen plants without crossing. (3) Use of the ipt-type MAT vector, pNPI30GFP, containing the 35S-ipt and 35S-R, genes, resulted in the regeneration of marker-free transgenic reice plants directly from infected scutellum tisues at high frequency within 1 mo. (4) Use of the rol-type MAT vector pNPI702, containing the rol genes and the 35S-R gene, produced transgenic marker-free plants of tobacco and snapdragon at high frequency without crossing. Our results show that the promoter of the ipt gene, the preculture periods of plant tissues and the culture medium are important factors to improve the generation efficiency of marker-free transgenic plants. We can rapidly produce marker-free transgenic plants without the production of ipt-shooty intermediates. Therefore, it is a most promising method to save time and work for the generation of marker-free transgenic plants in crops.     

Asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species

We describe here a practical system for generating selectable marker-free transgenic woody plants independent of sexual crossing. We previously reported that the GST-MAT vector system could produce marker-free transgenic tobacco plants containing a single-copy transgene at high frequency. The GST-MAT vector system consists of a DNA excision cassette of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the isopentenyltransferase gene from Agrobacterium tumefaciens has been inserted. In this study, we applied this new GST-MAT vector to hybrid aspen (Populus Sieboldii X Populus grandidentata), a model of vegetatively propagated plant species, to produce selectable marker-free transgenic woody plants. In the new GST-MAT vector, the chimeric ipt gene fused with a light-inducible rbcS promoter efficiently produced transgenic ipt-shooty with GUS activity from 38.0% of infected stems. Upon excision of the R and ipt genes between RS sites, regulated by the inducible promoter of the maize glutathione-S-transferase (GST-II-27) gene, 3 (21.4%) transgenic hybrid aspen plants with marker-free and normal phenotype were generated from 14 ipt-shooty lines within 2 months after cutting induction. These results suggest that the MAT-vector system might be useful for removing a selectable marker gene independent of sexual crossing in vegetatively propagated species.     

Transgene stacking in plants in the absence of sexual crossing

A transgene stacking system is a prerequisite for the introduction of multiple genes and for the modification of complex metabolic pathways in plants. We demonstrate here that the MAT-vector system previously used for generating marker-free transgenic plants is also an efficient and reliable transformation system for the repeated introduction of multiple transgenes independent of sexual crossing. We previously reported that the GST-MAT vector system, in which excision of the yeast site-specific recombination R/RS system is regulated by the maize GST-II-27 promoter, could generate marker-free transgenic plants containing a single transgene with high frequency. Here we show that the GST-MAT vector can be used successfully to introduce a second transgene (GFP) into a marker-free transgenic tobacco line containing single copies of the first transgenes (nptII and uidA genes). The transgene-stacked marker-free transgenic tobacco plants were generated from ca. 20% of excision-positive ipt-shooty explants within 5 months of Agrobacterium infection. The presence of uidA, nptII, GFP genes and the absence of the ipt gene were verified by PCR analyses. Furthermore, Southern blot analysis showed that no chromosomal rearrangements were introduced between the first and second transformations.     

Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis>

The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation [MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone- and selective agent-free MS medium, and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than ninety percent of the normal shoots were ipt ⁻, gus ⁻ but lacZ ⁺ as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene.     

Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress

In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)‐mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast‐targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non‐transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.     

Marker-Free Transgenic Chrysanthemum Obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation with Twin T-DNA Binary Vectors

In this study, a superbinary vector was constructed to evaluate the potential of a twin T-DNA system for generating selectable marker-free transgenic chrysanthemum plants. The first T-DNA of the pCAMBIA 1300 vector contained the hygromycin phosphotransferase (hpt) selectable marker gene, while the second T-DNA carried the β-glucuronidase gene (uidA) and featuring the gene of interest. The two T-DNA regions were placed adjacent to each other with no intervening region. This vector was then used to transform transversal thin cell layers (1–2 mm thick) of internodal stem segments of chrysanthemum via Agrobacterium-mediated transfer. Putative transgenic plants were obtained and analyzed for presence and integration of the transgene using polymerase chain reaction amplification and Southern blotting. The primary cotransformation frequency was calculated at 38.4%. A total of 17 hpt-resistant/gus-positive T0 plants were evaluated for segregation in the next generation (T1), and among those approximately 15.7% carried the transgene. Overall, the two T-DNA system appeared to be a useful approach to generate marker-free transgenic chrysanthemum plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance genes into the environment.     

Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system

We investigated the potential of a novel double T-DNA vector for generating marker-free transgenic plants. Co-transformation methods using a double T-DNA vector or using mixture of two Agrobacterium tumefaciens strains were compared, and showed that the double T-DNA vector method could produce marker-free transgenic tobacco (Nicotiana tabacum L.) plants more efficiently. A dual marker double T-DNA vector was then constructed by assembling the green fluorescent protein (GFP) gene mgfp5 and the neomycin phosphotransferase gene nptII into the same T-DNA. The frequency of co-transformants produced by this vector was 56.3%. Co-expression of mgfp5 and nptII was found in 28 out of 29 T₁ lines, and segregation of the reporter -glucuronidase gene, gusA, from mgfp5 to nptII was found in 12 out of 29 T₁ lines. Therefore, GFP could be used as a vital marker to improve the transformation efficiency and to easily monitor the segregation of marker genes, thus facilitating screening of marker-free progeny.     

Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker

The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.     

Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) with a linear <Emphasis Type="Italic">GFP</Emphasis> cassette transformation

The presence of selectable marker genes and vector backbone sequences has affected the safe assessment of transgenic plants. In this study, the ovary-drip method for directly generating vector- and selectable marker-free transgenic plants was described, by which maize was transformed with a linear GFP cassette (Ubi-GFP-nos). The key features of this method center on the complete removal of the styles and the subsequent application of a DNA solution directly to the ovaries. The movement of the exogenous DNA was monitored using fluorescein isothiocyanate-labeled DNA, which showed that the time taken by the exogenous DNA to enter the ovaries was shortened compared to that of the pollen-tube pathway. This led to an improved transformation frequency of 3.38% compared to 0.86% for the pollen-tube pathway as determined by PCR analysis. The use of 0.05% surfactant Silwet L-77 + 5% sucrose as a transformation solution further increased the transformation frequency to 6.47%. Southern blot analysis showed that the transgenic plants had low transgene copy number and simple integration pattern. Green fluorescence was observed in roots and immature embryos of transgenic plants by fluorescence microscopy. Progeny analysis showed that GFP insertions were inherited in T₁ generation. The ovary-drip method would become a favorable choice for directly generating vector- and marker-free transgenic maize expressing functional genes of agronomic interest.     

Alleviation of ultraviolet-C-induced oxidative damage through overexpression of cytosolic ascorbate peroxidase

Experiments were conducted to investigate the relationship between ultraviolet (UV) C-induced oxidative damage and the activity of ascorbate peroxidase (APX), using transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana) plants overexpressing cytosolic APX gene (apx1). Transgenic plants having 2.3 fold higher total APX activity, as compared to the wild type plants, showed normal morphological characters. Exposure of 70-day-old plants to fixed intensity UV-C radiation caused an increase in the malondialdehyde (MDA) content in wild type as well as transgenic plants. However, the wild type plants showed significantly higher (p < 0.05) lipid peroxidation as compared to the transgenic plants. Higher proline accumulation was recorded in transgenic plants as compared to the wild type plants, after 24 hours of UV-C exposure. Although the ascorbate content decreased continuously with increasing exposure to UV-C radiation, yet the wild type plants exhibited higher ascorbate levels than the transgenic plants. A marked difference in H₂O₂ content, between the wild type and transgenic plants, was consistently observed up to 20 hours of UV-C exposure. A direct correlation of ascorbate, MDA and H₂O₂ levels was recorded with the extent of oxidative stress, signifying that these could be used as potential bio-marker molecules for oxidative stress. The results clearly demonstrate that overexpression of cytosolic APX can protect tobacco plants from UV-C-induced oxidative damage.     

Auto-excision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT site-specific recombination system

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13–41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.     

Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar “KU50” was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19–21% was observed. Among the total number of ESP explants, 32–38% regained normal extended shoot phenotype and 88–96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.     

Generation of marker-free Bt transgenicindica rice and evaluation of its yellow stem borer resistance

We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borerScirpophaga incertulas (Lepidoptera: Pyralidae). The transgenicindica rice harbours a translational fusion of 2 differentBacillus thuringiensis (Bt) genes, namelycry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an eliteindica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the markerhpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3∶1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T₂ progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.     

Strategies for developing marker-free transgenic plants

The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops. It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies, co-transformation, site-specific recombinase-mediated excision, and non-selected transformation.     

<Emphasis Type="Italic">Rol</Emphasis> (root loci) gene as a positive selection marker to produce marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis>

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.     

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

Enhanced tolerance to methyl viologen‐induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD,APX and NDPK2 genes

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)‐induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress‐inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non‐transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV‐treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.     

Enhanced tolerance of transgenic sweetpotato plants that express both CuZnSOD and APX in chloroplasts to methyl viologen-mediated oxidative stress and chilling

Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with an enhanced tolerance to multiple environmental stresses were developed by expressing the genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of an oxidative stress-inducible SWPA2 promoter in the chloroplasts of sweetpotato plants (referred to as SSA plants). SSA plants were successfully generated by the particle bombardment method and confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 μM methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μM MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage compared to leaves of NT plants, which were almost destroyed. The expression of the introduced CuZnSOD and APX genes in leaves of SSA plants following MV treatment was significantly induced, thereby reflecting increased levels of SOD and APX in the chloroplasts. APX activity in chloroplast fractions isolated from SSA plants was approximately 15-fold higher than that in their counterparts from NT plants. SSA plants treated with a chilling stress consisting of 4°C for 24 h exhibited an attenuated decrease in photosynthetic activity (Fv/Fm) relative to NT plants; furthermore, after 12 h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants remained at a lower level of Fv/Fm activity. These results suggest that SSA plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied to the development of various other transgenic crops with an increased tolerance to multiple environmental stresses.