黑果枸杞高效植株再生与遗传转化体系的建立

为建立高效的黑果枸杞组织培养与遗传转化体系,本研究以黑果枸杞叶片、茎节、茎段为外植体,在含有不同浓度组合的NAA和BAP培养基上进行愈伤组织和不定芽诱导比较;选择黑果枸杞叶片为材料,采用农杆菌介导法,将带有绿色荧光蛋白基因(sGFP)的双元表达载体导入黑果枸杞,探讨侵染时间和抗生素浓度对遗传转化效果的影响。结果表明,黑果枸杞的最适植株再生体系为:以叶片为外植体,MS基本培养基中添加0.5 mg·L^-1NAA和0.3 mg·L^-1BAP,愈伤组织诱导率为88.56%,不定芽诱导率为54.54%,将不定芽置于1/2MS培养基中进行生根诱导,生根率为95%;稳定高效的遗传转化体系为:农杆菌侵染液OD₆₀₀为0.25,共培养3 d,卡那霉素(kanamycin)和羧苄西林(carbenicillin)浓度分别为25和250 mg·L^-1。愈伤组织和不定芽诱导时经卡那霉素和荧光标记双重选拔,愈伤组织阳性率为85.47%,出芽生根后经PCR鉴定,阳性转化率为29%;利用实时荧光定量PCR检测转基因植株,导入的外源sGFP基因均有较高的表达水平。本研究为利用基因工程技术对黑果枸杞进行品种改良提供了技术支撑。     

大白菜高效遗传转化体系的建立

研究了影响根癌农杆菌（Agrobacterium tumefaciens)LBA4404菌系介导的大白菜（Brassica campestris L.ssp.pekinensis)遗传转化转化的若干因素,建立了大白菜快速有效的遗传转化体系,获得了转基因抗性苗。结果表明,大白菜的不同基因型,苗龄,外植体类型,以及预培养与否,工程菌液浓度,侵染时间,共培养基的pH值和Co^2 ,卡那霉素,头孢霉素浓度等对转化频率都有一定的影响。最高转化频率可达5．74％。而不同共培养方式和附加乙酰丁时酮对转化效果影响不明显。抗性植株经PCR和Souhern blot检测表明,目的基因已整合进大白菜基因组中。     

抗生素对高粱茎尖再生的影响及再生体系的建立

研究羧苄青霉素、头孢霉素、潮霉素和卡那霉素4种抗生素对高粱茎尖再生的影响及再生体系的建立结果表明,MS培养基中添加甘氨酸、乙二胺四乙酸的铁络合物(Fe EDTA)和抗坏血酸等有机物有利于高粱茎尖的分化与生长。农杆菌介导的高粱茎尖遗传转化过程中羧苄青霉素为最理想抗生素,最佳浓度为2 5 0mg/ L。并初步确立了高粱茎尖农杆菌介导的遗传转化过程中抑菌和转化体的筛选策略     

番茄高效再生体系的优化

本文以自交高纯和番茄的子叶和下胚轴为材料,通过对其离体培养研究,筛选出诱导不定芽分化的最佳培养基:MS 6-BA1.0mg/L IAA0.1mg/L 3%蔗糖(子叶);MS 6-BA0.8mg/L IAA0.1mg/L 3%蔗糖(下胚轴);生根壮苗的最佳培养基为MS(无琼脂) IAA0.5mg/L 3%蔗糖。卡那霉素的敏感性实验确定了番茄在遗传转化中Kan的抗性筛选浓度:子叶为60mg/L,下胚轴为70mg/L。     

匍匐型地被菊再生及遗传转化体系的建立

为构建有效的地被菊再生和遗传转化体系,本研究以匍匐型地被菊雨花落英叶片为外植体,研究不同苗龄和激素配比对叶盘再生的影响;通过农杆菌介导法将外源基因导入野生型植株中,研究预培养、延迟培养、抗生素浓度等遗传转化条件对转化效率的影响。结果表明,雨花落英叶盘转基因最适再生条件为,以苗龄为30 d的组培苗幼嫩叶片为外植体,设置再生培养基配方为MS+6-BA 1.5 mg·L^-1+NAA 0.8 mg·L^-1,此时再生率高达91.11%,平均芽数为5.90;最优遗传转化组合为预培养2 d,侵染5 min,共培养2 d,延迟培养5 d,之后进行选择培养。羧苄青霉素在延迟培养阶段最适抑菌浓度为400 mg·L^-1;潮霉素叶盘筛选的最适选择压为8~10 mg·L^-1,生根筛选最适浓度为11 mg·L^-1;培养后期加入6-KT可以有效提高抗性芽的再生率;调节6-BA浓度为1.0 mg·L^-1,蔗糖浓度为40 g·L^-1,可以将得到的玻璃化苗转化为正常苗。分子鉴定结果表明,15株抗性苗中有10株阳性苗,阳性苗概率为67%;与野生型植株相比,9个株系的CmERF12基因均受到了明显的抑制表达。本研究建立了地被菊雨花落英再生和遗传转化体系,为进一步进行基因功能研究和遗传改良奠定了一定的理论基础。     

爬行卫矛再生体系建立及根癌农杆菌介导的遗传转化

以爬行卫矛(Euonymus fortunci var.radicans)无菌苗下胚轴为外植体,在附加不同浓度植物生长调节剂的MS基础培养基上诱导培养,通过器官直接发生途径获得再生植株.结果表明,0.5 mg/LBAP和0.01 mg/L NAA组合的诱导效果最佳,其不定芽再生率高达92%,外植体平均不定芽数目为4.2个.将含有雪花莲凝集素基因(Galanthus nivalis agglutinin,GNA)的植物表达载体pBCGm导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,通过农杆菌介导法遗传转化爬行卫矛的下胚轴,经PCR和PCR-Southern检测,GNA基因已整合到转化植株的基因组中.遗传转化过程中,外植体不经预培养,菌液浓度OD=0.6,共培养3 d,有利于获得最高遗传转化效率.     

抗生素对高粱茎尖再生的影响及再生体系的建立

研究羧苄青霉素、头孢霉素、潮霉素和卡那霉素4种抗生素对高粱茎尖再生的影响及再生体系的建立结果表明,MS培养基中添加甘氨酸、乙二胺四乙酸的铁络合物(Fe-EDTA)和抗坏血酸等有机物有利于高粱茎尖的分化与生长。农杆菌介导的高粱茎尖遗传转化过程中羧苄青霉素为最理想抗生素,最佳浓度为250mg／L。并初步确立了高粱茎尖农杆菌介导的遗传转化过程中抑菌和转化体的筛选策略。     

根癌农杆菌介导苜蓿遗传转化体系的建立

以建立的苜蓿高频再生体系为基础，利用GUS染色组织分析法，研究了影响根癌农杆菌(Agrobacterium tumefaciens Conn)LBA4404菌株介导的苜蓿（Medicago sativa L)遗传转化的若干因素，建立了苜蓿快速有效的遗传转化体系，获得了转BADH基因植株。苜蓿的不同基因型、附加乙酰丁香酮的浓度、预培养与否、菌液浓度、共培养时间和卡那霉素浓度等对转化频率都有一定影响。最高转化频率可达43.3％，抗性植株经PCR和PCR-Southern blot检测表明，目的基因已整合进苜蓿基因组中。     

川东獐牙菜体外高效再生体系的建立

为了构建川东獐牙菜(Swertia davidii Franch.)高效稳定的再生体系,以川东獐牙菜带叶茎尖、带芽茎段和叶片为材料,在单因素试验的基础上,通过完全组合及L₉(3⁴)正交试验,探究不同激素对其离体培养的影响。结果表明,川东獐牙菜3种外植体中,叶片适宜作为间接器官发生材料,在MS+2.0 mg·L^-1 6-BA+1.5 mg·L^-1 KT培养条件下,培养7 d便可见愈伤组织从切口处产生,培养30 d后即可分化出大量丛芽;不定芽在相同培养基中培养30 d后,增殖系数可达8.75。带芽茎段则适宜直接器官发生途径,其在MS+2.0 mg·L^-1 6-BA+1.0 mg·L^-1 NAA中培养7 d后,节上腋芽开始萌动,培养30 d后腋芽发生系数可达4.06;试管苗适宜的生根培养基为MS+NAA 0.05 mg·L^-1,培养30 d后即可获得再生植株,生根率为100%;生根苗经过炼苗,移栽30 d后成活率达90%以上。在川东獐牙菜的...     

苜蓿组织培养高频率再生体系的建立

苜蓿组织培养的研究早期主要集中在多年生紫花苜蓿上，Saunders等首先从愈伤组织上获得了再生植株，其它一些苜蓿也通过细胞和组织培养获得了再生植株。黄绍兴等获得了紫花苜蓿原生质体转基因植株。然而，国内还未见有适宜于基因转化的高频再生组织培养体系的报道。本实验的目的在于建立苜蓿高频再生组织培养体系，为今后的基因转化改良苜蓿性状打下基础。     

克新4号马铃薯高产施肥技术的研究

在浙江省马铃薯主产区丽水市,通过2年试验研究了克新4号马铃薯施肥措施对产量及相关性状的影响。试验表明,在当地习惯施肥基础上增施钾肥,提高了马铃薯产量、块茎淀粉含量、干物质含量和商品率,提高了叶面积系数、叶绿素含量和净同化率。同时试验表明,增施钾肥的不同处理效果不同。根据增产提质效果及实际效益,推荐施肥量为:N 120 kg/hm2、P2O580 kg/hm2、K2O 180 kg/hm2、农家肥104kg/hm2。     

Effect of excess zinc and arbuscular mycorrhizal fungus on bioproduction and trace element nutrition of Tomato (Solanum lycopersicum L. cv. Micro-Tom)

ABSTRACT

The effect of excess Zn and arbuscular mycorrhizal (AM) fungus on bioproduction and trace element nutrition were investigated in tomato. In a completely randomized factorial design, the experimental treatments – Zn addition at 0 (normal) and 300 (excess) mg Zn kg^?1 soil, and AM inoculation (non-AM and Rhizophagus irregularis) – were set up in a growth chamber for 10 weeks. Generally, AM effects on the available Zn, Mn, Cu, and Fe in the rhizosphere soil were in tandem with the effects in host tissues. Under normal Zn condition, AM enhanced Cu availability in the rhizosphere, optimized the Cu:Zn balance in shoots, and increased the host biomass production. Excess Zn reduced mycorrhizal colonization in AM plants and the total plant biomass in both AM and non-AM plants. Although AM decreased the Zn concentrations in soil and host tissues under excess Zn, the distortions in host TE balance were not significantly ameliorated by the fungus. While Zn in fruit was within the safety threshold, Mn deficiency in the fruit was observed under excess Zn, alongside increased root-to-fruit Fe and Cu translocations. Mycorrhizal reductions in soil and tissue Mn concentrations were considered a minus in terms of probable symbiont amelioration of Mn:Zn in-balance under excess Zn. Additional microbe(s) that can enhance Mn homeostasis might be helpful in tomato under elevated soil Zn.     

Genetic diversity of the African hexaploid species Solanum scabrum Mill. and Solanum nigrum L. (Solanaceae)

Two hexaploid species of Solanum sect. Solanum are present in Africa: Solanum scabrum and S. nigrum. Solanum scabrum is a widely cultivated species and is used as a leafy vegetable, as a source of medicine and as a source of ink dye. In previous studies a wide range of morphological diversity has been reported in this species and in some studies subspecies have been proposed. Subspecies are also recognized in S. nigrum. However, it has not been established whether or not the morphological differences are reflected at the genomic level. The present study applies AFLPs to study the genetic diversity in S. scabrum and its relationship to geographical provenance, morphological differences and the possible existence of subspecies within S. scabrum and S. nigrum. The data obtained were analyzed with cluster analysis (using UPGMA and NJ). The results indicate that the genetic variation within S. scabrum was higher within accessions than between accessions. Accessions did not cluster according to their geographical provenance, indicating that accessions from different geographical areas were not significantly different genetically. The clustering reflected neither morphological differences nor domestication status (cultivated or wild). The morphological differences exhibited by S. scabrum could be due to selection by farmers for different plant types. The AFLP derived clustering pattern did not segregate the subspecies recognized in S. scabrum and S. nigrum into separate subclusters.     

大白菜抽薹相关基因BrFLC1的KASP标记开发

为了提高大白菜耐抽薹品种的分子育种效率,本研究针对大白菜抽薹相关基因BrFLC1第6个内含子的第一个碱基(Pi6+1)G-A的SNP变异开发进行高通量检测的竞争性等位基因特异性PCR(KASP)标记。结果表明,开发的KASP标记BrFLC1-KASP1可有效将晚抽薹类型材料Y177-12(G)和早抽薹类型材料Y195-93(A)分为2组。利用BrFLC1-KASP1标记可以对57份大白菜材料的基因型进行有效鉴别,且鉴定结果与酶切扩增多态性标记(CAPS)G-MvaI以及直接测序法的鉴定结果完全一致。综上所述, BrFLC1-KASP1标记在大白菜材料中具有通用性,且具有准确率高、成本低、效率高的特点。本研究结果对大白菜耐抽薹性的分子标记辅助选择具有重要的育种实践价值。