In vitro asymbiotic germination and seedling development of Limodorum abortivum (Orchidaceae)

Abstract

Germination tests were carried out using immature seeds of Limodorum abortivum and applying in vitro techniques. The results proved that BM₁ culture medium is suitable to promote both germination and further growth stages. Details of the developmental pattern, and some micromorphological features, are described.     

Outbreeding and asymbiotic germination in the conservation of the endangered Italian endemic orchid Ophrys benacensis

Abstract

The Insubric bee orchid, Ophrys benacensis (Reisigl) O.&E. Danesch & F. Ehrend, occurs in fragmented populations only in northern Italy, and suffers from inbreeding depression. We found that pollen from a depressed population at Monte Barro, Lecco, did not fertilise plants of the same population, nor of a larger population at nearby Valmadrera. Fertilisation was successful at both sites when pollen from Valmadrera was used, although the proportion of seeds containing embryos was almost six times greater at Valmadrera. Embryos produced by both populations were equally likely to develop in vitro. Sowing seed on medium enriched with 50 mL L^?1 coconut milk more than doubled the germination rate with respect to a non‐enriched control (i.e. from 14.5% to 39.8%; p = 0.024, Student’s t‐test), whereas other complex organic media inhibited germination. We conclude that both pollen and ovules have inherent developmental problems that can be partially overcome by outbreeding with larger populations. Once seed is produced propagation is relatively easy: sufficient plantlets were produced to enlarge the Monte Barro population to 195 times its current size.     

同色兜兰的非共生萌发与快速繁殖研究

以授粉后不同发育时期的同色兜兰种子为材料,观察其形态特征和萌发过程,并探讨建立同色兜兰高效快繁体系的最佳条件。结果表明,种子发育中后期即授粉后210~240d为较适宜的采收期,授粉后210d的种子萌发率最高(达77.79%);1/4 MS和1/2MS为同色兜兰适宜的基本培养基,添加100mL/L椰乳或1g/L蛋白胨对种子萌发及原球茎生长和分化有明显的促进作用;添加1g/L活性炭对原球茎褐化有一定的抑制作用,但添加剂量不宜过大;添加香蕉汁和苹果汁对同色兜兰种子萌发和原球茎生长分化有抑制作用;暗处理对同色兜兰种子萌发无影响;分化后的原球茎在壮苗和生根培养基上培养120d即可得到4~5片叶、高3~5cm的同色兜兰健壮试管苗。     

In vitro culture of Chloraea gavilu Lindl., an endemic terrestrial orchid from Chile

Orchids are the second most diverse plant family, recognized for their importance as ornamental species; this has driven research development in propagation. One of the most common culture methodologies is in vitro asymbiotic germination, in which the nutritional conditions that provide orchids with a fungal partner are emulated. Although Chile possesses more than 60 terrestrial orchid species, in vitro cultivation protocols have only been developed for Chloraea crispa. In Southern Chile, Chloraea gavilu stands out due to its floral characteristics. We evaluate different explants and cultivation conditions for C. gavilu. The best germination and development results were achieved in the MM medium +0.1% yeast extract +1% sucrose +0.454 µmol l^?1 TDZ, using immature seeds of 24–30 days after pollination, which we cultivated into seedlings in order to be acclimatized and mycorrhized. In addition, induction of protocorm-like bodies was achieved from germinated seeds, using the same culture media as in the germination and development of immature C. gavilu seeds. This resulted in the successful asymbiotic germination of immature seeds, along with the micropropagation of a terrestrial, temperate orchid. We hope to use our protocol in the commercial production of Chilean orchid species as well as to propagate threatened species.     

Effects of fungicides and bactericides on orchid seed germination and shoot tip cultures in vitro

Amphotericin B, benomyl, gentamycin, nystatin, quintozene penicillin G, sodium omadine, and vancomycin singly and in several combinations have no deleterious effects on the germination of orchid seeds, but inhibit the growth in vitro of shoot tip explants.     

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

In vitro propagation of Paphiopedilum orchids

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.     

番木瓜的离体繁殖

建立番木瓜离体繁殖体系.用0.1%HgCl2溶液对番木瓜的新生嫩茎进行消毒,适宜的消毒时间为12min,1mm茎尖的成苗率达到87.6%.随蔗糖浓度的提高,番木瓜试管苗的株高显著降低,增殖系数显著增加.在附加IBA0.3mg/L的1/2MS培养基上新梢的生根率达到89.3%,试管苗大规模移栽的成活率达90%以上.基因型显著地影响番木瓜离体增殖的效率.     

History of orchid propagation: a mirror of the history of biotechnology

Part I Orchid seeds are nearly microscopic in size. Because of that, many fanciful theories were proposed for the origin of orchids. Almost 400 years separate the time when orchid seeds were seen for the first time and the development of a practical asymbiotic method for their germination. The seeds were first observed and drawn during the sixteenth century. Seedlings were first described and illustrated in 1804. The association between orchid and fungi was observed as early as 1824, while the requirement for mycorrhiza for seed germination was established in 1899. An asymbiotic method for orchid seed germination was developed in 1921. After Knudson’s media B and C were formulated, orchids growing and hybridization became widespread. Hybrids which early growers may not have even imagined became possible. Part II A commonly held view is that Prof. Georges Morel is the sole discoverer of orchid micropropagation and that he was the first to culture an orchid shoot tip in 1960. In fact, the first in vitro orchid propagation was carried out by Dr. Gavino Rotor in 1949. Hans Thomale was the first to culture an orchid shoot tip in 1956. The methods used by Morel to culture his shoot tips were developed by others many years before he adapted them to orchids. This review also traces the history of several techniques, additives, and peculiarities (agitated liquid cultures, coconut water, banana pulp, a patent and what appears to be an empty claim) which are associated with orchid micropropagation. A summary of plant hormone history is also outlined because micropropagation could not have been developed without phytohormones.

Re-establishment of the lady's slipper orchid (Cypripedium calceolus L.) in Britain

The Sainsbury Orchid Conservation Project (SOCP), based in the Micropropagation Unit of the Royal Botanic Gardens, Kew, has worked for many years with English Nature, and the Species Recovery Programme in particular, in raising plants of the lady's slipper orchid for re-establishment. This is a collaborative venture with the involvement of a large number of organizations and individuals at national and local level.Cypripedium calceolusis one of Britain's rarest plants. Thought at one time to be extinct, its decline is due to overcollection by botanists for herbarium specimens and by gardeners and it is found now on a single, fragile, natural site. It has been wardened since 1970s because of concerns about long-term safety and is protected under Schedule 8 of the Wildlife and Countryside Act 1981. Natural pollination has not been observed and hand pollination is carried out to ensure seed set. Some seed is scattered on site resulting in a few seedlings and the remaining capsules are sent to the Sainsbury Orchid Conservation Project. In addition, some plants of native origin are held in cultivation and molecular techniques are being carried out at Kew to ascertain how these are related. The best pollination strategy to increase genetic diversity is co-ordinated by English Nature and SOCP, as is the optimum collection time post-pollination to achieve maximum germination. Seedlings are propagated in the laboratory using immature green capsules sown on a range of nutrient media. No symbiotic fungus has yet been found for this species. Germination occurs after 6 weeks in the dark and the plants chilled when roots are well developed and shoots beginning to form. Plants potted up at Kew and near the native site have produced leaves and buds and extensive planting trials have now commenced. The main objective is to increase the number of localities whereCypripediumoccurs through re-establishment. As its decline is due to overcollection rather than habitat loss, many old sites have changed very little. Observation of continental populations is important in assessing suitability of potential sites as the extant clone may not be in optimum conditions. An initial planting trial on the wild site in winter 1989/90 resulted in a 75% survival rate of seedlings planted out. However, establishing seedlings is a long process and careful monitoring is required.     

In vitro propagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared with symbiotic germination

Using a common temperate herbaceous terrestrial orchid from Australia (Caladenia latifolia) this study investigated 19 asymbiotic media variations comprising four commonly used basal media [half‐strength Murashige and Skoog (½MS), Knudson C (KC), Pa5, and Vacin and Went (VW), with combinations of the plant growth regulators 6‐benzylaminopurine (BA) and α‐naphthalene acetic acid (NAA) or coconut water (CW) and compared their performance with germination on a standard symbiotic germination medium, oatmeal agar (OMA). Percentage germination of seeds every 2 weeks for a total of 8 weeks (five replicates per treatment), time to germination, and growth and development phases in seedlings were recorded. ½MS with 5% (v/v) fresh CW delivered 93% germination, with seedling vigour and development indistinguishable from OMA (95% germination). The same protocol was applied to a further ten species (including the endangered Caladenia huegelii), demonstrating high asymbiotic germination performance (60–93%) across a wide phylogenetic range of terrestrial orchid species. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 556–566.     

In vitro propagation of Dampiera diversifolia and Prostanthera rotundifolia

Shoot multiplication and root formation was induced in vitro from mature shoot explants of the woody ornamental plant species Dampiera diversifolia and Prostanthera rotundifolia. 0.5 M BAP+0.5 M kinetin in the absence of auxins gave the most prolific shoot multiplication in both species but there were qualitative and quantitative differences between species in rooting responses to auxin applications. A high percentage of rooted plants were successfully grown-on in pot culture.     

In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae)

BACKGROUND AND AIMS: Many Orchidaceous species are threatened globally by development and over-collection from their natural habitats for horticultural purposes. Artificial propagation from seeds is difficult in most terrestrial orchids native to temperate regions. Seed production is another limiting factor in the artificial propagation for these species because of the lessened probability of pollination and the destruction of fruit by insect larvae. Members of the genus Cephalanthera are distributed across Europe, Asia and North America. C. falcata is a temperate species of East Asia and an endangered species in Japan. As successful propagation from seeds of this species has never been reported, a reproducible method is described here for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro. METHODS: Effects of hand-pollination and bagging treatment of ovaries were examined. Young capsules were collected every 10 d from 50 d after pollination until 120 d after pollination. Immature seeds obtained from these capsules were cultured asymbiotically on modified Kano medium and ND medium. Seed viability was examined within TTC (2,3,5-triphenyl tetrazolium chloride) test solution and histological observations were made on viable seeds by paraffin embedding at each collection stage. KEY RESULTS AND CONCLUSIONS: Hand-pollination followed by bagging treatment of ovaries with aluminium foil was effective for insect control during fruit development, and successfully yielded capsules. Of the capsules, 74.5 % survived to full maturity. The highest frequency (39.8 %) of seed germination was obtained with seeds harvested 70 d after pollination. The frequency declined with progress of seed maturity on the mother plant. Minimal germination was observed with seeds harvested 100 d or later after pollination. Histological observation suggests that accumulation of such substances as lignin in the inner integument surrounding the embryo during seed maturation plays an important role in induction of dormancy.     

In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal explants: A globally endangered,high trade medicinal plant of Western Ghats

Nilgirianthus ciliatus is a globally endangered aromatic slender shrub of Western Ghats with extensive applications in Ayurveda. It is endangered due to its indiscriminate collection and overexploitation to meet the requirements of the pharmaceuticals. This study deals with the preservation of this endangered plant through in vitro nodal culture. Nodal explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) or 6-furfurylaminopurine individually for primary shoot induction. For multiple shoot induction, primary shoots were transferred onto MS medium containing BA individually or in combination with auxins. Clusters of multiple shoots (up to 24.3) occurred with highest frequency (93.2%) on MS medium fortified with BA (3 mg l^?1) and indole-3-acetic acid (0.1 mg l^?1). In vitro regenerated plantlets were rooted on half-strength MS medium with maximum rooting frequency (82.2%) obtained in the presence of indole-3-butyric acid (1.0 mg l^?1). The rooted plantlets were acclimatized to soil with 100% survival rate. Results of this study allowed us to develop an efficient regeneration system that will permit to carry out restoration programmes of N. ciliatus in Western Ghats. In future, this protocol will be an invaluable tool to produce synthetic seeds for cryopreservation and long-term conservation.     

The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L⁻¹ of BA and 0.036 mg L⁻¹ of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L⁻¹ IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.     

In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree

 A rapid clonal propagation system has been developed for Litsea cubeba. Following investigation of a range of cytokinins and a variety of explant sources (shoot tip, node, leaf and petiole) it was established that 6-benzyladenine with shoot tip explants gave optimal multiple-shoot induction. In vitro rooting on growth-regulator-free medium was possible and over 100 plantlets were successfully weaned to the glasshouse. Received: 11 July 1996 / Revision received: 24 December 1996 / Accepted: 22 March 1999     

In vitro propagation of Hedychium coronarium Koen. through axillary bud proliferation

This report describes an efficient and reproducible protocol for large-scale multiplication of Hedychium coronarium plantlets. Axillary bud explants were cultured on Murashige and Skoog medium supplemented with 3 mg/l benzylaminopurine, 3 mg/l kinetin (KIN), and 0.2 mg/l thidiazuron, yielding a maximum of 13.2 ± 0.3 number of shoots. Sub-culturing of shoots every 4 weeks on fresh multiplication medium yielded a consistent proliferation rate. Shoot clusters containing three to five shoots were successfully rooted in KIN (3 mg/l) and indole acetic acid (0.5 mg/l), yielding a maximum of 6.3 ± 0.5 number of roots. Plantlets grown in vitro were acclimatized and subsequently transferred to the field for phenotypic evaluation. Random amplified polymorphic DNA and inter-simple sequence repeat analysis has confirmed the genetic uniformity of in vitro plantlets up to 2 years. After 2 years, these plantlets were transplanted to field, and evaluation of phenotypic characteristics was done. This study is of high significance as these could be commercially utilized for large-scale production of true-to-type plantlets.     

离体条件下暴马丁香切开种子的萌发

分别以三种催芽处理10、20、40 d的暴马丁香种子进行离体培养试验,对种子做三种切割处理,中间切、两端切和完整种子。结果表明:采用切开种子的方法进行离体培养可提高种子发芽率,中间切种子的萌发最好,且在培养10 d后发芽率即达到最高值;经过40 d催芽处理的种子优于未经催芽和只进行20 d催芽处理的种子;切开种子以MS培养基附加5 mg/L BA或5 mg/LBA+0.1 mg/L IBA为最适培养基。     

Stimulatory effects of sodium and calcium hypochlorite, pre‐chilling and cytokinins on the germination of Cypripedium macranthos seed in vitro

Cypripedium macranthos is a wild orchid that is becoming endangered. Efficient methods for its propagation from seed, which is indispensable for conservation, production and breeding, have not been reported. The effects of sodium and calcium hypochlorite, pre‐chilling and cytokinins on the germination of seeds of Cypripedium macranthos Swartz were examined. The duration of treatment with a solution of hypochlorite prior to sowing was one of the critical factors that affected germination. Approximately 70% of seeds that had been treated with either a solution of NaClO that contained 0.5% available chlorine for 60 min or with one of Ca(ClO)₂ with 3.2% available chlorine for 7 h, germinated after 3 months of culture at 20°C, subsequent to 2 months chilling at 4°C. Chilling seeds at 4°C prior to culture at 20°C was another factor that stimulated germination. Even chilling for 2 weeks had a promotive effect on germination, and chilling for 2 months enhanced it most effectively: the frequency of germination was 67% after 3 months of culture at 20°C. However, the promotive effects of chilling on germination were reduced by holding seeds at 20°C for 3 and 6 weeks prior to chilling treatment. Germination of 58‐70% was achieved by the addition of 1 µ M cytokinin to the medium, while the frequency was only 17% in cytokinin‐free medium. We report a reproducible and efficient method for enhancing seed germination of C. macranthos , which involves treatment with hypochlorite prior to sowing, and the combination of chilling at 4°C prior to germination and exposure to a cytokinin.     

木薯离体培养与快速繁殖

选取3个木薯品种的腋芽作为外植体,分别接种于MS基本培养基上进行离体培养及快速繁殖。结果表明, 6-BA不同浓度对木薯品种不定芽诱导效果不一,在MS+6-BA 0.5~1.0mg/L中,3个品种均可诱导产生幼芽;在MS+6-BA 3.0mg/L中,3个品种的幼芽诱导率高达100%;MS+6-BA 3.0mg/L+NAA 0.1mg/L对继代效果较好;MS+NAA 0.1mg/L对生根效果最佳。