Invited review: Transformation of strawberry: The basis for translational genomics in Rosaceae

Summary Translational genomics is defined as the application of molecular-genetic principles derived from model systems to species of experimental or economic interest. The past 20 years of research in plant model systems such as Arabidopsis thaliana have relinquished vast amounts of information regarding gene function, the integration of genetic components into pathways, and the interrelationships between pathways to control form and function in plants and plant-products alike. At present, the challenge is to relate these paradigms to other species of economic or scientific interest. Apart from being an important and valuable crop, strawberry (Fragaria spp.) is a member of the Rosaceae, a plant family containing fruit, nut, ornamental and wood-bearing species. Strawberry is unique within the Rosaceae in that it is a rapidly growing herbaceous perennial with a small genome and the ability to thrive in a laboratory setting. Strawberry species may also be transformed and regenerated in a time scale of weeks or months instead of years. For these reasons, strawberry has been recognized as the translational genomics model for the Rosaceae family. This review summarizes and synthesizes the technical reports of strawberry regeneration and transformation, consolidating the large body of information regarding genetic modification of this important genus.     

Agrobacterium tumefaciens-mediated transformation of recalcitrant crops

The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general guidelines for the development of a transformation protocol are discussed.     

MiniMax,a new diminutive <Emphasis Type="Italic">Glycine max</Emphasis> genotype with a rapid life cycle,embryogenic potential and transformation capabilities

We developed Glycine max cv MiniMax (PI643148) that has a rapid life cycle, short stature and characteristic simple sequence repeat (SSR) markers that could make it useful for mutant screening, functional genomics, genetic mapping and other studies involving soybeans. We demonstrate that MiniMax is able to make somatic embryos (SEs) that rapidly develop into plantlets. Thus, the rapid cycling habit carries over into aspects of plant regeneration. Chimaeras (having transformed roots with untransformed aerial stocks) have been produced rapidly under non-axenic conditions using Agrobacterium rhizogenes-mediated transformation. Part of these experiments involved the engineering an enhanced green fluorescent protein (eGFP) reporter cassette outside the multi-cloning site of a plant expression vector, permitting non-invasive visual screening of the transformed roots. The rapid cycling growth habit of MiniMax, its ability to efficiently generate SEs and ability to be transformed should prove useful for basic aspects of G. max molecular and genetic research.     

Non-antibiotic,efficient selection for alfalfa genetic engineering

A selectable marker gene (SMG), usually conferring resistance to an antibiotic or herbicide, is generally introduced into the plant cells with the gene(s) for the trait of interest to allow only the cells that have integrated and express the foreign sequences to regenerate into a plant. The availability of several SMGs for each plant species is useful for both basic and applied research to combine several genes of interest in the same plant. A selection system based on gabaculine (3-amino-2,3-dihydrobenzoic acid) as the selective substance and the bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde aminotransferase (GSA-AT)] as the SMG was previously used for genetic transformation of tobacco. The hemL gene is a good candidate for a safe SMG, because GSA-AT is present in all plants and is likely involved in one metabolic step only, so that unintended effects of its overexpression in plants are not probable. In this work, we have compared this new selection system with the conventional, kanamycin-based system for alfalfa Agrobacterium-mediated transformation. The hemL and NptII genes were placed together into a T-DNA under the control of identical promoters and terminators. We show that the gabaculine-based system is more efficient than the conventional, kanamycin-based system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect of its expression was observed.     

Direct transformation of the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. by particle bombardment using immature thalli developing from spores

The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3'-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.     

植物瞬间表达系统与功能基因组学研究

结构基因组学和功能基因组学的发展使特定植物基因组和转录组序列的获取更为方便和快捷。随之而来的是对各种基因和调控序列的功能注释,探索植物生长和发育的遗传机理。表达和调控表达是遗传物质的自身语言和动态属性,因此通过植物细胞内表达来分析目标基因和序列的表达和调控行为是功能分析的主要立足点。除创造转基因植株外,近几年来植物细胞瞬间表达系统得到了广泛的使用,与基因重排、病毒诱导基因沉默和RNA干扰等新兴技术的结合使其在植物功能基因组研究中扮演了越来越重要的角色。     

Hypocotyl-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.     

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.     

Comparison of gene targeting efficiencies in two mosses suggests that it is a conserved feature of Bryophyte transformation

The moss, Physcomitrella patens, is a novel tool in plant functional genomics due to its exceptionally high gene targeting efficiency that is so far unique for plants. To determine if this high gene targeting efficiency is exclusive to P. patens or if it is a common feature to mosses, we estimated gene-targeting efficiency in another moss, Ceratodon purpureus. We transformed both mosses with replacement vectors corresponding to the adenine phosphoribosyl transferase (APT) reporter gene. We achieved a gene targeting efficiency of 20.8% for P. patens and 1.05% for C. purpureus. Our findings support the hypothesis that efficient gene targeting could be a general mechanism of Bryophyte transformation.     

Plant regeneration from embryogenic cell suspension cultures of <Emphasis Type="Italic">Lolium temulentum</Emphasis>

Summary Lolium temulentum L. (Darnel ryegrass) is a self-fertile and diploid grass species with a relatively short life cycle. We propose to use L. temulentum as a model system for genetic manipulation studies in forage and turf grasses, since most of the important grasses are outcrossing, require vernalization to flower, and in some cases are polyploid. As the first step to develop an efficient regeneration and transformation system, we performed a large-scale genotype screening for tissue culture responses using 46 L. temulentum accessions. Embryogenic callus formation frequency ranged from <1% to 11% across all accessions tested. Embryogenic calluses of a few responsive accessions were used to establish cell suspension cultures. The regeneration frequency of green plantlets from the established cell suspension ranged from 15% to 39%. After transferring the regenerants to the greenhouse, fertile plants were readily obtained without any vernalization treatment. This efficient plant regeneration system is being used for genetic transformation studies. With the development of genomics approaches for the improvement of forage and turf grasses, L. temulentum could serve as a model system for testing gene functions.     

Analysis of a <Emphasis Type="Italic">LEA</Emphasis> gene promoter via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the desiccation tolerant plant <Emphasis Type="Italic">Lindernia brevidens</Emphasis>

An Agrobacterium tumefaciens-based transformation procedure was developed for the desiccation tolerant species Lindernia brevidens. Leaf explants were infected with A. tumefaciens strain GV3101 harbouring a binary vector that carried the hygromycin resistance gene and an eGFP reporter gene under the control of a native dehydration responsive LEA promoter (Lb2745pro). PCR analysis of the selected hygromycin-resistant plants revealed that the transformation rates were high (14/14) and seeds were obtained from 13/14 of the transgenic lines. A combination of RNA gel blot and microscopic analyses demonstrated that eGFP expression was induced upon dehydration and ABA treatment. Comparison with existing procedures used to transform the well studied resurrection plant and close relative, Craterostigma plantagineum, revealed that the transformation process is both rapid and leads to the production of viable seed thus making L. brevidens a candidate species for functional genomics approaches to determine the genetic basis of desiccation tolerance.     

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.     

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.     

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.     

植物功能基因组学的研究策略

植物基因组学是一门研究植物基因组内基因与遗传信息是如何有机结合并如何决定其功能的一门科学。随着植物基因组计划的顺利进行 ,植物基因组学的研究已从结构基因组学转向功能基因组学。近年来 ,多采用高通量 (highthroughput,HTP)序列分析技术、大规模实验技术及计算机统计分析技术研究植物基因组功能。概述了植物功能基因组学的最新进展。     

Improvement of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in Hi-II maize (<Emphasis Type="Italic">Zea mays</Emphasis>) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.     

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.     

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。     

Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation

Cultivated strawberry (Fragaria ×ananassa) is a valuable crop, yet the absence of a rapid, high-throughput transgenic system has precluded meaningful application of biotechnology and translation of information from plant models to this crop. A new octoploid strawberry genetic line Laboratory Festival #9 has been identified, selected solely for its rapid regeneration and efficient transformation. Direct organogenesis has been achieved from all tissues tested, with rapidly-growing shoot initials visible in as few as 13 days. The conditions for optimal shoot regeneration, transformant selection, root generation, and plant acclimatization are presented. The progression from explant to plant in soil can be achieved in about 60 days. The development of transformation protocols in this rapid-cycling genotype allows high-throughput studies of gene function in the octoploid strawberry genetic background.