Temperature sensitive diphtheria toxin confers conditional male-sterility in Arabidopsis thaliana

A gene encoding a temperature-sensitive diphtheria toxin A chain (DTA) polypeptide was fused to the Arabidopsis thaliana tapetum-specific A9 promoter. Expression of the chimaeric gene in transgenic A. thaliana lines resulted in plants that were male-sterile, but female-fertile, when grown at 18 degrees C, and fully self fertile at 26 degrees C. No pollen grains were found on the anthers of transgenic plants grown at 18 degrees C, although aggregated pollen grains were found inside the anthers. Electron microscopy revealed discrete alterations in the tapetal cells of the male-sterile transgenic plants. The strength of the phenotype observed in segregants correlated with the level of expression of the gene and the copy number. The low frequency at which fully male-sterile plants were generated suggests that the temperature-sensitive DTA protein is disabled as a cytotoxin, relative to the wild-type protein activity.     

Enhanced seed phytosterol accumulation through expression of a modified HMG-CoA reductase

The regulation of phytosterol biosynthesis in seeds is of interest to biotechnologists because of the efficacy of dietary phytosterols in reducing blood cholesterol in humans. Mevalonate synthesis via 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a key step in phytosterol biosynthesis. HMG-CoA reductase is inactivated by phosphorylation by SNF1-related protein kinase 1 (SnRK1). With the aim of increasing seed phytosterol levels, transgenic tobacco plants were produced expressing a full-length Arabidopsis (Arabidopsis thaliana) HMG-CoA reductase gene (HMG1) coding sequence, a modified HMG1 sequence encoding a protein lacking the target serine residue for phosphorylation by SnRK1, or a chimaeric sequence encoding the N-terminal domain of the Arabidopsis HMG1 enzyme fused with the catalytic domain of yeast HMG-CoA reductase, which lacks an SnRK1 target site. All three transgenes (35S-AtHMG1, 35S-AtHMG1m and 35S-AtScHMG1) were under the control of a cauliflower mosaic virus 35S RNA promoter. Levels of seed phytosterols were up to 2.44-fold higher in plants transformed with the 35S-AtHMG1m gene than in the wild-type, and were significantly higher than in plants expressing 35S-AtHMG1 or 35S-AtScHMG1. In contrast, levels of phytosterols in leaves of plants transformed with the 35S-AtHMG1m gene were unchanged, suggesting that regulation of HMG-CoA reductase by SnRK1 is an important factor in seeds but not in leaves. A total of 11 independent transgenic lines expressing 35S-AtHMG1m or 35S-AtScHMG1 also showed an altered flower phenotype, comprising a compact floret, prolonged flowering, short, pale petals, a protruding style, short stamens, late anther development, little or no pollen production, premature flower abscission and poor seed set. Because of this phenotype, the modified HMG-CoA reductase gene would have to be expressed seed specifically if it were to be engineered into a crop plant for biotechnological purposes.     

Antisense-mediated suppression of transgene expression targeted specifically to pollen

A potential problem in the field release of transgenic plants is the spread of foreign gene products via pollen. Therefore, the use of the tomato pollen-specific lat52 gene promoter was investigated as a means of targeting antisense RNA to pollen without affecting transgene expression elsewhere in the plant. A transgenic tobacco line T115, which showed GUS expression in pollen, leaves and roots were retransformed with a construct containing the pollen-specific lat52 promoter driving the GUS encoding uid A gene in antisense orientation. From 24 independent transformants obtained, 19 showed a significant reduction in pollen GUS activity. Of these lines, four showed a reproducible antisense effect in pollen in the next generation, while it was shown in one line that GUS activity in leaves and roots was also unaffected. To ascertain the effectiveness of the antisense strategy to downregulate very high levels of pollen expression, a lat52-gus antisense construct was introduced into tobacco lines containing lat52-gus, which had pollen GUS activity of up to 250 times greater than in line T115. Results showed that 30 out of 34 independent lines exhibited a significant antisense effect in pollen, confirming the effectiveness of pollen-targeted antisense strategy to reduce undesirable expression in pollen independent of expression level in pollen.     

Engineering cytoplasmic male sterility via the chloroplast genome by expression of {beta}-ketothiolase

While investigating expression of the polydroxybutyrate pathway in transgenic chloroplasts, we addressed the specific role of beta-ketothiolase. Therefore, we expressed the phaA gene via the chloroplast genome. Prior attempts to express the phaA gene in transgenic plants were unsuccessful. We studied the effect of light regulation of the phaA gene using the psbA promoter and 5' untranslated region, and evaluated expression under different photoperiods. Stable transgene integration into the chloroplast genome and homoplasmy were confirmed by Southern analysis. The phaA gene was efficiently transcribed in all tissue types examined, including leaves, flowers, and anthers. Coomassie-stained gel and western blots confirmed hyperexpression of beta-ketothiolase in leaves and anthers, with proportionately high levels of enzyme activity. The transgenic lines were normal except for the male-sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Floral developmental studies revealed that transgenic lines showed an accelerated pattern of anther development, affecting their maturation, and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium, and vacuolation affected pollen grains and resulted in the irregular shape or collapsed phenotype. Reversibility of the male-sterile phenotype was observed under continuous illumination, resulting in viable pollen and copious amount of seeds. This study results in the first engineered cytoplasmic male-sterility system in plants, offers a new tool for transgene containment for both nuclear and organelle genomes, and provides an expedient mechanism for F(1) hybrid seed production.     

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.     

表达核糖核酸酶基因的雄性不育油菜的获得

从细菌Ｂａｃｉｌｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ＲＮａｓｅ（ｂａｒｎａｓｅ）基因,构建了ＴＡ－２９基因５＇调控区（－１３００－＋３）与ｂａｒｎａｓｅ基因的嵌合基因,通过农杆菌介导的遗传转化,获得了“双低”甘蓝型油菜“中双８２１”的转基因植株。转化植株与末转化植株在高度、生长速度、花器形态、花色等方面基本相同,但转化植株花丝短小、花药干瘪、没有花粉;自花授粉或以其为父本进行的异花授粉均不能结实,表现为完全的雄性不育。花药的横向解剖结构表明：转基因油菜雄性不育与绒毡层细胞的破坏有关     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

染色体重组对转基因大麦中gfp基因表达的作用

利用5种转绿色荧光蛋白基因(gfp)大麦不同株系及野生型大麦为材料,对不同转基因株系间以及转基因株系与野生型植株间进行杂交,分别对不同世代植株的根尖、花粉中gfp基因的表达量进行测定.结果表明,不同转基因株系间的根尖、花粉的gfp基因在表达量上存在差异,同一转基因材料的gfp基因表达存在组织差异;gfp基因在杂交后代中作为一个显性基因以孟德尔方式稳定遗传,不同染色体上的gfp基因重组有利于提高持基因表达.     

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

GFP‐tagged pollen to monitor pollen flow of transgenic plants

In this study, the pollen‐active LAT59 promoter from tomato was used to express a green fluorescent protein (GFP) encoding gene in Nicotiana tabacum (tobacco) pollen. This promoter is preferentially expressed in anthers and pollen. Pollen in transgenic plants segregated in a 1 : 1 Mendelian ratio, and the plants were polymerase chain reaction (PCR)‐positive. GFP‐tagged pollen was developed as a tool for tracking the movement of transgenic plant pollen in the environment. Specifically, it should be a useful tool for characterizing the spatial distribution patterns of transgenic pollen, to determine pollination mechanisms, to monitor the effects on nontarget organisms, and to monitor gene flow in field conditions.     

Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation

Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. ‘Hillary’ was 55.3% whereas that of cv. ‘Tenorio’ reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l⁻¹ allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.