沙田柚杂种胚性愈伤组织的诱导及遗传鉴定

【目的】沙田柚（Citrus grandis [L.] Osbeck cv.Shatian）是中国的特有品种，其愈伤组织难以诱导。为进一步进行细胞工程研究，本研究通过与其它品种杂交的方法获得杂种胚性愈伤组织。【方法】以沙田柚为母本，用体细胞杂种橘柚+无酸甜橙 ([C.reticulata Blanco× C.paradisi Macf.] cv.Nova+C.sinensis [L.] Osbeck cv.Succari)为父本进行杂交，90 d后将幼胚取出，在MT + ME（麦芽提取物，500 mg·L-1）和MT + GA3（1 mg·L-1）两种固体培养基上培养。【结果】幼胚培养约5个月左右，在两种培养基上从胚状体和幼小植株的下胚轴处都长出了可继代保存的胚性愈伤组织，经细胞流式仪和SSR鉴定，该胚性愈伤是三倍体杂种愈伤，继代保存两年后，在MT+乳糖（50 g·L-1）培养基上仍可以产生大量的胚状体，具有较强的胚状体发生能力。【结论】沙田柚与体细胞杂种杂交后，可以较容易地获得其杂种的胚性愈伤，对沙田柚种质资源的保存和利用具有重要价值。     

柑橘愈伤组织内源激素代谢与体细胞胚胎发育能力关系的研究Ⅰ．柑橘不同愈伤组织体细胞胚胎发生能力的比较

为了探明柑橘愈伤组织内源激素代谢与体细胞胚胎发生能力的关系，研究了长期继代保存的纽荷尔脐橙和伏令夏橙不同愈伤组织的体细胞胚胎发生。结果表明，无论是在加吸3％蔗糖的MT增殖培养基上，还是在加有30mL/L甘油的MT分化培养基上，纽荷尔脐橙的胚性愈伤组织和二倍体伏令夏橙愈伤组织的体细胞胚胎发生能力均较强，而纽荷尔脐橙的非胚性愈伤组织，四倍体和六倍体伏令夏橙愈伤组织的体细胞胚胎发生能力较弱。     

柑橘愈伤组织内源激素代谢与体细胞胚胎发生能力关系的研究Ⅰ.柑橘不同愈伤组织体细胞胚胎发生能力的比较

为了探明柑橘愈伤组织内源激素代谢与体细胞胚胎发生能力的关系，研究了长期继代保存的纽荷尔脐橙和伏令夏橙不同愈伤组织的体细胞胚胎发生.结果表明，无论是在加有3%蔗糖的MT增殖培养基上，还是在加有30 mL/L甘油的MT分化培养基上，纽荷尔脐橙的胚性愈伤组织和二倍体伏令夏橙愈伤组织的体细胞胚胎发生能力均较强，而纽荷尔脐橙的非胚性愈伤组织，四倍体和六倍体伏令夏橙愈伤组织的体细胞胚胎发生能力较弱.     

Study on Agrobacterium-Mediated Transformation of Pepper with Barnase and Cre Gene

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper （Capsicum annuum L.）. Chili pepper inbred lines （A, D, E, and I） were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR[MB（MS mineral＋vitamine B5）＋BA（6-Benzyladenine） 5.0 mg·L^-1 ＋IAA（indoleacetic acid） 1.0 mg·L^-1＋GA3（gibberellic acid） 1.0mg·L^-1＋sucrose 3%＋agar 6.5g·L^-1] for 2d. The explants were infected by Agrobacterium tumefaciens when their OD600（optical density at 600 nm）reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB＋BA5.0 mg·L^-1＋IAA 1.0 mg·L^-1 ＋GA3 1.0 mg·L^-1＋sucrose 3% ＋agar 6.5 g·L^-1＋AS （acetosyringone） 200μmol·L^-1, these cotyledons with petiole were cultured on selective differentiation medium in the media MT[MB medium supplemented with BA [5.0 mg·L^-1＋ IAA 1.0 mg·L^-1＋ GA3 1.0 mg·L^-1＋ AgNO3 5.0 mg·L^-1＋ CW （coconut water） 5% ＋ Km （kanamycin） 65 mg·L^-1＋ Cb （carbenicillin） 500 mg·L^-1＋ 3% sucrose ＋ agar 6.5 g·L^-1].The Kmr （kanamycin resistant） bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen germination experiments.     

Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg L⁻¹. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g L⁻¹ glutamine and 0.5 g L⁻¹ asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g L⁻¹ glutamine, 0.5 g L⁻¹ asparagine, and 6.0 g L⁻¹ agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.     

冰糖橙胚性愈伤组织的诱导与植株再生

以冰糖橙成熟果实中的未发育胚珠为外植体，培养诱导胚性愈伤组织并进行植株再生。结果表明：在不同的培养基和不同的培养条件下胚性愈伤组织和胚状体的发生频率不同；麦芽提取物和GA3有利于胚性愈伤组织的发生，暗培养有利于胚性愈伤组织的诱导；在MKBN(MT KT0．5mg／L BA0．5mg／L NAA0．1mg／L)培养养基上，胚状体的再生率达88．24％，以酸橙作砧木进行试管嫁接，嫁接成活率达88．89％。     

橡胶树热研88-13品种珠心易碎愈伤组织诱导及其胚状体发生

以橡胶树热研88-13品种的幼嫩种子的珠心组织为材料,诱导出胚性愈伤组织。对胚性愈伤组织诱导培养基、继代培养基中不同的影响因素如植物生长调节剂的种类和浓度、钙离子浓度等进行了研究,筛选到了合适的影响因素。经过连续5个月的继代选择培养,逐渐诱导出易碎的胚性愈伤组织。对易碎胚性愈伤组织进行了长期继代培养。组织学切片证明长期继代培养的愈伤组织维持了胚性的状态。取继代培养了2年多的橡胶树热研88-13品种的珠心易碎胚性愈伤组织诱导胚状体,得到了186个胚状体,正在诱导植株再生。     

油松胚性愈伤组织培养中褐化因素研究及增殖培养基的优化

【目的】调查油松Pinus tabulaeformis胚性愈伤组织增殖培养阶段不同因素对于褐化产生的影响,并结合增殖率对培养基进行调整优化以降低褐化率,提高利用率。【方法】以3个年份的9个细胞系的油松胚性愈伤组织为材料,利用单因素试验设计对放置不同数量愈伤块、pH、植物凝胶含量、糖种类和含量以及培养时间的褐化率和增殖率进行测定计算。采用正交试验选出最佳培养条件。【结果】单因素试验结果表明,褐化率在不同基因型间差异显著,且与增殖继代年龄显著正相关;在培养皿中放置6~7块愈伤组织、pH 5.8~6.0、培养基中添加2.0~3.0 g·L~(-1)植物凝胶、添加10 g·L~(-1)蔗糖能保证较低的褐化率和较高的增殖率,且蔗糖优于葡萄糖和麦芽糖。【结论】结合正交设计,90 mm培养皿中放置7块愈伤组织,培养基中添加2.5 g·L~(-1)植物凝胶、10 g·L~(-1)蔗糖,调节pH为5.9时,褐化率、增殖率和利用率最理想。     

长期继代培养过程中楸树愈伤组织的分化能力

以长期继代培养的楸树愈伤组织和新诱导的楸树愈伤组织为试验材料,利用石蜡切片比较分析它们的组织形态特征,并测定过氧化物酶（POD）、超氧化物歧化酶（SOD）和淀粉酶的活性,以及可溶性蛋白与可溶性糖的含量,以探究楸树愈伤组织长期继代培养后分化再生困难的形成原因。组织形态学观察分析发现,继代培养7 a的白色颗粒状愈伤组织细胞中淀粉粒含量丰富,分布均匀,仍为胚性愈伤组织;新诱导的浅绿色愈伤组织存在明显的细胞分化中心,有均匀分布的淀粉粒,为早期胚性愈伤组织;新诱导的黄白色致密愈伤组织为非胚性向胚性转化的愈伤组织,新诱导黄白色疏松愈伤组织为非胚性愈伤组织。生理生化指标测定分析发现,继代培养7 a的楸树胚性愈伤组织中可溶性糖和可溶性蛋白的含量均显著高于新诱导出的3种愈伤组织,但其同工酶活性却较低,表明在长期继代培养的过程中,楸树愈伤组织的生理生化代谢活性降低。由此推测生理生化代谢活性的下降可能是造成楸树胚性愈伤组织长期继代培养后分化再生困难的重要因素。     

Optimizing Culture System of Ri T-DNA Transformed Roots for Citrus grandis cv. Changshou Shatian You

Genetic transformation experiments of the different explants from Citrus grandis cv. Changshou Shatian You infected with Agrobacterium rhizogenes were carried out in darkness or in light. The optimizing culture system of Ri T-DNA transformed roots for C. grandis cv. Changshou Shatian You was constructed as follows: After the ventral wounded striations on the single activation cotyledon were inoculated by A. rhizogenes A4 (logarithmic period), they were cocultured at (25 ± 2)°C in darkness for 25–30 days; some transformed roots were generated from wounded striations of most cotyledons. The genetically transformed ratio is (83 ± 11)%. Axenic Ri T-DNA transformed roots (hairy roots) were harvested after five subcultures. Explants were activated on MT medium. The MS medium was used for subculture of transformed roots. Mass Ri T-DNA transformed roots in which the hormone was produced independently were harvested from this optimizing culture system. White, fresh Ri T-DNA transformed roots were (1.14 ± 0.07) cm long, (0.73 ± 0.04) mm wide, and the growth direction of transformed roots was negative geotropism.     

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.     

猕猴桃体外快繁条件优化及高效再生体系的建立

为进一步优化‘红阳’猕猴桃（Actinidia chinensis cv. Hongyang）组织培养育苗技术体系,采用‘红阳’猕猴桃无菌组培苗的茎尖和叶片为材料,以ZT 1.5 mg·L^-1为外源激素,筛选适宜的基本培养基,并研究不同外源激素及组合对‘红阳’猕猴桃间接、直接器官发生及植株再生的影响。结果表明,适合‘红阳’猕猴桃生长的基本培养基为OM。在OM + 2.0 mg·L^-1 6-BA + 0.5 mg·L^-1 NAA + 0.01 mg·L^-1 2, 4-D培养基中,叶片愈伤诱导率为100%,不定芽发生系数为3.68;而带叶茎尖在OM + 1.5 mg·L^-1 6-BA + 0.5 mg·L^-1 NAA + 1.0 mg·L^-1 KT培养基中培养60 d后,通过直接器官产生基茎丛生芽,其增殖系数可达7.65。试管苗适宜的生根培养基为1/2 OM + 0.5 mg·L^-1 NAA,60 d后平均不定根数为4.87,驯化移栽后成活率为90%以上。选择出了适宜‘红阳’猕猴桃生长的基本培养基,解决了组培苗叶片黄化、植株矮小和畸形的问题,且大幅提高了增殖系数。优化了‘红阳’猕猴桃的人工快繁体系,可为猕猴桃其他品种的研究提供参考。     

Somatic embryogenesis and plant regeneration in glandless upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Glandless upland cotton has an important economic value. Embryogenic calli and regenerated plants were obtained from the hypocotyl explants of glandless upland cotton seedlings, cultivar Jisheng1. The results indicated that somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. 2, 4-D was advantageous to induce cotton callus, but embryogenic callus could not be obtained on the 2, 4-D medium. Embryogenic calli were also not obtained on the MSB sold medium with the combination of IBA and BA. However, embryogenic calli were induced when the hypocotyl explants were cultured on the IBA and KT medium. More than 31% of the hypocotyl segments produced embryogenic calli when the MSB medium was supplemented with 1.0 mg·L⁻¹ IBA and 0.5 mg·L⁻¹ KT. Embryogenic calli with somatic embryos could be observed within three months. Somatic embryo germination and maturation occurred on the hormone-free MSB medium with 1.0 g·L⁻¹ Gln and 0.5 g·L⁻¹ Asn. A number of regenerated plants could be obtained in six months. In the present study, a simple and efficient system was established to induce a number of embryogenic calli and regenerate plantlets from hypocotyl explants.     

Effects of Chlorine Dioxide Gas on Postharvest Physiology and Storage Quality of Green Bell Pepper （Capsicum frutescens L. var. Longrum）

The effects of treatment of chlorine dioxide (ClO2) gas on postharvest physiology and preservation quality of green bell peppers were studied. Green bell peppers were collected in bags and treated with 0, 5, 10, 20, and 50 mg L-1 ClO2 gas at 10 ± 0.5℃ for over 40 d, and the changes in postharvest physiology and preservation quality of the peppers were evaluated during the storage. The inhibition of rot of the peppers was observed for all the tested ClO2 gas treatments. The rot rates of the treated samples were 50% lesser than those of the control after day 40 of storage. The highest inhibitory effect was obtained after 50 mg L-1 ClO2 gas treatment, where the peppers did not decay until day 30 and showed only one-fourth of the rot rate of the control at day 40 of storage. The respiratory activity of the peppers was significantly (P ＜ 0.05) inhibited by 20 and 50 mg L-1 ClO2 treatments, whereas no significant effects on respiratory activity were observed with 5 and 10 mg L-1ClO2 treatments (P＞ 0.05). Except for 50 mg L-1 ClO2, malondialdenyde (MDA) contents in the peppers treated with 5,10, or 20 mg L-1 ClO2 were not significantly (P＞0.05) different from those in the control. Degradation of chlorophyll in the peppers was delayed by 5 mg L-1 ClO2, but promoted by 10, 20, or 50 mg L-1 ClO2. The vitamin C content, titratable acidity,and total soluble solids of the peppers treated by all the tested ClO2 gas did not significantly change during the storage.The results suggested that ClO2 gas treatment effectively delayed the postharvest physiological transformation of green peppers, inhibited decay and respiration, maintained some nutritional and sensory quality, and retarded MDA accumulation.     

提高N6培养基钙浓度对玉米幼胚培养的影响

筛选试验结果表明 ,将 N6 培养基 Ca2 + 浓度从 1.13mmol/ L提高到 5 m mol/ L ,对玉米幼胚胚性愈伤组织的诱导、继代和分化具有促进作用。在一次继代过程中 ,Ca2 +浓度为 5 m mol/ L 的培养基上培养的胚性愈伤组织相对生长量比 1.13m mol/ L 的原培养基增加 0 .8倍。 Ca2 +浓度提高以后 ,在培养基中再添加钙调蛋白 (Ca M)抑制剂盐酸氯丙嗪 (CPZ) ,可抑制 Ca2 +的促进作用 ,说明钙增加后对玉米胚性愈伤组织诱导和继代的促进作用受Ca2 + / Ca M信息系统控制。用改进后的 N6 培养基 ,可提高生产上大面积推广的玉米自交系胚性愈伤组织的诱导率和继代生长量     

Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa

To study the growth effects of differing concentrations of bensulfuron-methyl on Chlorella pyrenoidosa and to evaluate the ecological risk, the effects of bensulfuron-methyl on the growth and the content change of chlorophyll and protein in Chlorella pyrenoidosa were studied through 96 h acute toxicity tests. Bensulfuron-methyl accelerated the growth of algae at lower concentrations （〈 1 mg L^-1） with content increase of chlorophyll or protein, and it inhibited the growth of algae at higher concentrations （〉 5 mg L^-1）. The content of chlorophyll or protein in algae cells reduced with the increasing concentration of bensulfuron-methyl, exhibiting the good concentration-effect relationship. The 96 h-EC50 of bensulfuronmethyl upon the algae was 15.7 mg L^-1 Bensulfuron-methyl has inhibiting effect on the growth of Chlorella pyrenoidosa and is low in toxicity.     

黄精的组织培养与植株再生

研究离体条件下黄精不同外植体的愈伤组织诱导及其植株再生。结果表明,黄精的根茎、嫩茎及幼嫩的组培叶片在MS+6-BA 2.0 mg.L-1+2,4-D 1.0 mg.L-1和MS+6-BA 2.0 mg.L-1+NAA 1.0mg.L-1的培养基中均诱导产生愈伤组织,并在MS+6-BA2.0 mg.L-1+2,4-D2.0 mg.L-1和MS+6-BA2.0 mg.L-1+NAA2.0 mg.L-1的培养基上继代增殖良好。黄精愈伤组织在MS+6-BA3.0 mg.L-1+IAA1.0 mg.L-1的培养基上可诱导出不定芽,出芽率达66.33%,平均每块愈伤组织可分化出5.2个芽。试管苗在MS附加IAA0.5 mg.L-1培养基上诱导生根,生根率达86.67%。     

长寿沙田柚不同外植体愈伤组织诱导的研究

[目的]对长寿沙田柚不同外植体愈伤组织的诱导进行研究。[方法]以长寿沙田柚无菌苗为材料,分别采用其上胚轴、下胚轴和子叶节段为外植体,研究不同外源激素对愈伤组织诱导的影响。[结果]诱导长寿沙田柚愈伤组织的外植体以下胚轴为佳,激素组合以1.0 mg/L 6-BA+1.0 mg/L 2,4-D的效果最好。[结论]为以后长寿沙田柚高效离体再生体系的建立及其遗传转化研究奠定了基础。     

掌叶覆盆子离体培养适宜外植体的筛选及脱分化条件研究

以安徽地区掌叶覆盆子（Rubus chingii Hu）为试验材料,MS培养基为基本的培养基,研究了在不同外植体和不同植物生长调节剂水平下对其愈伤形成率的影响和不同因素的抗氧化试验对其污染率与褐化率的影响,筛选出能稳定形成健康的愈伤组织的培养条件。结果表明,最适合掌叶覆盆子愈伤组织形成的外植体为腋芽;最佳外植体的表面灭菌条件为75 %乙醇溶液处理30 s和0.1 %HgCl₂溶液处理8 min;最优掌叶覆盆子愈伤形成的培养基为MS + 5.0 g·L ^-1 PVP + 0.5 mg·L ^-1 6-BA + 0.1 mg·L ^-1 NAA + 0.5 mg·L ^-1 GA₃ + 25 g·L ^-1蔗糖 + 6.0 g·L ^-1琼脂;最适合暗培养时间为7 d;最适合的抗氧化剂为PVP。在上述条件下,掌叶覆盆子的愈伤组织形成率为96.67%,且能基本抑制外植体的褐化现象。该研究结果为掌叶覆盆子的种苗快繁及后期开展覆盆子次生物质代谢途径的研究提供了材料基础。     

The Effects of the Chemical Components of Soil Salinity on Electrical Conductivity in the Region of the Delta Oasis of Weigan and Kuqa Rivers, China

In order to assess the effects of chemical properties of soil salinity on electrical conductivity of 1：5 soil/water extract （EC1：5）, the study focused on revealing the main chemical factors contributing to EC of soil extracts and their relative importance. The relationship between EC1：5 and the chemical properties of soil salinity in the delta oasis of Weigan and Kuqa rivers, China, were studied using path coefficient analysis, a path analysis method. We studied each key element affecting EC1：5 either directly or indirectly. The results obtained show that the salt content, total dissolved solids （TDS）, and the sum of the sodium ion concentration and the kalium ion concentration are the most influential factors on 1：5 soil/ water extract （EC1：5） in the 0-10 cm and the 30-50 cm soil layer. The results show that the sequence of direct path coefficients in the 0-10 cm and the 30-50 cm soil layers on soil conductivity is TDS→Na^＋ ＋ K^＋→Salt content→Ca^2＋→Cl-→the sodium dianion ratio （SDR）→pH→ SO4^2-→HCO3^-→Mg^2＋→the soluble sodium percentage （SSP） sodium absorption ratio （SAR） and TDS→Salt content→Na^＋ ＋ K^＋→Ca^2＋→SDR→Mg^2＋→HCO3^-→SSP→pH→SO4^2-→SAR→Cl^-. The salt content, chlorine ion, and SAR are the main factors affecting 1：5 soil/water extract （EC1：5） in the 10-30 centimeter soil layer. The order of direct path coefficients result is as follows： Salt content→Cl^-→SAR→SSP→TDS→Ca^2＋→Mg^2＋= SO4^2-→HCO3^-→pH→SDR→Na^- ＋ K^＋. Moreover, the effects of HCO3^-, pH were very weak. Though the direct path coefficients between EC1：5 and SAR, SO4^2- and Ca^2＋ were not high, influence of other chemical factors caused the coefficients to increase, making the summation of their direct and indirect path coefficients relatively high. The models of the different soil layers were structured separately. Evidences showed that multiple regression relations between EC1：5 and most of the primary factors had sound reliability and very good accuracy. The research results can serve as a reference to the scientific management amelioration and utilization of saline in the Delta Oasis of Weigan and Kuqa rivers.