针叶树遗传工程:微弹和农杆菌介导的基因转移及其在未来林业中的应用(英文)

林业生物技术的研究已经取得了许多重要的进展。遗传转化和转基因树的应用是当代林业研究中的重要领域。尽管微弹和农杆菌介导的转化已经被应用于重要的针叶树种,转基因植株仅仅在少数几个针叶树种上获得成功。大多数转基因针叶树来自微弹介导的转化,例如转基因的挪威云杉、白云杉、黑云杉和辐射松等。利用转基因技术可以改良树的形状、生长率、木材质量、抗虫性、抗除草剂能力和对不良环境的抵抗力。本文对遗传转化技术在这些领域中所取得的进展做了简短的综述。     

南林895杨抗旱耐盐基因DREB1C的转化

以美洲黑杨杂种优良无性系南林895杨为转基因受体材料,利用农杆菌介导法转化与逆境胁迫相关的DREB1C基因.经潮霉素筛选,共获得80株抗性植株,经PCR及RT-PCR检测有30株抗性植株呈阳性,初步证明DREB1C基因已整合到南林895杨基因组中.抗性试验结果初步表明,转基因植株的抗旱、耐盐性均有所提高.     

农杆菌介导的甜橙基因转化系统建立研究

以3个不同品种甜橙上胚轴为对象，进行了农杆菌介导的基因转化影响因素研究。研究结果表明：红心橙、柳橙、新会橙卡那选择浓度分别为140、100、80μg／mL；不同品种外植体达最高Gus瞬时表达阳性率所需的AS浓度不同，红心橙、柳橙、新会橙分别为150、150、100μmol／L；红心橙、柳橙、新会橙的最适预培养时间分别为2、2、3天，预培养基成分为不定芽分化培养基有利于外植体Gus阳性率的提高；实生苗苗龄以20～25天为宜。     

火炬松丛生芽诱导及植株再生

采用火炬松带子叶项芽为外植体建立了组织培养再生体系。结果表明,火炬松丛生芽的形成受诱导培养基中激素浓度及外植体年龄的影响较大。适合火炬松丛生芽诱导的培养基为改良GD 6-BA 4 ms／L NAA 0．02 mg/L(诱导率86．7％),截取外植体的最佳苗龄为14-21 d。已分化的丛生芽继代培养在无6-BA但附加0．5- 1．0 g/L活性炭或0．02-0．05 mg/L NAA的培养基上伸长很快。伸长的丛生芽接种在附加0．05 ms／L NAA的1／2改良GD培养基中,1个月后有12．5％产生不定根。     

微弹介导的火炬松遗传转化

本文建立了一个微弹介导的火炬松遗传转化系统。这个系统解决了火炬松遗传转化过程中存在的许多困难。运载抗虫基因的质粒载体经微弹转化法进入火炬松成熟合子胚,然后在添加了卡那霉素的培养基上从转化的成熟胚上诱导出有器官发生潜力的愈伤组织,再从转化的愈伤组织上产生转基因植株。利用这一系统生产的转基因植株已经被随机扩增技术、Southern杂交技术和虫试验所证实,并且转化的植株已在土壤中成活。图3表2参28。     

火炬松胚性愈伤组织诱导和植株再生的研究

火炬松胚性愈伤组织诱导和植株再生的研究唐巍欧阳藩（中国科学院化工冶金研究所生化工程国家重点实验室北京１０００８０）郭仲琛（中国科学院植物研究所北京１０００９３关键词火炬松,体细胞胚胎发生,植株再生本文于１９９６年１０月２８日收到。国家“８６３”资...     

四倍体刺槐转甜菜碱醛脱氢酶基因的研究

试验以所建立的四倍体刺槐高频再生体系为基础,通过农杆菌介导法转化甜菜碱醛脱氢酶(BADH)基因,以GUS染色组织分析法为依据探讨了影响转化效率的各种因素,建立了优化转化体系,结果如下:20mg·L~(-1)乙酞丁香酮可显著提高转化效率;菌液浓度OD_(600)值0.3～0.7、预培养2d为宜;转化后暗培养对转化效率没有影响。并在以上研究基础上,成功地建立了高效、可重复的遗传转化体系,选择培养基上头孢霉素400mg·L~(-1)可有效地抑制农杆菌;卡那霉素50mg·L~(-1)时愈伤组织的白化死亡率达96.4％。经PCR检测,外源基因已成功的整合到植株的基因组DNA中,获得了15个转基因株系。     

火炬松基因资源的育种值预测与选择

以引自美国的258个不同改良程度的火炬松家系作为试验材料,利用基于混合线性模型的 BLUP 方法对258个家系14年生树高和胸径的育种值进行预测。首先以不同的模型分别预测半同胞家系和全同胞家系的育种值,再通过构建亲缘关系矩阵预测所有个体的育种值,对各类群家系和个体育种值的差异进行分析。14年生时,在参试的各个类群中,入选率同为15%的情况下,各类群根据胸径育种值共选出优良家系39个;全同胞家系类群入选家系的胸径遗传增益最高,高于群体平均值1.40 cm,达8.56%。以配合选择方式选出优良单株77个,入选率为1%。入选的优良家系可从美国重复引种,为火炬松良种生产服务。根据火炬松核心-主群体育种系统的需要,入选的优良单株可用以充实第2代育种群体,其中10个进入核心育种群体,其余67个进入主群体。精确的 BLUP预测值和基于个体育种值的选择将提高遗传改良进程中的选择效率和遗传增益,但在利用这些先进的统计分析方法提升林木良种选育效率的同时,必须对育种方案与试验设计进行改进完善。当参试家系数较多时,可采用平衡不完全区组设计,把所有参试家系统一在一个试验中,科学安排田间试验,以获得最大的成效。     

抗生素对火炬松的器官发生植株再生的影响(英文)

本文研究了氨苄青霉素、羧苄青霉素和头孢霉素等3种抗生素对火炬松愈伤组织的生长和分化及不定芽生根的影响。结果表明,头孢霉素最有利于愈伤组织的诱导和生长,羧苄青霉素最有利于芽的分化,氨苄青霉素降低了不定芽的生根频率。所有试验的3种抗生素提高愈伤组织的形成和芽再生,但降低了芽的生根频率。这些结果表明,选择合适的抗生素对优化火炬松遗传转化体系有重要作用。图3表4参25。     

火炬松种子园经营管理技术的研究

本文从无性系管理、密度管理、土壤管理、花粉管理、树体管理、球果虫害管理等方面提出了火炬松种子园经营管理技术。     

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma…     

Genetic transformation of loblolly pine using mature zygotic embryo expiants byAgrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of β-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected withAgrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of β-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficientAgrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.     

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

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火炬松体细胞胚胎发生和干化体细胞的氧化物酶活性(英文)

培养于附加2,4-D、BA和KT的愈伤组织诱导培养基上的火炬松成熟合子胚在培养3-9周后形成白色、半透明、有光泽的粘性愈伤组织。这类愈伤组织形成于成熟合子胚的子叶,但当用NAA或者IBA代替愈伤组织诱导培养基中的2,4-D时,它的诱导频率明显降低。这种粘性愈伤组织在分化培养基上形成体细胞胚。体细胞胚经过去50μm ABA和8.5%PEG600处理后成为耐干化胚。扫描电镜观察表明,萌发处理36小时后,耐干化胚恢复到干化处理之前的状态且大小和形态正常,而不耐干化胚不能恢复到干化处理之前的状态且表面撕裂。过氧化物酶活性的分析结果表明,耐干化胚有更高的过氧化物酶活性。耐干化胚的高过氧化物酶活性可能与催化H2O2的分解和保护体细胞胚免受氧化的伤害有关。     

Effect of magnetic treatment on seed germination of loblolly pine (Pinus taeda L.)

Based on preliminary experiments, the speed of germination (SG) was in general increased for Pinus taeda L. seeds treated with a static magnetic field (B = 150 mT) for 10, 30 and 60 min. Negative impact was obtained for seeds treated with SMF for 24 h and 48 h. Mean germination time (MGT), the SG, and time required to obtain 10%, 25%, 50%, 75%, and 90% of seeds to germinate (T₁₀–T₉₀) were calculated. Results showed a reduction of the MGT and T₁₀–T₉₀ for seeds treated with SMF for 10, 30, and 60 min; therefore, the germination speed was increased. Among various magnetic treatments, loblolly pine seeds treated with S3 (SMF60 min) yielded the peak performance.     

Durability of selected mulches,their ability to control weeds,and influence growth of loblolly pine seedlings

Several mulches of natural, synthetic, or blends of natural and synthetic fibers were tested around newly planted loblolly pine (Pinus taeda L.) seedlings on a sheared and windrowed site in central Louisiana, USA. The vegetation was primarily winter annuals, some residual grasses and forbs, and sparse woody regrowth. Study 1 was rotary mowed just prior to planting in March 1992, and 35 mulches and an untreated check were established. In Study 2, 15 mulches and an untreated check were established in a 1-year-old rough in March 1993. In both studies, a single loblolly pine seedling formed each plot established in a randomized complete block design, with 10 blocks as replicates. Each block was planted with a separate open-pollinated loblolly pine family.Nearly all mulches had deteriorated to some extent after three growing seasons. Synthetic mulches were generally more durable than the natural or natural/synthetic mulches. Mulching eliminated the established vegetation and germinants, and vegetation did not readily reestablish following the deterioration of a mulch. The soil seed bank apparently was not sufficient to regenerate areas that were once covered with mulch and many of the natural materials deteriorated into a fibrous cover that acted like a natural litter layer. Both of these residual weed control effects -- insufficient soil seed bank and formation of a fibrous cover -- were important in stopping vegetation from reestablishing after a mulch had deteriorated. After three growing seasons, the loblolly pine seedlings generally grew better if mulches were used.     

Micropropagation of loblolly pine by somatic organogenesis andRAPD analysis of regenerated plantlets

IntroductionOrganogenesis and somatic embryogenesis haveben regarded as the in vitro system of choice withthe Pbtenhal for eventual mass propagation of superior and gen6tically engineering forest tree genotypesIn both conal6rous and hardwood (Gupts et al. 1991,Becwar et al. 1995). Somatic embryogenesis andorganeqenesis have been induced from more than 30ired speCies in conifers, but plant regeneration viaSOmahc embryogenesis and organogenesis remainsdffeutt cd a low rqeneration frequency (A…     

Preliminary studies on establishment of genetic transformation system in loblolly pine

A transformation system was established for loblolly pine (Pinus taeda L.) mature zygotic embryos usingAgrobacterium tumefaciens. The gene coding for the β-glucuronidase (GUS) gene was introduced into loblolly pine tissues and its transient expression was detected with histochemical staining. The influences of different genotypes.Agrobacterum concentrations. and cocultivation time on GUS expression and kanamycin resistant callus and shoot regeneration were investigated. The results showed that the highest GUS expression frequency (16.3%) and shoot regeneration frequency (78%) were obtained from genotype 9–1003 withAgrobacterium concentration decreased 9 times and cocultivation time of 56 hours respectively. GUS expression was obtained in all genotypes tested. The successful expression of the GUS gene in different genotypes suggested that it will be a useful transformation system for loblolly pine. (Responsible Editor: Chai Ruihai)     

Fertilization,weed control,and pine litter influence loblolly pine stem productivity and root development

Following site preparation, three cultural treatments and three open-pollinated loblolly pine (Pinus taeda L.) families were studied on a gently sloping Beauregard silt loam in central Louisiana. The treatments were: (1) fertilization (either broadcast application of 177 kg N and 151 kg P/ha or none); (2) herbicide application (either broadcast application of herbicides during the first through third growing seasons, and felling of a few, scattered volunteer hardwood trees greater than 2.5 cm dbh during the third growing season or none); and (3) litter application (either broadcast application of 37 Mg/ha (oven-dried weight) of pine straw over the plots to form a 10 to 15 cm layer or none). The subplot treatment was planting stock, where in November 1988, 28-week-old container-grown loblolly pine seedlings from three open-pollinated families were randomly assigned to planting locations. Through five growing seasons, fertilization and weed control with herbicides resulted in the greatest loblolly pine productivity, but the use of herbicides severely reduced other vegetation. Applying litter, which was less effective than herbicides as a weed control treatment, increased the presence of blackberry (Rubus spp.) when herbicides were not applied. Applying litter resulted in a decrease and fertilization resulted in an increase in the number and length of live lateral roots. Soil temperature was reduced by litter application. Treatment responses were not influenced by loblolly pine family.