Genetic engineering of millets: current status and future prospects

This review summarizes progress on the genetic transformation of millets and discusses the future prospects for the development of improved varieties. Only a limited number of studies have been carried out on genetic improvement of millets despite their nutritional importance in supplying minerals, calories and protein. Most genetic transformation studies of millets have been restricted to pearl millet and bahiagrass and most studies have been limited to the assessment of reporter and marker gene expression. Biolistic-mediated gene delivery has been frequently used for the transformation of millets but Agrobacterium-mediated transformation is still lagging. Improved transformation of millets, allied to relevant gene targets which may offer, for example, improved nutritional quality, resistance to abiotic and biotic stresses, and resistance to fungal infection will play important roles in millet improvement.     

促进植物高效遗传转化的发育调节因子及其在玉米中的应用进展*

高效遗传转化技术体系的建立对植物功能基因组学研究和作物新品种的培育均具有促进作用,目前,再生效率低下是限制许多植物高效遗传转化体系建立的主要技术屏障之一。随着对植物分生组织和体细胞胚形成过程研究的深入,鉴定到了一些关键调控基因,统称为发育调节因子。发育调节因子应用于植物遗传转化后,可以有效改善植物分生组织诱导和再生能力,为提高遗传转化效率提供了重要机遇。综述了7类发育调节因子在提高植物遗传转化效率中的研究进展,重点介绍了其中3类在促进玉米遗传转化中的应用,最后展望了建立植物高效遗传转化体系的发展方向。     

2,4-D和干燥处理对谷子成熟胚离体再生体系的影响

谷子离体再生体系不够稳定、转化效率低，已成为谷子功能基因研究和品种改良的瓶颈。为了建立谷子成熟胚稳定的离体再生体系，以当地高产优质的6个谷子品种成熟胚为外植体，以不同2,4-二氯苯氧乙酸（2,4-dichlorophenoxyacetic acid，2,4-D）浓度及对胚性愈伤的不同干燥处理时间为变量，通过单因素实验和正交实验考察各因素对谷子愈伤组织分化及成苗的影响。结果表明，晋谷21在2,4-D浓度为9 μmol·L-1、4 h干燥处理的条件下所建立的再生体系最好，分化率为64.35%，成苗率为29.06%。研究通过探索谷子组织培养的最适条件，为谷子高效稳定遗传转化体系的建立和利用基因工程手段进行品质改良提供了依据。     

A leaf-based regeneration and transformation system for maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.     

In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

Transformation ofBrassica oleracea L.: a critical review

Brassica oleracea is a highly polymorphic species encompassing a wide range of important vegetable and fodder crops. Gene transfer into cultivated forms of this species requires reproducible and efficient methods for genetic transformation and plant regeneration. In this review, we have collated the research experience on transformation ofB. oleracea to highlight the problems encountered. Most research effort has been directed at developingAgrobacterium-mediated transformation methods with relatively little emphasis to date on direct gene transfer techniques. Common procedures for the transformation ofB. oleracea have not emerged, due to the inherent variability between and amongst genotypes. Future progress would be facilitated by the use of genetically fixed material, such as double-haploid or inbred lines, to reduce variation of response within genotypes and would avoid the need for cultivar-specific transformation protocols if responsive lines amenable to crossing with cultivated forms could be identified. The principal difficulties relate to combining efficient plant regeneration with gene transfer. Methods that enhance bacterial virulence and increase the proportion of cells susceptible to transformation and competent for regeneration are discussed. Inefficient selection is a major cause of poor transformation frequencies inB. oleracea and has resulted in the regeneration of chimeric plants uponAgrobacterium tumefaciens-mediated transformation. Promising results have been obtained withAgrobacterium rhizogenes-mediated transformation but the impact of therol genes on flowering of primary transformants has not yet been fully assessed. Strategies to reduce the deleterious effects of therol genes on flowering are discussed. Few agronomically useful characters have been introduced, the majority of research having been confined to the introduction of marker and reporter genes; possible candidate genes are discussed.     

Phaseolus vulgaris — Recalcitrant potential

Since the ability to genetically engineer plants was established, researchers have modified a great number of plant species to satisfy agricultural, horticultural, industrial, medicinal or veterinary requirements. Almost thirty years after the first approaches to the genetic modification of pulse crops, it is possible to transform many grain legumes. However, one of the most important species for human nutrition, Phaseolus vulgaris, still lacks some practical tools for genomic research, such as routine genetic transformation. Its recalcitrance towards in vitro regeneration and rooting significantly hampers the possibilities of improvement of the common bean that suffers from many biotic and abiotic constraints. Thus, an efficient and reproducible system for regeneration of a whole plant is desired. Although noticeable progress has been made, the rate of recovery of transgenic lines is still low. Here, the current status of tissue culture and recent progress in transformation methodology are presented. Some major challenges and obstacles are discussed and some examples of their solutions are presented.     

Biotechnological applications in in vitro plant regeneration studies of broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">italica</Emphasis>), an important vegetable crop

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.     

河南项城贾庄和后高老家遗址炭化植物遗存揭示的仰韶时期的原始农业

豫东地区是史前中原文化系统、海岱文化系统等的中介地带,由于这里地处黄泛区且人口密集,史前遗址或被掩埋在地下数米深处,或位于岗地、堌堆之上,后期破坏严重,目前缺乏相关植物考古资料,对史前农业发展的认识并不清晰。本研究对河南项城市贾庄和后高老家遗址开展浮选,获取了仰韶时代中期的炭化植物遗存,其中炭化植物种子主要包括粟、黍、水稻三种农作物和狗尾草属、马唐属、稗属、黍亚科等野生植物,可食用野生植物的核壳来自菱属、芡实、柿属、栎属、桃属等。农作物及典型田间伴生杂草遗存的量化结果显示,两处遗址仰韶时代中期的农作物结构以粟、黍为主,水稻的比重很低,具备黄淮地区稻粟兼作农业的地域性和时代特征。多种可食用野生植物遗存显示了植物性食物资源的多样性。各类植物遗存的绝对数量和出土概率表明,农业在生业经济中占据主体地位,采集野生植物仍然是先民获取植物性食物资源的重要方式。贾庄和后高老家遗址炭化植物遗存分析结果为了解豫东地区仰韶时代原始农业发展状况提供了重要资料,对探索中华文明早期阶段的农业发展状况及其与文明演进的关系有重要意义。     

The Current Status of Culture Collections and Their Contribution to Biotechnology

Abstract

Cereals are the most important group of plants for human nutrition and animal feed. Partially due to the commercial value of crop plants, there has been an ever-increasing interest in using modern biotechnological methods for the improvement of the characteristics of cereals during the past decade. The rapid progress in molecular biology, plant cell culture techniques, and gene transfer technology has resulted in successful transformations of all the major cereals—maize, rice, wheat, and barley. This brings the biotechnological methods closer to the routine also in barley breeding. In this article, the current status of barley genetic engineering, including the patent situation, is reviewed. The needs, aims, and possible applications of genetic engineering in barley breeding are discussed.     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

小麦遗传转化研究进展

小麦作为最重要的3大禾谷作物之一,其离体培养具有很强的惰性,再生频率与水稻、玉米相比要低一些,目前大多通过对基因型和外植体的选择来达到植株的高频再生分化,因此其遗传转化就远远滞后于水稻和玉米,更不用说与其它双子叶植物相比了.重点综述了小麦转基因技术和外源基因在小麦中的遗传转化研究现状,其内容包括几种主要的小麦转基因方法和以基因枪法为主的各种转化技术对品质基因、抗除草剂基因和抗病等基因在小麦中的遗传转化研究进展,并对存在的一些问题进行了简要的论述.     

兰花组织培养和分子生物学研究进展

兰科植物是开花植物中最大的家族之一,其科研和经济价值越来越受到全世界的重视。兰花的组织培养近年来发展迅速,对兰花组织培养中原球茎的诱导和培养基选择的国内外研究进行了综述;并对近年来应用分子标记、转基因等分子生物学技术研究兰花的遗传多样性、系统分类和基因功能进行综述。     

棉花体细胞胚胎发生的研究进展

经过30多年的发展, 已经在多个不同的棉花品种中获得了体细胞胚, 并再生成苗。但由于体细胞胚胎发生往往受多种因素影响, 如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展, 分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述, 并对研究中存在的问题进行了讨论。     

Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops

Summary Immature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to rontine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures, of immature embryos or their cell suspensions, and cryoperserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months, of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristerms from seedlings of maize oat, sorghum, millet, wheat, and barley in an effort to develop a less genetype-dependent and efficient cereal regneration system that can be maintained in vitro for long pertiods of time without the need for cryopreservation. Furthermore, apical mesistem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.     

Forage and Turf Grass Biotechnology

Referee: Dr. Ian Ray, Plant Breeding and Genetics, Department of Agronomy & Horticulture, New Mexico State University, MSC 3Q, P.O. Box 30003, Las Cruces, NM 88003-8003 Forage and turf grasses are the backbone of sustainable agriculture and contribute extensively to the world economy. They play a major role in providing high quality and economical meat, milk, and fiber products and are important in soil conservation, environmental protection, and outdoor recreation. Conventional breeding contributed substantially to the genetic improvement of forage and turf grasses in the last century. The relatively new developments in genetic manipulation of these species open up opportunities for incorporating cellular and molecular techniques into grass improvement programs. For some commonly used forage and turf species, significant advances have been achieved in the following areas: (1) establishment of a tissue culture basis for the efficient regeneration of fertile and genetically stable plants, (2) generation of transgenic plants by biolistic transformation and direct gene transfer to protoplasts, (3) recovery of intergeneric somatic grass plants by protoplast fusion, (4) development of molecular markers for marker assisted selection, and (5) sequencing of expressed sequenced tags and the development of DNA array technologies for gene discovery. Although difficulties still exist in genetic manipulation of these recalcitrant monocot species, impressive progress has been made toward the generation of value-added novel grass germplasm incorporating traits such as improved forage quality. The joint efforts of molecular biologists and plant breeders make the available biotechnological methods a useful tool for accelerating forage and turf grass improvement.     

Inheritance of somatic embryogenesis and plantlet regeneration from primary (type 1) callus in maize

Summary Genetic factors controlling the differential expression of somatic embryogenesis and plant regeneration of maize from tissue culture were studied in two crosses. Inbred, hybrid, F2 and backcross generations developed from crossing maize inbred A188 with two commercially important inbred maize lines (B73 and Mo17) demonstrated genetic and environmental effects on somatic embryogenesis and plant regeneration when immature zygotic embryos were cultured on MS medium. Additive gene effects were more important in both crosses than dominant gene effects for precent somatic embryogenesis and percent or number of plants regenerated per embryo when generation means were analyzed. In backcross generations of each cross, cytoplasmic, maternal and/or paternal effects were significant for frequency of somatic embryos three weeks after culture as well as frequency, or number of plants regenerated per embryo, nine weeks after culture. Analysis of genetic variances suggests at least one gene (or block of genes) controls the expression of the frequency of somatic embryogenesis in these crosses. Differences in somatic embryogenesis and plant regeneration between B73 and Mo17 are discussed. This is Journal Paper No. 11,435 of the Purdue University Agricultural Experiment Station.     

Chilli peppers — A review on tissue culture and transgenesis

Biotechnology techniques involving plant tissue culture and recombinant DNA technologies are powerful tools that can complement conventional breeding and expedite Capsicum improvement. The rate of progress in Capsicum is relatively slower than other members of Solanaceae because of its high genotypic dependence and recalcitrant nature. Capsicum is a recalcitrant plant in terms of in vitro cell, tissue and organ differentiation, plant regeneration and genetic transformation which makes it difficult to apply recombinant DNA technologies aimed at genetic improvement against pests, diseases and abiotic stress. Despite this, application of tissue culture and genetic transformation have led to significant development in chilli pepper plants, and studies are underway to achieve the targets of pre-harvest improvement and post-harvest characterization for value addition to this crop. This review presents a consolidated account of in vitro propagation and focuses upon contemporary information on biotechnological advances made in Capsicum.     

普通小麦品种Alondra＇s遗传转化体系的建立

小麦（Triticum aestivum）幼胚愈伤组织的诱导和分化再生有高度依赖基因型特征。为了建立和优化Alondra’s的高效再生及遗传转化体系,为小麦遗传转化提供更多的受体基因型,以Alondra’s的幼胚为外植体,研究了培养基种类、不同激素配比等对其幼胚愈伤组织诱导及再生的影响。结果表明,在使用N6培养基时,添加3mg·L^-1的2,4-D并附加1000mg·L^-1的CH对愈伤组织的诱导效果较好;添加4mg·L^-1的ZT、不附加IAA对愈伤组织的分化效果最好。通过构建植物表达载体pCAMBIA1301-220.6,利用基因枪法将HYG基因导入Alondra’s幼胚愈伤组织中,以建立Alondra’s的高效遗传转化体系。结果在含100mg·L^-1潮霉素的选择培养基上进行筛选、分化,获得了30棵抗性植株。经PCR检测,其中5株为阳性转基因植株,转化率为0.5%。Alondra＇s遗传转化体系的建立丰富了小麦遗传转化的基因型,为小麦品种的转基因改良和在不同背景下研究基因的功能奠定了良好的基础。