中药“枳壳”的组织培养与植株再生（简报）

以酸橙的茎段为外植体,进行离体再生体系研究.结果表明,培养基MS + 6-BA 1.0 mg/L + IAA 0.5 mg/L + CH 400 mg/L适宜不定芽诱导;培养基MS + 6-BA 0.5 mg/L+ IAA 0.25 mg/L + CH 400 mg/L适宜不定芽增殖;培养基1/2MS + NAA 1.0 mg/L + IBA 2.0 mg/L生根效果较好,生根率达80%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.     

‘巴斗杏’组培快繁体系建立与耐盐植株筛选

以淮北黄里‘巴斗杏’茎段为外植体,MS为基本培养基,通过茎段诱导植株再生及进行耐盐筛选。结果表明:采用4、5月‘巴斗杏’茎段用0.1%升汞灭菌8 min较适宜;在含有1.0 mg/L 6-BA和0.1 mg/L IBA的MS培养基上茎段增殖较快,生长旺盛;较理想的生根培养基为MS+NAA 0.1 mg/L+IBA 0.2 mg/L,生根率达46.3%;‘巴斗杏’组培苗进行耐盐筛选的适宜盐浓度为0.4%～0.8%,筛选植株与对照相比差异显著。     

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。     

罗布麻离体培养及快繁技术的研究

采用野生罗布麻(Apocynum venetamL.)顶芽及芽下茎段为外植体进行离体培养及快繁技术优化的研究。结果表明:适宜罗布麻离体培养的基本培养基为MS,适宜外植体起始分化的培养基为:MS BA 1.8mg/L KT 0.5mg/L,分化率为88.9%以上;适宜茎段增殖的培养基为MS BA 2.0mg/L KT 0.5mg/L,繁殖系数最高达5.67倍,通过切段繁殖还可提高繁殖系数3倍;适宜生根的培养基为MS IBA 0.5mg/L NAA 0.02mg/L,生根率达90%以上。     

蝴蝶兰的组织培养和快速繁殖

通过诱导残败花梗上的休眠芽萌发,以萌发的幼叶和去茎尖的茎段为外植体进行组织培养,建立了蝴蝶兰(Phalaenopsis amabilis Bl.)的无菌繁殖体系,并筛选出最佳培养基组成.诱导休眠芽萌发的最佳培养基为不加任何激素的MS0培养基;原球茎诱导的适宜培养基为MS 3.0 mg·L-1 6-BA 0.5 mg·L-1 ZT 30 mg·L-1柠檬酸和MS 5.0 mg·L-1 6-BA 30 mg·L-1柠檬酸 30%椰乳(CM),其中茎段的诱导效果明显优于叶片,诱导率达95%;诱导无菌苗生根的最适培养基为1/4 MS 1.0 mg·L-1 6-BA 0.1 mg·L-1 NAA,生根率可达79%.     

携病毒马铃薯茎尖分化成苗与脱毒率检测

以携带病毒的‘夏波蒂’马铃薯无菌苗为材料,38℃/4h热处理4周,剥离带1个叶原基的茎尖,接种至不同浓度激素组合的24种MS固体培养基上,22℃/16h培养30d后统计茎尖的愈伤组织诱导率和分化成苗率;RT-PCR检测茎尖再生苗3种病毒(PVX、PVY、PLRV)和纺锤块茎类病毒(PSTVd)的脱毒率。结果表明:茎尖分化成苗最适培养基为MS+1.0mg/L ZT+0.2mg/L NAA+2.0mg/L GA3,愈伤组织诱导率为76.25%,分化成苗率为26.25%;再生苗3种病毒PVX、PVY和PLRV的脱毒率分别为69.4%、91.7%和100%,纺锤块茎类病毒PSTVd脱毒率仅为8.3%,二次茎尖剥离后脱毒率增加到20.8%。     

藜蒿的组织培养和快速繁殖研究

通过组织培养技术可以诱导藜蒿的幼叶和茎段产生愈伤组织和植株.研究表明,MS NAA 0.5 mg/L 6-BA 0.5 mg/L(蔗糖3%)最适合诱导藜蒿的叶片、茎段形成愈伤组织以及愈伤组织分化成再生植株,而MS NAA 0.5 mg/L最适合外植体诱导生根,1/2MS培养基对幼苗的生根作用十分明显.再生植株水培试验,筛选出了适合于藜蒿工厂化生产的营养液.     

三倍体毛白杨组织脱分化培养与植株体再生

采用毛白杨三倍体幼叶作为外植体进行组织培养,以MS为基本培养基获得了再生植株。从NAA和IAA与6-BA间进行的18个正交试验中,选择出适宜脱分化培养基MS+6-BA 0.5mg/L+NAA 0.1mg/L,对三倍体毛白杨愈伤组织诱导率为87.6%。5种生长素与6-BA配比的再分化培养基中,12MS+IAA 0.1mg/L+6-BA 0.1mg/L对不定芽的分化诱导率可达到68.5%;MS+6-BA 0.5mg/L+NAA 0.01mg/L的培养基可使单芽直接增殖出7.4个芽。在MS+IBA 1.0mg/L的生根培养基上,试管苗的生根率可达85.7%。     

太子参超低温脱毒及规模化组培育苗技术

以太子参茎尖为外植体,采用超低温去除病毒方法,通过组织培养研究了芽增殖诱导、丛生芽诱导、生根壮苗诱导的适合条件,以期形成太子参规模化育苗技术。结果表明：超低温处理1 h后,太子参脱毒率可达90%以上;规模化组培育苗最适宜配方为初代培养基为改良MS＋2.5 mg/L 6-BA＋0.5 mg/L IAA继代增殖培养基为改良MS＋1.2 mg/L KT＋0.5 mg/L NAA,壮苗生根培养基为改良MS＋0.2 mg/L KT＋1.5 mg/L DA-6＋10%蛋白粉。     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO₃ on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BA+TDZ+Ads+AgNO₃ for multiple shoot induction. AgNO₃ influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO₃ in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA₃ (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L.

We have developed a highly efficient three-stage protocol for plant regeneration in sunflower (Helianthus annuus L.) from embryonal cotyledons. This protocol uses phenylacetic acid (PAA) for both shoot-bud induction and the elongation of smaller buds. The medium used for inducing bud formation from the cotyledons was modified MS medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l PAA. Buds were elongated on MS medium supplemented either with only 0.2 mg/l gibberellic acid (GA₃) or with 0.2 mg/l GA₃ + 0.1 mg/l PAA + 0.3 mg/l BAP. The elongated shoots were then transferred onto rooting medium containing 1 mg/l PAA. The complete plantlets with well-developed roots were transferred to field conditions where they survived and set normal seeds. The induction of shoot buds from embryonal cotyledons was also observed on modified MS medium supplemented with 0.5-5 mg/l BAP in combination with 0.5-5 mg/l !-naphthaleneacetic acid (NAA). In this case, the formation of callus took place along with shoot-bud formation, which hindered further development of the latter. The presence of PAA with BAP in the primary bud induction medium promoted normal development and elongation of shoot buds.     

观音莲的组织培养研究

通过观音莲的茎尖培养获得无菌试管苗,研究了生长调节剂和椰乳(CM)组合对芽增殖的影响,探讨了生长调节剂和多效唑(MET)组合对生根的影响,并对移栽基质进行了筛选。结果表明:观音莲增殖的适合培养基为MS+6-BA 4.0 mg/L+NAA 0.2 mg/L+CM 15%~20%;适合观音莲生根的培养基为1/2MS+NAA 0.5 mg/L+MET 0.5 mg/L;最佳的移栽基质为珍珠岩,移栽成活率达100%。     

In vitro propagation of emetic nut Randia dumetorum (Lamb.)

An efficient protocol for in vitro shoot multiplication of Randia dumetorum (Emetic nut) has been developed. The seeds of R. dumetorum were germinated in vitro in MS medium in 5 weeks. Subsequent propagation using shoot tip as an explant was carried out in MS medium along with different concentrations and combinations of BAP (0.5-2.0) and NAA (0.0-2.0). Maximum shoot multiplication was obtained (12.7 shoots per shoot tip) in MS medium containing 1 mg/L BAP and 1 mg/L NAA. Micropropagated shoots were rooted in 1/2 MS medium supplemented with 1 mg/l IBA. This is the first report of in vitro plant propagation of R. dumetorum. In vitro grown plantlets showed a survival rate of 70% after 2 months of transplantation to natural environment.     

三倍体枇杷的茎尖培养及诱导生根

以三倍体枇杷为材料, 研究了不同消毒方式、MS培养基浓度、植物生长调节剂及浓度配比对茎尖培养及诱导生根的影响。结果表明, 初代培养时, 选择生长饱满、健壮的顶芽及适宜的消毒方式, 外植体剥离长度0.5-0.8 cm, 能显著提高茎尖培养的成活率; MS培养基浓度的变化对外植体的褐化没有明显的影响; 最适茎尖的启动培养基为MS＋1.0 mg·L-1 6-BA＋0.5 mg·L-1 NAA, 成活率高达84.8%; 最适组培苗生根培养基为1/2MS＋0.1 mg·L-1 NAA＋0.01 mg·L-1 IAA＋0.3%活性炭, 生根率达66.7%, 每株平均生根2.83条。该研究结果将为三倍体枇杷再生体系的建立及利用转基因技术对三倍体无籽枇杷进行遗传改良奠定基础。     

Induction of root and shoot formation from root meristems ofPetunia hybrida

Summary A procedure has been developed for the induction of root or shoot formation from root meristems of germinated seeds ofPetunia hybrida. Root formation was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine (BA) (0–0.5 mg/l) and naphtaleneacetic acid (NAA) (0.05–2.0 mg/l). Induction of predominantly shoot formation was obtained on MS medium containing the following combinations of hormones (in mg/l): 0.05–0.5 NAA and 0.25–2.0 BA. Complete plant formation was obtained after rooting of the shoots on MS medium supplemented with IAA (0–2.0 mg/l) or NAA (0-0.5 mg/l).     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.     

An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.)

We developed a novel large-scale micropropagation pathway for date palm (Phoenix dactylifera L.) based on organogenesis. We obtained organogenic stems from shoot tip explants of the Moroccan date palm cultivar Najda, and investigated shoot proliferation from these organogenic stems in vitro on various media; Beauchesne medium (BM) and Murashige and Skoog medium (MS) at full-strength, half-strength, and one-third-strength, containing various concentrations (0, 0.25, 0.5, and 1 mg/L) of 2-naphthoxyacetic acid (NOAA) and kinetin. The optimal medium during the multiplication phase was half-strength Murashige and Skoog medium (MS/2) supplemented with 0.5 mg/L NOAA and 0.5 mg/L kinetin (23.5 morphologically superior shoots per explant, with low vitrification rates). For the shoot elongation phase, shoots were transferred to the same proliferation medium, or to MS or MS/2 media without plant growth regulators (PGRs). Shoots elongated rapidly and showed a high rate of root formation on media supplemented with PGRs. For example, on MS/2 medium containing 1 mg/L NOAA and 1 mg/L kinetin, the average shoot length was 15.1 cm, the average number of roots per shoot was 6.2, and their average length was 3.4 cm. On PGR-free media, shoots were shorter with wider and greener leaves, and had fewer roots. The plantlets were transferred to a greenhouse for acclimation. The survival rate after 2 months was related to the medium used during the elongation phase; >90 % of shoots that were cultured on PGR-free media survived, while there was a poor survival rate of shoots that had been cultured on media containing PGRs.     

芨芨草愈伤组织器官发生及植株再生

芨芨草(Achnatherum splendens (Trin.) Nevski)种子消毒并在MS培养基上萌发获得无菌苗, 以幼苗的叶鞘和胚轴为外植体诱导愈伤组织, 经继代后进一步诱导不定芽及生根。研究结果表明, 诱导愈伤组织最适合的培养基为B5+1.5 mg.L-12,4-D+0.5 mg.L-1 NAA; 诱导芽分化较适合的培养基为B5+0.5 mg.L-1 6-BA +0.2 mg.L-1 NAA; 1/4 B5+1.0 mg.L-1 NAA+0.2 mg.L-1 IBA +1.0 g.L-1活性炭培养基则有利于芨芨草试管苗的生根。本实验建立了完整的芨芨草植株再生体系, 移栽成活率高。