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.     

高羊茅和其他羊茅植株再生与遗传转化研究进展

高羊茅、紫羊茅和草地羊茅均为很重要的多年生冷季型牧草与草坪草,生物技术在其品种改良中具有很大的应用潜力。30年来,三种羊茅的组织培养、胚性培养物的长期保存以及遗传转化等研究取得了较大进展,已建立起多种植株再生体系和遗传转化技术,但作为单子叶植物,这些草种的组织培养和转基因遗传改良也还存在一些问题。本文就以上几方面的内容进行了综述。     

Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

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.     

Is genetic engineering ever going to take off in forage,turf and bioenergy crop breeding?

Background

Genetic engineering offers the opportunity to generate unique genetic variation that is either absent in the sexually compatible gene pool or has very low heritability. The generation of transgenic plants, coupled with breeding, has led to the production of widely used transgenic cultivars in several major cash crops, such as maize, soybean, cotton and canola. The process for regulatory approval of genetically engineered crops is slow and subject to extensive political interference. The situation in forage grasses and legumes is more complicated.

Scope

Most widely grown forage, turf and bioenergy species (e.g. tall fescue, perennial ryegrass, switchgrass, alfalfa, white clover) are highly self-incompatible and outcrossing. Compared with inbreeding species, they have a high potential to pass their genes to adjacent plants. A major biosafety concern in these species is pollen-mediated transgene flow. Because human consumption is indirect, risk assessment of transgenic forage, turf and bioenergy species has focused on their environmental or ecological impacts. Although significant progress has been made in genetic modification of these species, commercialization of transgenic cultivars is very limited because of the stringent and costly regulatory requirements. To date, the only transgenic forage crop deregulated in the US is ‘Roundup Ready’ (RR) alfalfa. The approval process for RR alfalfa was complicated, involving several rounds of regulation, deregulation and re-regulation. Nevertheless, commercialization of RR alfalfa is an important step forward in regulatory approval of a perennial outcrossing forage crop. As additional transgenic forage, turf and bioenergy crops are generated and tested, different strategies have been developed to meet regulatory requirements. Recent progress in risk assessment and deregulation of transgenic forage and turf species is summarized and discussed.     

用RAPD分析多年生黑麦草品种间遗传多样性

多年生黑麦草是世界上应用最广泛的冷季型草坪草兼牧草之一,具有建植快速,分蘖能力强,适口性良好等特点。为了评价其品种间的遗传多样性,选取了多年生黑麦草的14个草坪型品种和2个意大利黑麦草品种用RAPD进行遗传多样性分析,并且对每个品种的几项坪用特性进行了观察。61个引物共扩增出408条带,多态条带比率(PPB)为89．95％。聚类分析结果表明,多年生黑麦草品种之间的遗传多样性较低,相似系数分布于44．55％～95．69％之间。一年生黑麦草的2个品种TopOne和A,vance没有单独聚成一类,而是与14个多年生黑麦草品种混合聚在一起,而且Avance与其他多年生黑麦草之间的相似系数较高。聚类结果与形态特征没有明确的对应关系。     

几种草坪草的引种栽培试验研究

通过对引种栽培的8种草坪草的物候期、覆盖度、再生速度、品质特性及抗逆性等方面进行分析研究和综合评价,筛选出适合北方温带环境条件下栽培的几种优良草坪草品种——新哥来德(Poa pratensis cv．Nugtade)、枪手股(P.pratensis cv．BlueChip)、午夜(P．pratensis cv．Midnight)、帕特(Agrostis tenuis cv．Putter)、贝克(碧西)(Festuca arundinacea cv．Plxie)等。对陕西关中草坪草的引种和适地栽植提供一定的理论依据。     

Tall fescue EST-SSR markers with transferability across several grass species

Tall fescue (Festuca arundinacea Schreb.) is a major cool season forage and turf grass in the temperate regions of the world. It is also a close relative of other important forage and turf grasses, including meadow fescue and the cultivated ryegrass species. Until now, no SSR markers have been developed from the tall fescue genome. We designed 157 EST-SSR primer pairs from tall fescue ESTs and tested them on 11 genotypes representing seven grass species. Nearly 92% of the primer pairs produced characteristic simple sequence repeat (SSR) bands in at least one species. A large proportion of the primer pairs produced clear reproducible bands in other grass species, with most success in the close taxonomic relatives of tall fescue. A high level of marker polymorphism was observed in the outcrossing species tall fescue and ryegrass (66%). The marker polymorphism in the self-pollinated species rice and wheat was low (43% and 38%, respectively). These SSR markers were useful in the evaluation of genetic relationships among the Festuca and Lolium species. Sequencing of selected PCR bands revealed that the nucleotide sequences of the forage grass genotypes were highly conserved. The two cereal species, particularly rice, had significantly different nucleotide sequences compared to the forage grasses. Our results indicate that the tall fescue EST-SSR markers are valuable genetic markers for the Festuca and Lolium genera. These are also potentially useful markers for comparative genomics among several grass species.Electronic Supplementary Material Supplementary material is available for this article at .     

Advanced phenotyping offers opportunities for improved breeding of forage and turf species

Background and Aims

Advanced phenotyping, i.e. the application of automated, high-throughput methods to characterize plant architecture and performance, has the potential to accelerate breeding progress but is far from being routinely used in current breeding approaches. In forage and turf improvement programmes, in particular, where breeding populations and cultivars are characterized by high genetic diversity and substantial genotype × environment interactions, precise and efficient phenotyping is essential to meet future challenges imposed by climate change, growing demand and declining resources.

Scope

This review highlights recent achievements in the establishment of phenotyping tools and platforms. Some of these tools have originally been established in remote sensing, some in precision agriculture, while others are laboratory-based imaging procedures. They quantify plant colour, spectral reflection, chlorophyll-fluorescence, temperature and other properties, from which traits such as biomass, architecture, photosynthetic efficiency, stomatal aperture or stress resistance can be derived. Applications of these methods in the context of forage and turf breeding are discussed.

Conclusions

Progress in cutting-edge molecular breeding tools is beginning to be matched by progress in automated non-destructive imaging methods. Joint application of precise phenotyping machinery and molecular tools in optimized breeding schemes will improve forage and turf breeding in the near future and will thereby contribute to amended performance of managed grassland agroecosystems.     

Floral inhibition in red fescue (Festuca rubra L.) through expression of a heterologous flowering repressor from Lolium

Extension of the vegetative growth phase through delay of flowering is an important goal in today's breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species.     

高羊茅和其他羊茅植株再生与遗传转化研究进展

高羊茅、紫羊茅和草地羊茅均为很重要的多年生冷季型牧草与草坪草,生物技术在其品种改良中具有很大的应用潜力.30年来,三种羊茅的组织培养、胚性培养物的长期保存以及遗传转化等研究取得了较大进展,已建立起多种植株再生体系和遗传转化技术,但作为单子叶植物,这些草种的组织培养和转基因遗传改良也还存在一些问题.本文就以上几方面的内容进行了综述.     

Characterization and practical use of self-compatibility in outcrossing grass species

BackgroundSelf-incompatibility (SI) systems prevent self-fertilization in several species of Poaceae, many of which are economically important forage, bioenergy and turf grasses. Self-incompatibility ensures cross-pollination and genetic diversity but restricts the ability to fix useful genetic variation. In most inbred crops it is possible to develop high-performing homozygous parental lines by self-pollination, which then enables the creation of F₁ hybrid varieties with higher performance, a phenomenon known as heterosis. The inability to fully exploit heterosis in outcrossing grasses is partially responsible for lower levels of improvement in breeding programmes compared with inbred crops. However, SI can be overcome in forage grasses to create self-compatible populations. This is generating interest in understanding the genetical basis of self-compatibility (SC), its significance for reproductive strategies and its exploitation for crop improvement, especially in the context of F₁ hybrid breeding.ScopeWe review the literature on SI and SC in outcrossing grass species. We review the currently available genomic tools and approaches used to discover and characterize novel SC sources. We discuss opportunities barely explored for outcrossing grasses that SC facilitates. Specifically, we discuss strategies for wide SC introgression in the context of the Lolium–Festuca complex and the use of SC to develop immortalized mapping populations for the dissection of a wide range of agronomically important traits. The germplasm available is a valuable practical resource and will aid understanding the basis of inbreeding depression and hybrid vigour in key temperate forage grass species.ConclusionsA better understanding of the genetic control of additional SC loci offers new insight into SI systems, their evolutionary origins and their reproductive significance. Heterozygous outcrossing grass species that can be readily selfed facilitate studies of heterosis. Moreover, SC introduction into a range of grass species will enable heterosis to be exploited in innovative ways in genetic improvement programmes.     

高温胁迫对两种冷季型草坪草生理生化特性的影响

为研究高温胁迫对冷季型草坪草生理生化指标的影响,通过盆栽试验测定了昼夜(38/28℃)两种温度下两种冷季型草坪草的5种生理生化指标(质膜透性、脯氨酸含量、过氧化物酶(POD)活性、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量)。结果表明:随着胁迫时间的延长,各品种叶片的相对电导率、丙二醛含量和游离脯氨酸含量均呈递增趋势,其增加幅度与胁迫时间呈正相关,胁迫后各指标相对于对照均有了显著的增加(P<0.05);过氧化物酶和超氧化物歧化酶活性则呈先上升后下降的趋势。高羊茅属3个品种的耐热性大于早熟禾属各品种的耐热性,其中高羊茅属的RebleⅣ和早熟禾属的Blue Sap-phire的耐热性优于种间其他品种。     

An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent validation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation.     

八种引种草坪植物的抗寒性比较研究

通过测定引种的八种草坪植物越冬存活率以及叶和越冬器官(根、根茎及分蘖节)质膜透性的变化、可溶性糖积累量、脯氨酸积累量等指标,得出:可溶性糖积累量是比较越冬器官抗寒性的有效生理生化指标,质膜透性的变化是比较叶器官抗寒性的有效生理生化指标;对八种引种草坪植物叶片进行过氧化物同工酶分析,得出过氧化物同工酶区带条数只能在一定程度上反映草坪植物抗寒性的大小,用其作为比较植物抗寒性的参照指标。     

Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula

Legumes are second only to grasses in worldwide economic importance, and understanding their molecular genetics is vital to the breeding of important grain and forage legumes. Over the past decade, Medicago truncatula has been selected as a model plant in which to study biological processes that are unique and pertinent to legumes, and that cannot easily be studied in Arabidopsis. Here, we discuss the most common tools for introducing and analyzing genetic mutations in M. truncatula. Because transformation and regeneration are still bottlenecks in studying a legume species, large-scale insertional mutagenesis poses a major challenge in M. truncatula. We discuss the tobacco retrotransposon Tnt1 as a viable and attractive option for introducing multiple independent insertions per plant for saturation mutagenesis.     

In vitro regeneration and genetic manipulation of grasses

Regeneration of plants from cultured cells is an important and essential component of plant biotechnology. Advances in the recovery of plants from cultured cells and protoplasts of grasses, and in genetic transformation provide challenging opportunities for the genetic manipulation and improvement of this most important group of food plants.     

呼伦贝尔草地野生禾本科牧草资源及资源评价

对呼伦贝尔草地野生禾本科牧草的属种组成、生长习性及饲用价值进行了研究。结果表明,呼伦贝尔草地现有禾本科牧草42属112种,这些禾本科牧草大多为多年生草本植物,适应典型草原和丘陵草甸草原生境,饲用价值高,适口性良好,是植被组成中的重要组成成分,生态利用前景广阔,是本地区重要的牧草资源。对其资源进行评价,为其合理开发利用提供理论依据。     

Flowering does not decrease vegetative competitiveness of Lolium perenne

The theory of life-history evolution commonly assumes a trade-off between sexual and vegetative reproduction. Hence, production of flowers and fruits should have measurable costs in terms of reduced vegetative growth. This trade-off may be meaningful for breeding of forage and turf grasses as reduced flowering could free resources and increase productivity. But if so, less-flowering cultivars might be more competitive and invade natural swards. We tested for costs of sexual reproduction on vegetative propagation and competitiveness of the perennial grass Lolium perenne, one of the most important forage and turf grasses worldwide. We used the differences in vernalisation requirement between northern and southern European provenances to manipulate the degree of flowering. Over three growing seasons, we counted the number of flower stems and measured the clone diameter. The vernalisation treatments were successful in producing clones with largely differing degrees of flowering. However, we found no negative correlation between flowering and vegetative propagation and competitiveness. Early and strongly flowering southern provenances showed less clonal growth and higher mortality, but within provenances the response of clone diameter to flowering was positive or neutral. We conclude that investment of resources into flowering has no measurable costs on vegetative propagation and competitiveness of L. perenne. The apparent lack of costs of sexual reproduction could be explained by bet-hedging strategy that is focused on survival and growth rather than reproductive effort in order to maximise the life-time fitness.     

Genetic and molecular basis of grass cell-wall biosynthesis and degradability. III. Towards a forage grass ideotype

Lignification of cell walls is the major factor controlling the digestibility of forage grasses. Thus far, from QTL analysis, about 15 locations involved in cell-wall lignification or digestibility have been identified in the maize genome, many of which colocalise with QTLs involved in corn borer susceptibility. Genetic diversity for enhancing cell-wall digestibility in maize must be identified in novel germplasm, but genetic engineering is also a relevant way both to design specific cell-wall characteristics for improved digestibility and to identify genes involved in these traits for further discovery of alleles of interest in grass germplasm.