Metabolie Engineering of Flower Color in Ornamental Plants

Abstract

Advances in the knowledge of plant pigment pathways at genetic, biochemical and molecular levels, and the establishment of genetic transformation methods for an increasing number of plant species have paved the way to genetic engineering of flower and plant color for ornamental purposes. From trial-and-error approaches based on the available few genes, the trend is now to comprehensively study the target species, genotypes and pigment pathways to precisely address specific breeding goals. This review examines the state of the art in this field, from the pioneering stages to fundamental and applied research on flavonoids, which is being extended to carotenoids and is also likely to involve other pigments in the near future.     

Problems and Challenges of Invasive Ornamental Plants and Molecular Tools to Control Their Spread

Abstract

Invasive plants, one of the most devastating ecological problems in the 21st century, cause an estimated $35 billion loss per year to the economy in the United States alone. More than 50% of all invasive plant species and 85% of invasive woody plant species were introduced originally for ornamental and landscape use. Because many non-native ornamentals are commercially important and widely utilized for various purposes, completely banning their use and prohibiting their import are unpractical solutions. On the other hand, currently used methods to control the spread of non-native plants are ineffective, expensive, or environmentally problematic. Recent advances in plant molecular biology and plant genetic transformation may enable us to create sterile cultivars of these non-native ornamental crops of high commercial value. The use of sterile cultivars should reduce or eliminate the undesirable spread of some non-native invasive plants into natural areas.     

Allelic Analysis for bar Gene from Different Rice Transformation Events

Abstract

Mechanism(s) of gene transformation and integration in rice (Oryza sauva L.) is/are not currently well understood. This research was conducted to determine whether a transgene is inserted into the rice genome specifically or randomly. Seven homozygous transgenic Taipei (T) 309 and Nipponbare plants with the bar transgene from different rice transformation events were crossed. The segregation of F2 and F3 populations from a total of 21 crosses was studied in a greenhouse and field to determine if the genes were allelic or non-allelic. Five genomic locations appeared to be involved among the seven transgenic plants. An additional 20 homozygous transgenic T309 plants, with the bar transgene from different transformation events, were crossed reciprocally with the previous seven plants. One hundred and fifteen crosses made during 1999 and 2000 were analyzed for allelism. In some combinations, the genes were allelic, but most of them were non-allelic, with two or more pairs of genes being expressed. Twenty loci among the 27 transgenic plants were involved and some plants had several inserted genes expressed. Genes in nine out of 27 transformed plants were allelic. We concluded that the functional foreign (bar) gene was restrictively/preferentially inserted into the rice genome in some cases and was not completely randomly inserted and expressed in the rice genome. If the mechanism(s) for preferential insertion were identified, rice researchers could possibly control insertion sites of transgenes to optimize gene expression.     

转基因技术在大豆育种中的应用

获得转基因植株是植物基因工程的基础,但是由于转化效率较低,在转基因的过程中通常需要一个有效筛选细胞和组织的手段,于是选择标记基因被广泛应用.然而,选择标记基因的使用,存在生物安全性和环境安全性隐患.因此,建立一个安全高效稳定的大豆转化体系,是改善大豆产量、品质、抗逆能力,培育新型大豆品种的保障.该文综述了新型标记基因在...     

Auxin,Cytokinin and Ab sei sic Acid

Abstract

Auxin, cytokinin, and abscisic acid (ABA) are important plant hormones that regulate many growth and developmental processes. In recent years, a number of genes involved in the metabolic and signaling pathways for auxin, cytokinin and ABA have been cloned and characterized. With organ-and tissue-specific or conditionally active gene promoters, it has become possible to manipulate concentrations of plant hormones in planta to create commercially desirable traits. Seedless fruit production and the extension of shelf life of green produce and ornamental plants are two successful examples of manipulating concentrations of these hormones in planta. In this review, we will focus our discussion on the effects of the over-or under-expression of genes involved in the biosynthetic and catabolic pathways of these hormones with an emphasis on their potential applications in ornamental crops.     

Transgenic Approaches to Disease Resistance in Ornamental Crops

Abstract

Viral, bacterial, and fungal diseases of ornamental plants cause major losses in productivity and quality. Chemical methods are available for control of fungal diseases, and to a lesser extent for bacterial diseases, but there are no economically effective chemical controls for viral diseases except to control vector species. Host plant resistance is an effective means of controlling plant diseases, and minimizing the necessity for the application of pesticides; however, there are many ornamentals in which no natural disease resistance is available. It is possible to introduce resistance derived from other species, or even from the pathogen itself, by genetic engineering. This allows the introduction of specific, or in some instances broad spectrum, disease resistance into plant genotypes that have been selected for desirable horticultural characters; in contrast, introduction of natural resistance by traditional breeding may take many cycles of breeding to combine disease resistance with desirable ornamental quality. This article briefly reviews existing work on transformation systems for ornamentals, and discusses the various approaches to introducing resistance to viral, bacterial, and fungal diseases, and to nematode infestations. These include pathogen-related proteins, R genes, and general pathogen resistance; anti-microbial peptides; expression of anti-pathogen antibodies; viral sequences; ribozymes; antiviral peptides; ribonucleases; and ribosome-inactivating proteins. Examples are given of application of these approaches to disease resistance in other types of crop and model plant systems, and actual or potential application to disease resistance in ornamentals. Future prospects for obtaining plants with multiple pest and disease resistances are discussed.     

Improvement of Horticultural and Ornamental Crops Through Transgenic Manipulation of the Phytochrome Family of Plant Photoreceptors

Abstract

Consumer demands drive continuous developments in the production of horticultural and ornamental crops. In addition to improvements in quality and nutritional content, crop producers must supply an increased variety of products to a year-round market. The availability of many horticultural and ornamental crop products is dependent on the timing of reproductive development. The time at which many plants initiate sexual or vegetative reproduction is governed by a number of interacting environmental factors such as daylength, light quality and temperature. Artificial manipulation of the growing environment is therefore frequently used to ensure production meets retailers' marketing programs, a strategy which can often result in high energy costs. An alternative approach involves the manipulation of genes encoding proteins responsible for perceiving and transducing environmental stimuli, in particular, the genes encoding the phytochrome family of plant photoreceptors. Alterations in the expression of genes encoding phytochromes can modulate not only the timing of reproductive development, but also plant architecture. Such approaches can therefore be used to modulate a variety of phenotypic traits such as height, lateral branching and harvest yield, while enabling growers to tailor crop reproduction to their marketing needs. In this review, we will discuss examples of crop improvement using transgenic manipulation of phytochrome expression, along with benefits and disadvantages of such approaches.     

转酸性蛋白酶pepB基因玉米的分子鉴定与农艺性状分析

通过分子生物学技术把酸性蛋白酶pepB基因转入受体玉米自交系基因组中,培育转酸性蛋白酶pepB基因玉米新品系。以耐盐基因badh作为筛选标记性基因,经过抗性筛选,对T1、T2、T3代转基因植株进行PCR检测,得到14个T1代转基因阳性植株,32个T2代转基因阳性植株和27个T3代转基因阳性植株。Southern杂交结果表明,外源酸性蛋白酶基因已经整合进玉米基因组中。RT-PCR结果表明,酸性蛋白酶基因在受体玉米中获得表达,获得外源酸性蛋白酶pepB基因遗传表达的T3代转基因玉米株系。通过对T2、T3代转基因玉米各品系农艺性状的分析结果表明,转基因玉米各品系在株高、茎粗、穗长等农艺性状上与受体亲本没有差异,但生育期均有不同程度的缩短。     

A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used “minimal gene cassettes” and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA from regenerated plants identified 15 T₀ plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T₁ to T₃ generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T₃ generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1Dy10 transgene were identified in the T₄ generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.     

Ethylene

Abstract

Ethylene is a simple gaseous hormone that is involved in many aspects of plant growth and development. Two physiological responses that are commonly controlled by ethylene, fruit ripening and flower senescence, have been given the most significant attention in terms of crop improvement using the tools of modern plant molecular biology and biotechnology. Fortunately, the wealth of research on ethylene biosynthesis and signaling pathway has been providing tools for the genetic control of ethylene effects. Today, manipulation of ethylene biosynthesis and signaling is one of the most promising approaches to improve crops through genetic engineering. In this review we will discuss current status and future directions of transgenic techniques to control ethylene effects with a primary focus on flower senescence.     

酢浆草属植物观赏性评价体系的建立与应用

通过运用层析分析法筛选出株型、株高、花色等14个酢浆草属（Oxalis）植物观赏性状评价指标,对随机选择的40个球根酢浆草属植物进行观赏性评价。结果表明：应用层次分析法,可以有效地从观赏性方面对酢浆草属植物进行评价。14个指标中,株型、花色、冠幅、花量所占权重值均较大（>0.09）,是影响酢浆草属植物观赏价值最重要的指标。在评分标准中,心形叶、白花、肉质叶、大花径分值较高,均为5分。为酢浆草属植物观赏盆栽品种（种）的选择、DUS测试指南性状指标的筛选、育种亲本选择提供参考依据。40个参评植物中观赏价值较高的有钝叶酢‘锂辉石’（O. obtuse ‘Kunzite’）、芙蓉酢‘晨光’（O. purpurea ‘Alba’）和迷糊酢（O. ambigua）等6种,其得分均>3.499,这几种植物观赏性高、适应性强,是极具开发和应用价值的优良植物材料。     

Phosphomannose isomerase,pmi, as a selectable marker gene for durum wheat transformation

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional methods. The development of transgenic plants requires the use of selectable marker genes, as the efficiency of plant transformation is less than optimal for many important species, especially for monocots such as durum wheat (Triticum turgidum var. durum). Many concerns have been expressed about the persistence of currently used marker genes in plants used for field cultivation. To sustain further progress in this area, alternative efficient selection methods are desirable. A recent development is the use of selective genes that give transformed cells a metabolic advantage (positive selection) compared to untransformed cells, which are slowly starved with a concomitant reduction in growth and viability. This selection strategy is in contrast to traditional negative selections during which the transgenic cells are able to survive on a selective medium whereas the non-transgenic cells are actively killed by the selective agent. We compared the ‘selection efficiency’ of a commonly used negative selection method that employs the Streptomyces hygroscopicus bar gene to confer resistance to the herbicide bialaphos, to a positive selection employing the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable gene and mannose as the selective agent. Calli derived from immature embryos of the durum wheat cultivar Svevo were bombarded separately with bar and pmi genes using a biolistic system. The integration and expression of the two genes in the T₀ generation were confirmed by PCR analysis with specific primers for each gene and the chlorophenol red assay, respectively. The selection efficiency, calculated as the number of expressing plants divided by the number of total regenerants, was higher when pmi was used as the selectable marker gene (90.1%) than when bar was used (26.4%). Thus, an efficient selection method for durum wheat transformation was established that obviates the use of herbicide résistance genes.     

利用RNAi方法构建MS26基因雄性不育植物表达载体及农杆菌介导玉米遗传转化研究

应用RNAi技术创制了花粉彻底败育的玉米雄性不育株系,为玉米杂交制种提供雄性不育的基础材料。构建玉米MS26 RNAi植物表达载体,其中,以玉米综31未成熟的幼胚组织为受体,利用农杆菌介导法将目的基因定向转入到玉米中,以甘露糖为选择剂进行筛选获得能够稳定遗传的转基因植株。通过Taqman探针法进行分子检测,获得18株单拷贝T0代转基因植株,碘-碘化钾染色分析结果显示,12株完全不育。在田间试验中,5个转化事件的所有T1代转基因植株在散粉期都表现雄性不育,且除此之外与野生型玉米对照植株之间没有其他形态不同。实时定量PCR结果显示,MS26 RNAi转基因玉米植株中MS26基因的表达量显著下调,由此可推断,MS26 RNAiT-DNA已经整合到玉米基因组中,并能引起完全的雄性不育。     

TaNAC14转基因小麦外源目标基因拷贝数及遗传稳定性分析

外源基因拷贝数是影响转基因植物遗传稳定性及其自身表达水平的重要因素。为了探析TaNAC14基因在小麦生长发育中的生物学功能,本研究通过农杆菌介导的遗传转化法获得了14个T0代TaNAC14转基因小麦植株,采用BASTA溶液涂抹和目标序列PCR检测相结合的方法,从14个转基因小麦植株中鉴定出9个阳性植株。通过TaqMan实时定量PCR方法,以小麦内源单拷贝基因Pinb为内参基因,对9个T0代转基因小麦阳性植株中外源目标基因拷贝数进行检测,结果表明,T0代转基因小麦再生植株中有5株为单拷贝,4株为双拷贝,其中单拷贝插入整合的比率接近55.6%。选取单拷贝转基因植株收获的种子进行种植,根据BASTA溶液涂抹鉴定对T0:1代小麦株系遗传情况进行分析,结果表明目标基因TaNAC14是可遗传的。此外,还对T1代转基因小麦目标基因表达水平进行测定,结果表明,与野生型JW1相比,各转基因小麦植株目的基因TaNAC14表达量均极显著增加。该研究结果为后续TaNAC14基因的功能研究奠定了基础。     

Inheritance of Cold Hardiness and Dehydrin Genes in Diploid Mapping Populations of Blueberry

Summary

Studies of herbaceous plants suggest that cold hardiness is a complex, quantitatively inherited trait. Although development of cold hardiness is an integral part of the life cycle of woody perennial plants, studies on the genetic control of cold hardiness in woody perennials are scarce. A better understanding of the genetic control of cold hardiness would be valuable for developing more effective strategies to increase cold hardiness and, hence, climatic adaptation of woody perennial crops. In blueberry, three major dehydrins of 65, 60, and 14 kDa have been found to increase with cold acclimation and decrease with deac-climation. A comparison of these dehydrin levels among various blueberry cultivars and selections has revealed their level of accumulation to be closely associated with cold hardiness level. Efforts are underway to isolate and map the dehydrin genes of blueberry utilizing blueberry populations that segregate for cold hardiness in order to determine if the dehydrin genes map to or co-segregate with QTLs controlling cold hardiness. Progress has been made toward this goal. Cold hardiness levels were determined for a portion of the blueberry mapping populations (derived from testcrosses of Vaccinium darrowi Camp X V. caesariense Mackenz. F₁s to another V. darrowi and another V. caesariense) using a laboratory controlled freeze-thaw regime, followed by visual assessment of injury to floral buds. As expected, the V. darrowi and V. caesariense parents were found to differ significantly in terms of cold hardiness levels (LT₅₀s of -13°C and -20°C, respectively). Mean cold hardiness level of F₁s (LT₅₀ of -14.7°C) was skewed toward the V. darrowi parents suggesting that cold hardiness is a partially recessive trait. The sequence of a 2.0 kb cDNA clone, which encodes the 60 kDa blueberry dehydrin, was used to map a dehydrin-related gene to current linkage group 12 of the V. caesariense testcross population. A preliminary comparison of the segregation pattern of the dehydrin-related gene to that of the cold hardiness trait suggests that the marker does not segregate with cold hardiness.     

Transgene Transfer to United States Commercial Rice Cultivars via Conventional Breeding Techniques

Abstract

Breeding programs can benefit from transfer of a foreign gene from one transgenic plant to commercial cultivars through continuous backcrossing, especially to cultivars in which it is difficult to transfer a foreign gene directly through biotechnology. In this study, two homogeneous transgenic plants, T-28 and T-64 (from Taipei 309), and one homogeneous transgenic plant, N-84 (from Nipponbare), were used as donors of Bar gene. Commercial cultivars, Cypress and Laffite, were used as the recurrent parents. Three to five backcrosses were made using the transgenic plants as donor parents and the commercial cultivars as recurrent parent. The results from selected progeny rows, and two-years of yield tests with selected lines, indicated that the target Bar gene could be transferred to lines from homozygous transformants in 2–3 years of backcrossing, giving lines similar to the recurrent parents in phenotype and yield potential.     

耐除草剂转基因玉米CM8401的获得及功能性状鉴定

耐草甘膦和耐草铵膦是转基因作物育种重要的目标性状。将耐草甘膦基因MC1-EPSPS构建到含有bar基因的植物表达载体pTF101.1中，通过农杆菌介导法转入玉米材料Hi-II中，从而获得兼具耐受草甘膦和草铵膦性状的转基因玉米材料 CM8401。目的基因PCR检测显示，MC1-EPSPS和bar基因稳定整合到玉米基因组中。目的蛋白试纸条检测结果显示，MC1-EPSPS蛋白和PAT蛋白在转基因玉米世代间中表达稳定。田间除草剂耐受性鉴定试验表明，转基因玉米CM8401对草甘膦和草铵膦都具有良好耐受性，可耐受4倍推荐中剂量的草甘膦和草铵膦。     

Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice

Background

The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation.

Findings

Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.

Conclusions

Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.