根癌农杆菌介导的玫瑰茄愈伤组织的遗传转化

以根癌农杆菌LBA4404和EHA105为供体菌株,对玫瑰茄愈伤组织进行了转化条件的研究,建立了一套玫瑰茄愈伤组织遗传转化体系。利用该转化体系获得了2个稳定表达新霉素磷酸转移酶活性的玫瑰茄转化细胞系。GUS活性组织化学检测和PCR扩增鉴定的结果表明,愈伤组织的转化率为4%。说明采用农杆菌介导法将外源基因经愈伤组织导入玫瑰茄细胞是可行的。     

产藏红花素栀子愈伤组织的诱导和筛选

为解决藏红花资源问题,研究了栀子愈伤组织的诱导,并首次应用目视法和HPLC 方法对产藏红花素细胞系进行了筛选。结果表明栀子叶子愈伤组织优化的诱导条件为: 在添加1mg/L 2,4-D和 0.25 mg/L 6-BA的MS 培养基上, 25±1℃和31.74 mmol/m2s光照强度下每天光照16小时,培养15d后可获得100%的诱导率。种子愈伤组织也易诱导,在添加0.5mg/L 2,4-D 和 0.25 mg/L 6-BA的MS培养基上,25±1℃全时暗培养15d后可获得100%的诱导率。首次应用建立的目视法和HPLC 方法, 从90株细胞系中筛选出细胞系seed4,其藏红花素1 的含量是0.348mg/g,生长较快、不易褐化,兼顾了此次生代谢产物的含量和细胞的生长速度,为采用植物细胞工程法解决藏红花素资源短缺问题提供了种质资源。     

表达HIV壳体蛋白转基因枸杞悬浮细胞的培养与鉴定

枸杞是我国珍贵的中药材,利用枸杞作为转基因材料具有易于遗传操作,生物性状稳定的优点。将携有人类免疫缺陷病毒I型（HIV-1）壳体蛋白基因的植物表达载体导入根瘤农杆菌EHA105中,并通过农杆菌侵染枸杞叶片,诱导产生抗性愈伤组织,利用抗性愈伤组织作为材料进行悬浮细胞的培养并对转基因枸杞悬浮细胞鉴定。PCR结果表明已获得遗传转化的转基因枸杞悬浮细胞系。免疫组织化学检测结果表明HIV壳体蛋白已在转基因枸杞悬浮细胞中表达。     

简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101/pHQ9,EHA 101/pHQ 10,EHA 101/pHQ T3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤/g愈伤外植体,抗性愈伤的分化频率平均高达85%。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3∶1。该系统的建立将有助于应用T-DNA标签法和基因打靶法进行水稻功能基因组的研究。     

缺氧胁迫法选育高产银杏内酯悬浮细胞系研究

以银杏优良品种的种子萌发的幼苗诱导愈伤组织,考察了不同培养基地对愈伤组织生长和银杏内酯产生的影响。采用缺氧胁迫小细胞团法从愈伤组织中共选育出7个高产悬浮细胞系,其合成银杏内酯的能力比选前的愈伤组织有了显著提高,其中细胞系MH－3培养周期18d,细胞生物量增加3．71倍,银杏内酯含量达到细胞干重的0．048％,比选育前提高了182．4％,为国内领先水平。并对MH－3在遥瓶培养时其生产银杏内酯能力的稳定性进行了研究。     

超声波辅助农杆菌介导CP基因转化番小瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV 外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV 菌株、美国夏威夷PRSV 菌株、泰国PRSV 菌株及日本PLDMV 菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset 的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS 6%蔗糖 1%葡萄糖,pH 5.7)调整至光密度OD_(600(?)m)=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml 无菌离心管装载胚性愈伤材料进行15s 的超声波处理,在80块被转化的胚性愈伤中获得21个CP 基因G 转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s 的超声波处理48块被转化的胚性愈伤中获得8个CP 基因B 转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP 基因转化上均表现出相似的效果。试验还表明:120mg/L 是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR 和Southern 印迹杂交进行了鉴定。     

抗除草剂转基因小麦愈伤组织的获得

小麦是重要的粮食作物,但在植物基因工程的研究中其进展是较缓慢的。除 Lorz.H 在1985年报导获得转基因愈伤组织外,迄今未见有更多的报导.我们在1989—1991年先后将抗除草剂 Basta 的 Bar 基因和萤火虫荧光素酶(Luc)基因转入小麦悬浮细胞系徐州211和(中3×科冬58)F_1的原生质体中,获得转基因的多细胞团和愈伤组织,并从(中3×科冬58)F_1的转基因抗性愈伤组织中得到根的分化。     

超声波辅助农杆菌介导CP基因转化番木瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法.分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株.试验着重在转化方法上进行探索.结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS+6%蔗糖+1%葡萄糖,pH 5.7)调整至光密度OD600nm=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率.用15m1无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系.上述操作方法用在两种CP基因转化上均表现出相似的效果.试验还表明120mg/L是卡那霉素抗性筛选的最佳浓度.抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系.所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定.     

植物激素和抗生素调节的转基因火炬松愈伤组织的生长和分化

包含质粒载体pBI121的农杆菌菌株LBA4404被用于转化火炬松的成熟合子胚。质粒载体pBI121含有胭脂碱合成酶基因的启动子驱动的新霉素磷酸转移酶基因（npt Ⅱ)和花椰菜花叶病毒的35S启动子驱动的GUS基因。器官发生的转基因愈伤组织和转基因的再生植株已经获得,并经GUS组织化学染色、聚合酶链式反应和Southern杂交分析证实。植物激素（BA/IBA）和抗生素对转基因愈伤组织的生长和分化的影响被研究。500mg/L羧苄青霉素和2mg/LBA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加54.2%,分化增加45.7%。500mg/L Claforan和2mg/L BA、0.5mg/L IBA的组合导致转基因愈伤组织的生长增加40.8%,分化增加38.7%。高浓度的植物激素和抗生素降低了转基因愈伤组织的生长和分化。实验结果有助于建立一个高效的农杆菌介导的火炬松遗传转化系统,也有助于未来针叶树的遗传转化研究。     

HIV-1衣壳蛋白在转基因枸杞中表达的免疫组织化学定位

目的:研究转基因植物中重组蛋白的细胞定位,有助于进一步了解转基因枸杞中HIV-1衣壳(CA)蛋白融合蛋白的分泌表达途径。方法:利用含有MA4-CA融合基因的农杆菌转化枸杞,转化植株获得再生。采用免疫组织化学方法对转基因枸杞表达的CA融合蛋白进行初步定位。结果:免疫组织化学定位表明,在转基因枸杞愈伤组织中,HIV-1CA主要在细胞浆、细胞壁和细胞间隙中表达。结论:免疫组织化学结果初步证明了CA融合蛋白在转基因枸杞中的表达分布。     

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.     

High anthocyanin accumulation in the dark by strawberry (Fragaria ananassa) callus

Fragaria ananassa (strawberry) callus, which produced high amounts of anthocyanin in the dark, was isolated from a cell line not producing anthocyanin. The isolated callus (FAR) was homogeneous and more than 90% of the cells were pigmented. The FAR callus accumulated more than 1000 g of anthocyanin per g fresh cell in the dark. Four different basal solid media were examined to maintain FAR callus: Though growth rate and anthocyanin concentration were different on each media, total anthocyanin production was about the same at 400 g anthocyanin/0.1 g fresh cell wt after 22 days. This FAR cell line could therefore be used for the industrial production of anthocyanin.     

Rosmarinic acid synthesis in transformed callus culture of Coleus blumei benth

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.     

Effect of Light and Activated Charcoal on Tracheary Element Differentiation in Callus Cultures of Pinus Radiata D. Don

Light has been found to increase the proportion of tracheary elements differentiating in callus cultures derived from xylem-parenchyma of Pinus radiata D. Don grown on an induction medium containing activated charcoal but no phytohormones. The differentiation rate increased from 20% when callus was grown in darkness to 45% when callus was grown with a 16 h or 24 h photoperiod. When callus was grown with a 16 h photoperiod, tracheary elements were observed 2 days after transfer of callus to the induction medium, as compared to 5 days when callus was cultured in darkness. The differentiation rate was also influenced by the concentration of activated charcoal added to the induction medium, the optimum concentration being 5 g l⁻¹. Exclusion of activated charcoal from the induction medium decreased the differentiation rate to 2%. The activities of the lignin-related enzymes L-phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase were significantly higher in cell cultures grown with a 16 h photoperiod as compared to when grown in darkness. The results show that light had a stimulating effect on tracheary element differentiation and the activities of lignin-related enzymes in P. radiata callus cultures. The new growth conditions markedly improve this cell culture system and make it particularly useful for functional gene testing and cell-wall analysis of in vitro grown tracheary elements of coniferous gymnosperms.     

Formation of Interfamilial Cell Symplast During Cell Co-Culture in Vitro

Two cell lines, white tobacco (Nicotiana tabacum) cell line and green carrot (Daucus carota) cell line, each with very distinct cellular structure markers by which the two cell lines could be identified at levels of callus cells, with light microscopy and electron microscopy, were established and used for interfamilial cell co-culture in which callus cells were well mixed and finely dispersed and treated with K+ hypotonic solution. Variegated interfamilial chimeral calli were observed after 10 to 15 days of co-culture. An isolation layer was formed and became thickened at the interface between the two attached unrelated callus cells and the heteroplastic cell wall complex was gradually established. Then the isolation layer became thinned and disappeared and plasmodesmata were formed secondarily within the thinned region and the interfamilial cell symplast was established. The wall in the region with isolation layer was about twice as thick as the fused cell wall of the symplastic. The developmental process of the interfamilial cell symplastic connection was discussed and it was suggested that (1) the nonspecific formation of isolation layer as initial adhesion between the two attached unrelated cells was the prerequisit for symplastic connection de novo formation, and (2) the specific cell recognition leading to disappearance or thickening to lignification or suberization of isolation layer and formation of plasmodesmata started within the isolation layer.     

离体细胞共培养中科间细胞共质体的形成

离体培养下选出的绿色胡萝卜（Ｄａｕｃｕｓ ｃａｒｏｔａ）细胞系和白色普通烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）细胞系,各自具有独特的细胞结构标志,在愈伤组织、光镜和电镜３ 个水平上均可区分。对两个细胞系进行分散、混合、Ｋ＋ 液低渗处理后在固体培养基上共培养,１０—１５ ｄ 后可观察到两种细胞的镶嵌生长。光镜和电镜下均观察到烟草细胞和胡萝卜细胞之间隔离层的存在与消失。在隔离层消失的区域可见到异种细胞间次生胞间连丝的形成,从而将独立的两个共质体连成一个统一的共质体。对科间细胞共质联系的建立过程进行了讨论,认为细胞接触后首先非特异粘连——以隔离层形成并适度加厚为标志,然后特异的细胞识别在隔离层中启动,从而导致隔离层或消失而重新建立共质联系或加厚、木质化、木栓化     

不同碳源对悬浮培养玫瑰茄细胞主要基质消耗的影响

采用了蔗糖、葡萄糖、可溶性淀粉3种不同碳源,考察了在摇瓶培养过程中,这3种碳源对维持悬浮培养过程中玫瑰茄细胞的生长及主要基质消耗的影响。结果表明,蔗糖和葡萄糖在这些方面的表现基本相同,而可溶性淀粉由于植物细胞对其没有有效的水解利用手段,因而不能支持植物细胞的生长,从基质消耗水平上来看,也明显慢于上述两类碳源。     

Variability of azadirachtin inAzarirachta indica (neem) and batch kinetics studies of cell suspension culture

Seeds of neem were collected from different parts of India and analyzed for their azadirachtin content by High Performance Liquid Chromatography (HPLC). In order to assess the effects of genotypic and geographical variation on azadirachtin content in cell cultures, callus development was attempted in the seeds containing high and low concentration of azadirachtin. The concentration of azadirachtin in callus cultures was significantly affected by the explant source. Seed kernels with higher azadirachtin content produced higher azadiractin content in callus cultures and lower azadirachtin content was seen in callus cultures produced from seed kernels with low azadiractin content. The protocol for development of elite stock culture ofAzadirachta indica was established with the objective of selecting a high azadirachtin-producing cell line. The highest azadirachtin-producing cell line was selected and the effects of different media and illumination conditions on growth and azadirachtin production were studied in shake flask suspension culture. Detailed batch growth kinetics was also established. These studies provided elite starter culture and associated protocols for cultivation ofA. indica plant cell culture in the bioreactor.     

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.