根癌农杆菌介导的马铃薯转化系统的优化

以马铃薯品种PB 04的叶片和茎段为外植体,对其再生和农杆菌介导的遗传转化体系进行了研究。分别比较不同转化条件(菌液浓度、感染时间、预培养及共培养时间)对马铃薯遗传转化效率的影响,以及选择压浓度和加入时间对抗性愈伤出芽的影响,结果表明:(1)在玉米素(ZT)2 m g/L,萘乙酸(NAA)0.01 m g/L条件下能有效地提高马铃薯愈伤组织分化率;(2)外植体经过2 d的预培养,在OD600=0.8的农杆菌菌液中侵染10 m in后,抗性愈伤诱导率最高。(3)头孢霉素(C ef)比羧苄青霉素(C ar)更适合用于马铃薯外植体的遗传转化。(4)农杆菌侵染后恢复培养5~7 d再加入卡那霉素(Km)50 m g/L能有效地提高外植体的转化率。     

黑果枸杞高效植株再生与遗传转化体系的建立

为建立高效的黑果枸杞组织培养与遗传转化体系,本研究以黑果枸杞叶片、茎节、茎段为外植体,在含有不同浓度组合的NAA和BAP培养基上进行愈伤组织和不定芽诱导比较;选择黑果枸杞叶片为材料,采用农杆菌介导法,将带有绿色荧光蛋白基因(sGFP)的双元表达载体导入黑果枸杞,探讨侵染时间和抗生素浓度对遗传转化效果的影响。结果表明,黑果枸杞的最适植株再生体系为:以叶片为外植体,MS基本培养基中添加0.5 mg·L^-1NAA和0.3 mg·L^-1BAP,愈伤组织诱导率为88.56%,不定芽诱导率为54.54%,将不定芽置于1/2MS培养基中进行生根诱导,生根率为95%;稳定高效的遗传转化体系为:农杆菌侵染液OD₆₀₀为0.25,共培养3 d,卡那霉素(kanamycin)和羧苄西林(carbenicillin)浓度分别为25和250 mg·L^-1。愈伤组织和不定芽诱导时经卡那霉素和荧光标记双重选拔,愈伤组织阳性率为85.47%,出芽生根后经PCR鉴定,阳性转化率为29%;利用实时荧光定量PCR检测转基因植株,导入的外源sGFP基因均有较高的表达水平。本研究为利用基因工程技术对黑果枸杞进行品种改良提供了技术支撑。     

牡丹组织培养研究进展

本文从牡丹组织培养对品种、外植体的选择、基本培养基类型、生长调节物质选择和组培苗的移栽等方面论述了牡丹组织培养的研究现状。牡丹的组织培养中,外植体多采用胚、腋芽、顶芽、茎尖、幼叶、叶柄和土芽(地下芽)等,并且培养经过低温诱导后的鳞芽,可显著提高萌发率;在愈伤组织诱导试验中,选择分化程度较低的材料(如土芽),容易脱分化,获得愈伤组织;在牡丹的增殖培养中,6-BA起到了不可或缺的作用;生根培养中常使用的生长调节物质有IBA、NAA和IAA。此外,还探讨了牡丹组织培养研究中存在的问题,以及未来的研究和发展方向。     

甘蓝型油菜抗病虫双价基因转化体系的建立

以甘蓝型油菜（Brassica napus L.）品种浙双758子叶柄外植体为受体,建立农杆菌介导转化体系,研究乙酰丁香酮、羧苄青霉素浓度和潮霉素筛选浓度等对农杆菌遗传转化效果的影响。结果发现,羧苄青霉素对农杆菌的抑制效果以500mg/L最佳,且对子叶柄外植体愈伤组织诱导和分化的影响最小,愈伤组织诱导率和芽再生率分别为75.2%和65.1%。5.0mg/L的潮霉素能完全抑制未转化再生植株的生长,使其最终褐化死亡,在此潮霉素筛选浓度下,获得了32株转化再生植株。共培养时培养基中添加100μmol/L AS的芽再生率为3.9%,显著高于未添加AS的芽再生率（2.0%）,说明共培养阶段添加酚类物质乙酰丁香酮有利于转化载体T-DNA的转化。部分转化再生植株经PCR和Southern杂交检测呈阳性,病虫害接种试验表明转化植株对菌核病和小菜蛾有较好的抗性。     

根癌农杆菌介导的甘蓝高效稳定的遗传转化系统的建立及对CpTI基因转化的研究

通过对影响根癌农杆菌转化的多种因素的比较和探索后，建立了一种以农杆菌介导的高效和稳定的甘蓝遗传转化系统。选择目前在生产上大量推广应用的6个优良甘蓝品种，用其无菌苗的下胚轴作为外植体，经《农杆菌LBA4404（含质粒pBin-TI-19-2)感染，在含卡那霉素50mg/L的抗性培养基上筛选，80％的外植体表现出抗性，形成愈伤组织并进一步分化成苗。每块愈伤组织最低成苗5株，最高可达22株。对部分抗性植株的总DNA进行Southern杂交，结果表明T－DNA上的CpTI基因已整合到甘蓝基因组中，外源基因的转化频率高达20％。通过抗小菜蛾实验发现，转基因植株未转基因植株有明显的抗性。     

Reduction of ferric iron by tumorous crown gall tissue is associated with cells modified by agro‐bacterium tumefaciens

Iron (Fe) uptake and use in plants is genetically controlled and physiological mechanisms such as Fe reduction are induced during Fe‐deficiency stress to make it available. Transfer of DNA into the cell genome by Agrobacterium tumefaciens alters physiological processes and causes undifferentiated growth. Tumor cells in sunflower (Helianthus annus L. cv. Mammoth Russian) show enhanced Fe reduction compared to normal stem tissue in a manner similar to root cells in plants that are genetically switched on or off to manage Fe acquisition. This study addresses whether alterations caused by the DNA transfer from A. tumefaciens result in Fe reduction or whether A. tumefaciens inoculum alone reduces Fe. Reduction of Fe was quantified from A. tumefaciens inoculum and from uninoculated or inoculated sunflower stem tissues daily over a 14‐day period. Neither A. tumefaciens inoculum nor uninoculated stem tissue alone activated massive Fe reduction. High rates of Fe reduction were associated with the proliferation of cells modified by A. tumefaciens. The mechanisms that transformed normal tissue to uncontrolled tumor growth appeared to be linked to active Fe reduction. These modified cells may provide a key to locating and understanding the genetic control of the Fe reduction process in plant cells. Our results suggest a critical role for Fe in development of tumorous tissues and raises the question of whether other tumor cells induce similar mechanisms for Fe acquisition.     

抗生素对高粱离体培养反应的影响

探讨了 5种抗生素对高粱离体培养反应的影响。在 3种抑菌抗生素中 ,羧苄青霉素和头孢霉素的抑菌效果较好。头孢霉素对高梁外植体离体培养的毒性比羧苄青霉素大 ,羧苄青霉素对愈伤组织的生长起促进作用 ,且只在高浓度才对分化有抑制作用。在农杆菌介导高梁遗传转化时 ,选用 2 5 0～ 5 0 0mg L的羧苄青霉素来抑菌是合适的。高梁不同基因型和外植体对潮霉素和卡那霉素的反应表现不同 ,但对潮霉素的反应比卡那霉素敏感。在筛选转化体时 ,以潮霉素较好 ,使用浓度对茎尖和幼胚以 2 5～ 5 0mg L为宜 ,对愈伤组织则以 5 0～ 75mg L为宜。     

紫花苜蓿组织培养体系的建立

通过外植体类型、生长素和细胞分裂素浓度等因素对紫花苜蓿愈伤组织诱导、分化影响的试验,建立了高效的紫花苜蓿组织培养体系,即以真叶为外植体,在MS+0.2mg/L2,4-D+0.01mg/LNAA+0.2mg/LKT培养基上进行愈伤组织的诱导,诱导率可达到100%,愈伤组织继代2～3次,然后在MS培养基上分化,分化率可达90%。分化苗转入1/2MS培养基进行生根,形成完整的组培苗。愈伤组织诱导的正交试验表明,2,4-D浓度是影响苜蓿愈伤组织诱导率的主要因素,其次是外植体类型、NAA浓度、6-BA浓度和KT浓度;对苜蓿愈伤褐化率影响较大的因素是外植体类型,其次是KT、2,4-D、6-BA、NAA浓度。外植体类型对愈伤组织诱导率和褐化率的影响均达到极显著水平(P0.01);生长素(2,4-D、NAA)对苜蓿愈伤组织的诱导作用大于细胞分裂素(6-BA、KT)。分化试验表明,紫花苜蓿愈伤组织分化的主要决定因素是愈伤诱导培养基的激素配比。     

In vitro propagation and germplasm cold-storage of fertile and male-sterile Allium trifoliatum subsp. hirsutum

Summary Procedures for in vitro clonal propagation and for cold-storage of propagules were developed for fertile and male-sterile genotypes of Allium trifoliatum subsp. hirsutum, var. hirsutum and var. sterile, respectively. Highest rate of shoot multiplication was achieved from basal leaf and umbel explants on a modified BDS medium supplemented with 9 mg/l benzyladenine. Naphthalene acetic acid reduced the propagation rate, and was not required for shoot multiplication. The resulting shoots were rooted in an indole butyric acid-supplemented medium, and bulbing occurred upon exposure to a 16 h photoperiod. The small dormant bulbs were transplanted into potting mixture and sprouted after termination of dormancy, resulting in phenotypically-normal plants. No significant differences, in either shoot regeneration or plant establishment, were found between fertile and male-sterile genotypes.Basal leaf explants of in vitro grown plantlets were stored at 4–6°C for up to 16 months. Standard medium, or modified media containing 0.4 to 10.0% sucrose or 1 to 10 mg/l paclobutrazol, were used in storage experiments. Eighty to 100% and 70% survival rates, were recorded after 8 and 16 months cold-storage, respectively, in a 10% sucrose medium. Following their transfer to standard culture conditions, these explants regenerated plants which were phenotypically similar to those developed in the potting mixture.Isozyme polymorphism of 13 proteins was used to assess the genetic stability of cold-stored explants. Considerable differences in zymograms were found between the fertile and the sterile varieties. However, no differences in isozyme profiles were detected between the control field-grown plants, and those which were established from in vitro-stored leaf base explants. The only exception were plantlets exposed to a high paclobutrazol concentration in storage. The latter exhibited a doubling in the alcohol dehydrogenase profile.Abbreviations ADH Alcohol dehydrogenase - BA benzyl adenine - IBPGR International Board for Plant Genetic Resources - MDH malate dehydrogenase - MS Murashige & Skoog medium - NAA naphthalene acetic acid     

阔叶猕猴桃抗生素敏感性及遗传转化的研究

以阔叶猕猴桃(Actinidia latifolia)叶片为外植体,研究了各种抗生素对形态分化和器官形成的影响,并以叶盘为受体,通过根癌农杆菌介导法获得了抗性愈伤组织和抗性芽,组织化学染色表明gus基因已在植物组织中表达。研究结果表明,对不定芽分化来说,头孢霉素(Cef)效果明显好于羧苄青霉素(Carb)。Cef浓度为300mg.L-1时效果最好,再生频率达100%,平均再生芽数达最大为9.76个芽/外植体。生根过程中高浓度的Cef(≥200mg.L-1)明显抑制生根,而Carb对生根没有显著影响。在卡那霉素(Kan)和Carb组合处理中,Carb对生根没有显著影响,但Kan明显抑制生根。随着Kan浓度的升高,生根率和平均根数迅速减少,当浓度升高到50mg.L-1时,生根完全被抑制。因此,农杆菌介导的阔叶猕猴桃遗传转化中愈伤组织和不定芽诱导过程宜选用300mg.L-1Cef来抑制农杆菌生长,Kan筛选的临界浓度为20mg.L-1。在抗性芽生根过程中Kan浓度应该控制在50mg.L-1以内,并改用Carb作为杀菌剂,生根效果会更好。该研究确定了农杆菌介导的阔叶猕猴桃叶片遗传转化中使用抗生素的种类和用量,为通过基因工程对阔叶猕猴桃进行遗传改良奠定了基础。     

川东獐牙菜体外高效再生体系的建立

为了构建川东獐牙菜(Swertia davidii Franch.)高效稳定的再生体系,以川东獐牙菜带叶茎尖、带芽茎段和叶片为材料,在单因素试验的基础上,通过完全组合及L₉(3⁴)正交试验,探究不同激素对其离体培养的影响。结果表明,川东獐牙菜3种外植体中,叶片适宜作为间接器官发生材料,在MS+2.0 mg·L^-1 6-BA+1.5 mg·L^-1 KT培养条件下,培养7 d便可见愈伤组织从切口处产生,培养30 d后即可分化出大量丛芽;不定芽在相同培养基中培养30 d后,增殖系数可达8.75。带芽茎段则适宜直接器官发生途径,其在MS +2.0 mg·L^-1 6-BA+1.0 mg·L^-1 NAA中培养7 d后,节上腋芽开始萌动,培养30 d后腋芽发生系数可达4.06;试管苗适宜的生根培养基为MS + NAA 0.05 mg·L^-1,培养30 d后即可获得再生植株,生根率为100%;生根苗经过炼苗,移栽30 d后成活率达90%以上。在川东獐牙菜的离体培养中,间接器官发生途径较直接器官发生途径效率更高。本研究通过叶愈伤组织-不定丛芽途径建立了川东獐牙菜高效再生体系,为保护其野生资源和种苗繁育提供了技术支撑,也为其遗传转化奠定了试验基础。     

梭梭(Haloxylon ammodendron)愈伤组织诱导及植株再生

以梭梭(Haloxylon ammodendron)无菌苗的茎尖、子叶、下胚轴为材料进行愈伤组织诱导,培养基中单独添加1~3mg/L的2,4-D或与0.5mg/L的6-BA配合使用,均能成功诱导出愈伤组织,但在愈伤组织诱导起始阶段,用含2,4-D 2.0mg/L、BA 0.5mg/L的培养基效果较好;在2,4-D1.0mg/L、BA 0.5mg/L的分化培养基上经过两次连续继代培养后,在含BA 0.5mg/LI、AA 1.0mg/L的培养基上愈伤组织生长状态最好,并从茎尖诱导的愈伤组织上获得了再生植株。     

安祖花愈伤组织诱导及其分化的正交试验设计

用正交设计法研究了不同激素处理及不同外植体对安祖花愈伤组织诱导及分化的影响。结果表明,在本试验设计中,外植体是诱导安祖花愈伤组织的主要因素;以茎段为外植体诱导愈伤组织的最佳培养基为MS+1.0mg/L NAA+2.0mg/L 6-BA;愈伤组织不定芽诱导的主要因素是6-BA;不定芽诱导分化的最佳培养基为1/2MS+0.5mg/L 2,4-D+1.0mg/L 6-BA。     

蔺草成熟种子愈伤组织诱导及植株再生研究

以"岗山3号"蔺草成熟种子作为外植体,研究了不同激素浓度及组合对愈伤组织诱导及植株再生的影响。结果表明,在2,4-D浓度为2.0mg/L,6-BA浓度为0.5mg/L时,蔺草种子的愈伤组织诱导率最高,为74.7%;在IAA浓度为0.5mg/L,KT浓度为2.0mg/L时,蔺草种子的愈伤组织分化率最高,为83.3%;IAA与KT的比值对再生芽和再生根形态影响较明显。     

Bulb organogenesis of Tulipa tarda in vitro cultures in relation to light environment

Here organogenesis of tarda tulip (Tulipa tarda Stapf.) from callus explants is presented. The callus tissue was cultivated on MS media containing 3% or 6% sucrose and either no addition of BAP (6-benzyl-aminopurine) or supplemented with 0.5 μM BAP. The cultures were maintained under a 16?h photoperiod under white, red or blue fluorescent light, at 20?±?2°C for 12 weeks. This study aimed to determine the most suitable light conditions and medium composition for seed-derived callus explants in order to obtain an efficient formation of adventitious bulbs. There were no differences between the spectra of light in differentiating adventitious bulbs. Explants cultured in darkness (control), on 0.5?µM BAP and 3% sucrose, formed the highest number of adventitious bulbs. The efficiency of adventitious organogenesis amounted to 36.6 bulbs per 1?g of callus tissue. The fresh weight of biomass, cultured in these conditions, increased within 12 weeks from 1 to 6.99?g. Supplementation with BAP of the medium containing 3% sucrose promoted the formation of bulbs, but in the case of the medium with 6% sucrose, BAP had an adverse influence under every type of light. The obtained results provide a useful protocol for the micropropagation of T. tarda, which can be used commercially for rapid and cost-effective production of the tulip.     

滇黄芩毛状根的诱导及其黄芩苷含量测定

本文利用发根农杆菌A grobacterizum rhizogenes1.2556感染滇黄芩再生苗的茎段和叶片,建立了毛状根培养及其植株再生体系。毛状根可直接从受伤的茎、叶外植体表面产生,在无外源激素的MS固体和液体培养基上自主生长,表现出典型的发根特征。毛状根茎段的诱导率较叶片高,最高可达到14.44%;经rolB基因PCR分析和甘露碱纸电泳检测,证明Ri质粒T-DNA已整合到滇黄芩基因组中并表达;毛状根在附加6-BA2mg/L和NAA0.2mg/L的MS固体培养基上直接诱导不定芽,并在MS培养基上生根,形成再生植株。获得的毛状根系经MS液体培养基培养30d后通过HPLC都能检测到黄芩苷,其中1个转化系黄芩苷含量为2.59%,是药材黄芩的0.20倍,而从3年单位时间黄芩苷生成量计算,毛状根是药材黄芩的7.18倍。本研究建立的毛状根培养体系,将对滇黄芩转基因技术的完善和利用毛状根生产黄芩苷的生物转化提供了实验基础。     

Growth Patterns of Jojoba Male and Female Leaf Callus with Various Ammonium and Nitrate Sources

Abstract

Male and female leaf discs of Jojoba [Simmondsia chinensis (Link) Schneider] were cultured on Murashige and Skoog (MS) media supplemented with various nitrate:ammonium ratio and phytohormones concentrations. For the optimum callus growth, hormonal concentrations were remained equal for both male and female leaf tissues i.e., 0.4 mg L^?1 2,4‐dichlorophenoxyaceticacid, 1.25 mg L^?1 6‐benzyladenine and 0.5 mgL^?1 kinetin. However, a statistically significant difference was observed when Murashige and Skoog media was supplemented with an additional nitrogen source. In female leaf tissue, maximum fresh and dry weights were recorded in Murashige and Skoog media supplemented with an additional source of NO₃ ^?:NH₄ ⁺ (60 mM) whereas in male leaf tissue this addition was inhibitory. This study suggests that nitrogen requirement may be different for optimum callus growth in both male and female leaf tissues.     

观赏凤梨胚性愈伤组织的诱导、高频再生及超微结构研究

以观赏凤梨‘名宝剑’的短缩茎为外植体,研究了不同激素及浓度组合对愈伤组织诱导和植株分化的影响,并通过形态学和组织细胞学观察了不同类型愈伤组织的超微结构和胚性愈伤组织的分化过程。结果表明：NAA对愈伤组织诱导的影响达到极显著水平（P=0.0019）,其中0.5mg/L NAA最有利于提高愈伤组织的诱导率和胚性愈伤组织的比...     

红比利时杜鹃愈伤悬浮颗粒瞬时转化体系构建及植株再生

为建立红比利时杜鹃花愈伤悬浮颗粒瞬时转化体系,以嫩叶为材料,探讨了愈伤组织诱导与增殖的条件,构建并评价了愈伤悬浮颗粒瞬时转化体系,同时诱导愈伤颗粒出芽、生根,并获得组培小苗。结果表明,在2.41 g·L^-1木本植物基本培养基(WPM)+20 g·L^-1蔗糖+10 g·L^-1麦芽糖+7.0 g·L^-1琼脂+0.050 mg·L^-1植物组培抗菌剂(PPM)为基本培养基的条件下,嫩叶愈伤组织诱导的生长调节剂最优方案为:0.3 mg·L^-1 2,4-二氯苯氧乙酸(2,4-D)+0.3 mg·L^-1 噻重氮苯基脲(TDZ),此时愈化率可达97.78%;愈伤组织继代增殖的生长调节剂方案为:0.61 mg·L^-12,4-二氧苯氧乙酸(2,4-D)+0.65 mg·L^-1 6-苄基腺嘌呤(6-BA)+1.37 mg·L^-1 TDZ,此时增殖率可达167%。将继代5次后呈疏松状的愈伤颗粒接种于液体继代培养基中,大约4~12 d后愈伤颗粒进入指数生长阶段,增殖率为45%。含有GUS基因的pRI 101-AN与含有GFP基因的pCAMBIA1301-GFP分别在农杆菌GV3101介导下转化处于指数生长期的愈伤悬浮颗粒,OD₆₀₀为0.6的农杆菌侵染液侵染15 min后,愈伤颗粒的GUS染色效率为26.28%,愈伤颗粒的GFP荧光表达效果明显。另外,以2.41 g·L^-1 WPM+7 g·L^-1琼脂+20 g·L^-1 蔗糖为基本培养基,添加2.0 mg·L^-1TDZ + 0.5 mg·L^-1 2,4-D时,可诱导愈伤组织不定芽;以1/2 Read为基本培养基,添加0.5 mg·L^-1 IBA+1 g·L^-1活性炭可诱导出芽的愈伤颗粒生根。本研究结果为进一步开展红比利时杜鹃花稳定遗传转化研究和转基因植株培育奠定了基础。     

影响根癌农杆菌介导的丰香草莓遗传转化因素分析

将构建的携带NPTⅡ和AP-D基因的植物表达载体pBI121导入根癌农杆菌EH105中后,用其对草莓主栽品种丰香叶盘进行遗传转化,探讨了卡那霉素(Kan)的浓度、菌液浓度、侵染时间、预培养时间、共培养时间和筛选方式等因素对转化率的影响。结果表明:丰香草莓适宜的转化条件是Kan筛选浓度为20 mg/L,预培养时间为2~3 d,菌液浓度OD6000.3~0.5,侵染时间10~20 min;共培养2~3 d后将外植体接到含有5 mg/L Kan和500 mg/L Carb的诱导培养基上进行培养,以后每次继代逐步提高Kan选择压力至终浓度20 mg/L(每次增加5 mg/L);或共培养2~3 d后将外植体接到含有500 mg/L Carb的诱导培养基上,培养2周后继代到含20 mg/L Kan和400 mg/L Carb的培养基上进行选择培养。根据PCR检测结果,抗性愈伤组织转化率高达18.5%。抗性愈伤组织经进一步诱导,最后获得了17株抗性植株。本文探讨了影响丰香草莓转化的多个因素,以期建立高效的遗传转化体系,为草莓属植物种质的遗传转化奠定基础。