The Application of Biotechnology to Orchids

This review provides an informative and broad overview of orchid biotechnology, addressing several important aspects such as molecular systematics, modern breeding, in vitro morphogenesis, protoplast culture, flowering control, flower color, somaclonal variation, orchid mycorrhiza, pathogen resistance, virus diagnosis and production of virus-free plants, functional genomics, genetic transformation, conservation biotechnology and pharmaceutical biotechnology. This resource will provide valuable insight to researchers who are involved in orchid biology and floriculture, using biotechnology to advance research objectives. Producing an improved orchid through biotechnology for industrial purposes or to serve as a model plant for pure and applied sciences is well within reach and many of the current techniques and systems are already employed at the commercial production level.     

Conservation of rare and endangered plants using in vitro methods

Summary Many botanic gardens now have tissue culture laboratories for the micropropagation of plants that are difficult to propagate by conventional horticultural techniques. In many cases the work centers on rare and endangered species. Examples of the use of different techniques including micropropagation, in vitro seed germination, dual culture with symbiotic fungi, and regeneration from callus are discussed with reference to their application to plant germplasm conservation. Presented in the Session-in-Depth Cell Culture of Endangered Species at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Light stimulation and darkness requirement for the symbiotic germination of Dactylorhiza majalis (Orchidaceae) in vitro

Germination percentage of Dactylorhiza majalis (Rchb.f.) Hunt & Summerh. (Orchidaceae) was increased by illuminating surface-sterilized, rinsed and incubated seeds with white light in 16 h photoperiods. Optimal exposures (10–14 days) raised germination from 40 to 75%. Longer light treatments resulted in reduced germination percentage and smaller seedlings. Only about 2% of the seeds could actually germinate in photoperiods; the germination of the rest was delayed by initial light and required about 14 days in constant darkness. Interruption of this darkness period with two consecutive photoperiods increased the germination percentage when the interruption occurred before day 8, but not when occurring later than that.     

Micropropagation of ‘Holy Basil’ (Ocimum sanctum Linn.) from young inflorescences of mature plants

In vitro micropropagation of holy basil (Ocimum sanctum L.), an Indian medicinal herb, has been accomplished on Murashige and Skoog (MS) medium utilizing young inflorescence explants. MS supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ) produced only non-morphogenetic callus. Direct multiple shoots differentiated within 2--3 weeks when explants were cultured on MS containing 6-benzyl aminopurine (BAP). Of the various levels of BAP tested, MS + BAP (1.0 mgl^–1) produced the maximum number of shoots. Incorporation of indole-3-acetic acid (IAA) (0.05 mgl^–1) along with BAP (1.0 mgl^–1) in the culture medium showed a marked increase in the number of shoots. About 92% of the in vitro regenerated shoots rooted on MS hormone-free medium within 2--3 weeks of culture and 85% of the micropropagated plantlets could be successfully established in soil, where they grew normally.     

阳春砂仁微繁殖技术研究

以阳春砂仁的芽块为外植体,在附加1~6mg/L 6-BA的MS培养基上,可实现丛生芽增殖。最佳启动和增殖培养基为MS+5mg/L BA+5ml/L 20%硫代硫酸钠,出苗率和增殖率分别为62.5%和5.36。采用1/2 MS+1mg/L IBA+0.5mg/L IAA进行离体生根,生根率可达94.6%。     

Strategies for ex situ conservation of Centaurea cineraria subsp. circae (Asteraceae), an endemic plant from Lazio (Italy)

Centaurea cineraria subsp. circae is an endemic plant with a distribution area limited to Circeo mountain (Lazio, Italy), whose population was estimated in a very low number of individuals. The aim of this work was to investigate ex situ conservation strategies such as achene collection and in vitro plant propagation, which will permit to carry out restoration programmes. The test carried out on the achenes demonstrated that only 5.5% of them were morphologically healthy. Seed germination tests showed that seeds do not display dormancy and that germination does not require pre-treatments. The higher germination rate (67.5%) was observed under a photoperiod of 12/12 h (light/dark) and temperature regime +20/+10°C. The in vitro studies demonstrated that micropropagation, acclimatization and the transfer outdoors of C. cineraria subsp. circae are not particularly difficult: 74% of shoot explants in a Murashige and Skoog (MS) medium added with 0.5 mg/l benzylaminopurine and 2 mg/l kinetin formed multiple shoots; 100% of shoots rooted in the MS medium added with 0.5 mg/l indole-3-butyric acid and over 90% survived the acclimatization phase. After been transferred outdoors, the totality of in vitro-propagated plants bloomed and appeared morphologically indistinguishable from wild plants. Preliminary chemical analyses showed a similar profile for in vitro-propagated and wild plants.     

Micropropagation of sugarcane (Saccharum spp.)

A method for callus induction, adventitious bud regeneration, shoot multiplication and rooting of in vitro formed shoots of Helianthus annuus L. var. Argentario is described. Hypocotyl and cotyledon explants formed callus on medium containing 2 mgl^–1 naphthalene acetic acid and 0.5 mgl^–1 benzyladenine. Adventitious buds were formed on hypocotyl segments on medium containing 0.5–2 mgl^–1 benzyladenine. The optimal level of sucrose concentration for shoot regeneration from hypocotyls was 1.5%. Multiplication from shoot apices was promoted by kinetin (2 mgl^–1) plus gibberellic acid (5 mgl^–1), benzyladenine (2 mgl^–1) plus gibberellic acid (10 mgl^–1) or at lower frequency by benzyladenine (1 mgl^–1). A general feature of the plantlets formed in vitro was the precocious flowering.     

The Effect of Various Culture Media on the Formation of Embryo-like Structures in Cultures Derived from Explants Taken from Mature Larix decidua

The effect of various collection dates and nine different culture media on the formation of ‘embryo-like structures’ (ELS) in cultures derived from explants taken from a 42-year-old Larix decidua tree was studied. The best medium was AFC, a medium low in NH₄⁻, NO₃ ⁺, Mg²⁺ and SO₄²⁻ but high in PO₄³⁻ compared with the concentration of these elements in the other media. On AFC, ELS production reached a peak with collections made in late summer during the period when needle primordia are being initiated. For the other media, collection date had a lesser effect on ELS initiation.     

Protoplast and leaf explant culture of Lycopersicon cheesmanii and salt tolerance of protoplast-derived calli

In vitro manipulation of Lycopersicon cheesmanii was attempted in order to evaluate its potential to improve salt tolerance of L. esculentum following somatic hybridisation of these two species. The main study concerned three populations of the typicum form (LCS) used for tissue culture and protoplast studies. The minor halophytic form (LCT) was used only for protoplast experiments. Mother plants were sown and propagated in vitro. Shoots (2-7) were obtained from explants (leaflets, petioles, cotyledons), through calli, when 3-4 mg l^–1 BA, kinetin or 2iP were used in association with auxin. IAA (0.2 and 1 mg l^–1 was the most efficient, while 2,4-D or NAA were sometimes inhibitory according to genotype and explant. Leaf protoplasts (2-5 × 10⁷/g of fresh weight) were obtained from LCS and LCT. For LCT, protoplast division declined after five subcultures of the plantlets that had been established from seeds. Protoplast-derived calli were obtained from all populations (maximum PE 30% for LCS). However, they failed to regenerate shoots. Protoplast-derived calli involved in salt tolerance tests showed that LCS17 line was the most tolerant.     

光照和温度对红花槭限制生长保存的影响

该文主要探讨了光照时间、光照强度、温度及昼夜温差等保存条件对卓越红花槭(Acer rubrum cv. ‘Somerset’)限制生长保存的影响。结果表明, 在为期182天的保存过程中, 离体材料前期以分化生长为主, 后期以营养生长为主, 并呈现一定的低温适应性。温度对离体材料生长的影响达极显著水平(P<0.01); 光照时间和光照强度影响持久, 二者交互作用达显著水平(P<0.05); 昼夜温差对平均出芽数和生根率都有显著影响。研究表明, 保存效果最好的条件是T3 (25°C, 12小时光照, 62.50 μmol·m ^-2·s ^-1)和T7 (25°C, 12小时光照, 31.25 μmol·m ^-2·s ^-1)处理组, 但最佳保存条件的选择标准并不唯一, 其核心是保证种质材料的分化能力。     

甜瓜属种间杂交中3种不同倍性资源的微繁

以3种不同倍性的甜瓜属种间杂交资源(双二倍体Cucumis hytivus、异源三倍体黄瓜和种间杂种F1)无菌苗的顶芽或腋芽为试材,比较它们再生能力的差异,寻求各自增殖的最适培养基,从而建立其离体繁殖体系.结果表明:倍性越高,增殖能力越小,增殖速度也最小(双二倍体种转接的间隔时间比其它两种材料长1～2周).双二倍体、异源三倍体黄瓜和种间杂种F1的最大繁殖系数分别为4.6、7.1和8.4;相对应的培养基分别为MS 0.05 mg·L-1TDZ、MS 2.2 mg·L-1 6-BA和MS 0.5 mg·L-1 6-BA).3种材料的生根结果基本一致,在1/2MS 0.2 mg·L-1IBA上的生根率均能达84.5%,驯化成活率达90%以上.同一材料的田间性状表现整齐一致,没有发现变异.     

Multiplication of the endangered Indian pitcher plant (Nepenthes khasiana) through enhanced axillary branching in vitro

Axenic seedling-derived two- to three-node stem segments of Nepenthes khasiana Hook.f. were successfully cultured on Woody Plant Medium containing 2.2 M benzyladenine to produce a 0.5–1.5 cm axillary shoot from each node in 7–8 weeks. The rapid growth along with the axillary branching of this shoot enabled amassing of 6–12 shoots during subculture. Excised shoots transferred to basal medium or rooted in medium containing 2.7 M naphthaleneacetic acid produced typical pitchers at leaf tips. Rooted plants were established in pots at 90–95% survival rate.Abbreviations AA ascorbic acid - AC activated charcoal - CA citric acid - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - KC Knudson-C (1946) basal medium - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & McCown 1980)     

Direct plant regeneration from different explants through micropropagation and determination of secondary metabolites in the critically endangered endemic Rhaponticoides mykalea

Direct plant regeneration from different explants, micropropagation and determination of secondary metabolites were studied in the critically endangered endemic Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab & Greuter. Seed germination was achieved by damaging the seed coat and cultivating the embryos on Woody Plant Medium (WPM), of which 40% germinated. The epicotyls and cotyledonary petioles of seedlings were used as initial explants and direct shoot regeneration was obtained on WPM containing 2.22 μM 6-benzyladenine (BA). WPM medium supplemented with 2.22 μM BA and 4.92 μM indole-3-butyric acid (IBA) significantly improved the production of multiple shoots, resulting in an average of 5.6 shoots per explants. The highest rooting of shoots (35.6%) was observed with WPM medium containing 19.68 μM IBA with 990 μM putrescine. Plantlets with well-developed roots were transferred to soil and acclimatised within a plant growth chamber. Acclimatised plants showed 100% survival rate and remained healthy. As a part of our study, the content of secondary metabolites in three tissue culture regenerated lines were determined by HPLC analysis. Chlorogenic acid, Quercetin and scutellarin were confirmed secondary metabolites of R. mykalea.     

Characterisation and pathogenicity of bacteria from shoot tips of the globe artichoke (Cynara scolymus L.)

Bacteria have been isolated from shoot tips of symptomless globe artichoke plants. These were identified as Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas spp., Serratia liquefaciens, Enterobacter agglomerans/Erwinia, Agrobacterium radiobacter, an unidentified member of Rhizobiaceae and another classified in the “corynebacteria” group. The most frequently isolated species was P. fluorescens, biovars II and III. The endogenous character of these bacteria was studied in plants growing in vitro and in the open field. P. fluorescens, P. marginalis, S. liquefaciens and E. agglomerans/Erwinia caused symptoms in plants growing in vitro, but only P. fluorescens biovar II and P. marginalis produced symptoms in plants growing in open fields. Differences in pathogenicity were observed on inoculated plants growing in vitro or in the open field. This suggests that several endophytic bacterial species may be responsible for the high levels of contaminants found during the micropropagation of globe artichoke.     

Micropropagation ofAcacia mearnsii

Summary A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 μEm⁻²·s⁻¹). Multiple shoot formation was promoted by BAP at 2 mg · liter⁻¹ (8.87μM) and higher combined with or without 0.01 mg · liter⁻¹ (0.049μM) IBA. Cytokinins at concentrations of less than 1 mg · liter⁻¹ combined with 0.01 to 0.1 mg · liter⁻¹ auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter⁻¹ BAP and 0.01 mg · liter⁻¹ IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter⁻¹[4.3μM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter⁻¹ (3.22μM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.     

云南火焰兰种子无菌萌发和试管育苗

对云南火焰兰进行无菌播种技术试验.结果表明,适宜萌动的培养基为1/4MS 椰子水20% 蛋白胨1 g L-1 蔗糖10 g L-1,播种30 d时萌动率可达93.1%;适宜萌发的培养基为VW 椰子水10%,培养75 d时萌发率达36.6%.盐浓度较高的MS培养基不适合种子萌发和原球茎生长,低盐的KC培养基上种子能较好地萌发和成苗;添加椰子水可明显提高种子萌动率,并有利于原球茎的生长和分化.光培养和暗培养对种子萌动的影响差异不明显,但暗培养3周会对种子后期发育不利.添加150 g L-1的苹果匀浆有利于幼苗生长.试管苗移栽成活率在95%以上.     

‘神马’菊花的离体保存及遗传稳定性

用添加不同蔗糖与甘露醇配比的培养基对切花菊品种‘神马’(Jinba)试管苗进行离体保存,并对保存材料再生后代的遗传稳定性进行了研究。结果表明,在温度(23±2)℃、光照强度2 000~3 000 lx、光照时间12 h/d的培养条件下,MS 0.3 mg.L-16-BA 0.1 mg.L-1NAA培养基中分别添加10 g.L-1蔗糖和10 g.L-1甘露醇、10 g.L-1蔗糖和20 g.L-1甘露醇、20 g.L-1蔗糖和20 g.L-1甘露醇或30 g.L-1蔗糖和10 g.L-1甘露醇均能使菊花试管苗连续保存12个月,其中10 g.L-1蔗糖和20 g.L-1甘露醇、20 g.L-1蔗糖和20 g.L-1甘露醇组合处理保存12个月后成活率较高,分别达到50.00%和60.00%,且均保持最高的增殖倍数2.67。保存12个月的试管苗在增殖、生根培养基上均能正常恢复生长,且其再生后代的田间生物学性状、过氧化物酶(POD)及酯酶(EST)同工酶酶谱、ISSR扩增图谱与对照株无差异,保持了良好的遗传稳定性,说明利用上述方法保存‘神马’种质是可行的。