苜蓿红豆草属间体细胞杂种的分子生物学鉴定

通过原生质体融合和培养获得苜蓿红豆草属间体细胞杂种植株。采用一种简便方法从杂种组织再生植株叶片、红豆草羟脯氨酸抗性系再生植株叶片和苜蓿根癌农杆菌702转化系愈伤组织提取DNA用于RAPD和Southern杂交分析。随机引物扩增结果显示两种亲本的RAPD多态具有明显差异。在所用20种随机引物中,6种产生较多的DNA片段。杂种组织具有两种亲本特有的DNA片段,但倾向于排除红豆草亲本的染色体,表明该杂种为非对称杂种,两种亲本染色体之间可能发生了重组。由于红豆草DNA的介入,杂种组织表现出较强的分化能力。分别利用RAPD扩增得到的OPA141000bp红豆草羟脯氨酸抗性系特异产物和OPA141600bp苜蓿根癌农杆菌702转化系特异产物为探针进行Southern分子杂交,证明杂种组织同时具有这两种DNA片段的同源序列。     

豆科植物原生质体培养再生植株名录

自1980年Kao和Michayluk首次获得豆科苜蓿的第一棵原生质体培养再生植株后,到1989年为止,据报道已在11属25个物种中取得了成功。此后又有小豆、豌豆、多年生野生大豆、刀豆等相继成功地获得原生质体再生植株。现将豆科植物原生质体再生植株成功的物种、取材组织及再生途径归纳如表,供参考。     

天蓝苜蓿原生质体培养再生植株

苜蓿属（Ｍｅｄｉｃａｇｏ）植物多为优质牧草,是进行原生质体培养研究较多的一个属。至今已有１０余种苜蓿属植物进行过原生质体培养的研究,多数获得了再生植株［１～６］。天蓝苜蓿是一种优质牧草、绿肥和药用植物。关于它的体外培养已有一些报道［５,７,８］,但原生质体培养尚未能再生植株。本文以悬浮系为材料进行了原生质体的分离与培养,并经胚状体发生和器官发生两种途径获得了再生植株。１　材料和方法天蓝苜蓿（ＭｅｄｉｃａｇｏｌｕｐｕｌｉｎａＬ．）种子采集于吉林省西部草原。干种子用浓硫酸浸泡３ｍｉｎ,升汞灭菌５ｍｉ…     

苜蓿根癌农杆菌转化系原生质体培养研究

首蒂是一种重要的豆科牧草。1980年,Kao等首次由首精叶向原生质体通过胚状体途径再生植株获得成功'",此后有关首请原生质体再生植株的报道已经涉及很多不同品种'。本工作对首带胭脂碱型农杆菌转化愈伤组织进行了原生质体培养,建立起一种简便有效的培养方法;并分析了碘乙酸胺对原生质体的作用,为融合实验打下了基础。1材料与方洁以本实验室得到的首精（Medicago。tiva）根瘤农杆菌（Agrohacteriumtumdeciens）702菌株转化系为材料。取2g转代12d的颗粒状黄绿色愈伤组织转人10mL酶液,于黑暗、25C、50r／min条件下游离6h。酶液组成为2…     

红豆草细胞悬浮培养中体细胞胚的形成

红豆草(Ooobrychis viciaefolia Scop.)为豆科蝶形花亚科红豆草属多年生草本植物。由于营养丰富,生活力强,抗旱,耐瘠薄,适合西北干旱地区种植,是农民喜爱的牧草。有的学者已对红豆草在组织培养中体细胞胚的形成     

红豆草与苜蓿原生质体融合再生属间体细胞杂种植株

通过PEG诱导红豆草抗羟脯氨酸细胞系原生质体与苜蓿根癌农杆菌702转化系原生质体融合,首次选择得到非对称属间体细胞杂种愈伤组织,并分化出17株小植株.PEG可诱导原生质体紧密粘连,使相连膜环化形成泡囊状结构,导致原生质体相互融合.融合诱导溶液中附加 5％～ 10％甘油可以明显提高异源融合频率.采用后滴原生质体法具有较好的结果.杂种再生芽的染色体数目处于30～60条之间,并包含红豆草的小染色体和苜蓿的2条具有2个明显缢痕的染色体.同工酶分析和胭脂碱合成酶活性检测,均证实其杂种特征.     

红豆草抗羟脯氨酸变异系原生质体培养后愈伤组织的抗性特征

通过ＮａＮ３诱变得到的红豆草抗羟脯氨酸变异系,在酶液中游离原生质体进行培养,获得再生植株。在含不同浓度ＮａＣｌ,羟脯氨酸或ＰＥＧ的ＭＳ培养基上,原生质体来源抗性愈伤组织中的游离脯氨酸含量在１周之内均急剧增加,随后开始下降,３周后接近正常水平。随着胁迫程度的提高,抗性愈伤组织中游离脯氨酸含量呈递增趋势,生长速度呈递减趋势。     

红豆草抗羟脯氨酸变异系原生质体培养后愈伤组织的抗性特征

通过NaN3诱变得到的红豆草抗羟脯氨酸（Hyp）变异系,在酶液中游离原生质体进行培养,获得再生植株。在含不同浓度NaCl、羟脯氨酸或PEG的MS培养基上,原生质体来源抗性意伤组织中的游离脯氨酸含量在1周之内均急剧增加,随后开始下降,3周后接近正常水平。随着胁迫程度的提高,抗性愈伤组织中游离脯氨酸含量呈递增趋势,生长速度呈递减趋势。     

红豆草和苜蓿的光合效率比较研究

红豆草,又名驴食草,是甘肃省近年广泛种植的豆科牧草。为了深入认识它的生长发育规律,揭示其光合生产的限制因素,我们于1986—1988连续三年在甘肃省陇中南部黄土丘陵区通渭县申家山试验场开展了红豆草光能利用率的研究。     

草坪草生物技术研究进展

概述了草坪草植株再生体系和遗传转化体系建立的方法和进展.通过愈伤组织培养、悬浮细胞培养和原生质体培养方法对草坪草的一些种已建立较为完善的植株再生体系.在建立再生体系的基础上,利用原生质体融合、农杆菌介导、基因枪和碳化硅纤维介导等转基因方法在一些草坪草种上建立了遗传转化体系并获得了有一定价值的转基因植株.最后,对草坪草转基因存在的问题和前景作了讨论.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

Somatic embryogenesis and plant regeneration in callus culture derived from immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson

Callus was induced on the wounded immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson (Berberidaceae) on a medium based on Murashige and Skoog's (1962) formula supplemented with 1 mg/l 2,4-dichlorophenoxy-acetic acid (2,4-D). Spontaneous embryoid formation occurred on the media containing low concentrations of 2,4-D (0.1–0.5 mg/l). These embryoids germinated in either MS or B5 medium containing 1 mg/l N⁶-benzyladenine and 1 mg/l gibberellic acid. The regenerated plantlets were successfully transferred to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GA₃ gibberellic acid - MS medium Murashige and Skoog's (1962) medium     

Protoplast culture and plant regeneration from Brassica carinata Braun

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4–6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.Abbreviations BA 6-Benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashiqe-Skoog     

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.     

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     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens Pall.

A reproducible release of viable protoplasts was obtained from friable calli of Astragalus adsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5 mg/l 2,4-D, 0.5 mg/l BA and 0.5 M glucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems showing a plating efficiency of 0.8±0.5% and 6.5±0.7%, respectively. Upon transfer to Murashige and Skoog (1962) medium (MS) with 1–2 mg/l BA, alone or in combination with NAA or 2,4-D at 0.1 mg/l, the protoplast-derived calli produced complete plantlets through somatic embryogenesis. The maximum percentage of calli producing somatic embryos (52.5± 2.2%) occurred on MS medium containing 0.1 mg/l NAA and 1 mg/l BA, whereas the maximum number of calli regenerating plantlets (64.7±6.2) was obtained on MS medium with 0.1 mg/l NAA and 2 mg/l BA. Received: 25 April 1997 / Revision received: August 1997 / Accepted: 2 September 1997     

从石刁柏原生质体培养再生植株

石刁柏,又名芦笋(Asparagus officinalisL.)是百合科天门冬属植物。其栽培品种含有丰富的维生素类及蛋白质。同时,石刁柏对于某些疾病有一定的药效,因此它已成为人们所喜爱的一种高级营养蔬菜。国外已有不少关于石刁柏试管苗繁殖的报告,但至今只有Bui Dang Ha等从石刁柏枝状叶分离的原生质体得到愈伤组织,并由此愈伤组织诱导获得了再生植株。此后,未见在石刁柏的原生质体培养方面再有新的工作。在本文中,我们利     

Plantlet regeneration from cotyledon,cotyledon petiole,and hypocotyl explants via somatic embryogenesis pathway in roquette (Eruca sativa Mill)

Abstract

A high-efficiency plant regeneration protocol based on somatic embryo formation for Huining Roquette, an interesting ecotype of Eruca sativa Mill, was established for future transgenic applications. On Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), alone or in combination with 6-benzylaminopurine (BA) or kinetin (KT), the cotyledon explants, cotyledon petioles, and hypocotyls all produced embryogenic callus (ECs) or somatic embryos (SEs) to different extents. After transferring onto hormone-free MS medium, the ECs or SEs from the different explants and media, all of them developed shoots with a frequency of 6–48%, and then produced roots with a frequency of 2–29%. As regards the probability of shoot differentiation, cotyledon explants appeared similar to hypocotyls, but superior to cotyledon petioles; 2,4-D + KT worked more effectively than 2,4-D alone and 2,4-D + BA for callus induction and shoot differentiation. The optimal hormone combinations for plant regeneration of cotyledon, cotyledon petiole, and hypocotyl explants were 1.0 mg/l 2,4-D + 0.1 mg/l KT, 0.8 mg/l 2,4-D + 0.3 mg/l BA, and 1.0 mg/l 2,4-D + 0.3 mg/l KT, respectively. MS medium with 60–80 g/l sucrose was the most effective for improving SE maturation and germination.