Increased tolerance to salt- and water-deficit stress in transgenic lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) by constitutive expression of LEA

A late embryogenesis abundant (LEA) protein gene, ME-leaN4, from rape (Brassica napus) was successfully introduced into lettuce (Lactuca sativa L.) using Agrobacterium tumefaciens-mediated transformation. Infection by Agrobacterium strain EHA101 containing the binary vector pIG121-LEA was applied. Six independent transgenic lettuce plants were generated as a result. Transgenic lettuce demonstrated enhanced growth ability as compared to non-transformed control plants under salt-stress and water-deficit stress conditions. After 10-day growth under 100 mM NaCl condition in the hydroponics, average plant length and fresh weight of transgenic lettuce were 2.8 cm and 2.5 g plant^–1, while control plants were only 0.2 cm and 0.3 g plant^–1, respectively. The increased tolerance was also reflected by delayed wilting of leaves caused by water-deficit stress. These results suggest that growth characteristics were improved in transgenic lettuce plants constitutively expressing the rape LEA gene in response to salt- and water-deficit stress. Byong-Jin Park, Zaochang Liu These two authors contributed equally to this work.     

Field Performance of Transgenic indica Hybrid Rice with Improved Cooking and Eating Quality by Down-regulation of Wx Gene Expression

High amylose content (AC) in rice endosperm is correlated with poor grain quality, particularly in indica hybrid rice. We have generated several homozygous transgenic parent lines of indica hybrid rice carrying an antisense Waxy (Wx) gene and demonstrated that the AC in seeds of these lines decreased dramatically. Two transgenic maintainer lines (L25B and L18B), derived from one of the key maintainer parents of an indica hybrid rice in China, Long-te-fu B (LTF-B), were selected and the antisense Wx gene was subsequently introgressed into the male-sterile counterpart, LTF-A, with the aim to generate improved indica hybrids. The indica hybrids derived from the selected transgenic male-sterile lines and restorer lines were tested for quality and agronomic performance under normal field conditions. Our results demonstrated that the reduction of AC in the homozygous transgenic maintainer lines stably passed down in five successive generations and the improved quality was also found in their relevant transgenic hybrids produced. The other two key characters of rice cooking and eating quality, the gel consistence (GC) and gelatinization temperature (GT), were also improved in the grains of both the transgenic maintainer lines and their relevant hybrids. In addition, no change was observed for most of the agronomic characters of the transgenic maintainer lines and the relevant transgenic hybrids. Although the grain weight of the transgenic line was reduced, the grain yield of the homozygous transgenic parent lines and the transgenic hybrids was similar when compared with that of the wild-type controls. These results suggest that transgenic approaches are an effective way to obtain rice lines with both improved qualities and high yield, especially for indica hybrid rice.     

Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation

Efficient procedures for regeneration and Agrobacterium-mediated transformation were established for Agrostis mongolica Roshev. and generated transgenic plants tolerant to drought and heat stresses using a regulatory gene from Arabidopsis, ABF3, which controls the ABA-dependent adaptive responses. The identification and selection of regenerable and reproducible callus type was a key factor for successful transformation. The transformation efficiency was 49.2% and gfp expression was detected in hygromycin-resistant calli and stem of putative transgenic plants. The result of Southern blot analysis showed that the ABF3 transgene was stably integrated into the genome of transgenic plants. Of the five transgenic lines analyzed, single transgene integration was observed in two lines and two copy integration was observed in three transgenic lines. Northern blot analysis confirmed that ubi::ABF3 was expressed in all transgenic lines. Transgenic plants exhibited neither growth inhibition nor visible vegetative phenotypic alternations. However, both transgenic and wild-type plants were highly sterile and did not flower during 3 years of growth period in the open field under subtropical Jeju Island climate. The stomata of the transgenic plants opened less than did stomata of the wild-type plants, and water content of the transgenic leaves remained about 3–4 fold higher than observed for wild-type leaves under drought stress. The transgenic plants showed about 2 fold higher survival rates under drought stress and about 3 fold higher survival rates under heat stress when compared to wild-type plants. Thus, overexpression of the Arabidopsis ABF3 gene results in enhancement of both drought and heat stress tolerance in Agrostis mongolica Roshev.     

Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H₂O₂ were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H₂O₂ metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants. Zhenqiang Lu and Dali Liu contributed equally.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Ectopic Expression of the Tomato <Emphasis Type="Italic">Mi-1</Emphasis> Gene Confers Resistance to Root Knot Nematodes in Lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis>)

The full genomic region of the root knot nematode (Meloidogyne spp.) resistance gene Mi-1 was cloned from tomato and transformed into lettuce to investigate its function in a heterologous system. Transgenic lettuce lines containing the Mi-1 gene were developed using Agrobacterium-mediated transformation. Ectopic expression of the Mi-1 gene was observed in transgenic lines, and resistance to root knot nematode was improved.     

Enhancement of flowering and branching phenotype in chrysanthemum by expression of <Emphasis Type="Italic">ipt</Emphasis> under the control of a 0.821 kb fragment of the <Emphasis Type="Italic">LEACO1</Emphasis> gene promoter

The cytokinin biosynthesis gene, isopentenyl transferase (ipt), under the control of an 821 bp fragment of the LEACO1 gene promoter (from Lycopersicon esculentum) was introduced into Dendranthema × grandiflorium ‘Iridon’ (chrysanthemum). LEACO1_0.821kb-ipt transgenic lines grown in the vegetative state, exhibited a range of phenotypic changes including increased branching and reduced internode lengths. LEACO1_0.821kb-ipt transgenic lines grown in the generative state, exhibited increased flower bud count that ranged from 3.8- to 6.7-times the number produced by wild-type plants. Dramatic increases in flower number were associated with a delay of flower bud development and a decrease in flower bud diameter. RT-PCR analysis indicated differences in ipt gene expression between individual transgenic lines that exhibited a range of phenotypes. Within an individual transgenic line, RT-PCR analysis revealed changes in ipt gene expression at different stages of generative shoot development. Expression of ipt in transgenic lines correlated well with high concentrations of the sum total to bioactive cytokinins plus the glucosides and phosphate derivatives of these species, under both vegetative and generative growth conditions. In general, transgenic lines accumulated higher concentrations of both storage-form cytokinins (O-glucosides) and deactivated-form cytokinins (N-glucosides) in generative shoots of than in vegetative shoots. Based on the range of phenotypes observed in various transgenic chrysanthemum lines, we conclude that the LEACO1 _0.821kb -ipt gene appears to have great potential for use in ornamental crop improvement. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enhancement of resistance to aphids by introducing the snowdrop lectin genegna into maize plants

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through theAgrobacterium tumefaciens- mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that thegna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.     

RNAi介导珠子参PjCAS基因沉默对皂苷合成的影响

该研究利用Gateway技术构建珠子参环阿屯醇合成酶基因(Panax japonicus cycloartenol synthase,PjCAS)的RNAi表达载体,利用农杆菌介导转化在珠子参细胞中成功实现了PjCAS 的RNA干扰;采用实时荧光定量PCR分析珠子参皂苷生物合成途径中关键酶基因的表达情况,同时检测转基因细胞中皂苷和植物甾醇含量的变化,探讨PjCAS基因对珠子参皂苷合成的调控作用。结果表明：(1)成功获得PjCAS基因的RNAi片段,并成功构建了PjCAS基因RNAi载体pHellsgate PjCAS。(2)经农杆菌遗传转化,获得6株实现PjCAS基因RNA干扰的转基因阳性细胞系。(3)与普通细胞系相比,转基因细胞系中PjCAS基因的表达量大约下降了85%,同时与珠子参皂苷合成直接相关的关键酶基因PjDS、PjAS表达量最高分别上调了90%和150%。(4)转基因细胞系中6种单体皂苷的含量均显著高于对照组,其中达玛烷型单体皂苷Re、Rb1、Rd和齐墩果烷型单体皂苷R0、IV、IVa的平均含量比普通珠子参细胞系分别提高了28%、49%、40%、36%、59%、50%。说明珠子参皂苷含量的变化受PjCAS基因的间接调控。(5)6株转基因细胞系中植物甾醇含量较对照显著降低了53%~73%。研究发现,沉默PjCAS基因可促进珠子参皂苷合成的关键酶基因PjDS、PjAS显著上调表达,并提高转PjCAS基因细胞系中单体皂苷的含量,从而促进了珠子参皂苷合成量的显著增加,证明通过抑制植物甾醇合成通路关键基因PjCAS的表达可以有效降低植物甾醇合成支路的代谢通量,使更多的代谢流朝着珠子参皂苷合成方向流动,最终促进了珠子参皂苷的生物合成。     

Engineering tocopherol biosynthetic pathway in lettuce

In order to increase tocopherol content, genes encoding Arabidopsis homogentisate phytyltransferase (HPT) and γ-tocopherol methyltransferase (γ-TMT) were constitutively over-expressed in lettuce (Lactuca sativa L. var. logifolia), alone or in combination. Over-expression of hpt could increase total tocopherol content, while over-expression of γ-tmt could shift tocopherol composition in favor of α-tocopherol. Transgenic lettuce lines expressing both hpt and γ-tmt produced significantly higher amount of tocopherol and elevated α-/γ-tocopherol ratio compared with non-transgenic control and transgenic lines harboring a single gene (hpt or γ-tmt). The best line produced eight times more tocopherol than the non-transgenic control and more than twice than hpt single-gene transgenic line.     

Rapid attainment of a doubled haploid line from transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants by means of anther culture

Summary We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.     

The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L.

Male-sterile lines were generated in oilseed mustard (Brassica juncea) with a cytotoxic gene (barnase) in conjunction with either of two tapetum-specific promoters, TA29 and A9. Several transformation vectors based on different promoter and marker gene combinations were developed and tested for their efficacy in generating agronomically viable male-sterile lines. Use of strong constitutive promoters (e.g. CaMV 35S or its double-enhancer variant) to express the marker gene (bar) in barnase constructs generated male-sterile plants at an extremely low frequency with most plants showing abnormalities in vegetative morphology, poor female fertility, low seed germination frequencies and/or distortion in segregation ratios of transgenes. Such abnormalities were considerably reduced on using weaker promoters (e.g. nos) to drive the marker gene (nptII) in barnase constructs and could therefore be attributed to leaky expression of the barnase gene under enhancing effects of strong constitutive promoters. We show that the use of a Spacer DNA fragment between the barnase gene (driven by a tapetum-specific promoter) and the CaMV 35S promoter-driven bar gene insulates tissue-specific expression of the barnase gene over all developmental stages of transgenic plants and significantly enhances recovery of agronomically viable male-sterile lines. All TA29-barnase male-sterile lines containing the Spacer DNA fragment exhibited normal morphology, growth and seed set on backcrossing as observed for wild-type plants. Around 75% of single-copy events tested further also showed proper segregation of the marker gene/male-sterile phenotype among backcross progeny. Constructs based on the use of Spacer DNA fragments as insulators could be successfully used to alleviate limitations associated with transformation of plant systems using cytotoxic genes for development of agronomically viable male-sterile lines in crop plants and for cell/tissue ablation studies in general.     

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM₁-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.)

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed‐specific overexpression of AtGCHI caused significant increases of up to 150‐fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 μg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.     

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

转鞭毛蛋白基因水稻细菌性条斑病抗性研究

该研究从生防菌枯草芽胞杆菌Bs-916中克隆了鞭毛蛋白基因,利用转基因载体pCAMBIA1300转入水稻,筛选得到98株阳性转基因植株。分子检测结果表明,有12个转基因株系可检测到目的基因的表达。随后抗病性鉴定表明,有3个转基因株系对水稻细菌性条斑病具有较高的抗性。该研究为目前水稻抗细菌性条斑病转基因研究拓宽了可应用基因资源的范围。