Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina

Hoffmannseggella cinnabarina has not been found in the wild for the last 70?yr in the State of S?o Paulo and, therefore, wild populations of this native orchid are thought to be extinct. This investigation studied seed storage at a low temperature, in vitro germination, and seedling development of H. cinnabarina in order to establish an optimized protocol for propagation, and thus assure species conservation. Seeds of different ages were incubated on Knudson C (KC), Murashige and Skoog, and Vacin and Went media with or without 1???M of N⁶-benzyladenine (BA) and exposed to either 12 or 16?h of light (30???mol?m^?2?s^?1 at 26?±?2°C). Seed surface sterilization was deleterious to 3-mo-old seeds and severely reduced the viability of the 4-mo-old seeds. More mature seeds were not affected by the sterilization procedure. In general, the germination of 4-, 5-, 6-, and 7-mo-old seeds increased when BA was added to the culture medium especially under 16?h of light. Germination rates were highest with 8- and 9-mo-old seeds, and application of BA failed to enhance germination rates further. Developmental studies revealed that this cytokinin reduced seed and protocorm mortality rates; however, protocorm development was negatively affected in its presence. Seedling development was more pronounced when KC medium with 16?h of light was used. Long-term seed storage at 4°C did not provide promising results. The protocol described in this study proved to be efficient and relevant to in vitro seed germination and initial development of H. cinnabarina, and thus will contribute to conservation of this orchid species.     

纹瓣兰的无菌播种与试管成苗

纹瓣兰作为一种野生的兰花资源有其重要的保护及保存意义,本试验以纹瓣兰的种子作为外植体对其进行无菌播种,并成功诱导出再生植株.试验结果表明,在添加椰子水和6-BA的1/2MS培养基中纹瓣兰的种子可以在较短的时间内形成原球茎,其中以添加6-BA 2.0 mg/L+10%椰子水诱导效果最佳.添加6-BA 0.5 mg/L+NAA 0.2 mg/L的1/2MS培养基最有利于纹瓣兰芽的分化,在分化培养50 d时其分化率可达到100%,且芽生长健壮.当生根培养基为1/2MS+NAA 0.1 mg/L时,纹瓣兰的生根率达95%;生根培养30 d后移栽,幼苗成活率达90%以上.     

Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

Continuing loss of native orchid habitat has lead to an increased emphasis on orchid conservation. Major obstacles in the production of native orchid seedlings for use in conservation have been: (1) development of efficient and reliable seed germination protocols and (2) an understanding of early seedling growth and development. Effects of six asymbiotic media (Modified Lucke, Murashige & Skoog, Lindemann, Vacin & Went, Malmgren Modified, Knudson C), four exogenous cytokinins (BA, Zea, Kin, 2-iP), and three photoperiods (0/24, 16/8, 24/0 h L/D) were examined on seed germination and early protocorm development of Habenaria macroceratitis, a rare native Florida terrestrial orchid. Finally, the effects of three photoperiods (8/16, 12/12, 16/8 h L/D) on in vitro seedling development were examined. Percent seed germination was highest on both LM and KC after seven weeks culture (LM = 89.1%, KC = 89.2%); however, protocorm development was enhanced on MM after both seven and 16 weeks. Both zeatin and kinetin at 1 μM enhanced seed germination (Zea = 58.1%, Kin = 47.2%). Final percent seed germination (91.7%) and protocorm development (Stage 4) was increased in the absence of light (0/24 h L/D). In␣vitro seedlings cultured under 8/16 h L/D conditions produced the highest number of tubers per seedling (1.06) with the greatest tuber (42.7 μg) and shoot (fwt = 69.5 μg) biomass and tuber diameter (3.1 mm).     

Asymbiotic seed germination,in vitro seedling development,and symbiotic acclimatization of the Mexican threatened orchid Stanhopea tigrina

Plant Cell, Tissue and Organ Culture (PCTOC) - Stanhopea tigrina commonly known in Mexico as “cabeza de víbora” or “calaverita”, is an endemic orchid from the mountain...     

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.     

濒危植物盘龙参种子的非共生萌发及种苗的快繁研究

以盘龙参种子为材料,筛选离体条件下适宜种子非共生萌发、种苗快繁的培养基。结果表明:盘龙参种子在无植物生长物质的培养基中能够萌发,但不能发育成苗;在含有1.0mg·L-1 KT、0.1mg·L-1 IAA和0.1mg·L-1 GA3的培养基中萌发,并能进一步发育形成种苗;在较高浓度生长素(1.2mg·L-1 NAA)的培养基中不能萌发。种苗在较高浓度细胞分裂素和较低浓度生长素配比的培养基中能够增殖,最高增殖系数可达到2.8,转入壮苗生长培养基中培养80d后可以移栽温室。最适增殖培养基为1/2MS+12.0mg·L-1 6-BA+0.1mg·L-1 NAA+10.0mg·L-1腺嘌呤;最适壮苗生根培养基为1/2MS+1.0mg·L-1 KT+0.1mg·L-1 IAA+10.0mg·L-1腺嘌呤。     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

The effects of methyl jasmonate on sunflower (Helianthus annuus L.) seed germination and seedling development

Some effects of methyl jasmonate (Me-Ja) on sunflower (Helianthus annuus L.) seed germination and seedling development are described and compared with those of ABA. Both growth regulators have very similar action. They inhibit germination, but high concentrations of O₂ in the atmosphere suppress this inhibitory action. Depending on the concentration, Me-Ja inhibits root and hypcotyl growth, however the root is more sensitive to Me-Ja than to ABA. Me-Ja also strongly reduces oxygen uptake during germination and inhibits chlorophyll biosynthesis in isolated cotyledons.     

Asymbiotic seed germination,in vitro seedling development,and greenhouse acclimatization of the threatened terrestrial orchid <Emphasis Type="Italic">Bletia purpurea</Emphasis>

Procedures for asymbiotic seed germination and seedling acclimatization were developed for Bletia purpurea, a threatened North America native terrestrial orchid. Six asymbiotic orchid seed germination media (Knudson C, PhytoTechnology Orchid Seed Sowing Medium, Malmgren Modified Terrestrial Orchid Medium, Vacin &; Went Modified Orchid Medium, ½-strengh Murashige &; Skoog, and BM-1 Terrestrial Orchid Medium) were examined for their effectiveness in promoting seed germination and protocorm development of B. purpurea in either a 0/24 h or 16/8 h L/D photoperiod. Germination occurred regardless of medium or photoperiod treatment. However, advanced seedling development (Stage 6) only occurred on Vacin &; Went Modified Orchid Medium in the 16/8 h L/D photoperiod. Further effects of photoperiod on in vitro seedling development were also examined. Shoot length, leaf width, root number and length, and fresh weight and dry weight in the 16/8 h L/D photoperiod were all significantly different when compared to the 8/16 h and 12/12 L/D photoperiods. In vitro seedlings were readily acclimatized to greenhouse conditions. Seedlings showed high survival all potting media. Seedlings acclimatized in Fafard Mix 4 potting medium developed significantly longer roots. Corm formation occurred regardless of potting media used.     

Hoary cress (Cardaria draba (L.) Desv.) seed germination ecology,longevity and seedling emergence

The effects of gibberellic acid (GA₃), potassium nitrate (KNO₃), prechilling, temperature, salt stress and osmotic potential on seed germination and sowing depth on seedling emergence and burial depth on seed viability of hoary cress (Cardaria draba (L.) Desv.), were studied in a series of laboratory, glasshouse and outdoor experiments. The optimal temperature for hoary cress seed germination was 20°C, both in light/dark and darkness regimes. Seed germination of hoary cress at 400 ppm concentration of GA₃ in a light/dark regime was maximal. Potassium nitrate concentrations increased the percentage of germination in comparison with the control treatment. Increasing the duration of dry prechilling to 30 and 45 days promoted the seed germination of hoary cress. Germination of hoary cress markedly decreased as salt and drought stress increased. Seed germination of hoary cress occurred at a range of pH from 3 to 11. Seedling emergence significantly decreased as planting depth increased. Total seed viability decreased with increasing burial depth. The maximum increase in mortality occurred in seeds that were buried at 5‐cm depth.     

Cultural requirements for in vitro seed germination, protocorm growth and seedling development of Geodorum densiflorum (Lam.) Schltr

Effects of different nutrient solutions, organic supplements and plant growth regulators on in vitro seed germination and protocorm development of Geodorum densiflorum (Lam.) Schltr. were studied. Seed germination was very high (up to 96%) in all the basal media, with Knudson's C and half-strength Murashige & Skoog being slightly more productive than Vacin & Went. Application of organic supplements and NAA had little effect on germination, but BAP proved inhibitory. After germination, protocorms exhibited a clear preference for peptone and NAA for much faster growth, while BAP resulted in stunted growth. Beside normal development, disorganisation of protocorms, followed by callusing occurred in presence of peptone and NAA. The calli were compact with limited growth and frequently regenerated protocorm like bodies. Development of seedlings was preceded by an intermediary rhizome phase. Growth of rhizomes was slow in the plant growth regulator free medium and about 15 months of culture was required for seedling formation. However, it was possible to hasten the process by 8-10 months with the employment of NAA, which also enhanced the number of seedlings per protocorm through axillary branching. Combined application of high BAP and low NAA was also useful for high rate of seedling formation.     

Hormonal regulation of germination and early seedling development in Acer pseudoplatanus (L.)

Summary Dormancy of intact sycamore (Acer pseudoplatanus) seeds was broken by chilling (5°C) for several weeks in moist conditions. Treatment of unchilled seeds with kinetin induced some germination, but gibberellin was ineffective. This stimulation by kinetin was not suppressed by the added presence of abscisic acid during incubation.The chilling requirement of intact seeds was eliminated by removal of the testa, and the naked embryos developed with no morphological abnormalities. During early growth of isolated embryos in the light, two distinct developmental processes were recognised. One involved initial elongation of the radicle accompanied by geotropic curvature and was stimulated by kinetin but not by gibberellin, while the other involved unrolling of the cotyledons, which was accelerated by gibberellin but much less by kinetin. Abscisic acid strongly suppressed both developmental processes when applied alone, inhibited cotyledon expansion in the presence of gibberellin, but failed to overcome the promotory effects of kinetin on radicle growth. Experiments with CCC indicated that under natural conditions the unrolling of the cotyledons is dependent upon endogenous gibberellin. Radicle growth of isolated embryos was unimpaired by incubation in the dark, but cotyledon expansion of water incubated embryos was poor, and although it was accelerated by gibberellin, the responses in all treatments were slower than in the corresponding light grown samples.It is suggested that endogenous cytokinins are primary factors in the initiation of radicle growth, while gibberellins are important in cotyledon expansion. Abscisic acid appears to have an inhibitory role in both processes, and the interactions of these regulators in the control of germination and development are discussed.     

Field studies on seed germination and seedling development in Zostera marina L.

Seeds of Zostera marina L. were collected in July 1978 and July 1979 and planted within two weeks in sediment-filled containers in shallow water at Northwest Creek, New York. The containers were retrieved at periodic intervals over the next 12 months and the seeds and seedlings examined. Between 76 and 93% of the recovered seeds germinated; primarily during October–December, 3–4 months after release. Seedling growth and development occurred during the autumn and spring, but not in winter. A progressive loss of seedlings was observed during the winter and spring, however, and none survived past May.Three stages of seedling development were distinguished. Stage 1 represented germination including emergence and straightening of the cotyledon. Stage 2 seedlings exhibited continued growth of the cotyledon and variable, but often extensive, elongation (2–50 mm) of the axial hypocotyl. The plumule remained undeveloped (length ? 3 mm), and there were no hairs on the basal hypocotyl. Stage 3 seedlings showed the same range in axial hypocotyl length, but they were distinguished by the onset of plumule growth and hypocotyl hair formation.     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

细茎石斛的快速繁殖和试管开花诱导

1植物名称细茎石斛[Dendrobiummoniliforme(L.)Sw.]。2材料类别野生植株的成熟种子。3培养条件种子萌发培养基:(1)1/2MS+NAA0.5mg·L-1(单位下同);增殖分化培养基:(2)1/2MS+NAA0.4;生根壮苗培养基:(3)1/2MS+NAA0.2+6-BA2.0;试管开花预处理培养基:(4)1/2MS+TDZ0.05,(5)1/2MS+PP3330.40+ABA1.0,预处理90d;试管开花培养基:(6)MS(1/6N,2P,2K)+PP3331.0+TDZ0.1。每个处理20株,重复3次,150d内统计花芽诱导率(花芽数/植株数)和正常花开放率(正常花开放数/植株数)。以上培养基均附加4%蔗糖、10%(W/V)香蕉汁、8g·L-1琼脂、0.1g·…     

Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand

Symbiotic seed germination of Pecteilis susannae (L.) Rafin was investigated using 11 fungal isolates recovered from roots of four Thai terrestrial orchids (P. susannae, Eulophia spectabilis, Paphiopedilum bellatulum and Spathoglottis affinis). Seed germination and protocorm development were evaluated up to 133 days after sowing. Protocorm development was most advanced, up to stage 5 (elongation of the first leaf), when seeds were cultured with 4 Epulorhiza isolates obtained from roots of P. susannae (CMU-Aug 028, 4.3%, CMU-Aug 007, 4.2%, and CMU-Aug 013, 2.2%) and E. spectabilis (CMU-STE 014, 3.9%). Moreover, stage 4 protocorm development (emergence of the first leaf) occurred with fungal isolates CMU-STE 011, 5.7%, (Epulorhiza sp.) and CMU-AU 212, 4.3%, (Tulasnella sp.) obtained from roots of E. spectabilis and S. affinis respectively. When seed was incubated without fungi (control), development was limited to stage 3 of protocorm development (appearance of promeristem). This is the first report of protocorm stage 5 development in P. susannae using compatible fungal symbionts. Optimization of seed germination and seedling fitness will assist the conservation and propagation of this orchid species and other terrestrial orchids in Thailand.     

盐胁迫对盐生植物黄花补血草种子萌发和幼苗生长的影响

盐生植物黄花补血草广泛分布于我国西北地区、东北西部以及华北北部,对改良盐碱土壤具有重要的生态作用。以黄花补血草(Limonium aureum(L.)Hill)为材料,研究分析了不同浓度NaCl胁迫对其种子萌发和幼苗生长产生的抑制效应及作用机制。结果表明:低浓度NaCl(25 mmol/L和50 mmol/L)处理不影响黄花补血草种子萌发和幼苗生长,25 mmol/L NaCl甚至促进了根生长,而高浓度NaCl(100 mmol/L和150 mmol/L)处理明显抑制种子萌发及幼苗生长。利用荧光探针的检测结果表明,NaCl处理的幼苗根中过氧化氢(H2O2)和一氧化氮(NO)含量明显高于对照水平。碘化丙啶(PI)染色结合激光共聚焦显微镜观察及检测相对电导率结果显示,高浓度NaCl处理抑制了幼苗根尖伸长区细胞的伸长生长,增加了细胞膜的通透性,对根细胞造成了明显的伤害。此外,高浓度NaCl处理诱导叶片丙二醛(MDA)含量显著升高。以上结果说明,黄花补血草对低浓度的盐具有一定的耐盐性,但高浓度盐降低了种子的萌发率,使幼苗根中H2O2产生增加,抑制根尖伸长区细胞的伸长生长,对根、叶造成明显氧化损伤,从而抑制黄花补血草幼苗的生长。