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.     

Colorado potato beetle resistant somatic hybrid potato plants produced via protoplast electrofusion

Leptines are natural glycoalkaloids found only in certain selections of the wild potato speciesSolanum chacoense. These rare glycoalkaloids have been identified to be phytochemical defensive agents against insect herbivores such as the Colorado potato beetle (CPB). In an attempt to introduce this CPB resistance into the cultivated potatoS. tuberosum, interspecific somatic hybrid plants were developed between a dihaploid ofS. tuberosum and a high leptine-producing germplasm selection ofS. chacoense. The somatic hybrid was fused using protoplast electrofusion and regeneration techniques. Selection of interspecies fusion cell lines was based on hybrid vigor in protoplast-callus (p-callus) growth, on shoot regeneration from p-calli, and on characteristic appearance of anthocyanin pigment. This selection system was highly efficient and 12 of 13 fully regenerated plants were identified as somatic interspecies hybrids, as determined by the analyses of morphologic biochemical, and isozyme markers. In vitro insect bioassays demonstrated that the hybrids averaged a threefold reduction in leaf consumption by the CPB when compared to cultivated potatoes.     

Evolutionary implications of matK indels in Poaceae

Insertion/deletion events (indels) and nucleotide substitutions at the extreme 3' end of the chloroplast gene matK have been identified that distinguish certain major lineages of grasses. A 1-bp (base pair) deletion creating a shift in the open reading frame (ORF) and a point mutation support the positions of Streptochaeta and Anomochloa as the two most basal lineages in Poaceae. Another 1-bp deletion resulting in early termination of the ORF is unique to Ehrharta, a member of the taxonomically disputable tribe Ehrharteae. A 6-bp insertion supports monophyly of subfamilies Panicoideae, Arundinoideae, Centothecoideae, and Chloridoideae (PACC). This marker appears useful in defining PACC clade members and may have potential in providing insight into the sister-group relationship between PACC and other lineages. Alignment of deduced amino acid sequences from bryophytes, gymnosperms, and angiosperms shows that this region is relatively conserved, but variation is notably higher in Poaceae. The evolutionary implications of these changes in grasses and other plant families are addressed.     

RAPD技术在植物遗传育种研究中的应用进展

RAPD技术是一种随机扩增多态性DNA的方法,操作简单、快捷且经济,可从分子水平提供直接的遗传证据。RAPD技术在植物遗传育种中的应用如下：1）遗传图谱的构建;2）分子标记辅助选择育种;3）外源染色体片段的鉴定和标记;4）遗传关系与遗传多样性的研究;5）体细胞杂种的鉴定。     

植物体细胞不对称杂交研究进展

介绍了近代的植物体细胞杂交研究发展历程,进一步综述了近年来植物不对称体细胞杂交和植物配子一体细胞杂交方面的研究进展以及存在的困难。     

Long-duration,high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 m in diameter; NE callus consists of long tubular cells averaging 52 m in width and 355 m in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - Trp L-tryptophan - E embryogenic - NE non-embryogenic     

Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens

Production of transgenic alfalfa plants by Agrobacterium-mediated transformation requires Agrobacterium infection and regeneration from tissue culture. Variation in alfalfa (Medicago sativa L.) germplasm for resistance to oncogenic and disarmed strains of A. tumefaciens (Smith & Townsend) Conn was tested in plant populations representing the nine distinct sources of alfalfa germplasm introduced into North America and used to develop modern varieties. For each of the virulent strains there was a positive correlation (p=0.05) of resistance to tumorigenesis with the trait for fall dormancy. There was also a significant correlation between plants selected for ineffective nodulation and resistance to tumorigenesis suggesting that the genetic loci required for successful symbiosis are also involved in tumorigenesis. Tissue explants of seedlings from the nine diversity groups were tested for transformation by three disarmed strains containing a plasmid with the scorable marker -glucuronidase. The strong correlation between dormancy and resistance to oncogenic strains was not observed with disarmed strains. However, there was a strong germplasm-strain interaction or transformation and embryogenesis in a highly embryogenic genotype. Thus, transformation at the whole plant level is germplasm dependent while in tissue culture the bacterial strain used is the critical variable for successful transformation.Abbreviations pTi tumor-inducing plasmid - GUS -glucuronidase     

Genetic engineering of grass cell wall polysaccharides for biorefining

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass‐derived lignocellulose processing industry.     

Deciphering the evolutionary interplay between subgenomes following polyploidy: A paleogenomics approach in grasses

How did plant species emerge from their most recent common ancestors (MRCAs) 250 million years ago? Modern plant genomes help to address such key questions in unveiling precise species genealogies. The field of paleogenomics is undergoing a paradigm shift for investigating species evolution from the study of ancestral genomes from extinct species to deciphering the evolutionary forces (in terms of duplication, fusion, fission, deletion, and translocation) that drove present‐day plant diversity (in terms of chromosome/gene number and genome size). In this review, inferred ancestral karyotype genomes are shown to be powerful tools to (1) unravel the past history of extant species by recovering the variations of ancestral genomic compartments and (2) accelerate translational research by facilitating the transfer of genomic information from model systems to species of agronomic interest.     

Polarization predicts the pattern of cellularization in cereal endosperm

Summary The endosperm of cereal grains develops as a multinucleate mass of wall-less cytoplasm (syncytium) that lines the periphery of the central cell before becoming cellular. The pattern of initial wall formation is precisely oriented and is followed by a round of precisely oriented formative cell division that gives rise to initials for the two tissues of endosperm. The initial anticlinal walls form at boundaries of nuclear-cytoplasmic domains (NCDs) defined by radial microtubules emanating from nuclei in the syncytium. Polarized growth of the NCDs in axes perpendicular to the embryo sac wall and centripetal elongation of the anticlinal walls results in a single layer of open ended alveoli overtopped by the remaining syncytial cytoplasm. This arboreal stage, so named because the elongate nucleate columns of cytoplasm resemble an orchard of trees, predicts the division polarity of the imminent formative division. Mitosis occurs as a wave which, like polarization, moves in both directions from ventral to dorsal. Spindles are oriented parallel to the long axis of the alveoli and cell plates give rise to periclinal walls. The outer daughter nuclei (aleurone initials) are thus completely enclosed by walls and the inner nuclei (starchy endosperm initials) are in alveoli adjacent to the central vacuole.     

Role of oxidative stress in cereal protoplast recalcitrance

Cereal leaf protoplasts are often extremely unstable in culture and usually lyse within 24 hours. Using the thiobarbituric acid test and the ferrous thiocyanate test we have shown that corn (Zea mays L. cv. Market Beauty) and wheat (Triticum aestivum L. cv. Benito) leaf protoplasts accumulate peroxides and peroxide degradation products during culture. This increase correlated with an increase in lipoxygenase activity. On the other hand, enzymes involved in detoxification of peroxides such as catalase and peroxidase decreased during culture. The occurrence of lipid peroxidation in leaf protoplasts is likely to be a consequence of a temporary imbalance in the enzymes involved in oxygen metabolism. It has previously been shown that the lipoxygenase inhibitor n-propyl gallate stabilizes the protoplasts in culture and so peroxidation is likely to be the cause of leaf protoplast instability. Protoplasts obtained from suspension cultures are stable in culture and do not undergo lipid peroxidation. This stability is due to a decrease in lipoxygenase activity and increases in catalase and peroxidase activity after protoplast isolation.Abbreviations MDA malonaldehyde - 2,4-D 2,4-dichlorophenoxyacetic acid - PVP polyvinylpolypyrrolidone Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Wide hybridization experiments in cereals

Summary Wide hybridization is a useful tool in plant breeding, but little is known about its possible range. For the cereals, wheat, barley and rye, this was tested with 15 different species of the Poaceae and Panicoideae. Embryo formation could be obtained with Agropyron repens, Alopecurus agrestis, Dactylis glomerata, Festuca glauca, Hordeum bulbosum, Lolium perenne, Pennisetum americanum, and Zea mays. As well, haploid as diploid embryos occurred. New embryo culture techniques should enable these embryos to grow to plants.     

转基因植物中的标记基因研究新进展

文章综述了转基因植物中标记基因研究的新进展,主要包括以下3个方面：第一是采用共转化、位点特异性重组和转座子等技术对传统抗性标记基因进行消除,以利于对同一作物进行多次转基因操作;第二是完善各种已应用的以糖类代谢酶基因、耐胁迫酶类基因和绿色荧光蛋白基因等为安全标记基因的转化体系,并大力研究、开发潜在的汞离子还原酶基因、叶绿体合成关键酶基因等作为安全标记基因;第三是着力发展无标记基因、无载体骨架的简单高效转化体系。此外,还展望了安全标记的应用前景。     

Inheritance of organelle genomes in citrus somatic cybrids

Restriction fragment length polymorphisms (RFLPs) were used for the characterization of citrus organelle inheritance in somatic cybrids produced during six different citrus protoplast fusions. All the cybrids in this work inherited their mitochondrial genome from the embryogenic fusion partner (callus or cell suspension). In some of the combinations, non-parental bands were observed among the mitochondrial configurations. In contrast, the cybrids inherited plastid DNA from either the embryogenic or the nonembryogenic (leaf) fusion partner. The relative abundance of organelle DNAs in the embryogenic and leaf cells was in accordance with these inheritance patterns. Stochastic processes may therefore influence the outcome of somatic cell fusions with respect to organelle genomes.     

Inhibition of different strains of enteropathogens in a lactic-fermenting cereal gruel

Twenty-eight strains of enteropathogens, including Campylobacter, Salmonella, Shigella, enterotoxigenic Escherichica coli (ETEC), Staphylococcus and Bacillus were added to cereal gruels prepared from low-tannin sorghum and inoculated with a lactic acid starter culture. Campylobacter strains were not detectable after 6 h, and Salmonella, Shigella and Staphylococcus strains not after 12 h. No viable cells of Bacillus strains were detected after 16 h of fermentation and the ETEC strains were completely inhibited after 24 h. No strain variability was observed within the different genera. In control gruels (no starter culture added), all the enteropathogens increased in number during incubation at 32°C except for the Campylobacter strains which decreased after 12 h of incubation.R. Kingamkono is with the Tanzania Food and Nutrition Centre, Dar-es-Salaam, Tanzania. E. Sjögren and B. Kaijser are with the Department of Clinical Bacteriology, Göteborg University, Gothenburg, Sweden. U. Svanberg is with the Chalmers University of Technology, c/o SIK, Box 5401, 5-402 29 Gothenburg, Sweden     

Advances in research of Achnatherum inebrians-Epichloë endophyte symbionts

有关醉马草(Achnatherum inebrians)内生真菌(Epichloë gansuensis, E. inebrians)共生体的研究, 代表了我国禾草内生真菌研究领域的重要方向, 使中国的醉马草-内生真菌与美国的苇状羊茅(Festuca arundinacea)-内生真菌(E. coenophiana)和新西兰的多年生黑麦草(Lolium perenne)-内生真菌(E. festucae var. lolii)成为禾草内生真菌国际三大研究分支。该文综述了近30年来对醉马草内生真菌共生体的系统研究, 包括: 内生真菌的分布、带菌率、检测方法、多样性, 内生真菌提高宿主的抗旱、耐寒、耐盐碱、耐重金属、抗虫、抗病等抗逆性及其机理, 共生体产生的生物碱等次生代谢物, 对草食动物的毒性, 及其在草地生态系统中的作用等。研究者实验证实了醉马草本身无毒, 只有当内生真菌与醉马草共生并产生麦角新碱和麦角酰胺等麦角类生物碱后才能导致采食醉马草家畜中毒。文章展望了醉马草内生真菌基因组学和功能分析, 利用杀菌剂杀死内生真菌进行醉马草脱毒, 利用无毒内生真菌菌株进行饲用醉马草新品种选育, 利用有毒醉马草内生真菌共生体进行抗虫防鸟的机场绿化新品种选育及生物源农药与医药开发等。     

Plant highly repeated satellite DNA: Molecular evolution,distribution and use for identification of hybrids

Relationships among genomes are often revealed by the occurrence of common or related satellite DNA (satDNA) types. A typical satDNA characterized by specific sites for one (or more) restriction endonuclease(s) is called ‘restriction satellite DNA’. Restriction satDNA comprises ‐ in addition to transposons and retrotransposable elements ‐ often highly repeated genome components present in most higher plants. Large arrays of satDNA elements are concentrated at subtelo‐meric and/or centromeric regions (intermingled with other retrotransposon‐derived elements), however, they can be also located as large intercalating blocks along the chromosome. The head‐to‐tail tandemly arranged repeat units (monomers) of satDNA mostly exhibit lengths of 160 to 180 bp or 320 to 370 bp, but other lengths were also found in plants. In particular, in interspecific hybrids between more distantly related species, which often exist only after polyploidization, the individual repetitive DNA of the crossing partners contribute to recombination and rearrangement processes in the hybrids, thereby stimulating genome evolution. Here, we concentrate on the possible origin, molecular evolution, organization and distribution of highly repeated satDNA in various higher plants with emphasis on hybrids and allopolyploids.     

Immunoreactivity in mammals of two typical plant glyco-epitopes,core alpha(1,3)-fucose and core xylose

The presence of nonmammalian core alpha(1,3)-fucose and core xylose glyco-epitopes on glycans N-linked to therapeutic glycoproteins produced in plants has raised the question of their immunogenicity in human therapy. We address this question by studying the distribution of these N-glycans in pea, rice, and maize (which are the crops intended for the production of therapeutic proteins) and by reinvestigating their immunogenicity in rodents. We found that immunization with a model glycoprotein, horseradish peroxidase, elicits in C57BL/6 mice and rats the production of antibodies (Abs) specific for core alpha(1,3)-fucose and core xylose epitopes. Furthermore, we demonstrated that about 50% of nonallergic blood donors contains in their sera Abs specific for core xylose, whereas 25% have Abs against core alpha(1,3)-fucose. These Abs probably result from sensitization to environmental antigens. Although the immunological significance of these data is too speculative at the moment, the presence of such Abs might introduce some limitations to the use of plant-derived biopharmaceutical glycoproteins, such as an accelerated clearance during human therapy.     

应用胚挽救技术获得三倍体柑橘植株

以3个柑橘异源四倍体体细胞杂种(即四倍体粗柠檬与哈姆林甜橙体细胞杂种,简称"HR";酸柚与粗柠檬体细胞杂种,简称"SR";墨西哥来檬与伏令夏甜橙体细胞杂种,简称"KV")为父本,分别与二倍体单胚性沙田柚进行有性杂交,在有性胚还没有完全败育以前,通过胚抢救技术进行三倍体植株培养(以四季柚花粉亲本为对照)。结果表明,处理的杂交组合直接成苗率极显著低于对照(P0.01),其中又以沙田柚×HR组合成苗率相对较高,达10.1%;较适合瘪籽沙田柚幼胚离体培养的培养基是MT+GA31.0mg/L+蜂皇浆200mg/L+水解乳蛋白250mg/L+蔗糖30g/L+琼脂0.7%;流式细胞分析仪检测结果显示,本试验成功获得了柑橘三倍体植株。