Inheritance of marker and target genes in seed and vegetative progenies of transgenic tobacco plants carrying the buckwheat serine protease inhibitor gene

The buckwheat serine protease inhibitor (BWI-1) target gene (ISP) was expressed under the control of the constitutive 35S promoter of the cauliflower mosaic virus was expressed in transgenic tobacco plants and conferred antibacterial resistance. A stable and linked inheritance and expression of the marker nptII and target genes were observed in a random sample of independent transgenic tobacco plants after longterm propagation by nodal segments or multiple (for 1.5 years) regenerations under nonselective conditions; the transgene insert was preserved in the T1 seed progeny. Transgenic plants displayed numerous alterations in microsporogenesis. A loss of kanamycin (Km) resistance was accompanied by a loss of antibacterial activity in two lines. Segregation was observed for Km resistance in line C7 and for seedling size in line C22.     

微量元素营养对桉树无性系苗木叶绿素及荧光参数影响

研究了不同微量元素营养液,对栽培的三种桉树无性系苗木叶绿素及荧光参数变化。结果表明:各无性系苗木在全部测试性状的差异表现出极显著。在六个测试的微量元素因子中,Fe(B)显著地影响苗木的Fo、Fm、Fv/Fo、Fv/Fm,Zn(C)显著地影响Fo、Fv/Fm,Mn(D)显著地影响叶绿素含量(SPAD值),Mo(F)显著地影响叶绿素含量、Fo,B(G)显著地影响叶绿素含量Fo、Fv/Fo、Fv/Fm。相关分析表明Fm与Fo、Fv/Fo与Fo、叶绿素含量与Fo、Fm、Fv/Fm与Fv/Fo均存在显著的相关性。随着微量元素Fe、Zn、Cu、Mo、B浓度增大,Fo、Fv/Fm均呈增大趋势,但在不利的高浓度环境下,Fv/Fm降低。对三个无性系叶绿素荧光比较,U₆与DH_32-29具有高的Fo、Fm和低的Fv/Fm、Fv/Fo,DH_201-2却具有低的Fo、Fm和高的Fv/Fm、Fv/Fo。对微量元素浓度变化,DH_32-29较敏感。因此,叶绿素荧光参数可作为诊断植物微量元素营养状况的指标之一。     

Unsuccessful search for DNA transfer from transgenic plants to bacteria in the intestine of the tobacco horn worm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

DNA transfer from transgenic plants to native intestinal bacteria and introduced Acinetobacter BD413 was assessed in the gut of the tobacco horn worm (Manduca sexta). The marker was kanamycin resistance gene (nptII), and tobacco carrying the nptII gene in the chloroplasts served as the donor. We detected neither whole gene transfer to native bacteria, nor transfer of fragments of nptII to Acinetobacter, using a marker exchange assay. This negative result was attributed to a heat-labile activity that degraded DNA in the feces, probably DNAase. Nevertheless, a few intact leaf cells survived transit through the gut, and DNA extracted from feces did transform Acinetobacter, albeit at lower frequencies than DNA extracted from leaves.     

Fertile transgenic Indica rice plants obtained by electroporation of the seed embryo cells

We have obtained fertile transgenic plants of Indica rice variety IR36, by using electroporation to transfer the neomycin phosphotransferase II (nptII) gene into cells of mature embryos. Resistant calli were selected in the presence of 30 g/ml G418. Nearly thirty transgenic plants were regenerated within three months after transformation. Many of them yielded seeds following self-pollination. Data from molecular analysis and enzyme assay proved that the foreign gene was stably integrated into the genome of resistant calli, R0 and R1 plants, and also expressed. Mendelian segregation of the nptII gene was observed in R1 progeny plants.Abbreviations NOS nopaline synthase - NPTII and nptII neomycin phosphotransferase II - OCS octopine synthase - Km kanamycin     

Growth and photosynthetic activity of micropropagated strawberry plants inoculated with endomycorrhizal fungi (AMF) and growing under drought stress

The plants produced by in vitro methods are free of any microflora contrary to natural systems where plants are colonized by symbiotic fungi. The present paper reports the experiments carried out to evaluate the role of arbuscular endomycorrhizal fungi in development of micropropagated strawberries and their photosynthetic activity (measured by chlorophyll fluorescence) under drought conditions. Mycorrhization strongly affected growth and tolerance to water deficiency of the plants cultivated in greenhouse. Wilting of not-mycorrhized plants was accompanied by drastic increase of Fo and Tfm and decrease of Fm. At the same time, the value of these parameters for mycorrhized plants did not change. Drastic decrease in the value of parameters Fv/Fm, Fv/Fo and Fo/Fm for plants without AMF appeared at the end of dry period. Rise of Fs and decrease Rfd was noted only for not-mycorrhized plants. The plants colonized by fungi, fully recovered their photosynthetic activity when watering was restored.     

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l^–1 -naphthaleneacetic acid (NAA) and 1.0 mg l^–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml^–1 Km, 200 g ml^–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T₁ seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.     

A comparative analysis of wild and laboratory grown Laminaria abyssalis (Phaeophyta) using modulated fluorescence

Laminaria abyssalis fronds were either collected at the Brazilian costal area - 40 meters below sea level - or grown in the laboratory. The photochemical yield as defined by the Fv/Fm and the Fo - the dark fluorescence level when all PSII centers are open - varied with the distance from the stipe to the tip of the blade in wild grown fronds while it stayed constant in the laboratory grown plants. The chlorophyll a/c ratio levels decreased in the wild fronds from 12 (near the stipe) to 6 near the top. The chlorophyll c content increased from 0.8 to near 1.7 mg cm^–2 in the wild fronds. The laboratory fronds did not show variations in their chlorophyll contents. The wild fronds pattern changed after 2 months kept in the laboratory, producing similar results to those grown in the laboratory. The results indicate that the levels of the antenna complex in the wild fronds increase from the stipe to the top of the blade, in a fashion similar of the sun/shade leaves. Also, results show, that this alga is able to adapt itself to new light conditions, possibly increasing its level of antenna complex and photosynthetic units.Abbreviations PSII Photosystem II - Fo Chlorophyll fluorescence when all PSII are opened - Fm Chlorophyll Fluorescence when all PSII are closed - Fv Variable Fluorescence (Fm-Fo) - Fv/Fm Quantum Yield for Photochemistry     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 × 10⁶-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 × 10³ genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR). Received: 20 May 1997 / Accepted: 17 October 1997     

Electroporation of immature maize zygotic embryos and regeneration of transgenic plants

Using the pulse-discharging electroporation system HPES-3, we have transferred the neomycin phosphotransferase II (nptII) gene and -glucuronidase (gus) gene into mechanically-woulded immature zygotic embryo cells of an elite local maize cultivar Huanong Supersweet No. 42 and have produced transgenic maize plants. DNA hybridization and NPTII dot assay showed that the foreign genes were integrated into the genomes and expressed stably in the cells of the transgenic calluses and plants.     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

超表达拟南芥2-烯醛还原酶基因对烟草抗旱性的作用机理分析

为研究是否可以利用2-烯醛还原酶(AER)来清除活性氧下游的醛自由基达到提高植物的抗旱性,以超表达拟南芥AER基因烟草和野生型烟草(SR)为研究材料,利用干旱胁迫处理进行抗旱性分析,测定了干旱胁迫及复水后各个烟草株系的生物量、光合速率、叶绿素荧光参数、叶绿素含量、MDA和H2O2含量等指标。结果显示:(1)干旱胁迫下,转基因烟草株系的生物量、叶绿素含量、净光合速率、PSⅡ最大光化学效率及H2O2的清除能力均显著高于对照;(2)复水之后,烟草植株的各项生理指标都得到一定程度的恢复,而转基因株系相比于野生型恢复迅速,恢复能力更强。研究认为,超表达AER基因可以通过清除活性氧及其下游醛自由基来提高烟草的抗旱能力。     

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.     

Changes of Photosystem Ⅱ and Respiratory Enzyme Activity in Transgenic Tobacco Enriched BADH Gene

The transient characteristics of chlorophyll fluorescence induction, the activities of respiratory enzymes (malate dehydrogenase, isocitrate dehydrogenese, suecinate dehydrogenase and cytochrome C oxidase) and the activities of photorespiratory enzymes (hydroxypyruvate reductase, glycolate oxidase and catalase) in the transgenic tobacco (Nicotiana tabacum L. ), in which betaine aldehyde dehydrogenase (BADH) gene had been introduced, were determined and compared with the parent plants. The results showed that the Fy/Fo, Fv/Fm and Fd/Fs of the transgenic plant had no changes; the activities of ma]ate dehydrogenase, isocitric dehydrogenase and succinic dehydrogenase in tricarboxylic acid cycle had a slight increase, and the activity of eytochrome C oxidase in the terminal oxidative pathway had a significant increase. The activities of hydroxypyruvate reductase, glycol]ic oxidase and catalase in the photorespiratory pathway had a marked increase. The possible significance on these changes was discussed.     

Arbuscular mycorrhizae improves low temperature stress in maize via alterations in host water status and photosynthesis

The effect of arbuscular mycorrhizal (AM) fungus, Glomus etunicatum, on growth, water status, chlorophyll concentration and photosynthesis in maize (Zea mays L.) plants was investigated in pot culture under low temperature stress. The maize plants were placed in a sand and soil mixture at 25°C for 7 weeks, and then subjected to 5°C, 15°C and 25°C for 1 week. Low temperature stress decreased AM root colonization. AM symbiosis stimulated plant growth and had higher root dry weight at all temperature treatments. Mycorrhizal plants had better water status than corresponding non-mycorrhizal plants, and significant differences were found in water conservation (WC) and water use efficiency (WUE) regardless of temperature treatments. AM colonization increased the concentrations of chlorophyll a, chlorophyll b and chlorophyll a + b. The maximal fluorescence (Fm), maximum quantum efficiency of PSII primary photochemistry (Fv/Fm) and potential photochemical efficiency (Fv/Fo) were higher, but primary fluorescence (Fo) was lower in AM plants compared with non-AM plants. AM inoculation notably increased net photosynthetic rate (Pn) and transpiration rate (E) of maize plants. Mycorrhizal plants had higher stomatal conductance (g_s) than non-mycorrhizal plants with significant difference only at 5°C. Intercellular CO₂ concentration (Ci) was lower in mycorrhizal than that in non-mycorrhizal plants, especially under low temperature stress. The results indicated that AM symbiosis protect maize plants against low temperature stress through improving the water status and photosynthetic capacity.     

Effects of Cadmium Stress on Leaf Chlorophyll Fluorescence and Photosynthesis of Elsholtzia argyi—A Cadmium Accumulating Plant

A hydroponic experiment was conducted to investigate the effects of cadmium (Cd) on chlorophyll fluorescence and photosynthetic parameters on a Cd accumulating plant of Elsholtzia argyi. Four weeks-seedlings of E. argyi were treated with 0 (CK) 5, 10, 15, 20, 25, 30, 40, 50 and 100 μmol L^?1 Cd for 21days. Fv/Fo, Fv/Fm, qP, ΦPSП, ETR and Fv′/Fm′ were significantly increased under low Cd (5–15 μmol L^?1 for Fv/Fo, Fv/Fm and qP, 5–10 μmol L^?1 for ΦPSП, ETR and Fv′/Fm′) stress, and these parameters were similar to control under Cd ≤ 50μmol L^?1. All above parameters were significantly decreased at 100 μmol L^?1 Cd. Compared with control, Pn was significantly (P < 0.05) increased under 5–30 μmol L^?1 Cd. However, 50 and 100 μmol L^?1 Cd significantly (P < 0.05) reduced it. Gs and Tr were substantially decreased at 50–100 and 40–100 μmol L^?1 Cd, respectively. Ci was significantly increased at 50 and 100 μmol L^?1 Cd. High Cd-induced decrease of Pn is not only connected to stomatal limitation but also to the inhibition of Fv/Fo, Fv/Fm, ΦPSП, qP, ETR and increase of NPQ. Maintain chlorophyll fluorescence and photosynthesis parameters under its Cd tolerance threshold were one of tolerance mechanisms in E. argyi.     

水分胁迫对牛心朴子叶片光合色素及叶绿素荧光的影响

研究了水分胁迫对牛心朴子叶片光合色素及叶绿素荧光动力学参数的影响。结果表明,在长期的水分胁迫中,牛心朴子叶片的叶绿素a(Chl a)、叶绿素b(Chl b)和类胡萝卜素(Car)含量没有下降或下降不明显。直到处理末期才显著下降;叶片叶绿素荧光动力学参数Fo、Fm、Fv、Fv／Fm变化不大,在处理末期各处理Fo降低,轻度、重度水分胁迫的Fm、Fv、Fv／Fm升高。说明K期水分胁迫后牛心朴子的光合功能受到影响,但牛心朴子仍表现出较强的适应干旱的能力。     

Photoprotective Effects of D1 Protein Turnover and the Lutein Cycle on Three Ephemeral Plants under Heat Stress

To clarify the characteristics of photoinhibition and the primary defense mechanisms of ephemeral plant leaves against photodestruction under high temperature stress, inhibitors and the technology to determine chlorophyll fluorescence were used to explore the protective effects of D1 protein turnover and the lutein cycle in the high temperature stress of the leaves of three ephemeral plants. The results showed that the maximum light conversion efficiency (Fv/Fm) of the ephemeral plant leaves decreased, and the initial fluorescence (Fo) increased under 35°C ± 1°C heat stress for 1–4 h or on sunny days in the summer. Both Fv/Fm and Fo could be recovered after 8 h of darkness or afternoon weakening of the external temperature. Streptomycin sulfate (SM) or dithiothreitol (DTT) accelerated the decrease of Fv/Fm and the photochemical quenching coefficient (qP) in the leaves of three ephemeral plants at high temperature, and the decrease was greater in the SM than in the DTT treatment. When the high temperature stress was prolonged, the Y(II) values of light energy distribution parameters of PSII decreased, and the Y(NPQ) and Y(NO) values increased gradually in all the treatment groups of the three ephemeral plants. The results showed that the leaves of the three ephemeral plants had their own highly advanced mechanisms to protect against photodamage, which inhibited the turnover of D1 protein and xanthophyll cycle. This can damage the PSII reaction center in the leaves of the three ephemeral plants under high temperature. The protective effect of D1 protein turnover on heat stress in Erodium oxyrrhynchum and Senecio subdentatus was greater than that of the lutein cycle, while the protective effect of lutein cycle was greater than that of D1 protein turnover in Heliotropium acutiflorum subjected to heat damage.     

茉莉酸甲酯对干旱及复水条件下烤烟幼苗叶绿素荧光特性的影响

以烤烟品种“龙江911”为试验材料,研究了干旱及复水过程中外源茉莉酸甲酯(MeJA)对移栽后烤烟幼苗叶绿素含量和叶绿素荧光特性的影响.结果表明： 干旱下烤烟幼苗叶绿素含量、PSⅡ反应中心完全关闭时荧光产量(F_m)、PSⅡ潜在活性(F_v/F_o)、最大光化学效率(F_v/F_m)、实际光化学效率(Ф_PSⅡ)、表观电子传递速率(ETR)和光化学猝灭系数(q_P)下降,而初始荧光(F_o)和非光化学猝灭系数(q_N)升高,0.2和0.5 mmol·L^-1的外源MeJA明显减缓了干旱下烤烟幼苗F_v/F_m、F_v/F_o、Ф_PSⅡ、ETR、q_P的下降和q_N的上升,而1.0 mmol·L^-1 MeJA效果不明显.复水后,烤烟幼苗各项叶绿素荧光指标均有明显恢复,并且MeJA处理后的幼苗恢复更明显.表明外源MeJA减轻了干旱胁迫下烤烟叶片叶绿素的分解,对PSII反应中心起到一定的保护作用,提高了电子传递速率,降低了干旱胁迫对烤烟幼苗的伤害,并且复水后叶绿素含量和叶绿素荧光参数能迅速恢复,从而保证了经干旱胁迫后烤烟幼苗能迅速缓苗.     

An Arabidopsis thaliana ABC transporter that confers kanamycin resistance in transgenic plants does not endow resistance to Escherichia coli

Concerns have been raised about potential horizontal gene transfer (HGT) of antibiotic resistance markers (ARMs) from transgenic plants to bacteria of medical and environmental importance. All ARMs used in transgenic plants have been bacterial in origin, but it has been recently shown that an Arabidopsis thaliana ABC transporter, Atwbc19, confers kanamycin resistance when overexpressed in transgenic plants. Atwbc19 was evaluated for its ability to transfer kanamycin resistance to Escherichia coli, a kanamycin‐sensitive model bacterium, under simulated HGT, staged by subcloning Atwbc19 under the control of a bacterial promoter, genetically transforming to kanamycin‐sensitive bacteria, and assessing if resistance was conferred as compared with bacteria harbouring nptII, the standard kanamycin resistance gene used to produce transgenic plants. NptII provided much greater resistance than Atwbc19 and was significantly different from the no‐plasmid control at low concentrations. Atwbc19 was not significantly different from the no‐plasmid control at higher concentrations. Even though HGT risks are considered low with nptII, Atwbc19 should have even lower risks, as its encoded protein is possibly mistargeted in bacteria.     

Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme

The human lysozyme gene, which is assembled by the stepwise ligation of chemically synthesized oligonucleotides, was introduced into tobacco (Nicotiana tabacum cv `SR1') by the Agrobacterium-mediated method. The introduced human lysozyme gene was highly expressed under the control of the cauliflower mosaic virus 35S promoter, and the gene product accumulated in the transgenic tobacco plants. The transgenic tobacco plants showed enhanced resistance against the fungus Erysiphe cichoracearum – both conidia formation and mycelial growth were reduced, and the size of the colony was diminished. Microscopic observation revealed that the transgenic tobacco plants carried the resistant phenotype, analogous to that of the resistant cultivar `Kokubu' which had been selected by conventional breeding. Growth of the phytopathogenic bacterium Pseudomonas syringae pv. tabaci was also strongly retarded in the transgenic tobacco, and the chlorotic halo of the disease symptom was reduced to 17% of that observed in the wild-type tobacco. Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against both fungal and bacterial diseases. Received: 9 September 1996 / Revision received: January 9 1997 / Accepted: 20 February 1997