基因工程方法抗甜菜丛根病病毒育种的研究(Ⅰ)甜菜坏死黄脉病毒外壳蛋白基因转化甜菜及植株再生

采用含有甜菜坏死黄脉病毒外壳蛋白(BNYVV CP)基因的农杆菌, 分别以不同甜菜品系的叶柄、下胚轴及子叶为外植体材料进行基因转化研究.含有BNYVV CP 基因和卡那霉素抗性筛选基因(Kanr)的农杆菌与甜菜组织不同外植体共培养后,经过诱导分化培养,在含有卡那霉素(kanamycin)的培养基上,从叶柄及下胚轴分别直接诱导出抗卡那霉素再生芽,而从子叶上只诱导出紧密型绿色愈伤组织,未分化出不定芽.从叶柄及下胚轴诱导再生芽的诱导培养基分别为:MS+BA0.5+TIBA0.2+3%蔗糖+Kan200+Cb500、MS+BA0. 15+NAA0.2+3%蔗糖+Kan100+Cb500.甜菜再生植株诱导频率与甜菜品系、外植体类型、苗龄、温度、TIBA、预处理等因素有关.再生植株生根培养基为:MS+IBA2.0+2%蔗糖+Kan50.     

二步培养和AgNO3对甘蓝型油菜子叶和下胚轴芽再生的影响

以甘蓝型油菜（Brassica napus L．）品种浙双758为材料,研究二步培养及添加AgNO3对甘蓝型油菜子叶和下胚轴外植体离体再生的影响。外植体先在含0．5—1．5mg／L2,4-D的MS培养基上预培养3d或7d诱导愈伤组织的产生,再转到含有3mg／LBA和0．15mg／LNAA及添加或不添加2．5mg／LAgNO3的分化培养基上诱导芽的分化。结果表明,外植体愈伤组织诱导率和芽再生率与2,4-D浓度、预培养时间和AgNO3密切相关;分化培养基中添加银离子可显著增加不定芽的再生频率;二步培养及添加AgNO3可使半子叶、完整子叶和下胚轴外植体芽再生频率分别达到了96．1％,96．7％和96．7％。     

甜菜转基因植株抗性表现及种子获得

用农杆菌介导方法，将甜菜坏死黄脉病毒外壳蛋白（BNYVV CP）基因转化甜菜不同品系叶柄外植体。以卡那霉素作为筛选标记基因，获得抗卡那霉素再生植株。再生植株经PCR扩增，获得目的基因大小的DNA片段，初步证明为转基因植株。部分转基因TO代植株在病土中实验表明，其抗病性明显高于对照。将移栽田间所获得的转基因甜菜母根进行单株套罩采种，获得不同品系单株自交种。     

花生组织培养及高频率植株再生

以14个花生品种为材料,对花生组织培养及植株再生进行了研究。将花生成熟胚幼叶、子叶和下胚轴外植体接种到添加0—2mg／LNAA和0～10mg／LBAP的MSB,（MS无机盐＋B5有机）培养基上诱导愈伤,再转到添加0～10mg／LBAP的分化培养基上诱导不定芽。结果表明,诱导培养基中添加的激素及其浓度对以后在分化培养基上不定芽分化有重要作用,以添加1mg／LNAA和6mg／LBAP最利于不定芽分化;分化培养基中添加4mg／LBAP利于不定芽伸长及植株再生;幼叶外植体分化不定芽频率明显高于子叶和下胚轴;不同基因型不定芽分化率存在明显差异,白沙1016和花育23幼叶外植体获得了较高的不定芽分化率,分别达到91．8％和88．5％。再生苗经生根培养和驯化后,移栽花盆可正常开花结果。     

激素对甜菜愈伤组织不定芽再生的调节作用

2，4－D能促进胚珠愈伤组织形成，但对不定芽再生有抑制作用，而IAA无此抑制作用。6BA和KT单独或组合使用在胚珠分化芽的愈伤组织诱导上，是不可缺少的。胚珠意伤组织不定芽可在附加IAA与KT（或再附加6BA）的诱导培养基上一步形成。0．5mg／L的6BA与NAA或IAA在一定浓度下的组合能诱导叶柄产生愈伤组织，分化培养后获得了再生芽。无论胚珠或叶柄，愈伤组织不定芽的再生，主要与前期诱导培营基的激素组合与浓度有关。     

甜菜组织培养中外植体褐变影响因素的研究

通过对组织培养中甜菜品系的染色体倍数性、无茵苗的苗龄、外植体部位及生理状态、培养基成分、光照条件的实验发现，这些因素均对甜菜组织培养过程中外植体褐变有影响。实验结果表明：二倍体品系制成的外值体在相同条件下要比四倍体品系的外植体发生褐变的时间延长，而且二倍体褐变的频率也低于四倍体：超过6周的无菌苗的外植体极易褐变；下胚轴、胚轴、叶柄出现褐变的时间早于叶片；NAA浓度超过O．2mg/L褐变率明显增高；光照时间超过16h的外植体很快褐变，光照强度在3000-4000k时，外植体虽然有不同程度的褐变．但再生率较好。     

转马铃薯Y病毒复制酶基因烟草的获得及抗病性分析

重组克隆ＤＮＡ用ＫｐｎＩ／ＢａｍＨＩ双酶切得到马铃薯Ｙ病毒ＮＩｂ基因，将其插入质粒ｐＲＯＫ２相应切点中构成植物表达载体。用重组质粒ｐＲＯＫ２转化农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａ－ｃｉｅｎｓ）ＬＢＡ４４０４菌株，叶盘法将ＮＩｂ基因转入烤烟品种ＮＣ８９的染色体，获得抗卡那霉素的转化再生植株。经抗性筛选、ＰＣＲ检测、无性扩繁和大量重复抗病鉴定，结果表明，转化烟草植株ＤＮＡ中整合了外源目的基因，且表现抵抗２０μｇ／ｍｌＰＶＹＮ的侵染，ＥＬＩＳＡ分析认为抗性植株无病毒累积，初步筛选出对ＰＶＹＮ侵染具有较高抗性的转基因烟草植株。     

甜菜远缘杂交胚培养及植株再生

以甜菜远缘杂交胚为外植体进行离体培养时，在ＭＳ培养基上，适合胚继续生长，分化的激素浓度为０．５ｍｇ／Ｌ，ＮＡＡ，０．５ｍｇ／Ｌ６ＢＡ。胚的离体时期至关重要，初步认为以授粉６～８天的远缘杂交胚的离体再生诱导率最高，同时比较了正，反远缘杂交的受精效果和诱导率，存在明显差异。     

几丁质酶基因在烟草栽培品种中的表达

利用多聚酶链式反应（ＰＣＲ）对来源于一种生物杀虫剂（ｂａｃｕｌｏｖｉｒｕｓ）的几丁质酶基因进行体外扩增，并插入载体质粒ｐＲＯＫ２中，然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）ＸＬ１Ｂｌｕｅ。用重组质粒ｐＲＯＫ２ＤＮＡ转化植物转基因载体——土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉａｔｕｍｅｆａｃｉｅｎｓ）ＬＢＡ４４０４菌株。借助于土壤农杆菌侵染烟草叶圆片，将目的基因导入。通过组织培养诱导生芽和生根，获得烟草再生植株。利用含卡那霉素的培养基对再生烟株进行初步筛选。进一步ＰＣＲ和Ｗｅｓｔｅｒｎ印迹检测结果表明：转基因烟草中有几丁质酶基因和蛋白的表达。初步检测结果表明：转基因烟草具有较高的几丁质酶活性。     

亚麻高效再生体系的优化研究

为了优化亚麻再生体系,对基因型、外植体的选择部位、基本培养基、植物生长调节剂等影响亚麻植株再生的因素进行了优化,建立了亚麻下胚轴高效组织培养再生体系.结果表明,亚麻种子消毒采用75%乙醇处理3min,再用0.1%升汞灭菌3min效果最好;亚麻下胚轴的下部是最佳外植体;在Y4培养基下（MS1＋NAA0.02mg/L＋6-BA1mg/L）,亚麻愈伤组织的诱导率和分化率最高,分别达到98.89%和95.56%;最佳生根培养基是1/2MS＋NAA0.001mg/L,生根率达90%;同时可以看出,不同基因型再生能力不同.与派克斯和华星009相比,再生能力最强的品种是中亚2号.通过再生体系优化使亚麻愈伤组织的诱导率和分化率以及再生植株的生根率都有所提高.     

两种抗生素对甜菜离体叶柄分化的影响

在培养基中添加不同浓度的卡那霉素和壮观霉素，观察抗生素对甜菜叶柄分化再生的影响。结果表明：一定浓度的卡那霉素和壮观霉素对甜菜叶柄的分化均具有抑制作用，当卡那霉素浓度为50mg/L时，叶柄分化率明显变化，当浓度达到100mg/L时，分化率为1．4％。当壮观霉素浓度为40mg/L和50mg／L时，分化率为5．7％和3．7％，当浓度为60mg/L时，基本无绿芽分化，统计学分析显示：壮观霉素的抑制作用要强于卡那霉素。结合实验结果讨论了这两种抗生素作为甜菜遗传转化研究的筛选标记的适宜浓度。     

细菌几丁质酶基因转化烟草的研究

为了提高烤烟品种K 32 6对真菌病害的抗性 ,以烟草再生植株的叶片为受体 ,采用农杆菌介导法将细菌几丁质酶基因导入烟草 ,获得了抗卡那霉素的转化植株 ,经PCR检测 ,初步证明了细菌几丁质酶基因已整合到烟草的基因组中 ;同时研究了再生烟草叶片的耐卡那霉素水平、受体预培养对转化的影响以及促进转化植株的生根等。结果表明 :培养基中卡那霉素浓度为 5 0mg/L时 ,能完全抑制烟草叶片的再生 ;烟草叶片预培养 2d的转化率提高 ;添加 0 .2mg/LIAA(吲哚乙酸 )能显著地提高转化植株芽梢的生根率。     

甜菜组织培养直接器官发生和植株再生

根据甜菜营养生长的特点，通过在MS培养基中添加植物激素BA和NAA或IBA，建立了由茎节和叶子两种外植体的直接器官发生再生植株的技术。     

In situ regeneration of rice (Oryza sativa L.) callus immobilized in polyurethane foam

An in situ regeneration system for rice calli comprised of a callus growth stage and two regeneration stages was developed. After the first stage of regeneration, the medium is changed and the calli are immobilized in polyurethane foam supports, in each of which 3-5 regenerated plantlets develop from the immobilized calli during the second stage. While no significant change in callus size was observed during the first stage of regeneration, in the second stage callus enlargement and shoot regeneration predominated. In the light of these findings, calli were immobilized in the second stage after medium exchange. The use of 10-mm support cubes with an average pore size of 3.6 mm resulted in the most efficient immobilization and in situ regeneration. Medium exchange after 15 d gave the largest number of support cubes with shoots. When rice calli were cultivated in support cubes placed in 60 ml second-stage medium in a 500-ml flask, the immobilization ratio was 83%, and 82% of the support cubes contained 3-5 regenerated plantlets after 25 d. The shoot lengths of the regenerated plantlets obtained from the in situ regeneration culture were longer than those from a suspension culture. When support cubes with 3-5 regenerated plantlets were transferred from the flask to 1 4 MS solid medium supplemented with 10 g/l sorbitol and 5 g/l sucrose, the regenerated plantlets developed quickly into plants with a length above 10 cm after 10 d.     

培养条件对紫苏下胚轴外植体再生的影响

以紫苏下胚轴为材料,研究紫苏外植体芽再生的影响因素。结果表明：苗龄、黑暗时间、激素浓度及配比对紫苏芽再生均有较大影响。取13d苗龄的下胚轴先在分化培养基（MS＋3.0mg/L BA＋0.3mg/L NAA）黑暗预培养8d后转为14h/d光照条件培养可获得较高的再生频率。诱导芽苗生根最佳培养基为1/2MS＋1.0mg/L BA＋0.3mg/L IAA。     

Enhancement of the solid-liquid extraction of sucrose from sugar beet (Beta vulgaris) by pulsed electric fields

A systematic study of the impact of pulsed electric fields (PEF) parameters (1–7 kV/cm, 5–40 pulses, specific energy of 0.006–0.19 kJ/kg per pulse, pulse frequency of 1–10 Hz, pulse width of 2–5 μs, square and exponential decay pulses) on the kinetics of the sucrose extraction from sugar beet at different temperatures (20–70 °C) has been carried out in this investigation. The efficiency of the solid-liquid extraction was independent of the frequency, as well as of the pulse width, and the pulse shape at 7 kV/cm, and it was influenced by the electric field strength applied and by the temperature of the extracting medium. Sucrose yield increased with both field strength, time of extraction, and temperature. The effect of the field strength was higher the lower the temperature. The application of 20 pulses at 7 kV/cm (3.9 kJ/kg) increased the maximum yield by 7 and 1.6 times, compared to non-PEF-treated samples, at 20 and 40 °C, respectively. A mathematical expression was generated, which enabled to evaluate the influence of the electric field strength (from 0 to 7 kV/cm) and temperature (from 20 to 70 °C) on the sucrose extraction efficiency and the extracting time in a solid-liquid PEF-assisted sucrose extraction process. Based on this equation, for 80%-sucrose extraction in 60 min, the temperature could be reduced from 70 °C to 40 °C, when 20 pulses of 7 kV/cm were applied.     

Sucrose accumulation and osmotic potentials in sugar beet at increasing levels of potassium nutrition

In a pot experiment sugar beet Beta vulgaris L. ssp. vulgaris, cv. Kawetina, was grown on alluvial soil (21 mg exchangeable K 100 g^?1) containing 0 (K₁), 20 (K₂), 40 (K₃) and 60mg fertiliser K 100 g^?1 soil (K₄). The plants were sown on 15 March 1983 and harvested on 23 June, 14 July and 30 August 1983. At final harvest root dry weight/plant had reached 269 g in K₁ and between 304 and 310 g in K₂-K₄. Sucrose values were 15% (K₁) and 17.3–17.7% of root fresh weight (K₂-K₄) respectively. The osmotic potential of the storage root decreased with increasing K nutrition and time. At final harvest ψ, was between ?1.83 (K₁) and ?2.26 MPa (K₄), 75–79% thereof being contributed by sucrose. Sucrose concentrations in press sap increased from 340–400 mM on 23 June to 475 (K₁) and 540–570 mM (K₂-K₄) at the end of August. In the same period K concentrations declined from 40–100 to 10–35 mM K⁺. The sucrose, K and Mg concentrations measured in the press sap were lower than those calculated from sucrose, K, Mg and moisture content of the storage root. This indicates that press sap from thawed storage root tissue is not fully representative. Betaine, analysed only at final harvest, significantly increased with increasing K concentrations in the storage root (r=0.83) and a significant linear regression was found between betaine and sucrose accumulation (r=0.57). This is consistent with the role of betaine as a cytosolic osmoticum for sugar beet storage tissue. Other solutes in the cytosol may also contribute to osmoregulation as sucrose accumulates in the vacuoles.     

甘蔗新品种桂糖48号的组织培养技术研究

甘蔗组织培养技术是加速繁殖新品种和加快新品种大面积推广的主要手段,不同品种的离体培养中其培养基中激素配比存在差异,因此筛选合适培养基是提高品种快繁效率的关键。本文以桂糖48号为材料,以MS基本培养基加外源激素浓度梯度进行试验处理设计,对品种的愈伤组织诱导、愈伤分化、试管苗增殖和生根阶段进行培养基筛选试验,以其筛选出最合适桂糖48号的培养基配方。试验结果表明:诱导愈伤组织培养基为MS+2,4-D 3.0 mg/L;愈伤组织分化培养基为MS+BA 1.0 mg/L;初代增殖培养基为MS+BA 2.0 mg/L+NAA 0.1 mg/L+GA 0.5 g/L;继代培养基为MS+BA 2.0 mg/L+NAA 0.1mg/L;生根培养基为1/2 MS+NAA 12 mg/L。在该培养基下,生根最好,苗绿,比较高,长势好,对加快甘蔗新品种桂糖48号在广西大面积推广起到积极的作用。     

An Efficient Regeneration System and Optimization of the Transformation from the Cotyledonary Node of Jute (Corchorus capsularis L.)

The purpose of this study was to use cotyledonary nodes as explants to establish an efficient regeneration protocol for jute (Corchorus capsularis L.) on Murashige and Skoog (MS) basal medium. Research into the different growth regulators using the orthogonal design L16 (4⁵) revealed that the best shoot induction medium is MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6BA and 0.25 mg/L NAA. The average number of shoots per explant and the explant induction rate were 9.8 and 100%. After 3 weeks, 2–3 cm shoots were rooting on 1/2 MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6-BA + 0.2 mg/L NAA. Moreover, we optimized Agrobacterium-mediated transformation using the GUS gene transient expression system. The best condition for obtaining higher transformation rate consisted of the use of fresh explants to which 100 μM acetosyringone was added for a co-culture time of 10 min, the OD value of Agrobacterium liquid is 0.5 at 600 nm. These data provide an important basis for the application of other trait gene in the improvement of jute fiber quality.