In Vitro Selection and Identification of Drought-Tolerant Mutants in Sweetpotato

In vitro selection of drought-tolerant mutants in sweetpotato cv. Lizixiang was studied by using PEG6000 as selection stress. Embryogenic suspension cultures were cultured in MS medium containing 0-35% PEG6000 and 2 mg L-1 2, 4-dichlorophenoxyacetic acid (2, 4-D). The results indicated that 30%PEG6000 can be used for the optimal selection stress of drought-tolerance. Embryogenic suspension cultures irradiated with 80 Gy gamma-ray were cultured in MS medium containing 30 % PEG6000 and 2 mg L-1 2,4-D and 20 drought-tolerant cell aggregates were obtained. These cell aggregates were transferred to solid MS medium supplemented with 2 mg L-1 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 1 mg L-1 abscisic acid (ABA), resulting in the germination of somatic embryos. In this study a total of 18 regenerated plants were obtained. The regenerated plants were transplanted in a greenhouse and 11 lines were formed. The analysis on drought treatment of seedlings, water retaining capacity of leaves and coefficient of drought-tolerance showed that 3 lines had significant drought-tolerance in comparison with the control plants.     

利用正交设计优化百子莲农杆菌转化条件的初探

利用正交试验设计,以百子莲胚性愈伤组织为受体材料,通过GUS染色确定遗传转化率,对农杆菌介导的百子莲遗传转化体系进行了优化。结果表明,百子莲遗传转化优化体系为：愈伤组织预培养7 d、菌液浓度（OD600）为0.9、侵染时间为5 min、预培养培养基中乙酰丁香酮（AS）的浓度为50μmol.L-1、侵染菌液中乙酰丁香酮（AS）的浓度为100μmol.L-1。在优化的转化条件下,可提高农杆菌EHA105菌株介导的百子莲愈伤组织遗传转化率。     

Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

Immature embryos from three elite Guizhou waxy maize inbred lines (W21019, B7, and QCL5036) were evaluated for theirability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid (2,4-D). The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect (P＜0.05) on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 (B7 and QCL5036) to 3.0 mg L-1 (W21019). The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1of 6-benzylaminopurine (6-BA). 6-BA in the medium significantly promoted the regeneration of embryogenic callus.Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenie callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.     

Using the Phosphomannose Isomerase (PMI) Gene from Saccharomyces cerevisiae for Selection in Rice Transformation

The phosphomannose isomerase (PMI) gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice (Oryza sativa L.) via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. β- Glucuronidase (GUS) activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.     

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.     

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.     

玉竹愈伤组织诱导及其细胞悬浮培养体系的建立

以玉竹叶片和根状茎为外植体,研究植物生长调节剂、碳源和pH对玉竹愈伤组织诱导的影响以及植物生长调节剂对愈伤组织增殖的影响,通过正交试验初步建立并优化玉竹愈伤细胞的悬浮培养体系。结果表明,叶片和根状茎愈伤组织诱导的最佳培养基均为MS+1.0mg·L~(-1)?6-BA+0.5mg·L~(-1)?NAA+30%蔗糖,培养基最佳pH5.6,叶片和根状茎愈伤组织增殖的最佳植物生长调节剂组合均为2.0mg·L~(-1)?6-BA+0.8mg·L~(-1)NAA,且根状茎愈伤组织的诱导率和增殖倍数均明显高于叶片。根状茎愈伤细胞悬浮培养的最佳接种量80g·L-1,最佳植物生长调节剂组合1.5mg·L~(-1)6-BA+0.6mg·L~(-1)?NAA,细胞生长曲线与多糖含量变化曲线均呈"S"形,细胞最佳继代周期16d,多糖含量在培养14d时最高,培养基pH呈先下降后上升然后趋于稳定趋势。     

萘乙酸对短穗鱼尾葵耐寒性的影响

为研究不同浓度萘乙酸(NAA)溶液对短穗鱼尾葵(Caryota mitis)幼苗耐寒性的影响,用0、25、50mg·L-1和100mg·L-1 4种浓度的NAA溶液喷施短穗鱼尾葵叶片。分别经过25、5℃和25℃各24h变温冷胁迫处理后,研究短穗鱼尾葵叶片在变温冷胁迫过程中的超氧化物歧化酶(SOD)、过氧化物酶(POD)、丙二醛(MDA)、游离脯氨酸(f-pro)、可溶性蛋白和叶绿素生理指标的动态变化规律,并对25℃恢复24h后的短穗鱼尾葵幼苗上述耐寒指标采用隶属函数法综合评价,结果表明,4种浓度下隶属函数值分别是0.35、0.67、0.53和0.29,其中25mg·L-1的NAA溶液喷施处理对提高短穗鱼尾葵的耐寒性效果最好。     

Effects of Auxins and Media on Callus Induction of Chinese Spring Wheat( Triticum aestivum L.)

The effects of auxins and media on callus induction from the mature and immature embryos of Chinese spring wheat(Triticum aestivum L.) varieties were investigated. It was found that genotype, medium, auxin source and concentration had the significant effects on the induction of embryogenic callus, explants germination and the increment of callus fresh weight. For immature embryos cultured on MS medium, 2 mg L-1of 2, 4-D was optimal, and the highest frequency of embryogenic callus (33.50%) was observed. For the mature embryos on N6 medium, 4 mg L-1 of 2, 4-D was optimal. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. These results indicated that 2, 4, 5-T was superior to 2, 4-D and NAA in the culture of immature embryos. This is the first report about the effect of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail.     

水稻类病变突变体spl5胚性悬浮细胞系的构建

类病变突变体（lesion mimic mutant）是一类研究细胞死亡与抗病机制的有效工具。试验以水稻类病变突变体spl5为材料,构建了spl5突变体的胚性悬浮细胞系。结果表明诱导spl5突变体愈伤组织的最佳2,4-D浓度为2 mg·L-1,疏松的Ⅱ型胚性愈伤组织经悬浮继代培养3～4个月后可获得理想的胚性悬浮细胞系,FDA-PI染色结果表明该细胞悬浮体系活力高。水稻类病变突变体spl5胚性悬浮细胞系的建立可为进一步研究spl5突变体细胞死亡及其与病原菌互作的机制奠定基础。     

Somatic Embryogenesis in Lily Bulb Scale Cultures

Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.     

抗生素对玉米愈伤组织生长和分化的影响

以继代培养的玉米胚性愈伤组织为材料,研究了不同质量浓度的卡那霉素(Kan)、遗传霉素(G418)和头孢霉素(Cef)对胚性愈伤组织继代生长和分化的影响.结果表明:(1)当质量浓度大于3 mg.L-1时,Kan对愈伤组织的生长具有一定抑制作用,其抑制作用在不同继代次数和不同基因型间具有明显差异;Kan明显降低了愈伤组织的分化出苗率;(2)G418具有显著抑制愈伤组织生长的负向效应.当G418的质量浓度大于25 mg.L-1时,愈伤组织就丧失了再生能力;(3)Cef质量浓度为100 mg.L-1时,对愈伤组织的生长无显著影响,随着质量浓度的增加,对愈伤组织的生长开始具有抑制作用,Cef对愈伤组织生长的抑制作用具有一定的累加效应.Cef对愈伤组织的分化再生能力具有一定的正向效应,但其临界质量浓度在基因型间和继代次数间存在差异.     

小麦成熟胚再生体系及基因枪转化的初步研究

以小麦品种晋麦2148的成熟胚为外植体,消毒后用解剖刀刨碎,接种于MS+VB5+11.2g·L-1葡萄糖+2mg·L-12,4D+8g·L-1琼脂糖的培养基中诱导愈伤组织,将诱导3周的成熟胚愈伤组织接种到胚状体成熟培养基(MS+VB5+11.2g·L-1葡萄糖+0.2mg·L-1IAA+8g·L-1琼脂糖)中培养4-8周,然后转接到再生培养基(1/2MS+VB5+30g·L-1蔗糖+8g·L-1琼脂糖)中进行再生成苗.接种1200块成熟胚,得到1123块愈伤组织,最终形成258株再生苗,再生率为21.5%.在建立了再生体系的基础上,用基因枪介导法将GUS基因导入成熟胚愈伤组织,Xgluc染色表明,GUS基因已经在小麦成熟胚愈伤组织中表达.将100块愈伤组织用Xgluc染色,29块愈伤组织出现肉眼可见的蓝色小点.     

木薯脆性胚性愈伤组织原生质体培养与植株再生

【目的】建立有效的木薯原生质体再生体系,为原生质体融合以及原生质体转化等研究奠定技术基础。【方法】酶解木薯品种TMS60444 的脆性胚性愈伤组织（FEC）的悬浮系,分离原生质体的产量最高达3.5×106个/g,FDA检测其活性约90%。用TM2G培养基以液体浅层法分别在5×105个/mL和2×105个/mL密度下培养,培养过程中前30 d用0.3 mol•L-1 TM2G新鲜培养基每10 d更换1次,此后每10 d用0.25 mol•L-1 TM2G新鲜培养基更换。原生质体培养45 d后即可挑出1-2 mm大小的愈伤组织分别在MSN培养基上分化胚、CMM上促胚成熟、CEM上茎伸长、MS上生根。【结果】在5×105个/mL的原生质体培养密度下,长出的都是致密型愈伤组织（能分化胚状体）,而在2×105个/mL的密度下培养长出的有致密型愈伤组织,也有空泡型愈伤组织（不能分化胚状体）。本试验共挑出1 479个致密细胞团,分化获得757个子叶胚,已再生完整植株186棵。【结论】本研究分离的原生质体产量和活性有较大提高,对前人所指出的瓶颈问题有所改进,原生质体再生植株效率有较大提高。     

中华猕猴桃华优遗传转化系统建立研究

为了更好的利用遗传转化技术进行猕猴桃品种改良研究,以中华猕猴桃华优组培苗幼嫩叶片为材料, 研究了外植体分化不定芽再生途径所需的培养条件及转化效率提高方法。结果表明,在叶片不定芽诱导培养基中加 入浓度为0.75~1.0 mg/L 的TDZ 时,叶片愈伤发生率达85.7%~90.1%,不定芽发生率达41.8%~44.6%;在TDZ 1.0 mg/L 基础上,补充0.1~0.4 mg/L IAA 可使叶片愈伤发生率提高到96.7%~100%,不定芽发生率提高到67.4%~71.9 %; 在农杆菌侵染液与共培养基中同时加入75~100 滋mol/L 浓度的AS,可使GUS 瞬时表达率及卡那抗性芽发生率分别 提高至27.9%、14.5%;外植体被农杆菌侵染前,的预培养2~3 d有利于提高GUS 表达率与卡那抗性芽发生率。GUS 染色与PCR 扩增检测结果显示,Shiva A基因已转化至中华猕猴桃华优转基因植株。     

Plant Regeneration from In Vitro Cultured Hypocotyl Explants of Euonymus japonicus Cu zhi

Adventitious shoots were successfully regenerated from hypocotyl explants of in vitro cultures of Euonymus japonicus Cu zhi. Hypocotyl slices were cultured on Murashige and Skoog （MS） and B5 basal medium supplemented with varied concentration of different plant growth-regulators, e.g., α-naphthaleneacetic acid （NAA） and indole-3-butyric acid （IBA） in combination with 6-benzylaminopurine （6-BA） and kinetin. The study showed that shoots could be directly regenerated from hypocotyl explants without the intervening callus phase; MS medium was more suitable for adventitious shoots regeneration. The ability of hypocotyls segments to produce shoots varied depending upon their position on the seedlings. The highest regeneration rate was obtained with hypocotyl segments near to the cotyledon cultured on MS basal medium supplemented with 1.5 mg L^-1 6-BA and 0.05 mg L^-1 NAA （63.64%）. The regenerated shoots were readily elongated on the same medium as used for multiplication and rooted on half-strength MS basal medium supplemented with 1.0 mg L^-1 IBA and 100 mg L^-1 activated carbon. After being transferred to greenhouse conditions, 96% of the plantlets were successfully acclimatized. This regeneration system is applied for genetic transformation now.     

南瓜中环丙基甘氨酸和环丙基丙氨酸提取工艺研究

为了进一步提升南瓜的经济价值,通过对南瓜中环丙基甘氨酸和环丙基丙氨酸两种非蛋白质氨基酸提取工艺中温度、提取时间、料液比、磺基水杨酸浓度等影响因素的试验分析,确定南瓜中环丙基甘氨酸和环丙基丙氨酸的最佳提取条件:提取温度为80℃,磺基水杨酸浓度为1%,提取时间为3h,料液比为1:30,并采用氨基酸分析仪对南瓜中这两种氨基酸含量进行了测定;确定南瓜中环丙基甘氨酸和环丙基丙氨酸含量分别为96.48mg·100g-1和162.60mg·100g-1。     

四倍体葡萄诱导技术的研究

 以秋水仙素为诱变剂，利用茎段浸泡和秋水仙素培养基两种方法对二倍体京秀和红地球2个品种的葡萄(Vitis. vinifera)组培苗进行诱导，并采用流式分析仪对诱变植株的细胞DNA含量进行鉴定。结果表明，茎段浸泡秋水仙素法诱导四倍体葡萄的效果优于秋水仙素培养基法。在低浓度秋水仙素（2000 mg•L-1或低于2000 mg•L-1）溶液中浸泡时间短于48 h的所有处理，仅在继代2~5次后获得了纯合四倍体植株；在秋水仙素浓度2000 mg•L-1对茎段浸泡3 ~4 d或3000 mg•L-1 和4000 mg•L-1对茎段浸泡2~4 d，在茎段扦插的当代就获得了纯合四倍体植株，诱变四倍体植株的适宜条件为在秋水仙素3000 mg• L-1 茎段浸泡3~4 d，纯合四倍体植株获得的比率为16.7~23.3%。     

离子束照射甘薯胚性细胞团获得突变体

用0~100Gy12C5+和0~200Gy4He2+分别照射甘薯品种栗子香的胚性悬浮细胞,用0~100Gy4He2+照射甘薯品种玉丰的胚性细胞团,以研究甘薯细胞水平离子束辐射的诱变效应。确定了甘薯胚性悬浮细胞适宜的12C5+和4He2+辐射剂量,分别为30~50Gy和70~100Gy;甘薯胚性细胞团适宜的4He2+辐射剂量为50~70Gy。得到大量再生植株,辐射后代的顶叶形、叶形、顶叶色、叶脉色、脉基色、顶端茸毛等性状出现了广泛的变异。     

巴东红三叶胚性细胞悬浮系的建立

以红三叶(Trifolium pratense)野生品种巴东红三叶的叶片为外植体,在MB-1至MB-9的固体培养基上诱导愈伤组织,筛选出最佳培养基MB-9.愈伤组织形成以后,均经MB-9固体培养基上继代培养3～4次后,获得胚性愈伤组织,然后转至改良的MB-9液体培养基中,经强化培养两个月后,获得胚性细胞悬浮系.结果表明,缩短换液时间,每隔3～4d换液1次,可加快胚性细胞悬浮系的建立.