苦荞胚性愈伤组织诱导与植株再生研究

以苦荞子叶和下胚轴为外植体,进行了不同浓度激素组合的MS和SH固体培养基对胚性愈伤组织诱导及植株再生的研究。结果发现,MS培养基比SH培养基更有利于胚性愈伤组织诱导;2,4-D是诱导愈伤组织的有效激素,KT能有效促进胚状体的形成;下胚轴和子叶都能有效诱导出胚性愈伤组织和再生植株。下胚轴在MS 1.5mg·L-12,4-D 1.5mg·L-1BA培养基,子叶在MS 2mg·L-12,4-D 0.5~1.5mg·L-1BA上能高效诱导出愈伤组织;愈伤组织在MS 2mg·L-12,4-D 0.1mg·L-1KT培养基中继代,能有效诱导胚性愈伤组织;来自下胚轴的胚性愈伤组织在1/2MS 2.0mg·L-1BA 0.5mg·L-1KT 0.1mg·L-1NAA培养基上能够高频再生出芽,来自子叶的胚性愈伤组织在1/2MS 1.0mg·L-1BA 0.1mg·L-1KT 0.1mg·L-1NAA培养基上芽诱导率较高;MS 1mg·L-1NAA是适宜的再生苗生根培养基。     

甜高粱再生体系的建立

利用甜高粱成熟种子和成熟胚作为外植体,通过愈伤组织再生途径建立了植株再生体系。结果表明,成熟种子在添加1.38 g·L^-1脯氨酸、500 mg·L^-1水解酪蛋白和3.0 mg·L^-1 2,4-D的MS培养基上可以较快地诱导出生长状态良好的愈伤组织;成熟胚在添加1.38 g·L^-1脯氨酸、500mg·L^-1水解酪蛋白、2.5 mg·L^-1 2,4-D和0.1 mg·L^-1KT的MS培养基上也能诱导出生长良好的愈伤组织。将愈伤组织转移到MS＋1 mg.L^-1 IAA＋0.5 mg·L^-1 6-BA培养基上诱导芽,然后再转移到MS＋3 mg·L^-1IBA培养基上生根后,可发育成为完整的植株。     

多花黄精诱导愈伤组织与不定芽培养基筛选

以多花黄精Polygonatum cyrtonema的根状茎为外植体,通过L9(34)正交试验比较不同植物生长调节剂及其组合对多花黄精愈伤组织诱导的影响,筛选最适生长调节剂配方,同时筛选通过器官发生方式直接成芽较为合适的培养基。结果表明,含有2,4-D和KT的培养基诱导愈伤组织效果显著,多花黄精愈伤组织诱导的最适培养基为MS + 6-BA 2.0 mg·L-1 + 2,4-D 0.5 mg·L-1 + NAA 0.1 mg·L-1 + KT 1.0 mg·L-1,该培养基的愈伤组织诱导率可达39.10%;培养基MS + 6-BA 4.0 mg·L-1 + NAA 0.2 mg·L-1可诱导多花黄精根状茎直接产生不定芽。     

银杏愈伤组织诱导的多因子正交试验研究

采用正交试验设计法,研究了NAA、KT、2,4-D、蔗糖浓度和不同外植体类型等因素对银杏愈伤组织诱导的影响。结果表明:不同外植体类型对银杏愈伤组织诱导率影响最大,KT和NAA其次,2,4-D和蔗糖浓度最小。银杏愈伤组织诱导最佳培养基为MS+NAA 0.5 mg·L-1+KT 0.5 mg·L-1+蔗糖40 g·L-1,最佳外植体为茎段,其愈伤组织诱导率可达100%。     

小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

甜高粱再生体系的建立

利用甜高粱成熟种子和成熟胚作为外植体, 通过愈伤组织再生途径建立了植株再生体系。结果表明, 成熟种子在添加1.38 g.L-1脯氨酸、500 mg.L-1水解酪蛋白和3.0 mg.L-1 2, 4-D的MS培养基上可以较快地诱导出生长状态良好的愈伤组织;成熟胚在添加1.38 g.L-1脯氨酸、500 mg.L-1水解酪蛋白、2.5 mg.L-1 2, 4-D和0.1 mg.L-1KT的MS培养基上也能诱导出生长良好的愈伤组织。将愈伤组织转移到MS+1 mg.L-1 IAA + 0.5 mg.L-1 6-BA培养基上诱导芽, 然后再转移到MS+3 mg.L- 1IBA培养基上生根后, 可发育成为完整的植株。     

虎杖的组织培养与快速繁殖

以虎杖茎段、叶柄、叶片为外植体探讨了愈伤组织诱导、分化和植株再生的条件,筛选出茎段生长培养基为1/2MS+BA1.0mg·L-1+KT0.5mg·L-1+NAA0.2mg·L-1,茎段、叶柄和叶片外植体愈伤组织诱导培养基为MS+BA1.0～2.0mg·L-1+KT0.2～0.5mg·L-1+NAA0.2～0.5mg·L-1或MS+BA2.0～3.0mg·L-1+KT0.2～0.5mg·L-1+2,4-D0.5mg·L-1;丛生芽诱导培养基为MS+BA2.0mg·L-1+KT0.5mg·L-1+IBA0.2mg·L-1+LH1000;不定根及根状茎诱导培养基为1/2MS+IBA0.2mg·L-1.     

蓝花楹组织培养与快速繁殖研究

以蓝花楹(Jacaranda mimosifolia Humb.et Bonpl.)胚轴为外植体进行组织培养和快繁体系建立的研究。结果表明,蓝花楹种子经40℃-45℃温水浸泡后发芽率较高,达到55.7%。蓝花楹不定芽和愈伤组织诱导的最适培养基分别为MS+6-BA2.0 mg L-1+NAA 0.1 mg L-1+2,4-D 0.1 mg L-1和M S+6-BA 0.5 mg L-1+NAA 1.0 mg L-1+2,4-D 1.0 mg L-1。不定芽和愈伤组织增殖的最适培养基分别为改良MS培养基+6-BA 0.5 mg L-1+NAA 0.5 mg L-1+IBA 0.5 mg L-1和MS+6-BA 1.0 mg L-1+NAA 0.5 mg L-1+ZT 3.0 mg L-1。愈伤组织分化最适培养基为M S+BA 1.0 mg L-1+NAA 0.5 mg L-1+2,4-D 0.5 mg L-1。最适生根培养基为1/2MS+蔗糖20 g L-1+NAA 0.1 mg L-1+活性炭2.0 g L-1,生根率达78.3%。     

留兰香玻璃苗愈伤组织化再生正常植株

植物名称:留兰香(Mentha spicata)。材料类别:玻璃苗梢。培养条件:以MS为基本培养基,诱导愈伤组织添加(1)2,4一D1.0mg/L(单位下同);(2)2,4-D2.0;(3)2,4-D3.0;(4)2,4-D4.0;(5)2,4-D5.0;(6)2,4-D1.0 KT0.1。嫩梢分化培养基为(2),(6)和(7)BA2.0 IBA0.2。生根培养基为(8)1/2MS IBA0.5。培养基含蔗糖3％(生根培     

结缕草再生体系的研究

以结缕草品种‘Zenith’(Zoysia japonica‘Zenith’)的种子为外植体,在附加1.0 mg.L-12,4-D和0.1mg.L-16-BA或2.0 mg.L-12,4-D的MS培养基上培养,愈伤组织诱导率较高,分别达到100%和92%。在添加了2.0 mg.L-12,4-D或1.0 mg.L-12,4-D和0.1 mg.L-16-BA的MS培养基上继代培养可诱导出胚性愈伤组织,诱导率分别为3.26%和14.52%。胚性愈伤组织在含0.01mg.L-12,4-D的1/2 MS分化培养基上的分化率和生根率都达到100%。     

一种改进的水稻成熟胚愈伤组织高效基因转化系统

以成熟胚愈伤组织为材料的农杆菌介导水稻转化法虽已建立, 但转化频率仍有待提高。本文以粳稻(Oryz a sativa)品种(中花10号和中花11号)的成熟胚诱导的愈伤组织为受体材料, 对组织培养体系及影响遗传转化的因素进行优化, 建立了一套改进的农杆菌介导的水稻高效遗传转化系统。农杆菌菌株为EHA105, 质粒载体是pUN1301/ OsRAA1, 其中含有标记基因GUS 和筛选基因HPT。愈伤组织诱导培养基为NBD2 (NB+2 mg·L-12,4-D), 继代培养基为NBD0.5, 预分化与分化培养基为RE1 (MS+1 mg·L-16-BA + 0.25 mg·L-1 NAA + 0.5 mg·L-1 KT + 0.2 mg·L-1 ZT)和RE2 (MS+ 1 mg·L-1 6-BA + 0.5 mg·L-1 NAA+ 0.5 mg·L-1 KT + 0.2 mg·L-1 ZT)。另外, 还分析了影响T-DNA转移的多种因素, 如外植体种类、愈伤组织预培养基和愈伤组织继代次数等。采用优化的转化程序, 水稻愈伤组织转化率和植株转化率可达70%以上。     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

珍稀濒危植物香果树胚性细胞悬浮系的建立和植株再生

以香果树(Emmenopterys henryi)未成熟种子为试材, 探讨不同的接种量、基本培养基、糖浓度及植物生长调节物质等对体细胞胚胎生长的影响, 建立稳定的香果树胚性细胞悬浮培养与植株再生体系。结果表明: 悬浮培养条件下, 最适接种量为2%(鲜重); 较适合的基本培养基为MS; 蔗糖浓度为1%时容易使球形胚聚合体愈伤化, 浓度为3%和6%适合球形胚聚合体增殖, 浓度为9%则容易使球形胚聚合体褐化; 添加0.5 mg.L-16-BA 和0.5 mg.L-1 NAA的MS液体培养基, 当初始蔗糖浓度为3%, 然后逐步提高到6%则有利于香果树各个发育阶段的同步化; 子叶胚转到不含任何植物生长调节物质的MS固体培养基中可以长成正常植株。     

扁蓿豆体细胞胚的诱导和植株再生

扁蓿豆实生苗的根、下胚轴、子叶、叶片和叶柄外植体,在含２,４—Ｄ２—０．２５ｍｇＬ－１与ＫＴ０．２５－２ｍｇＬ－１及２,４—Ｄ０．５ｍｇＬ－１与ＺＴ０．５ｍｇＬ－１或ＢＡＰ０．５ｍｇＬ－１与ＮＡＡ０．０５ｍｇＬ－１的ＭＳ琼脂培养基上均可产生愈伤组织．愈伤组织在含２,４—Ｄ０．５—０．１ｍｇＬ－１与ＫＴ０．５—０．１ｍｇＬ－１或ＢＡＰ０．２５＋ＮＡＡ０．０５ｍｇＬ－１的ＭＳ培养基上可诱导分化出体细胞胚．体细胞胚在无激素的培养基上发育成完整植株．用海藻酸钠包襄体细胞胚制成人工种子,其发芽率和植株转换率分别为９５％和５３％．     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Somatic embryogenesis and plant regeneration from leaf callus of Ocimum basilicum L

A effective protocol for complete plant regeneration via somatic embryogenesis has been developed for Ocimum basilicum L. Callus was initiated from leaf explant of young plant on supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 mg l(-1), 3% sucrose and 0.9% agar. The calli showed differentiation of globular structure embryos when transferred to MS medium containing 2,4-D 0.5 mg l(-1) and BAP 1.0 mg l(-1). The maximum globular structure embryos were further enlarged and produced somatic embryos in MS basal medium supplemented with BAP 1.0 mg l(-1)+NAA 1.0 mg l(-1) + KN 0.5 mg l(-1). Continued formation of globular embryo and germination of embryos occurred in this medium. Complete plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/l 2,4-D and 0.2–0.5 mg/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/l 2iPA and 0.2 mg/l NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/l BAP and 0.5 mg/l NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA 3-indoleacetic acid - 2iPA N⁶-(²-isopentenyl) adenosine - BAP 6-benzylaminopurine - KT kinetin - CH casein hydrolysate     

新疆雪莲体细胞胚胎发生

通过体细胞胚胎发生途径实现了新疆雪莲（Saussurea involucrata Kar．et Kir．）的植株再生。选用新疆雪莲子叶为外植体,接种于MS＋0．5mg·L^-12,4-D＋0．05—1mg·L^-1BA的固体培养基上,进行愈伤组织的诱导。从第1次继代培养的愈伤组织中挑选出黄绿色、颗粒状、质地致密的腔陛愈伤组织,转移到含0．05—0．1mg·L^-1 2,4-D的MS液体培养基中进行悬浮培养,20天后可分化产生大量球形胚。继代过程中相继加入PEG和GA3,可以促进体细胞胚的分化和生长。体细胞胚在含有5mg·L^-1 GA3的MS固体培养基上,可发育成完整的植株。