盾叶薯蓣组织培养技术的优化

以盾叶薯蓣的根状茎、茎段、叶柄、幼叶为材料,进行愈伤组织诱导、分化及再生植株形成的研究。结果表明：盾叶薯蓣不同外植体均能诱导出愈伤组织,其中茎段愈伤组织的诱导率最高;不同激素配比的培养基对愈伤组织的形成有很大的影响：以LS为基本培养基,2,4-D浓度为4．0mg／L、6-BA浓度为1．0mg／L的激素配比诱导率最高,达62．5％;以改良MS为基本培养基,2,4-D浓度为2．0mg／L、6-BA浓度为0．5mg／L的激素配比诱导率最高,达71．4％。筛选到优化的分化培养基为改良MS附加2．0mg／L的6-BA和0．5mg／L的Vc,且能直接诱导出根,并形成完整植株。     

盾叶薯蓣类原球茎的离体诱导及快繁体系的建立

为解决盾叶薯蓣离体培养中试管苗移栽困难的难题,以盾叶薯蓣带腋芽的茎段为外植体,借助正交试验设计方法,离体诱导出类原球茎并建立了类原球茎微繁殖技术体系。结果表明:以带腋芽茎段为外植体诱导致密愈伤组织的适宜培养基为MS+6-BA2.0mg/L+NAA0.4mg/L+KT0.6mg/L+蔗糖3%;类原球茎诱导和增殖培养基为:MS+6-BA4.0mg/L+KT1.0mg/L+蔗糖6%;类原球茎生根培养基:1/2MS+NAA 0.3mg/L+IAA 0.8mg/L+活性炭0.3%+蔗糖1.5%。经该途径诱导得到的生根类原球茎植株经炼苗后移栽的成活率可达到90%以上。     

扁桃愈伤组织诱导及分化的研究

以扁桃优良品种'Naporeil'的茎段、叶片和花药作为外殖体,分别对其进行愈伤组织诱导和分化研究,以筛选愈伤组织的最佳诱导增殖培养基、分化培养基和生根培养基.结果表明,该品种以茎段、花药作为外殖体最易诱导获得愈伤组织,叶片不适宜作为外殖体诱导愈伤组织;愈伤组织的最佳诱导增殖培养基均为B5+0.5 mg/L 2,4-D+0.2 mg/L 6-BA+1.0 mg/L NAA,愈伤组织诱导率为100%,增殖倍数最高可达7倍;茎段愈伤组织的分化培养基为MS+0.2 mg/L NAA+0.8 mg/L 6-BA+0.5 mg/L ZT,分化率为71%;花药愈伤组织未见分化.由茎段愈伤组织再分化获得的不定芽在1/2MS+0.5 mg/L IBA培养基上诱导生根,并给以黑暗预处理可使生根率达80%以上.     

白刺花胚性愈伤组织诱导及体细胞胚发生和萌发

探讨不同因素对白刺花下胚轴、子叶2种外植体胚性愈伤组织诱导及体细胞胚发生和萌发的影响。以B5和MS为基本培养基,研究2,4-D、6-BA和TDZ对白刺花下胚轴和子叶胚性愈伤组织的诱导;在MS培养基上添加不同浓度2,4-D,研究胚性愈伤组织增殖情况;采用ABA,探究对体细胞胚发生的影响。结果表明:下胚轴比子叶更易诱导胚性愈伤组织,筛选出2种外植最佳的胚性愈伤组织诱导培养基均为MS+2.0 mg/L 2,4-D+0.5 mg/L TDZ+0.5 mg/L 6-BA,胚性愈伤组织诱导率分别为77.3%和41.0%。15.0 mg/L ABA、0.2 mg/L 2,4-D和2.0 mg/L 6-BA有利于体细胞胚发生,1/3MS+0.2 mg/L NAA+0.1 mg/L 6-BA+2.0 g/L活性炭+25 g/L蔗糖+7 g/L琼脂的培养基可使体细胞胚萌发率达80%以上,再生植株移栽成活率高达90%。白刺花外植体种类及培养基类型均会影响胚性愈伤组织的诱导,其中下胚轴诱导效果优于子叶;MS培养基较适合启动细胞脱分化形成愈伤组织,2,4-D对胚性愈伤组织的增殖保持有调控作用,ABA有利于体细胞胚的发生。     

冬凌草离体培养体系的建立及主要次生代谢产物的测定

以冬凌草叶片为外植体,研究不同浓度激素组合对冬凌草愈伤组织诱导及植株再生的影响,并对不同外植体(茎、叶)诱导愈伤、芽的分化能力及再生植株内主要次生代谢产物的含量进行了比较研究。结果表明:在MS 2.0 mg/L 6-BA 1.0 mg/L NAA培养基上诱导愈伤组织效果较好;在MS 2.0 mg/L 6-BA的培养基上诱导芽的效果较好;叶片和茎段在愈伤诱导培养基上均能产生大量的愈伤组织,但其再分化能力以茎段最好;再生苗生根培养基以0.3 mg/L IBA最好;以叶为外植体诱导的再生植株中冬凌草甲素、迷迭香酸的含量均高于以茎为外植体诱导的再生植株。     

商陆离体再生体系的构建

目的:建立商陆离体再生体系。方法:选取商陆的幼茎、茎节、叶片、叶柄和顶芽为外植体,以MS作为基本培养基,通过添加不同浓度配比的植物生长调节剂分别进行愈伤组织、丛生芽和生根诱导,筛选商陆离体再生体系方案。结果:顶芽和幼茎为外植体诱导的愈伤组织出愈时间早,愈伤组织质量高,以培养基MS+6-BA0.5 mg/L+2.4-D 0.5 mg/L的诱导率最高,达到100%;其中,只有以顶芽产生的愈伤组织才能分化出丛生芽,芽分化培养基为MS+6-BA 2.0 mg/L+NAA 0.25 mg/L,诱导率为98%;诱导生根的适宜培养基为1/2 MS+NAA 0.3 mg/L,诱导率达100%。结论:建立和完善了商陆离体再生体系方案,为商陆遗传转化体系的构建奠定了基础。     

草麻黄植株再生体系的建立

目的:以草麻黄的子叶为材料,建立草麻黄植株再生体系.方法:采用组织培养的方法进行了愈伤组织诱导、愈伤组织分化和不定芽生根研究.结果:MS+2,4-D 2.0mg/1+6-BA 1.0mg/l为诱导子叶形成具有分化能力愈伤组织的理想培养基;MS+2,4-D 1.5mg/l+6-BA 1.5mg/l是愈伤组织的最佳继代培养基;MS+IAA 0.2mg/l+TDZ 2.0mg/l是愈伤组织不定芽分化的最佳培养基,分化率为75%;试管苗生根培养基为MS+2,4-D 1.0mg/l.结论:建立了草麻黄子叶再生系统,为开发和保护麻黄野生资源提供一定的材料来源和技术方法.     

激素对四裂红景天愈伤组织诱导及其继代培养的影响

以四裂红景天种子和幼茎为材料,应用正交试验进行其愈伤组织诱导及其继代培养的研究.结果表明:种子是诱导四裂红景天愈伤组织的理想外植体;诱导愈伤组织的最佳培养基配方为MS培养基加入6-苄基腺嘌呤(6-BA)3.0 mg/L和α-萘乙酸(NAA)2.0 mg/L和2,4-二硝基苯酸(2,4-D)1.0 mg/L,诱导率为87.1 %.愈伤组织继代培养的最佳培养基配方为MS 6-BA 3.0 mg/L NAA 1.0 mg/L 2,4-D 1.0 mg/L.细胞培养周期为30 d,21 d生物量(干质量)达到最大为13.72 g/L.     

盾叶薯蓣胚乳再生体系的建立及其染色体倍性鉴定

以盾叶薯蓣（Dioscorea zingiberensis）的胚乳为外植体,研究了不同植物生长调节剂对胚乳愈伤组织诱导及植株再生的影响,并鉴定了再生植株。结果表明：愈伤组织诱导形成的适宜培养基为MS＋2.0mg·L^–12,4-D＋0.5mg·L^–16-BA,不定芽分化的适宜培养基为MS＋2.0mg·L^–16-BA＋0.1mg·L^–1NAA,生根的适宜培养基为1/2MS＋0.3mg·L^–1NAA;再生植株炼苗移栽后,成活率可达80%;对获得的再生植株腋芽生长点进行染色体制片观察,发现染色体数目为20的细胞占观察细胞总数的10%,染色体数目为21–29的细胞占16%,染色体数为30的细胞占74%;获得了三倍体植株。     

如意皇后粗肋草愈伤组织及丛生芽诱导培养基优化

本研究以粗肋草‘Red Valentine’带侧芽的根茎为外植体,研究不同培养基类型、不同外源激素及浓度、不同转接周期对其愈伤组织及丛生芽诱导的影响,进而优化如意愈伤组织和丛生芽诱导培养基。结果表明:最佳愈伤组织诱导培养基为MS+0.5 mg/L TDZ+2.0 mg/L 2,4-D和1/2MS+0.5 mg/L TDZ+2.5 mg/L 2,4-D;最佳愈伤组织分化培养基为1/2 MS+0.4 mg/L TDZ,最佳转接周期为30 d;最佳丛生芽诱导培养基为5.0 mg/L6-BA+0.2 mg/L NAA和1/2 MS+0.5 mg/L TDZ,最佳转接周期为30 d。优化培养基后愈伤组织及丛生芽诱导效率更高。本研究通过设计优化试验,筛选出了愈伤组织诱导及丛生芽诱导的最佳培养基,为粗肋草属植物的快速繁殖及规模化生产提供了一定的理论指导。     

杯山药零余子愈伤组织诱导及植株再生的研究

对怀山药（Ｄｉｏｓｃｏｒｅａ ｏｐｐｏｓｉｔａ）零余子愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：⑴在不同激素组合的培养基上怀山药零余子均能产生愈作组织,而且具有一次成苗的能力。ＢＡ２ｍｇ／Ｌ＋ＮＡＡ２ｍｇ／Ｌ的培养基对诱导愈伤组织最有利,其出愈率达１００％;⑵在愈伤组织的分化中,ＢＡ１ｍｇ／Ｌ＋ＮＡＡ１ｍｇ／Ｌ的激素组合是最佳的,其分化率为６３．６％,且多形成丛生芽;⑶再生植株     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Analysis of quantitative trait loci which contribute to anther culturability in rice (Oryza sativa L.)

Anther culturability of rice is significantly different between indica and japonica varieties. A doubled haploid (DH) population was established via anther culture of an indica/japonica hybrid on SK₃ medium, which had been shown particularly suitable for anther culture of indica/japonica hybrids. For analyzing the quantitative trait loci (QTLs) responsible for anther culturability, anthers of the DH lines were again cultured with SK₃ medium and parameters for four traits representing the anther culturability were surveyed and analyzed with the molecular map constructed from the same DH population. The parameters for four major traits were as follows: callus induction frequency (CI), green plantlet differentiation frequency (GPD), albino plantlet differentiation frequency (APD), and green plantlet yield frequency (GPY). All four traits displayed continuous distributions among the DH lines. The correlation coefficients between these traits were also tested and showed that there was no relationship between callus induction and green plantlet differentiation frequencies, but both showed strong positive correlation with the frequency of green plantlet yield. For callus induction frequency, five QTLs were identified on chromosomes 6, 7, 8, 10 and 12. Two QTLs for green plantlet differentiation frequency were located on chromosomes 1 and 9. There was a major QTL for albino plantlet differentiation frequency on chromosome 9. No independent QTL was found for green plantlet yield frequency. The results may be useful in the selection of parents with high response to anther culture for rice haploid breeding and in the establishment of permanent DH populations for molecular mapping.     

高羊茅组织培养再生体系及GUS基因瞬间表达研究

以成熟种子为外值体,对高羊茅纰织培养和植株再生体系进行了优化,分析了不同浓度2．4-D、6-BA和激动素对高羊茅愈伤组织诱导和愈伤组织分化成苗的影响．结果表明：9．0mg／L 2．4-L)对愈伤组织的诱导效果最佳．0．2mg／L激动素是愈伤组织分化成苗的最适浓度．二者的诱导率和分化率分别达到68．08%和45．83％。在愈伤组织继代培养基中附加1．0mg／L 2．4-D、0．5mg／L 6-BA和1．25mg／L CuSO4;有利于胚性愈伤组织的形成,可以明显促进愈伤组织分化。同时．采用基因枪法将GUS基因导入高羊茅愈伤组织中,通过组织化学染色检测到了GUS瞬间表达活性;并对影响CUS基因瞬间表达的因素进行了分析．以期为提高基因枪法遗传转化效率提供参考。     

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.)

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3–5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).     

盾叶薯蓣试管株芽的诱导

以盾叶薯蓣(Dioscoreazingiberensis)试管植株为材料,选取带芽茎段为外植体,转接到株芽诱导培养基上15d后,原茎段基部开始产生株芽突起,30d后每一茎段可产生3-5个已生根的株芽,株芽诱导率为100%,株芽诱导数为180个/40株,其移栽成活率可达90%以上。株芽形成的适宜培养条件:温度为26±2℃,光照时间14hd-1,光照强度为1500-2000lx;适宜培养基组成为:MS+6-BA4.0mgL-1+IBA1.0mgL-1+蔗糖6%-9%+活性炭0.5%+琼脂7%。离体诱导的盾叶薯蓣试管株芽能直接发育为新植株,为盾叶薯蓣的快繁提供了一种新的方法。     

党参的离体培养及植株再生的研究

在附加激素的MS培养基上,培养党参下胚轴和无菌芽切段,诱导产生愈伤组织并且再生植株。经过两年多(15个世代)的继代培养,建立了党参体细胞无性系。实验结果表明:(1)培养基MS 0.4mg/L 2,4-D 0.8mg/L Kt 2.0mg/L IAA对愈伤组织诱导及继代培养,MS 0.2mg/L 6-BA诱导外植体产生丛芽和愈伤组织再分化,MS 0.5mg/L NAA 0.2mg/L 6-BA及MS 0.2mg/L NAA诱导生根效果最好。(2)愈伤组织再分化经过胚状体途径。     

曼陀罗茎段愈伤组织诱导和再生植株的研究

本试验以曼陀罗茎段为外植体,在附加不同植物激素组合的培养基中对愈伤组织的诱导和植株再生进行研究。结果表明:采用修改的MS培养基(除去甘氨酸,维生素B1含量增加至0.5mg/L,pH5.5)附加2mg/L2,4-D可由曼陀罗茎段诱导大量胚性愈伤组织;愈伤组织继代选用0.5mg/L2,4-D为宜;不定芽的诱导采用MS培养基(20g蔗糖,8g琼脂,0.1g水解干酪素) 6-BA(0.5mg/L);幼苗进一步转接至1/2MS IBA(0.2mg/L)生根培养基中,可完成曼陀罗茎段愈伤组织诱导和再生植株的组织培养过程。