麻类作物组织培养及遗传转化研究进展

概述了国内外红麻等麻类作物快速繁殖、花药培养、原生质体培养、体细胞胚发生、器官发生等几个方面的组织培养以及遗传转化的研究进展,并对存在的问题进行了讨论,提出了进一步研究的一些见解,以期为促进麻类组织培养及遗传转化研究提供科学依据。     

SAAT: sonication-assisted Agrobacterium-mediated transformation

Plant transformation via Agrobacterium can be limited by both host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue. Described here is a new and efficient Agrobacterium-based transformation technology that overcomes these barriers and enhances DNA transfer in such diverse plant groups as dicots, monocots, and gymnosperms. This new technology, called sonication-assisted Agrobacterium-mediated transformation (SAAT), involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Scanning electron and light microscopy reveal that SAAT treatment produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easy access to internal plant tissues. Unlike other transformation methods, this system has the potential to transform meristematic tissue buried under several cell layers. SAAT increases transient transformation efficiency in several different plant tissues including leaf tissue, immature cotyledons, somatic and zygotic embryos, roots, stems, shoot apices, embryogenic suspension cells and whole seedlings. A 100- to 1400-fold increase in transient - glucuronid ase expression has been demonstrated in various tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize. Stable transformation of both soybean and Ohio buckeye has been obtained using SAAT of embryogenic suspension culture tissues. For soybean, SAAT treatment was necessary to obtain stable transformation with this tissue     

香蕉转基因研究进展

香蕉转基因研究逐渐成为国内外植物分子生物学研究的热点。人们从 2 0世纪 60年代开始就进行了香蕉的组织培养 ,70年代茎尖培养成功 ,近 1 0年来相继在微繁殖、单克隆抗体、分子生物学和遗传转化等方面开展了工作。香蕉转化受体系统已从茎尖发展到胚状体和愈伤组织受体系统 ,遗传转化方法也在不断改进 ,从单一的方法发展为根癌农杆菌感染法与基因枪法的结合。从香蕉组织培养、遗传转化受体系统、基因转化方法、筛选方法的研究等方面进行综述 ,并提出了一些看法。     

Ornamental Chrysanthemums: Improvement by Biotechnology

The in vitro tissue culture and micropropagation of chrysanthemums, important floricultural (cut-flower) and ornamental (pot and garden) plants, have been well studied. An increase in genetic transformation studies aimed at improving aesthetic and growth characteristics of the plants has been hampered by low transformation efficiencies and genotype dependence of protocols. As a result chrysanthemum regeneration studies have once again emerged as an essential complement of transformation studies. This review highlights the impact that biotechnology has had on the improvement of chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.     

观赏兰科植物组培快繁及遗传转化的研究进展

兰花作为一种高档花卉,近年来其组培快繁和基因工程研究取得了比较大的进展。综述的观赏兰科植物组织培养内容包括外植体、培养基及培养方式等,遗传转化内容包括靶材料、选择标记基因、报告基因、启动子和转化方法等并总结了兰科植物基因工程研究的成果、最新进展及存在的问题 。     

Hypoxia,melanocytes and melanoma – survival and tumor development in the permissive microenvironment of the skin

The tissue microenvironment plays a critical role in cell survival and growth and can contribute to cell transformation and tumor development. Cellular interactions with the stroma and with other cells provide key signals that control cellular arrest or division, survival or death, and entrance or exit from a quiescent state. Together, these decisions are essential for maintenance of tissue homeostasis. Tissue oxygenation is an important component of the microenvironment that can acutely alter the behavior of a cell through the direct regulation of genes involved in cell survival, apoptosis, glucose metabolism, and angiogenesis. Loss of tissue homeostasis due to, for example, oncogene activation leads to the disruption of these signals and eventually can lead to cell transformation and tumor development. Here we review the role of tissue oxygenation, and in particular physiologic skin hypoxia, on cell survival and senescence and how it contributes to melanocyte transformation and melanoma development.     

An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants

A protocol for Agrobacterium-mediated transformation with mannose selection was developed for cotyledon petiole, hypocotyl and leaf explants of tomato (Lycopersicon esculentum L. Mill). More than 400 transgenic plants from three tomato varieties were selected with 1% mannose in combination with 0.1–0.5% glucose. Average transformation frequencies ranged from 2.0 to 15.5% depending on the construct, genotype and type of tissue used for transformation. The highest transformation rate was obtained for hypocotyl explants from tomato variety SG048. The ploidy levels of 264 independent transgenic events and 233 non-transgenic plants regenerated from tissue culture were assessed by flow cytometry. The incidence of polyploids within the total population of transgenic plants varied from 10 to 78% and was not significantly different from the non-transgenic population. The greatest variation in the proportion of polyploids was observed in plants derived from different explant types, both in transgenic and non-transgenic regenerants, across three studied genotypes. Transgenic and non-transgenic plants regenerated from leaves included the highest number of normal diploid plants (82–100%), followed by cotyledon petiole-derived plants (63–78%). Transgenic plants produced from hypocotyls contained 22–58% diploids depending on the genotype used in transformation. Results described in this study demonstrate that, although transformation frequencies for leaf tissue are still lower under current protocols, the high percentage of diploids obtained make leaf tissue an attractive transformation target.Abbreviations BAP Benzylaminopurine - MS Murashige-Skoog - MsCHI Medicago sativa chalcone isomerase - PMI Phosphomannose isomerase     

84K杨愈伤组织再生体系和直接分化再生体系遗传转化的比较性研究

植物转基因必须以建立良好的植物转化受体系统为基础,而良好的受体系统必须以具有高频率的转化率及较强的再生能力为前提。实验以84K杨叶片为材料研究其在不同培养条件、不同种类及浓度的植物激素对其脱分化及再分化的影响,并建立了两种成熟的组织再生系统,得出组织培养体系与遗传转化体系的受体系统在激素配比方面存在一定差距,所以在此基础上进行了PCK1、PCK2两个基因的愈伤途径和不定芽途径途径的遗传转化,并对两种遗传转化体系进行了对比性分析并总结出两种体系的优缺点。     

In Vivo and in Vitro Experimental Analysis of Lens Regeneration in Larval Xenopus laevis

After lentectomy of larval Xenopus laevis , the outer cornea undergoes tissue transformation resulting in formation of a new lens. This lens regeneration is triggered and sustained by neural retina. In the present study, lens-forming transformation of the outer cornea was completed in vitro when the outer cornea was cultured within the lentectomized eye-cup. Well-differentiated lens fiber cells, which showed positive immunofluorescence for total crystallins, were also formed when the outer cornea was cultivated with the retina. No lens tissue was formed when the cornea was cultured alone. Lens-forming transformation, originating from the cornea three and five days after lentectomy, completely regressed when the tissue was isolated in vitro . Fom the present and previous findings, we concluded that, the interaction of corneal cells with the retina plays a decisive role in lens regeneration in situ .     

甘蓝型油菜的遗传转化

甘蓝型油菜的各种外植体经遗传转化、组织培养后可以再生为转基因植株,但再生频率会因外植体的基因型、年龄、培养基添加成分和农杆菌共培养的不同而发生变化。转化方法包括农杆菌介导转化、基因枪法、花粉介导法、PEG介导法等,其应用前景非常广阔。甘蓝型油菜的遗传转化在其品质改良、抗逆性提高、雄性不育系的获得和一些特殊性状方面都取得了很大成就。简要介绍甘蓝型油菜的再生体系建立、转化方法及所取得的部分成就。     

Phytochrome in Cultured Wild Carrot Tissue: II. Dark Transformations

Disappearance and transformation of phytochrome in light-treated, dark-grown cultured tissue of wild carrot (Daucus carota L.) have been analyzed. Evidence is presented for the existence of two populations of far red-absorbing phytochromes: one which is subject to reversion, and a second which undergoes loss of photoreversibility. Phytochrome levels and transformation characteristics have been compared for several strains and developmental stages of cultured wild carrot tissue.     

Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were inoculated with 1 ml of diluted (OD_600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin^?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin^?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration. Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997     

The myth of plant transformation

Technology development is innovative to many aspects of basic and applied plant transgenic science. Plant genetic engineering has opened new avenues to modify crops, and provided new solutions to solve specific needs. Development of procedures in cell biology to regenerate plants from single cells or organized tissue, and the discovery of novel techniques to transfer genes to plant cells provided the prerequisite for the practical use of genetic engineering in crop modification and improvement. Plant transformation technology has become an adaptable platform for cultivar improvement as well as for studying gene function in plants. This success represents the climax of years of efforts in tissue culture improvement, in transformation techniques and in genetic engineering. Plant transformation vectors and methodologies have been improved to increase the efficiency of transformation and to achieve stable expression of transgenes in plants. This review provides a comprehensive discussion of important issues related to plant transformation as well as advances made in transformation techniques during three decades.     

根癌农杆菌转化紫草的研究

紫草 (ＬｉｔｈｏｓｐｅｒｍｕｍｅｒｙｔｈｒｏｒｈｉｚｏｎＳｉｅｂ .ｅｔＺｕｃｃ)是传统中药。其根部含有萘醌类化合物—紫草素及其衍生物 ,具有显著的抗菌、抗炎、抗癌以及促进伤口愈合等生理活性。紫草素同时也是一种名贵化妆品染料。科学家对紫草的研究兴趣是基于其资源的缺乏及紫草植物本身所具有的一些特点 ;如 :紫草素及其衍生物的颜色特性可凭借肉眼观察 ,紫草素及其衍生物只在紫草的根部积累 ,紫草素合成的次生代谢途径受多种酶和外界条件 (光照 ,营养等 )的调节等。紫草细胞培养 (Ｆｕｊｉｔａ等 ,1983;叶和春等 ,1991)可以产…     

An Improved Tissue Culture System for Embryogenic Callus Production and Plant Regeneration in Switchgrass (Panicum virgatum L.)

The increased emphasis on research of dedicated biomass and biofuel crops begs for biotechnology method improvements. For switchgrass (Panicum virgatum L.), one limitation is inefficient tissue culture and transformation systems. The objectives of this study were to investigate the utility of a new medium described here, LP9, for the production and maintenance of switchgrass callus and its regeneration, which also enables genetic transformation. LP9 medium is not based on Murashige and Skoog (MS) medium, the basal medium that all published switchgrass transformation has been performed. We demonstrate an efficient tissue culture system for switchgrass Alamo 2, which yields increased viability of callus and the ability to maintain callus for a duration of over 6 months. This longevity gives a greater useful callus lifetime than for published switchgrass MS-based media. This increased longevity enables greater potential efficiency and throughput for a transformation pipeline. Callus produced on LP9 is categorized as type II callus, which is more friable and easier to multiply, maintain and transfer than type I callus obtained from previously described tissue culture systems.     

甘露糖正向筛选体系在巨尾桉遗传转化中的应用

为了研究甘露糖正向筛选体系在巨尾桉遗传转化过程中的有效性,构建了以6-磷酸甘露糖异构酶（6-phosphomannose isomerase,PMI）为筛选标记的pCAMBIA1301植物表达载体,并将该载体通过农杆菌介导的遗传转化转入木本植物巨尾桉中。将获得的阳性植株通过氯酚红（chlorophenol red,CPR）法及PCR检测,桉树遗传转化的阳性率达到26.09%。另外,通过正交试验优化法,对巨尾桉组培快繁体系建立过程中不同浓度激素配比进行了研究,建立起良好的巨尾桉组织培养再生体系,由甘露糖筛选敏感性测试,获得了巨尾桉筛选临界浓度,蔗糖与甘露糖比例为19∶11,优化了巨尾桉遗传转化体系,为今后巨尾桉组织培养与遗传转化研究提供了重要的参考依据。     

大豆转基因体系的研究进展

从大豆的转基因方法和受体系统两个方面概述大豆转基因体系的最新研究进展,并讨论了大豆遗传转化的主要障碍及可能的解决途径。作者认为,以根癌农杆菌介导大豆子叶节和基因枪轰击大豆未成熟子叶是较有效的大豆遗传化系统。目前,在大豆遗传转化研究中存在着大豆组织培养体系有待于进一步完善、遗传转化率较低、重复性差、大豆受体基因型单一等问题,建立新的、高效和稳定的大豆组织培养体系,提高生产上栽培大豆品种的组织培养能力,改遗传转化现有的单一基因为多基因,可能是解决上述问题的有效途径。     

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11–15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.     

3-D finite element modelling of facial soft tissue and preliminary application in orthodontics

Prediction of soft tissue aesthetics is important for achieving an optimal outcome in orthodontic treatment planning. Previously, applicable procedures were mainly restricted to 2-D profile prediction. In this study, a generic 3-D finite element (FE) model of the craniofacial soft and hard tissue was constructed, and individualisation of the generic model based on cone beam CT data and mathematical transformation was investigated. The result indicated that patient-specific 3-D facial FE model including different layers of soft tissue could be obtained through mathematical model transformation. Average deviation between the transformed model and the real reconstructed one was 0.47?±?0.77?mm and 0.75?±?0.84?mm in soft and hard tissue, respectively. With boundary condition defined according to treatment plan, such FE model could be used to predict the result of orthodontic treatment on facial soft tissue.     

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.