Transfer of tolerance to different Potato virus Y (PVY) isolates from Nicotiana africana Merxm. to Nicotiana tabacum L.

The wild species Nicotiana africana is a source of Potato virus Y (PVY) resistance. Tolerance to PVY was transferred from N. africana to Nicotiana tabacum by two backcrosses with variety 'BP-210'. Successive generations were obtained by selfing. Cytological investigations covered counts of mitotic chromosomes, study of meiotic configurations and pollen viability. The number of mitotic chromosomes varied according to generations. In pollen mother cells, the number of univalents decreased with advancing generations. Stable 48-chromosome lines were found in the BC₂F₅. There was an increase of the percentage of PVY tolerant plants with each of the advancing generations. Challenged with two PVY isolates, the breeding lines showed higher levels of tolerance than conventional N. tabacum cultivars. There was a conspicuous absence of necrotic responses in the N. africana -derived materials. These stable breeding lines carrying N. africana- derived tolerance factors to different strains and isolates of PVY are the source of a very valuable germplasm to be used for breeding.     

Ecological aspects of transgenic sugar beet: transfer and expression of herbicide resistance in hybrids with wild beets

Summary An increasing number of genetically engineered cultivars of several crops is being experimentally released into the environment. In future, crops with new transgenic traits will probably play an important role in agricultural practice. The long-term effect of transgenes on community ecology will depend on the distribution and establishment of transgenic plants in the wild, on the sexual transfer of their new genes to the environment and on the potential ecological impact of the transgenic trait. The starting point was the use of transgenic sugar beet lines, Beta vulgaris subspec. vulgaris var. altissima DÖLL (Helm 1957), with transgenes coding for rhizomania and herbicide (BASTA^®) resistance. The first two questions to answer were: Can the transgenes be transferred via pollen to wild beets, Beta vulgaris subspec. maritima (L.) ARCANG. or cultivated relatives such as red beet or spinach beet and are they expressed in the hybrids? Can transgenes be monitored in young Beta vulgaris-hybrids? The experimental transfer of transgenes was conducted in 1993 at a field location in northern Germany. The beets were hand-pollinated with transgenic pollen. In a non destructive biotest, the hybrid seedlings were tested for herbicide resistance. Transgenic plants showed no noxious phenotypic effects whereas control plants developed leaf necroses. All herbicide resistant hybrids within the biotest were assumed to be transgenic.     

Overwintering of genetically modified sugar beet, Beta vulgaris L. subsp. vulgaris, as a source for dispersal of transgenic pollen

The potential impact of transgenic crops on community ecology will depend on the distribution and establishment of the new transgenic traits, on the sexual transfer of their new genes to the environment (Bartsch &; Pohl-Orf, 1996) and on the potential ecological impact of the transgenic trait. Flowering and pollen dispersal is important for outcrossing of the genetically engineered trait. For a biennial plant, like the cultivars of Beta vulgaris L., overwintering is normally necessary to become generative and to produce pollen and seeds (Abe et al., 1997), which usually does not happen with sugar beet as a field crop harvested in autumn (Longden 1989). The starting point for the project was a transgenic sugar beet, Beta vulgaris L. subsp. vulgaris (Lange et al., 1998), with rhizomania and herbicide ( Basta®, Liberty®) resistance. Cold tolerance is one of the most important factors for survival of sugar beet in Central- and North-Europe. Among other ways, spreading of transgenic traits into weed beet (Boudry et al., 1993) or wild beet can occur if genetically engineered – biennial – plants survive the winter, flower in spring and spread their pollen. Field experiments were performed with transgenic breeding lines and their hybrids, transgenic and non-transgenic hybrids with Swiss chard and three conventional beet cultivars to evaluate winter survival rates at seven different field sites. We could show that survival of sugar beet – transgenic as well as conventional ones – in Germany and at the Dutch border is possible. Survival rates were well correlated with temperature data and were unexpectedly high. Differences between sugar beet hybrids and breeding lines could be detected but not within different breeding lines or hybrids. There were no differences detectable between transgenic and non-transgenic plants. The data are crucial for the risk assessment of the release of transgenic sugar beet and are the basis for further experiments towards outcrossing and establishment.     

Characteristics of Pollen from Transgenic Lines of Apple Carrying the Exogenous CpTI Gene

It is fundamental for gene transformation and ecosystem hazard evaluation to study the pollen characteristics of transgenic plants. In this research,the characteristics of pollen from 7-or 8-year-old transgenic apple plants carrying an exogenous Cp TI gene were analyzed. The results showed that there was no significant difference in terms of size,morphology,or exine ornamentation between the pollen of the transgenic plants and the non-transgenic control. However,the transgenic plants had more abnormal pollen grains. Of the 13 transgenic lines tested,12 had a significantly lower amount of pollen and six exhibited a significantly lower germination rate when cultured in vitro. The pollen viability of three transgenic lines was determined,with two showing significantly lower viability than the control. The transgenic Gala apple pollen grains germinated normally via controlled pollination on Fuji apple stigmas. However,the pollen tubes extended relatively slowly during the middle and late development stages,and another 8 h were needed to reach the ovules compared with the control. The gibberellic acid concentration in transgenic Gala apple flowers was lower than in the non-transgenic control during all development stages tested. The abscisic acid concentration in the transgenic flowers was lower during the pink stage,and higher during the ball and fully open stages. Microscopic observation of the anther structure showed no difference. The tapetum of the pollen sac wall in transgenic plants decomposed late and affected pollen grain development,which could be one of the reasons for the lower number of pollen grains and poor viability in the transgenic plants.     

转双价抗虫基因BmkIT-Chitinase玉米株系的获得

通过超声波辅助花粉介导法,将双价抗虫基因BmkIT-Chitinase分别导入以玉米自交系昌7-2及郑58的花粉为受体的不同基因型的自交系中。本研究共处理玉米雌穗1072穗,获得T0代种子1563粒,经卡那霉素初筛,T1代～T4代PCR及SouthernBlot杂交分子跟踪检测共获得20个转化株系,田间抗虫性鉴定表明共有16个转化株系与对照在抗虫性方面有显著差异,且此抗性随着各代稳定遗传。农艺性状调查结果表明,所获得的转基因玉米株系中大部分材料的农艺性状与对照无显著差异,除了N55材料及N20-1材料。N55材料的穗位高度与对照相比略低6±0.5cm,而穗粒数增加75±5粒。而N20-1材料百粒重增加5±0.5g。因此,转入此双价抗虫基因对玉米农艺性状影响不是很大。经过分子检测、田间抗虫性鉴定及农艺性状调查我们最终选育了9个转双价抗虫基因昌7-2自交系优良株系,6个郑58转双价抗虫基因自交系优良株系。     

Heat stress affects vegetative and reproductive performance and trait correlations in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

High ambient temperature has adverse effects on plant vegetative and reproductive development and reduces crop yield. To better understand the importance of male and female fertility for tomato fruit set ability under high temperature conditions and to test whether heat tolerance levels among and between reproductive and vegetative traits of genotypes correlate with each other, 13 tomato cultivars were subjected to long-term moderate heat (LTMH) or short-term heat shock (STHS), depending on the trait that was evaluated. LTMH caused significant decrease in performance of nearly all reproductive traits, i.e. pollen viability, pollen number, female fertility, seeded-fruit set and flower number per inflorescence, but not in inflorescence number. Considerable variation was found among cultivars, both under control and LTMH conditions. The cultivars Nagcarlang, Saladette and Malintka 101 produced a higher percentage of viable pollen under LTMH. For fruit set under LTMH condition, only cultivars that had been previously reported as being heat-tolerant produced fruits with seeds. STHS negatively affected vegetative traits concerning seedling survival and membrane stability. Correlation analysis revealed relationships between various traits within the control and heat treatments, but not between the two. Under heat stress fruit set was positively correlated with pollen viability, as well as with flower number per inflorescence. However, no significant correlations were found between vegetative and reproductive traits. Our data highlight the prominent role of pollen viability for tomato fertility under LTMH growth conditions. The observed variation in thermotolerance among different cultivars offers the possibility to decipher underlying physiological and genetic mechanisms.     

Comparative Study of CaMsrB2 Gene Containing Drought‐Tolerant Transgenic Rice (Oryza sativa L.) and Non‐Transgenic Counterpart

Concerns regarding the safety of transgenic foods have been raised because of possibility of undesirable effects development during genetic engineering. Analysis of phenotypic traits can increase the likelihoods of identifying those unintended effects in dietary composition of the GM crops. Objective of this study was to compare the transgenic lines with their non‐transgenic counterpart. Different vegetative and reproductive traits as well as antioxidant properties were considered to evaluate the transgenic (HV8 and HV23) lines containing CaMsrB2 gene and their non‐transgenic (Ilmi) parent line. Grain size and weight, seed germination, root length, root and shoot dry weight, length and width of flag leaf, plant height, and ligule, stamen and carpel length were not significantly different. Onset and completion of heading in each line occurred almost during the same period. The antioxidant properties in terms of DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity and polyphenol content were not statistically different under same treatment condition. The results suggested that the transgenic rice lines containing CaMsrB2 gene were equivalent to their non‐transgenic counterpart without any visible unintended effects.     

New inheritance studies related to Potato Virus Y (PVY) resistance in Capsicum annuum L. ‘Serrano Criollo de Morelos-334’

Summary C. annuum L. ‘Serrano Criollo de Morelos-334’ (SCM-334) is a well-known source of resistance against pepper diseases, including Potato Virus Y (PVY). In this work, inheritance studies involving ‘SCM-334’ and related to PVY resistance were described. For this purpose, ‘SCM-334’ derived lines were used, and plant material from the cross between ‘SCM-334’ and ‘Yolo Wonder’ (YW) was employed as well. Five PVY isolates were used for the inoculations. The first resistance described is a monogenic and recessive gene, controlling a specific-isolate resistance, and provisionally named as pvr1 ⁴. The second one is related to the inheritance of systemic necrotic symptoms after PVY inoculation. In this case a codominant gene is proposed and expressed only when the Pvr4 gene is not present.     

棉花转基因材料的获得及主要农艺性状变异分析

 利用不同棉花受体材料进行花粉管通道法遗传转化研究。通过卡那霉素鉴定、抗虫性鉴定和PCR分子生物学鉴定,证明已成功将Bt基因转化到泗棉3号、辽棉15、中棉所19、中棉所29母本、中棉所35、中棉所36等棉花材料中。通过对转基因材料后代与非转基因材料的铃重、衣分和纤维品质等差异分析,发现花粉管通道转基因后代材料存在广泛的变异。     

The effects of high-temperature stress on the germination of pollen grains of upland cotton during square development

The reproductive stage of flowering plants is sensitive to high-temperature stresses. High temperature is a major factor influencing pollen grain viability in upland cotton (Gossypium hirsutum). The objective of this study was to identify the relationship between cotton pollen germination percentage and temperature by assaying the pollen germination of four upland cotton cultivars in vitro at different temperatures during the blooming period. The results showed that in vitro pollen germination percentage was related to the culture temperature of pollen germination and the temperature of the square development process. High temperature affected pollen development and germination, and high-temperature tolerance differed among the cotton cultivars. The pollen germination percentage decreased rapidly with changes in the culture temperature from 30 to 39 °C. A culture temperature of 35 °C might be a critical temperature for the pollen viability transition and could be used to screen cotton cultivars that have pollen grains with high-temperature resistance. Before the high-temperature stage, cultivars with rates of decrease in the percentage of pollen germination of less than 41 % at 35 °C relative to the rates at 30 °C might be considered as high-temperature tolerance cultivars, and cultivars with rates of decrease in the percentage of pollen germination greater than 41 % might be considered as susceptible cultivars. The high-temperature stress for pollen grain germination in vitro was greater than 30 °C, and the high-temperature stress for square development might be greater than 33 °C. Boll retention was significant; it was positively correlated with the pollen germination percentage and negatively correlated with temperature during the high-temperature stage. This study provided a method for rapidly screening cultivars (lines) with high-temperature tolerance pollen in upland cotton breeding.     

Early selection for extreme resistance to potato virus Y and tobacco etch virus in potato using a β-glucuronidase-tagged virus

The Ry_sto gene from Solanum stoloniferum introduced into potato cultivars (Solanum tuberosum L. ssp. tuberosum) confers resistance to potato virus A, potato virus V and potato virus Y (PVY). In addition to PVY, tobacco etch virus (TEV) and a TEV construct that encodes β‐glucuronidase (TEV‐GUS) were inoculated to determine the inheritance of resistance to these viruses in progenies obtained from potato cultivars containing the Ry_sto gene. While cultivars ‘Karlena’ and ‘Delikat’ were susceptible, ‘Bettina’ and clone 927eY were resistant to PVY, TEV and TEV‐GUS, as determined by enzyme linked immunosorbent assay, biotest and GUS assay, respectively. The segregation ratios obtained from the progenies of ‘Bettina’בDelikat’ and 816eY בKarlena’ indicate that resistances to PVY and TEV are governed by one dominant gene or two genes tightly linked in coupling phase. Evidently, Ry_sto confers broad spectrum resistance to potyviruses. TEV resistance could be reliably detected 4 days after inoculation with the TEV‐GUS construct by GUS assay. Therefore, the GUS‐tagged TEV construct can be used for early selection for resistances based on the gene Ry_sto or closely linked genes.     

Cajanus platycarpus (Benth.) Maesen as the donor of new pigeonpea cytoplasmic male sterile (CMS) system

Cajanus platycarpus, a distantly related wild species, was successfully crossed with cultivated pigeonpea using embryo rescue and tissue culture techniques. Advance generation lines showed a range of desirable characters including cytoplasmic male sterility. A range of pigeonpea cultivars restored fertility and was maintained by a few lines including cultivar ICPL 85010. Clasmogamous flowers were observed in the male sterile lines. In such flowers anthers did not form di-adlephous bundle. Cytological analysis revealed that meiosis proceeded normally till the tetrad stage in those anthers with pollen grains. After which many of the pollen grains turned sterile. In the anthers with pollen grains, dehiscence was not observed, thus creating functional sterility. In many other anthers, pollen mother cells (PMCs) were not formed at all, giving rise to sepalous anthers. In conclusion two mechanisms of male sterility existed, one was premeiotic, where PMCs did not form and in the second, although PMCs gave rise to pollen grains, they were either partially or totally sterile accompanied by non-dehiscence of anther wall.     

花粉特异性核糖核酸酶基因诱导籼稻雄性不育及其遗传研究

以水稻愈伤组织和悬浮细胞系作为基因枪转化的外植体,把水稻花粉特异性基因PS1启动子与barnase构成的嵌合基因导入籼稻,获得籼稻五个品种Basmati 1、青油占、胜优2号,明恢63,新山占29的转基因植株.试验以两个质粒PS1- barnase和pILTAB227共转化的方法,以潮霉素B作为筛选因子,选择抗性愈伤及再生植株.获得的barnase转基因植株的育性比未转化的对照明显降低,表现为部分不育和完全不育.除barnase阳性转化株平均株高比对照有所降低外,营养器官发育正常,雌性可育.完全不育的植株花粉粒畸形,不能被I-KI溶液染色,但它们与正常植株杂交能够获得杂交种子.转基因植株的Sonthern杂交分析表明,bamase基因普遍为多拷贝整合.对转基因植株白交及与非转化株测交后代以点杂交分析bamase转基因的遗传行为,发现转基因的遗传符合1～3个插入位点的孟德尔遗传模式.     

Genetics of the viability of pollen grain produced at low temperatures inLycopersicon Mill.

Summary The different viabilities of pollen produced at low temperatures in intra- and inter-specific crosses of tomato were studied. Cultivars Red Top, Moneymaker, and Marroqui were crossed with cultivar E-15 and these four cultivars were hybridized with lines of the wild speciesL. pimpinellifolium PE-13,L. parviflorum PE-52,L. pennellii PE-47, andL. hirsutum PE-37 and PE-41. A six-generation family of the Moneymaker x PE-47 cross was obtained to carry out a more detailed genetical study of pollen grain viability at low temperatures. Pollen grain viability was evaluated during the winter via acetocarmine staining. When the parents were compared with their F₁, the intra-specific tomato crosses showed dominance to better-quality pollen, theL. esculentum x L. pimpinellifolium inter-specific crosses showed positive heterosis, while the crosses ofL. esculentum with the tolerant speciesL. pennellii andL. hirsutum showed intermediate inheritance. However, in theL. esculentum x L. pennellii family, the dominance and the non-allelic interactions (homozygosis x homozygosis) were the most important factors, so that dominance to better viability at low temperatures appeared to be the general mode of inheritance. Genetical control of pollen grain viability at low temperatures seemed to be polygenetic.     

Over-expression of Gastrodia anti-fungal protein enhances Verticillium wilt resistance in coloured cotton

Currently there are no adequate control measures for the cotton fungal diseases Verticillium wilt and Fusarium wilt, which are important factors limiting yield under certain conditions. In this study the gene encoding a Gastrodia anti‐fungal protein was introduced into three cultivars of coloured cotton using the method of pollen‐tube pathway transformation, with the purpose of obtaining transgenic plants with improved resistance to wilt. Of the 121 herbicide‐resistant cotton plants two, LB‐5‐8 and ZB‐1‐49, were scored as transgenic based on Southern blot, RT‐PCR analysis and in vitro anti‐fungal activity assay. Field analysis demonstrated that the transgenic lines LB‐5‐8 and ZB‐1‐49 possess an increased resistance to wilt. After 2 years of breeding, the progeny of LB‐5‐8 and ZB‐1‐49 lines still showed a stable and strong resistance to Verticillium wilt. Lines with high levels of resistance to Verticillium wilt obtained from the present study may be widely planted and help to reduce the future impact of cotton wilt on cotton production resulting in increased yields.     

Identification and gene cloning of a male‐sterile mutant Oswbc11 in rice

Commercial application of nuclear genetic male sterility has been improved recently by a novel technique, Seed Production Technology (SPT), which incorporates transgenic maintainer lines capable of propagating non‐transgenic nuclear male‐sterile lines for use as female parents in hybrid production. Here, we identified a rice nuclear male‐sterile mutant, Oswbc11, with abnormal pollen development and lipid transport. We finely mapped the Oswbc11 gene into a 12.5‐kb region on chromosome 10 and found one candidate gene, which had a base substitution (C to T) resulting in a premature stop codon and was functionally confirmed by CRISPR/Cas9 technology. Gene OsWBC11 encodes a sub‐family member of adenosine triphosphate‐binding cassette transporter, which participates in the active transport of a wide range of molecules across membranes. Moreover, the agronomic traits of Oswbc11 mutants showed no significant differences compared to the wild‐type control except for the seed setting rate. These results indicated that Oswbc11 gene could be used in rice hybrid breeding as a recessive nuclear male‐sterile gene combined with CRISPR/Cas9 and the SPT technology and applied in different rice varieties.     

大丽花花粉活力及离体萌发研究

为筛选大丽花花粉活力有效的测定方法,了解大丽花不同品种的花粉活力,以大丽花栽培品种为试验材料,采用4种方法检测大丽花花粉活力,在此基础上,采用单因素试验和正交设计试验分别对大丽花花粉的离体萌发培养条件及培养基组分进行优化,得到最优方案后进一步检测、比较不同品种之间的花粉活力。研究结果显示,TTC染色法不能使花粉着色,I₂-KI和孢粉染色法不能有效区分有活力和无活力花粉,离体萌发法效果良好,可准确直观地反映大丽花花粉活力状况, 是测定大丽花花粉活力的有效方法。当培养条件为pH 6.0、温度25℃、培养时间2.5 h时,花粉萌发率最高。培养基组分对大丽花花粉萌发的影响程度依次为PEG>蔗糖>硼酸,实际最佳处理组合为A₃B₄C₂,即PEG4000 25 g/L、蔗糖60 g/L、硼酸50 mg/L的处理组合下,大丽花花粉萌发率最高达62.1%。采用上述获得的最优方案检测22个大丽花品种的花粉活力,花粉萌发率为11.75%~78.72%,不同品种的花粉萌发率差异大,其中,‘兰花公主’的花粉萌发率最低,‘波彻儿’最高。大丽花品种的花粉活力多样性丰富,所测定的22个大丽花品种有14个品种正常可育,杂交时可用作父本;8个品种为半不育或低不育,杂交时更适合作母本。     

Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide

In this paper, we described the breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer (SSB) and herbicide by conventional crossing of two transgenic parental lines transformed independently with different genes. Two stable transgenic rice lines used as donor parents were developed, one was Zhongguo91 which contained cry1Ab gene (for insect resistance) and bar gene (for tolerance of herbicide), and the other was Yujing6 which contained Xa21 gene (resistance to bacterial blight). The elite restorer line Hui773 was used as recipient and crossed with the two stable transgenic rice lines. Then five successive backcrosses were made using Hui773 as recurrent parent. Two rice elite restorers, T773-1 expressing cry1Ab and bar genes and T773-2 expressing Xa21 gene, were obtained, which were confirmed by PCR analysis and testing selectable marker genes in the hybrid progenies. The cross was made between T773-1 and T773-2 to select stable restorer line carrying Xa21, cry1Ab and bar genes. Finally, we obtained transgenic restorer line T773 with good agronomic traits and obvious multiple resistance to Xanthomonas oryzae pv. oryzae, striped stem borer (Chilo suppressalis) and herbicide. The hybrid F₁ generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis. Our results indicate that it is feasible to develop transgenic hybrid rice cultivar through breeding transgenic restorer lines.     

马铃薯Y病毒siRNA介导抗病基因工程研究进展

马铃薯病毒病是危害马铃薯生产的重要因素,尤其马铃薯Y病毒(Potato virus Y,PVY)严重影响马铃薯产量和品质,为防治带来很大困难。近年来,通过基因工程技术培育抗病毒转基因植株为抵抗PVY侵染提供有效途径。简述了目前PVY病毒siRNA介导抗病基因工程的主要方法策略,包括PVY不同功能基因介导的病毒抗性差异、影响PVY抗病毒基因工程效率的主要因素以及PVY不同株系联合抗病基因工程研究概况,分析了这些方法存在问题及发展趋势,旨在为更好地控制PVY危害提供借鉴。