The influence of thidiazuron on shoot regeneration from leaf explants of fifteen cultivars of Rhododendron

The influence of cytokinin thidiazuron (TDZ) and auxin indole-3-acetic acid (IAA) on in vitro shoot organogenesis of fifteen Rhododendron genotypes was investigated and a protocol for high frequency adventitious shoot regeneration from leaf explants was developed. High genotypic variation was observed and regeneration frequencies ranged from 0 to 100 %. Genotype Ovation had the highest number of shoots (26.4 per explant) after 12 weeks on medium with 0.57 μM IAA and 1.20 μM TDZ, but only 65 % of explants regenerated. Catawbiense Grandiflorum had 17.7 shoots per explant and 75 % regeneration on medium with 5.70 μM IAA and 0.45 μM TDZ and Van Werden Poelman had 14.3 shoots per explant and 100 % regeneration on medium with 0 57 μM IAA and 0.45 μM TDZ.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

麻疯树种子二萜类化学成分的研究

为深入了解麻疯树种子的化学成分,有必要对其具有生物活性的二萜类成分进行研究.该研究利用正相硅胶、ODS、制备液相等色谱分离方法,从麻疯树种子的乙醇提取物中共分离得到了6个二萜类化合物,并采用荧光偏振技术对化合物进行蛋白激酶C(PKC)抑制活性的测定.结果表明:根据化合物的理化性质、MS和NMR,并参考相关文献,这6个二...     

Direct somatic embryogenesis and shoot organogenesis from leaf explants of Primulina tabacum

An efficient propagation system via somatic embryogenesis and shoot organogenesis and plant regeneration system for endangered species Primulina tabacum Hance was established. Thidiazuron (TDZ) was the key plant growth regulator for inducing somatic embryogenesis and kinetin (KIN) and 6-benzylaminopurine (BAP) were the key cytokinins for inducing shoot organogenesis from leaf explants. TDZ combined with BAP or KIN in the induction Murashige and Skoog medium induced both somatic embryos and adventitious shoots. Leaf explants with abaxial site in contact with the medium induced less somatic embryos or adventitious shoots compared to inversely placed leaf explants and the optimum pH was 6.5–7.0. Secondary somatic embryos or adventitious shoot could be induced from primary somatic embryos using TDZ and BAP. Shoots developed adventitious roots on rooting medium containing 0.5 μM indole-3-butyric acid and 0.2 % activated carbon. Over 90 % of plantlets survived following acclimatization and transfer to potting mixture (sand:Vermiculite:limestone; 1:2:1).     

High frequency shoot regeneration from leaf explants of muskmelon

Efficient in vitro plant regeneration systems are critical for many purposes including plant transformation. Current regeneration systems for melon (Cucumis melo L.) plants generally utilize cotyledon explants; regeneration from melon leaves has received limited attention. We investigated several factors that influence regeneration from melon leaves including: genotype growth conditions and age of the source plant, leaf age, explant orientation, gelling agent, and the addition of silver nitrate and sulfonylurea herbicide to the culture media. Critical factors that influenced regeneration were preculture conditions of the donor plants, leaf size, and the use of silver nitrate and Phytagel in the medium. The best results were obtained with 3–4 cm diam leaves excised from pot grown greenhouse or growth chamber plants cultured on MS medium with 5 M IAA, 5 M BA, 1 M ABA, 30 M silver nitrate and 2.6 g l^-1 Phytagel. Low concentratons of sulfonylurea herbicide (0.25 mg l^-1 DPX-M 6316) also enhanced regeneration. Under optimized conditions 80–100% of the explants regenerated, with 10–100 shoots per explantAbbreviations ABA abscisic acid - BA benzyladenine - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA naphthalene acetic acid     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L.

A two-step protocol for the induction of shoots from Alstroemeria leaf explants has been developed. Leaf explants with stem node tissue attached were incubated on shoot induction medium for 10 days, and then transferred to regeneration medium. Shoots from the area adjacent to the region between the leaf base and node tissue regenerated within 3 weeks after transfer to the regeneration medium, without a callus phase. The best induction was obtained with Murashige and Skoog medium containing 10 μm thidiazuron and 0.5 μm indole butyric acid. The regeneration medium contained 2.2 μm 6-benzylaminopurine. After several subcultures of the leaf explants with induced shoots, normal plantlets with rhizome were formed. In Alstroemeria, the percentage of responding leaf explants is more important than the number of shoots regenerated per leaf explant, because rhizome formation is the most important factor for micropropagation. The effect of other compounds in the induction medium, including glucose, sucrose, silver nitrate, and ancymidol, on regeneration was also investigated. Received: 14 June 1996 / Revision received: 27 September 1996 / Accepted: 20 October 1996     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

Mannitol and thidiazuron improve in vitro shoot regeneration from spearmint and peppermint leaf disks

In vitro shoot organogenesis of peppermint and spearmint was obtained from leaf disks. Regeneration occurred within six weeks of placement in culture. Best results were obtained when explants were cultured for two weeks onto Murashige and Skoog medium supplemented with 300 mM mannitol, 2.0 μM 6-benzyladenine and 2.0 μM indole-3-butyric acid, and then transferred on a medium without mannitol and containing 0.5 μM α-naphthaleneacetic acid, 9.0 μM 6-benzyladenine and 0.5 μM thidiazuron. Using these culture conditions, percentages of regeneration were 78% for peppermint and 49% for spearmint. Because of its efficiency, this leaf disk regeneration method could be a suitable tool for genetic transformation with Agrobacterium tumefaciens. This revised version was published online in June 2006 with corrections to the Cover Date.     

小桐子果壳提取物杀虫活性的生物测定

以水、乙醇、正丁醇、乙酸乙酯、氯仿和石油醚为溶剂,采用冷浸法对小桐子果壳进行粗提,并分别测定各粗提物对豌豆长管蚜和菜青虫的毒力,从中筛选出毒力最强的粗提物进一步对菜青虫进行作用方式的测定.6种溶剂粗提物对豌豆长管蚜和菜青虫的毒力测试结果表明:三氯甲烷提取物和乙醇提取物的活性显著高于其它溶剂提取物,且两种提取物对豌豆长管...     

Adventitious shoot regeneration from leaf explants of Gypsophila paniculata L.

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering. Received: 25 July 1998 / Revision received: 11 November 1996 / Accepted: 30 November 1996     

Direct plant regeneration from leaf explants of Plumbago species

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 M 6-benzylaminopurine (BA), 1.4 M indole-3-acetic acid (IAA), 370 M adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 M indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.     

Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars

Protocols were developed to optimize adventitious shoot regeneration from four southern highbush blueberry cultivars. Leaf explants from 6 week-old shoots of the four cultivars were excised and cultured on woody plant medium each containing thidiazuron (4.54 or 9.08 μM), zeatin (18.2 μM), or zeatin riboside (5.7 or 11.4 μM) either separately or in combination with α-naphthaleneacetic acid at 2.69 μM. Optimum medium for shoot regeneration was genotype-dependent. Efficient regeneration was obtained at frequencies of 88.9% for ‘Jewel’, 87.8% for ‘Emerald’, 53.3% for ‘Jubilee’ and 87.8% for ‘Biloxi’. Leaf explants of newly developed shoots from the cultures having undergone five subcultures had higher regeneration frequencies than those having undergone two subcultures. Regenerated shoots, 80–100% for each cultivar, rooted in 8 weeks after transplantation to soil. The regeneration systems described have potential use in genetic transformation of southern highbush blueberry cultivars.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

小桐子低温诱导查耳酮合酶基因的克隆及其表达分析

为了解查耳酮合酶在小桐子(Jatropha curcas)抗冷性形成中的作用, 基于小桐子低温锻炼转录组和数字基因表达谱数据, 克隆了低温新诱导表达的小桐子查耳酮合酶基因(JcCHS), 并分析了该基因的表达特性和功能。结果表明, JcCHS 基因的cDNA全长为1386 bp, 包含完整开放阅读框(ORF) 1170 bp, 编码389个氨基酸, JcCHS的理论分子量为42.2 kDa、等电点为6.53, 与蓖麻CHS 蛋白序列的相似性高达93.6%, 具有III 型聚酮合酶家族保守的查耳酮合酶/ 对苯乙烯合酶结构域。半定量RTPCR分析表明, JcCHS 在小桐子各组织中都有表达, 其中根的表达量较高。JcCHS 基因的表达能在一定程度上提高重组酵母菌的低温抵抗能力, 这说明JcCHS 基因可能参与了小桐子的抗低温响应。     

Direct shoot regeneration from leaf and internode explants of Aloysia polystachya [GRIS.] mold. (Verbenaceae)

Summary Adventitious bud regeneration from leaf and internode explants of Aloysia polystachya was achieved. Shoots from nodal segments grown in vitro were cut into pieces and used as sources of explants. Organogenesis was induced from both explants cultured on quarter-strength Murashige and Skoog (MS) semisolid medium (plus sucrose 5 g l⁻¹) containing different combinations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 116 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD), 14-h photoperiod, and at a temperature of 27±2°C. The type of explant markedly influenced organogenesis and growth of the regenerated shoots. The regeneration frequencies were higher with leaf explants, while the number of shoots formed per responsive explant was greater with internode explants. However, the growth of regenerated shoots from internodes was seriously affected by vitrification. The number of shoots produced per responsive leaf explant increased from one to seven as the percentage of leaf explants producing shoots increased from 20 to more than 80%. NAA at 0.05 μM in combination with BA at 0.5μM induced the highest regeneration rate (87±8.8%) after 20 d of culture, yielding 5.9±0.8 shoots per responsive leaf explant. Histological examination confirmed the occurrence of direct organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium without plant growth regulators for 30 d. Finally, the elongated shoots were rooted by pre-treatment in an aqueous solution of NAA at 500 μM for 2 h and transferred to 1/4 MS. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. An experimental field plot with 2-yr-old in vitro-regenerated plants was established.     

Direct shoot formation and plant regeneration from cotyledon explants of rapid-cycling Brassica rapa

Summary An in vitro culture system for direct shoot regeneration from cotyledon explants of rapid-cycling Brassica rapa was developed. Cotyledons from 3-d-old seedlings, when cultured on Murashige and Skoog (MS) medium supplemented with 20 μM N⁶-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA), regenerated shoots directly at a frequency of 20%. The addition of 2 μM aminoethoxyvinylglycine (AVG) to this medium increased shoot regeneration to 33%, but silver nitrate drastically inhibited shoot regeneration. Shoot regeneration occurred directly, at the petiolar cut ends of cotyledonary explants, between 10 to 17 d in culture. The highest percentage of regeneration (33%) was obtained from 3-d-old seedlings. NAA was the most effective auxin for root induction and development, with 49% of shoots producing roots after 2 wk on medium containing 1.0 μM NAA. Regenerated plantlets were grown to maturity in pots containing peat moss and vermiculite (1:1). These plants were morphologically normal and fertile. With this protocol, over 100 independently derived, flowering R₀ plants were obtained from 40 regenerating cotyledonary explants within 40 d after culture initiation.