草坪草-高羊茅遗传育种研究进展

随着我国草坪业的快速发展 ,草坪草遗传育种工作越来越受到人们的关注。简要介绍了高羊茅的育种历史和现状 ,并对高羊茅植株再生体系的建立及遗传转化方面的研究进展进行了综述。此外 ,还提出了我国高羊茅育种的几点建议。     

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。     

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.     

农杆菌介导的高羊茅高效遗传转化和转基因植株再生

用带有质粒pDBA121(含hpt基因和bar基因)的农杆菌EHA 105转化高羊茅(Festucaarundinacea Schreb.)胚性悬浮细胞,建立了可重复的、高效的农杆菌介导的高羊茅遗传转化系统.商业用的除草剂Basta直接用于转化细胞的筛选.基因型、受体材料的类型、培养基成分和筛选剂影响农杆菌介导的转化频率.悬浮细胞的农杆菌转化效率为每克悬浮细胞再生2.85～10.9株转基因植株,大大高于基因枪法的高羊茅转化效率(2～5株).经PCR分析和Southern杂交检测表明,bar基因已整合进入高羊茅基因组,转基因植株Basta喷洒试验表明bar基因已成功地实现高水平的表达.此转化系统的建立为高效地将外源有用基因导入高羊茅并高效稳定地表达奠定了基础.     

富营养化水体灌溉对高羊茅生理生态特征的影响

通过采用两处不同浓度富营养化水体对高羊茅进行灌溉,研究了高羊茅生理生态特征响应。结果表明：稀释后的富营养化水体能显著促进高羊茅根系生长,当稀释浓度大于50%时,不利于株高的增长。富营养化水体的各个浓度对高羊茅叶绿素各项指标无显著影响。各富营养化水体稀释后灌溉高羊茅,对其游离脯氨酸、可溶性糖、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性的影响与对照相比差异不显著,外环水体能显著促进高羊茅过氧化物酶（POD）活性增加,稀释浓度为75%能使高羊茅过氧化氢酶（CAT）活性显著增加。综上分析表明,富营养化水体稀释后灌溉高羊茅是可行的,且生活区污水更适合用于高羊茅的灌溉。     

内生真菌感染对宿主植物高羊茅锌耐受性的影响

以感染内生真菌(Neotyphodium coenophialum)和未感染内生真菌的高羊茅(Festuca arundinacea Schreb.)为实验材料,在营养液中加入ZnSO4进行锌胁迫实验,分析内生真菌对宿主植物锌耐受性的影响。与未感染内生真菌的植株相比,内生真菌感染对高羊茅的总生物量没有显著增益作用,但增加了分蘖数和叶片延伸生长累积值。内生真菌感染降低了高羊茅中Zn2+的总含量,改变了锌在高羊茅中的分配,增加叶鞘中锌的含量,减少叶片中锌的含量。在高锌浓度下,内生真菌感染对净光合速率的变化没有影响,但是显著提高了其宿主的PSⅡ光化学效率(Fv/Fm)。总体来看,内生真菌感染改善宿主高羊茅的锌耐受性。     

高羊茅对高温的生理生态响应

分析了昼/夜38℃/30℃高温下2种高羊茅草坪草(凌志和交战Ⅱ)与狗牙根叶片的相对含水量、叶绿素含量、细胞膜脂过氧化、抗氧化系统以及光合作用等生理生态指标的变化.结果表明:随高温时间的延长,2种高羊茅的相对含水量、叶绿素含量、净光合速率和最大光能转化效率均呈降低趋势,而且凌志高羊茅的平均降幅小于交战Ⅱ高羊茅.经过9d的高温处理,凌志高羊茅叶片超氧化物歧化酶、过氧化物酶和过氧化氢酶的平均活性比交战Ⅱ高羊茅分别高19.7%、17.9%和17.7%,而凌志和交战Ⅱ高羊茅的净光合速率分别下降了60.7%和81.9%.与交战Ⅱ高羊茅相比,凌志高羊茅具有更高的光化学转换功能,有利于减轻高温对光合器官的损伤,使其保持较高的净光合速率.在整个高温处理过程中,狗牙根的各项生理生态指标随时间变化的差异不显著.狗牙根、凌志高羊茅和交战Ⅱ高羊茅对高温胁迫的适应能力由强到弱.     

保水剂对煤矸石基质上高羊茅生长及营养吸收的影响

为探索保水剂对煤矸石废弃地上高羊茅生长及营养特征的影响,按煤矸石与土壤质量比设3种基质梯度,每种基质分别添加3种质量的保水剂,设对照(CK),共10种处理。观测和分析不同处理下高羊茅的生长高度、生物量、营养特征及SPAD值(相对叶绿素含量),结果显示:保水剂主要影响高羊茅地上部分的生物量,而煤矸石与土壤的质量比主要影响地下部分的生物量;煤矸石与土壤质量比相同条件下,添加1 g/kg的保水剂即可促进高羊茅的生长,添加2 g/kg保水剂能使高羊茅尽快适应基质环境,使其日均生长率在出苗后第2周达到峰值。煤矸石基质中添加保水剂能促使高羊茅将煤矸石基质中的营养元素从植株地下部分向地上部分转移,供植株生长需要,基质中添加1 g/kg保水剂时高羊茅对磷的吸收效果最好。此外,添加保水剂能使叶片的SPAD值增加。综合比较不同处理下高羊茅的株高、生物量、营养特征及叶片的SPAD值认为,处理6(煤矸石与土壤质量比为750:250、基质中添加2 g保水剂)与处理8(煤矸石与土壤质量比为500:500、基质中添加1 g保水剂)是适合煤矸石废弃地上高羊茅生长的较好配比。     

翦股颖和高羊茅对铜、铅吸收及耐受性

测定了高羊茅和翦股颖两种草坪植物在Cu、Pb污染下发芽率、叶绿素、净光合速率和SOD的变化,以及两种草对Cu、Pb的吸收.结果表明, 重金属污染对高羊茅种子的萌发产生显著影响.高浓度Pb污染以及Pb、Cu复合污染对高羊茅的叶绿素影响很大,Cu单独污染对高羊茅叶绿素的抑制作用较小.翦股颖的叶绿素变化不显著.在3个Pb处理及350 mg·kg^-1 Cu处理下,翦股颖的SOD活性均高于对照,而只有在700 mg·kg^-1Pb 和350 mg·kg^-1 Cu+1 100 mg·kg^-1 Pb处理下高羊茅的SOD活性才升高.高羊茅的净光合速率受Cu污染影响较小,而受Pb污染影响较大.在Cu、Pb单独污染条件下,翦股颖净光合速率均呈下降趋势.从生理角度来看,翦股颖对Cu、Pb污染的耐受性强于高羊茅,而高羊茅对Cu污染的耐受性强于Pb.高羊茅Cu吸收量大,但Cu吸收量不随污染浓度升高而升高.翦股颖对土壤Pb的吸收能力较强, 且Pb吸收量随污染浓度的增加而增加.翦股颖和高羊茅可分别用于Pb和Cu污染土壤的治理.     

盐生境下硅对坪用高羊茅生物学特性的影响

干旱半干旱区草坪绿地的长期灌溉容易引发土壤次生盐渍化,提高草坪草在盐生境下的生长发育能力是应对土壤次生盐渍化的主要途径之一.采用盆栽试验研究了盐生境下硅对坪用高羊茅(Festuca arundinaea)生物学特性的影响.结果表明,向盐生境土壤中添加不同浓度硅均提前高羊茅出苗时间2d,加快出苗速率,增加出苗总数,提高保苗率,且总出苗率和保苗率随着硅浓度增加而显著增大(P＜0.05),这说明盐生境下向土壤添加硅改善了幼苗完全死亡的现象.盐生境下硅显著增加了高羊茅叶长,株高和分蘖数,但对叶宽影响不显著,说明硅能够促进高羊茅生长,但对草坪绿地的质地影响不大.虽然硅能显著增加高羊茅总生物量(P＜0.05),但较低浓度时促进其茎叶生长,高浓度促进其根系生长.硅在高羊茅体内的沉积量随施入硅增加而增大,但其茎叶和根系内硅含量不超过3.0％,且根系内硅含量约为茎叶内硅含量的2倍.结果显示,硅提高了坪用高羊茅在盐生境下的适应能力,这为以后草坪绿地管理中应对土壤潜在盐渍化的问题提供了一定的科学依据.     

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Tall fescue genomic SSR markers: development and transferability across multiple grass species

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT) _n repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

草坪草生物技术研究进展

概述了草坪草植株再生体系和遗传转化体系建立的方法和进展.通过愈伤组织培养、悬浮细胞培养和原生质体培养方法对草坪草的一些种已建立较为完善的植株再生体系.在建立再生体系的基础上,利用原生质体融合、农杆菌介导、基因枪和碳化硅纤维介导等转基因方法在一些草坪草种上建立了遗传转化体系并获得了有一定价值的转基因植株.最后,对草坪草转基因存在的问题和前景作了讨论.     

Genetic diversity in a world germplasm collection of tall fescue

Festuca arundinacea Schreb., commonly known as tall fescue, is a major forage crop in temperate regions. Recently, a molecular analysis of different accessions of a world germplasm collection of tall fescue has demonstrated that it contains different species from the genus Festuca and allowed their rapid classification into the three major morphotypes (Continental, Mediterranean and Rhizomatous). In this study, we explored the genetic diversity of 161 accessions of Festuca species from 29 countries, including 28 accessions of INTA (Argentina), by analyzing 15 polymorphic SSR markers by capillary electrophoresis. These molecular markers allowed us to detect a total of 214 alleles. The number of alleles per locus varied between 5 and 24, and the values of polymorphic information content ranged from 0.627 to 0.840. In addition, the accessions analyzed by flow cytometry showed different ploidy levels (diploid, tetraploid, hexaploid and octaploid), placing in evidence that the world germplasm collection consisted of multiple species, as previously suggested. Interestingly, almost all accessions of INTA germplasm collection were true hexaploid tall fescue, belonging to two eco-geographic races (Continental and Mediterranean). Finally, the data presented revealed an ample genetic diversity of tall fescue showing the importance of preserving the INTA collection for future breeding programs.     

Molecular characterisation and interpretation of genetic diversity within globally distributed germplasm collections of tall fescue (Festuca arundinacea Schreb.) and meadow fescue (F. pratensis Huds.)

Allohexaploid tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum [Schreb.] Darbysh.) is an agriculturally important grass cultivated for pasture and turf world-wide. Genetic improvement of tall fescue could benefit from the use of non-domesticated germplasm to diversify breeding populations through the incorporation of novel and superior allele content. However, such potential germplasm must first be characterised, as three major morphotypes (Continental, Mediterranean and rhizomatous) with varying degrees of hybrid interfertility are commonly described within this species. As hexaploid tall fescue is also a member of a polyploid species complex that contains tetraploid, octoploid and decaploid taxa, it is also possible that germplasm collections may have inadvertently sampled some of these sub-species. In this study, 1,040 accessions from the publicly available United States Department of Agriculture tall fescue and meadow fescue germplasm collections were investigated. Sequence of the chloroplast genome-located matK gene and the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) permitted attribution of accessions to the three previously known morphotypes and also revealed the presence of tall fescue sub-species of varying ploidy levels, as well as other closely related species. The majority of accessions were, however, identified as Continental hexaploid tall fescue. Analysis using 34 simple sequence repeat markers was able to further investigate the level of genetic diversity within each hexaploid tall fescue morphotype group. At least two genetically distinct sub-groups of Continental hexaploid tall fescue were identified which are probably associated with palaeogeographic range expansion of this morphotype. This work has comprehensively characterised a large and complex germplasm collection and has identified genetically diverse accessions which may potentially contribute valuable alleles at agronomic loci for tall fescue cultivar improvement programs.     

草坪植物遗传转化的研究进展

从草坪植物组织培养与植株再生体系的建立、转基因的研究现状以及转基因育种的应用前景和存在的问题入手,综述了近年来草坪植物遗传转化的研究进展,并对草坪植物遗传转化的几种主要方法作了较为详细的阐述。