Infection of Spathoglottis plicata (Orchidaceae) seeds by mycorrhizal fungus

Spathoglottis plicata seeds were encapsulated in 4-mm-diameter capsules of alginate-chitosan or alginate-gelatin and infected with the mycorrhizal fungus Rhizoctonia AM9. The encapsulated seeds were placed directly on Rhizoctonia culture. About 66% of the seeds encapsulated in sucrose-free chitosan-alginate established a symbiotic relationship with the mycorrhizal fungus after co-culturing for 2 weeks. The highest percentage of infection observed was about 84%. Addition of sucrose or using gelatin-alginate for encapsulation reduced the percentage of infection by about half. The growth of Rhizoctonia AM9 in sucrose-free alginate, chitosan and gelatin was found to be minimal. The advantages of germinating orchid seeds, encapsulated in sucrose-free polymers, through mycorrhizal infection is discussed. Received: 19 February 1998 / Revision received: 8 May 1998 / Accepted: 20 May 1998     

Micropropagation of mature wych elm (<Emphasis Type="Italic">Ulmus glabra</Emphasis> Huds.)

Explants of mature vigorous donor trees of wych elm (Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.Abbreviations DED: Dutch elm disease - BAP: 6-Benzylaminopurine - IBA: Indole-3-butyric acid - TDZ: Thidiazuron - WPM: Woody Plant Medium - DM: Dubovský Minimal Medium Communicated by D. Bartels     

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N ⁶-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.     

Micropropagation of Elaeocarpus robustus Roxb.

Multiple shoots were obtained from shoot tips and nodal explants of 20-year-old trees of Elaeocarpus robustus on Murashige and Skoog's medium supplemented with 0.5 mg l^–1 each of BA and Kn. Explants taken from in vitro-proliferated shoots subsequently produced multiple shoots when cultured on the same basal medium containing 0.5 mg l^–1 each of BA and Kn. Repeated subculture resulted in rapid shoot multiplication at an average rate of 10 new shoots per subculture. The addition of CM (10%) and CH (100 mg l^–1) to the medium enhanced the number of shoots up to 20 per subculture and increased the length of shoots. In vitro-raised shoots were rooted on half-strength MS medium containing 1.0 mg l^–1 IBA and 0.5 mg l^–1 IAA. Following transplantation in the field 85% of the plantlets survived and grew uniformly. Received: 17 March 1995 / Revision received: 30 December 1997 / Accepted: 9 January 1998     

分蘖洋葱试管鳞茎微繁技术研究

以MS BA0.4mg/L NAA0.1mg/L为起始培养基,将获得的分蘖洋葱丛生芽进行试管微鳞茎诱导实验,结果表明：离体条件下,温度和光周期对鳞茎的形成起着决定性的作用,提高培养基的pH值、增加蔗糖浓度对试管苗鳞茎化也有重要的作用。     

Plant regeneration in <Emphasis Type="Italic">Arachis stenosperma</Emphasis> Krapov. and W. C. Gregory from roots and calluses derived from leaflets of <Emphasis Type="Italic">in vitro</Emphasis> plants

Seed explants of A. stenosperma were cultured on MS medium supplemented with 6-benzylaminopurine with the aim of rescuing nonviable accessions stored in seed bank conditions. The regeneration potential of leaf explants from in vitro plants derived from embryonic axes was studied by using whole leaflets and leaflet segments. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine and naphthalene acetic acid. Indirect organogenesis was observed in response to 6-benzylaminopurine, either alone or in association with naphthalene acetic acid, in both explant types. Media supplemented with naphthalene acetic acid as the sole growth regulator induced rhizogenesis in whole leaflets and leaflet segments, with subsequent shoot production directly from the roots.     

生姜脱菌快繁研究进展

生姜是重要的经济作物 ,可用作香料、药物和调味品 ,它主要通过地下根状茎繁殖。自 1 977年生姜脱菌组培获得成功以来 ,在外植体的选择、培养基和培养条件等脱菌快繁技术上都有较大改进 ,培养效率明显提高 ,平均每个芽可增殖 7.7个芽 ,移栽成活率高达 95 %以上。就生姜脱菌快繁技术的发展及其应用前景 ,诸如创造遗传变异、离体种质保存、脱菌种子包衣和离体小块茎生产等作了综述。     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

An alternative propagation method ofAnanas through nodule culture

A micropropagation scheme forAnanas comosus Merr. was developed using nodule culture. Nodules were induced from leaf-base or chopped shoot-base explants on modified half-strength MS medium supplemented with 2.69-5.37 M NAA and 4.44 M BA and could be maintained long-term as nodules. The nodules proliferated into more nodules when chopped into pieces of 1–3 mm and placed onto the same medium. They regenerated shoots when transferred to medium supplemented with 0.54–10.74 M NAA and 0.44–8.88 M BA. The regeneration capacity of nodules is higher than that of direct regeneration or callus. Maximum regeneration was obtained from culture medium containing 0.54 M NAA and 0.44 M BA, where shoots could be observed as early as within 2 weeks. Many shoots formed roots in the same medium in which they were regenerated after 10 subcultures, but the best rooting occurred in medium containing 0.54 M NAA and 0.44 M BA. Rooted plantlets ofA. comosus Merr. could be routinely produced at 6-week intervals.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Direct and callus-mediated protocorm-like body induction from shoot-tips of <Emphasis Type="Italic">Dendrobium chrysotoxum</Emphasis> Lindl. (Orchidaceae)

An efficient method of mass propagation of Dendrobium chrysotoxum Lindl. was developed using a shoot-tip culture system. Both direct and callus-mediated formation of protocorm-like bodies (PLBs) occurred from the basal cut surface of explants. Frequency of callusing was best in the presence of 2 μM thidiazuron (TDZ) or N⁶-benzylaminopurine (BAP). The callus exhibited complete hormone autonomy for growth and differentiation of PLBs and was maintained for 18 months without any exogenous growth regulators, an aspect important for minimising somaclonal variation. However, the rate of callus growth and PLB formation varied with application of cytokinin and auxin. In addition, the callus exhibited a differential sensitivity to the exogenous cytokinins. While BAP promoted callus growth and PLB differentiation, TDZ was inhibitory to callus mediated PLB formation and caused extensive necrosis of callus. Although α-naphthaleneacetic acid (NAA) had no significant effect on the induction of callus, subsequent growth was best in its presence. Using a 3-month subculture period, a 69-fold increase in callus weight was achieved with 0.5 μM NAA, while as many as 133 PLBs could be obtained per 100 mg callus in the presence of 1 μM NAA. For direct PLB formation, the optimum cytokinin dosage was dependent upon the type of cytokinin used. While TDZ was most effective at a concentration of 1 μM (15 PLBs per explant), for similar PLB yield the application of 8 μM BAP was essential.     

Micropropagation of Dendrocalamus asper by shoot proliferation using seeds

An efficient and reproducible procedure for the large-scale propagation of Dendrocalamus asper is described. High-frequency direct shoot proliferation was induced in aseptic seed cultures of D. asper on modified Murashige and Skoog's (1962) medium supplemented with 1.0–10.0 mg/l benzyladenine (BA). Multiple shoots (1–25) were formed within 5 weeks of seed culture without root formation. The shoot-forming capacity of seeds was influenced by the BA concentration in the medium. Proliferating shoot cultures were established by repeatedly subculturing shoots in propagules of 3 shoots each. A multiplication rate of 15–16 fold was achieved on MS medium +3.0 mg/l BA. Roots were formed on excised propagules of 3–5 shoots when transferred to MS medium containing 10.0 mg/l indole-3-butyric acid (IBA) or 3.0 mg/l 1-naphthaleneacetic acid (NAA). Plantlets were hardened, acclimatized and established in soil, where they exhibited normal growth. Received: 10 April 1998 / Revision received: 18 November 1998 / Accepted: 12 December 1998     

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l⁻¹ each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l⁻¹ each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.     

Endogenous hormonal levels and growth of dark-incubated shoots of Catasetum fimbriatum

Apical shoots and Lateral buds of the epiphytic orchid Catasetum fimbriatum give rise to rootless etiolated stolons, when cultured in the presence of light and then transferred to the dark. The stolons are characterized by fast and continuous apical longitudinal growth. Measurements of four endogenous cytokinin, indole-3-acetic acid (IAA) and abscisic acid (ABA) levels in etiolated shoots and light-grown plants were low. However, after transfer of green plants to the dark, cytokinin Levels increased 3- and 7-fold by 10 and 30 days of incubation, respectively. IAA levels also increased significantly, but the increase was not as great as for cytokinins. A similar trend was observed in the roots. A close relationship seems to exist between both cytokinin accumulation and the formation of etiolated stolons. Variations in ABA levels were practically inconspicuous. The presence of paclobutrazol in the medium, a potent inhibitor of gibberellin synthesis, strongly inhibited etiolated and non-etiolated longitudinal shoot growth, although no apparent effect was observed on apical meristem activity.     

大野芋种子形成丛生芽的微繁殖

大野芋(Colocasiagigantea)的成熟种子(褐色)和未成熟的种子(淡黄色)在1/2MS培养基上均能萌发,种子萌发率最高达50%,种子没有休眠期。在室温下,种子在1/4MS 1%蔗糖培养基上,寿命约可达1年。种子在MS BA2mg·L-1 IAA0.25mg·L-1的培养基上,产生丛生芽。增值率1∶4/60d。生根培养基为MS NAA0.3mg·L-1,生根率达95%以上。通过诱导大野芋种子产生丛生芽,建成了快繁无性系,并成功地实现了种子的离体保存。本研究工作的完成,对于芋头的这一野生近缘种的保存和利用、芋头品种的改良,均具有较大意义。     

世界流行切花—重瓣丝石竹茎尖培养及微繁殖技术研究

重瓣丝石竹茎尖培养快繁技术的研究结果表明:适宜的芽增殖培养基为MS+A1.0～2.0mg/L+NAA0.2～1.0mg/L或MS+IBA0.5～1.0mg/L;生根培养基为MS+NAA0.03～0.05mg/L;白糖代替蔗糖对菌的增殖和生根无影响;液体培养基可获得和琼脂培养基相同的增殖效果;不同材料的增殖效果有明显的差异。     

灯盏细辛的组织培养与快速繁殖

本研究以野生高含量灯盏花的叶片为外植体,在7个不同浓度NAA和BA组合的MS培养基上进行愈伤组织诱导、不定芽分化和增殖及生根的培养,确定了灯盏花快繁体系的最适培养条件:(1)初代培养基:(MS 6-BA)0.5 mg·L-1 NAA0.1 mg·L-1;(2)丛生芽增殖培养基:(MS 6-BA)2 mg·L-1 NAA0.7 mg·L-1;(3)生根培养基:(2/3MS NAA)0.5 mg·L-1 IBA0.8.     

Micropropagation of nodal explants from adult trees of Cleistanthus collinus

The micropropagation of adult Cleistanthus collinus was accomplished. The nodal segments from terminal twigs of a 15-year-old tree and basal sprouts of a comparable chronological age were used for initiating shoot bud cultures. Washing explants with sterile mixtures of citric acid (520.5 μM) and PVP 40 (3.75 μM) three to four times controlled the leaching of brown inhibitory substances into the establishment medium. Axillary shoots proliferated best on MS medium containing citric acid (104.1 μM), and PVP 40 (12.5 or 25 μM) supplemented with 0.44 μM BA. The number of new shoots from nodal segments of explants placed on MS medium supplemented with 0.44 μM BA increased when the remaining lengths of nodal segments were transferred to fresh medium after the longer microshoots were harvested. The microshoots derived from basal sprouts rooted best (50%) when treated with 11.4 mM IAA for 2 min, whereas only 40% of the microshoots derived from terminal twigs produced roots after a 2-min exposure to 28.5 mM IAA. The placement of BA-soaked agar cubes on the apex-decapitated shoots controlled shoot-tip necrosis considerably. In general, explants from basal sprouts were more suitable than terminal twig explants for the micropropagation of adult trees of C. collinus. Received: 26 February 1997 / Revision received: 13 September 1997 / Accepted: 29 September 1997     

Direct somatic embryogenesis using leaf explants and short term storage of synseeds in Spathoglottis plicata Blume

Plant Cell, Tissue and Organ Culture (PCTOC) - Spathoglottis plicata Blume is a vulnerable orchid species in various parts of the world, and the conventional propagation provides limited success to...     

In vitro morphogenesis patterns from shoot apices of sugar cane are determined by light and type of growth regulator

The regeneration patterns of shoot apices derived from in vitro plants of four varieties of sugar cane in response to different growth regulators and light were evaluated. The cellular origin of the regeneration processes was also investigated. Explants cultivated on medium supplemented with NAA and incubated under light showed direct bud regeneration from cells of external layers of the ground parenchyma of the stem. Explants cultivated in the dark on medium supplemented with low concentrations of picloram (PIC) or 2,4D (4.0 and 4.5 μM, respectively) showed callus formation derived from the ground parenchyma of stem and development of preembryogenic masses derived from bundle sheath cells facing the phloem tissue of immature leaves. Somatic embryos at further developmental stages were visible following transfer to medium devoid of growth regulators and incubation under light. When incubated under light since the begining of the experiment, explants cultivated in the presence of higher PIC or 2,4D concentrations (40 and 22.6 μM, respectively) first displayed direct organogenesis from external layers of the ground parenchyma of the stem, followed by the development of organogenic calluses. Preembryogenic masses were also observed from bundle sheath cells of immature leaves. However, in contrast to the cultures pre-incubated in darkness for 30 days, the subsequent stages of embryo development were not detected. The regeneration efficiency of calluses induced by 2,4D and PIC was generally increased following desiccation in laminar flow or incubation on medium solidified with phytagel.     

巴西橡胶成龄无性系茎段的试管微繁技术研究

巴西橡胶成龄无性系茎段组织培养在生产中有很大的潜在应用价值,但一直是橡胶树组织培养的难点,至今国内外鲜有成功的报道.本研究以在中国热带地区大面积推广种植的巴西橡胶树优良品种热研7-33-97不同生长时期成龄树茎段为外植体,进行无性系试管微繁技术研究.研究结果表明不同生长时期的茎段在自然条件中受污染程度不同,茎段真菌污染率为稳定期＞淡绿期＞古铜期,细菌污染率为淡绿期＞稳定期＞古铜期.不同生长时期的成龄树茎段需要通过不同的灭菌方法才能显著提高外植体的利用率,外植体灭菌方法显著影响橡胶成龄树茎段的组织培养效率;与稳定期相比,古铜期和淡绿期茎段在组织培养过程中诱导丛生芽萌芽快、萌芽多,是较优的外植体材料;古铜期和淡绿期茎段在激素配比为4.0～5.0 mg/L6-BA+0.5 mg/L GA3的条件下能很好的诱导抽出丛生芽;丛生芽在激素配比为2.0 mg/L 6-BA+0.5 mg/LNAA,1.0 mg/t 6-BA+1.0 mg/L KT+0.5 mg/L NAA或0.5 mg/L 6-BA+1.5 mg/L KT+0.5 mg/L NAA的条件下能很好的诱导丛生芽抽出健壮的芽条;丛生芽抽出的健壮芽条在激素配比为0.5 mg/L IBA+0.5 mg/L IAA的条件下能较好的抽根成苗.外植体生长时期、激素种类和浓度配比都是影响巴西橡胶成龄无性系茎段的试管微繁的重要因素.