Somatic Fusion for Breeding of Tetraploid Potatoes

From two tetraploid, one Transformed tetraploid, one triploid and 11 dihaploid clones of Solanum tuberosum somatic hybrids were produced by polyethylene glycol mediated somatic fusion. The inter-dihaploid clones comprised clones of agronomic value, homozygous doubled monohaploids, and in vitro selected clones resistant t0 Fusarium or Phytophthora toxins. Presumptive hybrids were enriched at the callus Stage in vitro by using differentiating media and by growth characteristics; further identification was performed by chromosome counting in vitro shoots and by isozyme analysis of in vitro plants. Final analysis was made from morphological characteristic of plant and tuber phenotypes. From 15 different combinations, 6009 plantlets have been regenerated. From five combinations, 310 reentrants were checked for hybrid nature by morphology and cytology and 88 by peroxidase and esterase isozyme analyses. Amongst these, from two combinations, a total of 17 different hybrids were confirmed by all methods. The procedures described are general enough to allow genome combination of interdihaploids resulting in tetraploids of practical breeding value.     

Factors affecting the production and regeneration of protoplasts: the case of the mycorrhizal fungus Tulasnella calospora from roots of orchids

Protoplast isolation is relevant for many different applications and has been principally used in proceduresnvolving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia （3 d） of Tulasnella calospora were digested for 6 h at 30℃ in a mixture of 1.2 mol·L-1 MgSO_4 ＋ 10 mmoI·L-1 K2HPO4 as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase： more than 106 protoplasts mL-1 were obtained. When collected 3y density gradient centrifugation, the concentration of protoplasts can reach 107-108 protoplasts mL-1, an amount suitable enough for experiments of transformation in fungi. For every 10_5 protoplasts, about 15-25 protoplasts can egenerate after 24-36 h cultivation in a liquid medium and after 8-10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora solate.     

Production and testing of plants regenerated from protoplasts of photoperiod sensitive genic male sterile rice (Oryza sativa L.)

Plants were regenerated from protoplasts isolated from embryonic suspension cultures of N5047S, a photoperiod sensitive genic male sterile (PGMS) Japonica rice line. Flow cytometric analyses of nuclear DNA content identified some tetraploid regenerates whose agronomic traits could be distinguished from diploid regenerates. Pollen and female fertility of diploid protoplast-derived clones grown under different light and temperature conditions was compared. A promising PGMS protoplast clone, ZAU11S, was developed from these clones. Its male sterility was confirmed as a photoperiod × temperature interaction type. This revised version was published online in July 2006 with corrections to the Cover Date.     

从萝卜下胚轴再生完整植株

以萝卜无菌幼苗下胚轴为外植体,研究了不同基因型萝卜品种,不同浓度的NAA、BA、蔗糖、AgNO3对植株再生的影响。基本培养基(BM)为MS无机盐 Gly2mgL 肌醇2mgL 烟酸05mgL VB101mgL 琼脂68gL。试验证明萝卜离体再生主要受基因型和BA、NAA浓度影响。从自交系小红头下胚轴愈伤组织产生的绿色子叶状团块在提高BA、NAA浓度的培养基中大量增殖;降低激素浓度,尤其是BA浓度后,这些子叶状簇生团块发育成大量无根苗,叶片正常。无根苗在添加不同种类的生长素的培养基中生根,并成功地移植入田间。本实验也对AgNO3的作用进行了研究和讨论。     

球孢白僵菌原生质体获得和再生恢复培养条件的研究

对球孢白僵菌菌龄、β-巯基乙醇、溶壁酶、渗透压稳定剂和琼脂粉含量等条件对球孢白僵菌原生质体的获得和再生恢复培养的影响进行了研究,建立了球孢白僵菌原生质体获得和再生恢复培养的最佳体系。原生质体获得的最佳菌龄为培养24~28 h的菌丝体,最佳β-巯基乙醇浓度为0.01 mol/L,最佳酶为真菌溶壁酶,最佳渗透压稳定剂为0.7 mol/L NaCl,原生质体获得频率高达99%;原生质体再生恢复时培养基中最佳渗透压稳定剂为0.7 mol/L葡萄糖,最佳琼脂粉质量分数为0.75%,再生频率达57%。     

顶头孢霉原生质体制备、再生及带vgb基因的质粒DNA转化条件的研究

对头孢菌素C的工业生产菌种顶头孢霉(Cephalosporium acremonium)原生质体形成和再生条件进行了研究。优化后的制备及再生条件为:在蛋白胨固体培养基上培养110~120 h的菌丝体,30℃条件下经1%蜗牛酶和1%纤维素酶混合液酶解3.5 h,原生质体得率最高可达2.377×107个/mL;在以KC(0.6 mol/L KCl+25mmol/L CaCl2.2H2O)为渗透压稳定剂的再生培养基上进行培养,再生率可达40%。用pMK4-LV质粒通过电击法对原生质体进行转化,成功得到了重组子,转化率约为10个转化子/μg质粒DNA。转化子的PCR鉴定结果表明,外源基因已转入顶头孢霉基因组。     

播种深度对高粱出苗和幼苗生长的影响

为了明确高粱的适宜播种深度,观察不同高粱品种对不同播种深度的耐深播差异,研究了8 种播种深度对3 个高粱品种出苗和幼苗生长的影响,出苗时记载不同播种深度下高粱的出苗速度和出苗率,出苗结束后,调查其地上部分幼苗生长量、胚轴长度和种子根长度。结果表明：随着覆土厚度的增加幼苗出苗速度明显减慢。在播深3 cm、5 cm和7 cm处理下,各品种从播种至出苗需要5 天,在播深8 cm、10 cm、12 cm 和13 cm 处理下,需要5~7 天,在播深17 cm 处理下为7~11 天。在播深12 cm、13 cm 和17 cm处理下‘晋中0822’较其他2 个品种从播种至出苗所需时间早2~4 天。不同播深处理下高粱完成出苗所需要时间也不尽相同。在播深3 cm、5 cm和7 cm处理下,各品种完成出苗所需时间为9 天,在播深8 cm、10 cm、12 cm和13 cm处理下,需要9~13 天,在播深17 cm处理下需13 天。品种‘晋中0822’在播深8 cm、10 cm、12 cm和13 cm处理下9 天完成出苗,较其他2 个品种早。播种深度对3 个高粱品种的出苗率亦有显著影响。随着播种深度的增加,3 个高粱品种的平均出苗率不断降低。3 cm和5 cm为高粱最适播种深度。在播深3 cm、5 cm、7 cm、8c m、10cm和12 cm处理下品种‘晋中0822’出苗率显著高于其他品种。此外,播种深度对幼苗的生长发育也有显著影响。随着播种深度增加,3 个高粱品种的地上部分幼苗生长量和种根长显著降低,而胚轴长度显著增加。在3cm和5 cm播深处理下幼苗生长量、种根长和胚轴长与其他各处理差异显著。在播深7 cm、8 cm、10 cm、12 cm、13 cm和17 cm处理下‘晋中0822’品种各指标与其他品种差异显著。     

‘冬枣’子代种子萌发及下胚轴诱导愈伤组织研究

为实施‘冬枣’遗传改良方案,以采自山西柳林自然授粉获得的‘冬枣’子代种子为材料,测量种子横、纵径、种仁率等基本形态指标,并进一步研究种子萌发最适条件和下胚轴诱导愈伤组织适宜培养基。结果表明：（1）‘冬枣’子代种子横径为(8.62±0.05) mm,纵径为(17.95±0.05) mm,千粒重为(54.27±4.62) g,种仁率为(64.17±3.82)%,‘冬枣’属于种仁率较高的枣树品种。（2）种仁在含有GA3 0.4 g/L的萌发培养基中预处理24 h后接种到萌发培养基上,最有利于种仁萌发,萌发率可达100%;液体萌发培养基稍好于固体萌发培养基,但未达显著水平。（3）下胚轴诱导愈伤组织的适宜培养基为1/2MS+ TDZ 1.0 mg/L+蔗糖30 g/L,诱导率为(60.00±3.02)%。     

有斑百合多倍体诱导及鉴定

为了建立野生有斑百合多基因型种子离体多倍体诱导方法及快速、准确和高效的多倍体鉴定技术,本研究在离体培养条件下,以野生有斑百合预培养7 d的无菌萌动种子(2n=2x=24)为材料,用不同含量的秋水仙素溶液进行不同时间的诱变处理。以种子下胚轴明显膨大作为变异植株早期鉴定的标准,通过流式细胞仪检测细胞核DNA含量,并结合染色体计数法对诱导后的植株进行倍性鉴定,探讨在相同的种子萌动状态下,不同含量、不同浸泡时间的秋水仙素诱变效果,并初步建立了种子离体多倍体诱导和鉴定技术流程。结果表明:0.10%的秋水仙素溶液可有效地诱导野生有斑百合萌动种子的染色体加倍,其中以浸泡处理36 h的诱导效果最好,诱导率达44.43%;与二倍体植株相比,四倍体植株表现出叶片肥厚且增宽、叶色深绿、叶表面轻微褶皱、叶柄粗壮、生长缓慢等特征。研究还发现,植物外部形态变化不仅在二倍体和四倍体之间存在,而且在不同基因型的四倍体之间也存在差异。本研究利用下胚轴的膨大作为早期筛选多倍体的有效指标,同时结合直接测定细胞中DNA含量和计数染色体数可快速有效地培育出野生有斑百合同源四倍体植株。本研究建立的离体多倍体诱导流程除可应用于有斑百合种子多倍体的诱导外,也可为其他野生百合种子离体染色体加倍提供参考。      

甘蓝CMS下胚轴和子叶的离体培养与植株再生研究

【目的】探索一种利用甘蓝胞质雄性不育系(CMS)下胚轴和子叶进行快速扩繁的技术,为利用CMS大量繁制甘蓝杂交种提供一条新途径。【方法】以甘蓝CMS 05-2-10为试材,将苗龄5~7 d无菌苗的下胚轴和子叶切下,接种于MS培养基上,45~50 d后统计再生芽数,比较6-BA与NAA不同质量浓度配比对不定芽的诱导情况;将诱导出的不定芽接种于添加不同质量浓度(0,0.1,0.3,0.5 mg/L)NAA的MS生根培养基上,20 d后比较生根率和根的长势。【结果】在对下胚轴和子叶进行不定芽诱导时,MS培养基中分别添加1.0和4.0 mg/L的6-BA对下胚轴、子叶的诱导效果较好,其芽再生频率和分化系数分别为83.3%,53.0%和3.6,3.0。在MS培养基中添加1.0mg/L 6-BA+0.2 mg/L NAA,其对下胚轴不定芽的诱导效果较好,芽再生频率为80.7%,分化系数为3.4,褐化率为1.5%;在MS培养基中添加4.0 mg/L 6-BA+0.3 mg/L NAA,其对子叶不定芽的诱导效果较好,芽再生频率为50.0%,分化系数为3.2,褐化率为3.4%;二者的褐化率均明显低于仅添加6-BA的处理。在MS生根培养基中添加0.3 mg/L NAA时,不定芽的生根效果较好,生根率达100%,且根系生长健壮。【结论】利用甘蓝胞质雄性不育系CMS 05-2-10的下胚轴和子叶作为外植体进行不定芽的诱导,对于下胚轴,其适宜培养基为MS+1.0 mg/L6-BA+30 g/L蔗糖+8 g/L琼脂;对于子叶,其适宜培养基为MS+4.0 mg/L 6-BA+30 g/L蔗糖+8 g/L琼脂。不定芽诱导生根的最适培养基为MS+0.3 mg/L NAA+30 g/L蔗糖+8 g/L琼脂。