Recalcitrant Crop Research Articles

Induction of organogenesis and somatic embryogenesis in seedling explants of some novel genotypes of chickpea (Cicer arietinum L.) — A recalcitrant crop

Induction of organogenesis and somatic embryogenesis in seedling explants of some novel genotypes of chickpea (Cicer arietinum L.) — A recalcitrant crop

Banana (Musa spp.) as a model to study the meristem proteome: Acclimation to osmotic stress

Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using non-parametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop.

Molecular markers and doubled haploids in European plant breeding programmes

The breeding companies and laboratories involved in this article cover a wide range of crops grown in the temperate climate zone: small grain cereals, oilseed crops, forage crops, turf, vegetables and potato. Speed and efficiency are becoming increasingly important in variety breeding and doubled haploids (DH) and genetic markers are important biotechnological tools to accelerate materials to market. Collaborative research between universities, research institutions and breeding companies has resulted in the routine use of DH technology and molecular markers in practical breeding of barley, wheat and rapeseed. DH populations have been established not only for barley, wheat and rapeseed, but for rye, oat and triticale, where DH technology is less developed. A driver here is the value of the crop e.g. although wheat is less responsive to DH production the value of the end product makes the effort worthwhile. Simple and rapid DNA extraction methods used in high-throughput marker assisted selection (MAS) systems are essential for routine use of markers. MAS is used both to monitor the presence of genes of interest and also to monitor the genetic background. DH technology in forage, turf and vegetables is still in progress and the practical use of markers in all crops is limited by access to trait linked markers. Collaboration and technology transfer with universities, research institutions and breeding companies is essential for the improvement of both DH protocols in recalcitrant crops and marker technology in all crops.

Efficient and rapid in vitro plant regeneration system for Indian cultivars of chickpea (Cicer arietinum L.)

Lacking of an efficient regeneration protocol for the recalcitrant crop chickpea is a limiting factor for adapting genetic engineering approaches for its improvement. The present study describes a rapid and efficient method for multiple shoot regeneration for three Indian cultivars, B115, C235, ICCV89314, using single cotyledons with half embryos as explant. Modified MS medium with 1.5 mg l−1 6-benzyladenine (BA) and 0.04 mg l−1 α-naphthaleneacetic acid (NAA) induced a maximum of 26 shoots from a single explant after 20 days of culture. When cultured in modified MS medium containing 0.2 mg l−1 indole-3-acetic acid (IAA), 80% of the shoots from each regenerating explant elongated in another 20–25 days. Following a root-grafting protocol, 90–95% of the elongated shoots survived in soil which subsequently produced seeds. The regeneration process from explant preparation to complete plants took 55–60 days. The presently optimized rapid regeneration method holds promise for facilitating the deployment of agronomically important components through genetic transformation for betterment of this important food crop.

Fusarium Wilt of Banana Is Caused by Several Pathogens Referred to as Fusarium oxysporum f. sp. cubense

ABSTRACT Fusarium wilt of banana (also known as Panama disease) is caused by Fusarium oxysporum f. sp. cubense. Where susceptible cultivars are grown, management is limited to the use of pathogen-free planting stock and clean soils. Resistant genotypes exist for some applications, but resistance is still needed in other situations. Progress has been made with this recalcitrant crop by traditional and nontraditional improvement programs. The disease was first reported in Australia in 1876, but did the greatest damage in export plantations in the western tropics before 1960. A new variant, tropical race 4, threatens the trades that are now based on Cavendish cultivars, and other locally important types such as the plantains. Phylogenetic studies indicate that F. oxysporum f. sp. cubense had several independent evolutionary origins. The significance of these results and the future impact of this disease are discussed.

A Study of in vitro Regeneration in Relation to Doses of Growth Regulators in Hybrids of Upland Cotton

The frequency of in vitro callus induction and plant regeneration is influenced by several factors, including composition of culture medium, explant source, and the genotype. Crosses between regenerable and non-regenerable upland cotton cultivars were evaluated for hybrid vigour towards regeneration responses, which is consequential in recalcitrant crop species like cotton where regeneration is limited only to a few cultivars. The results indicated that regenerable and non-regenerable parental cultivars had similar potential of producing callus, but differed in producing callus weight and embryogenic calli. Mean performance of crosses, regarding callus induction, callus weight, callus growth rate, percent embryo induction, and percentage of germinating embryos, deviated considerably from the performance of their parents, signifying the presence of hybrid vigour for the expression of these traits. Magnitude of hybrid vigour varied across hormonal levels. Genetic component was evident for all the traits although of lower magnitude. The results indicated that genetic component in the phenotypic expression of callus growth, percentages of embryo induction and germinating embryos was higher than that of callus induction, callus weight and percentage of embryogenic calli. Hormonal concentration in the media had affect on the degree of gene expression responsible for regeneration in upland cotton. Over, partial- and additive-dominance types of gene effects were apparent in the expression of these traits. Genotype × growth regulator level interaction caused considerable variation in the expression of regeneration responses, suggesting that determination of specific level of growth regulator concentration in the medium was necessary for a particular genotype to obtain optimum response. Genotype × explant source interaction was, however, relatively less important. Differences among genotypes for percent embryo induction were clearly evident.

Identification of highly regenerative plants within sugar beet (Beta vulgaris L.) breeding lines for molecular breeding

Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration response, line FC607 also had the highest mean number (8.3±1.1) of shoots per explant. Individual plants within each line exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs.

Wheat transformation: current technology and applications to grain development and composition

Transgenesis is a powerful research tool that can be adapted to investigate many aspects of gene function. It has been used widely in model plants such as Arabidopsis, tobacco and rice but until recently, bottlenecks in DNA-delivery and tissue culture meant that it could not be used routinely for wheat research. However, many aspects of grain development and composition are unique to wheat and cannot be easily investigated in model species. Over the last decade, progress in biolistic- and Agrobacterium-mediated DNA delivery, reduction in genotype-dependency in wheat tissue culture and in the development of a range of supplementary technologies has enabled its application in this traditionally recalcitrant crop. The use of genetic modification has already made a significant impact on our understanding of interactions between high molecular weight glutenin subunits and their individual contribution to dough strength. As candidate genes become available the application of genetic transformation is set to play a major part in the elucidation of their function in determining other important grain traits such as starch and lipid composition, dietary fibre composition and grain texture.

Agrobacterium-mediated transformation of two high oleic sunflower (helianthus annuus l) genotypes: Assessment and optimization of important parameters

The establishment of an efficient Agrobacterium-mediated transformation protocol for a recalcitrant crop like high oleic sunflower genotypes, cv. Capella and SWSR2 inbred line, is presented in this paper. The protocol requires the identification and optimization of parameters affecting T-DNA delivery and plant regeneration. The basis for identifying the most appropriate conditions for transformation was the fluorometric, histochemical GUS activity coupled with plant cell vitality. Agrobacterium tumefaciens-mediated transformation was performed using strain LBA4404 for cv. Capella and strain GV3101 for the inbred line. Both strains harboring the pBI121 plasmid with the gus gene were under the control of 35S promoter. The flourometric GUS activity was increased in cv. Capella and SWSR2 inbred line 1.9 and 1.6 fold, respectively, relative to transformation without inducers, by optimizing the bacterial density to OD₆₀₀=1.0, by splitting shoot apices and by using MS medium as a co-cultivation medium supplemented with 200 μM acetosyringone. Pre-culturing the explants for three days on SIM2 medium before the bacterial inoculation followed by co-cultivation for 72 h increased the percentage of the GUS expression to 40% and 30% in cv. Capella and SWSR2 inbred line, respectively. It also improved the regeneration percentage as well as the vitality of cells.

Recent advances in wheat transformation

Since the first report of wheat transformation in the early 1990s, genetic engineering of wheat has evolved rapidly. Several laboratories worldwide have reported the production of fertile transgenic wheat plants using a variety of methods. While there are several innovative and promising approaches for wheat transformation using different explants as targets for transformation, different methods of transformation, and different selection schemes, the most common approach to wheat transformation is the bombardment of tissue derived from immature embryos followed by selection based on resistance to the bar gene. Even with all these successful reports, hurdles still exist for this recalcitrant crop. Of these hurdles, low transformation rates, tools for transgene expression, and transgene silencing in subsequent generations are probably the most critical. This review will provide an overview of wheat transformation in the past decade, addressing both positive and negative factors that effect transformation while highlighting the successes of the past and prospects for the future.

CONTROL OF ETHYLENE AND USE OF POLYAMINES CAN OPTIMISE THE CONDITIONS FOR SOMATIC EMBRYOGENESIS IN COCONUT (COCOS NUCIFERA L.) AND PAPAYA (CARICA PAPAYA L.)

The protective conditions under which callus cultures are grown to prevent contamination and tissue desiccation cause the accumulation of ethylene, which, in turn may reduce growth and prevent somatic embryogenesis. To demonstrate the importance of ethylene a study was undertaken on somatic embryogenic coconut (Cocus nucifera L.) and papaya (Carica papaya L.) callus cultures incubated with a number of medium additives which could either reduce production (aminoethoxyvinylglycine; AVG), protect from (silver thiosulphate; STS) or help combat ethylene-induced stress (polyamines). Increases in the ethylene concentration above coconut cultures reduced callus growth and somatic embryogenesis. AVG (2 mu M) and STS (3 mu M) improved somatic embryogenesis by 100% and 67%, respectively. Furthermore, coconut somatic embryogenesis was promoted (100%) by the addition of the polyamines, putrescine (7.5 mM) and spermine (1 mu M) to the media. Similarly, papaya callus cultures grew better in the presence of STS (ca. 50 mu M) as well as being promoted into somatic embryogenesis by STS. Furthermore, STS (ca. 1 mu M) aided somatic embryo proliferation, maturation and germination. Better control of ethylene and the reduction of culture stress can increase both the quantity and quality of tissue available for genetic engineering of otherwise recalcitrant tropical fruit crops such as coconut and papaya.

The effect of exogenously-applied phytohormones on gene transfer efficiency in sugarbeet ( Beta vulgaris L.)

Cellular competence for regeneration and transformation has been monitored in a recalcitrant crop, i.e. sugarbeet ( Beta vulgaris L.). Explants for Agrobacterium inoculation were taken from the transition zone between hypocotyl and cotyledonary petioles. De novo generation of adventitious shoots was very efficient (60%–70%) from these cotyledonary node explants. Regeneration could not be further stimulated by manipulation of cytokinin type or concentration. The mode of regeneration was direct, without a callus phase. Dedifferentiation, as induced by a preculture period on 2,4-D, gave significantly higher gene transfer frequencies, but regeneration was irreversibly impaired. NAA mimicked the effect of 2,4-D on gene transfer. However, explants retained their regenerative capacity upon pretreatment with NAA. Using the procedure described here, transgenic sugarbeet plants were obtained after selecting on 250 mg/l kanamycin at a frequency of 1%.

Philosophy and practice of variety-independent gene transfer into recalcitrant crops

Commercialization and marketing of agricultural products derived from recombinant DNA technologies is now becoming a reality. Genetically engineered tomatoes are poised to appear in supermarket shelves in the United States sometime next year, with other crops, including cotton, maize, soybean, rapeseed, potatoes, cucurbids, raspberries and others, scheduled for release 1 to 2 yr later (Hamilton and Ellis, 1992). One of the major breakthroughs responsible for the rapid creation of commercializable products has been the development of gene transfer methods capable of introducing foreign DNA into elite germplasm. This review examines some of the principles and applications of such gene transfer technologies in relation to conventional alternatives that are limited by cell culture, host, and genotype. Advantages and disadvantages of various gene transfer methods will be discussed. Special emphasis will be given to particle bombardment, as methods based on this technology paved the way for the engineering of a number of important agronomic species.

Biological ballistics-no longer a shot in the dark

Established methods of genetic transformation, such asAgrobacterium transfection and DNA uptake by protoplasts have not been successfully applied to some of the world’s major crops. This article reviews the evolution of microprojectile bombardment, from its inception to establishment at the method of choice for transformation of otherwise recalcitrant crops such as maize, wheat and barley. The potential of microprojectile bombardment, as a universal method of transformation, is discussed in the context of the wide range of species transformed, together with the transformation of plastid genomes and the contribution of this technology beyond the boundaries of the plant kingdom.

Genetic engineering: An additional tool for plant improvement

Advances in gene transfer technologies have enabled the production of both monocot and dicot transgenic plants. With the biolistic method, genes can be transferred in recalcitrant crop plants and forest trees, independent of their genotype. Inexpensive methods for both stable and transient gene transfers - ultrasonication, direct DNA insertion during imbibition using somatic embryos, and silicon carbide fibres - have been developed. The frequency of Agrobacterium-mediated transformation rates of cloned genes can be enhanced in plant cells. The analysis of molecular markers (RFLPs, RAPDs, DNA fingerprints) can accomplish the characterization, gene mapping and identification and certification and patent protection of cultivars. With PCR, selective amplification of a specific DNA segment from a small amount of an organism’s total DNA can be used toidentify transgenic cultivars. The expression of a target gene can be inhibited with antisense RNA. So far, a limited number of genes have been identified and cloned with genetic engineering. With specific gene transfers, many goals such as biological control of insect pests and fungi, male sterility, virus resistance, improving seed protein, and production of transgenic plants as “bioreactors” can be accomplished. T-DNA mutagenesis may lead to learning more about the genetic control of plant development and morphogenesis, and isolation of useful mutants. Before genetic engineering becomes a reliable tool of plant breeding, more attention is needed to explore: (a) new plant genetic resources in order toidentify and clone new genes, (b) fate of selective and scorable marker genes, and (c) field evaluation of transgenes in transgenic plants.

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.

ワサビ種子の発芽に関する研究

Germination of Wasabia japonia Matsum. seeds, typical recalcitrant crop seeds in Japan, was investigated. Wasabia seeds have deep dormancy, but during storage the dormancy weakens. In eight months after harvest, the dormancy almost all disappears, resulting in the heavy sprouting of stored seeds.Optimum germination temperature is 15°C. In deep dormant period, 20°C or higher temperature cannot induce full germination. At 10°C, germination speed is very slow, though the similar germination percentage can be attained as in 15°C.GA4 is more effective than GA3 in breaking the dormancy. Germination percentage is higher and mean germinating days is smaller in GA4 than in GA3.BA and thiourea are effective to some extent for dormancy breaking. Combination of GA3 and BA shows about as the same effectiveness as GA4.Decoated seeds show almost perfect germination even in the deep dormant period, showing the cause of dormancy is mainly due to seedcoat.

Aseptic culture techniques for banana and plantain improvement

There is an urgent need to identify or to produce ‘Black Sigatoka’ diseasetolerant or -resistant cooking and dessert bananas. Since bananas are perhaps the most conspicuously sterile of all cultivated fruits, breeding of resistant stock is fraught with great difficulties. An overview is provided of the potential value that may be derived from the use of aseptic culture techniques for generating and/or multiplying specific pathogen-tolerant clones. Special emphasis is given to the principles underlying various strategies and to the several levels of sophisticated methods presently available or that still need to be further developed before substantive practical benefits accrue. While the stance adopted in this paper is conservative, so-called tissue culture approaches to banana breeding and improvement may well serve as a model not only forMusa but for other recalcitrant crop plants.