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.

Errata

New Phytologist 145 (2000), 1–3
In the January 2000 issue of New Phytologist , we published the forum article entitled 'Cadmium for all meals – plants with an unusual appetite' by U. Krämer. Since its publication, it has been brought to our attention that an error was introduced during typesetting. In the text several instances of concentrations are erroneously presented as mg⁻¹: these should be kg mg⁻¹.
New Phytologist 145 (2000), 367–421
In the March 2000 issue of New Phytologist , we published the Tansley review entitled 'Numerical and physical properties of orchid seeds and their biological implications' by J. Arditti and A. K. A. Ghani. Since its publication, it has been brought to our attention that an error was introduced during typesetting. On p. 391, Eqn 5, the horizontal line above the right side of the equation should form a square root sign.
We apologize to the authors and to our readers for these mistakes.     

In vitro seed culture and seedling development of Calopogon tuberosus

A major obstacle to native orchid production is difficulty in seed germination. Culture media and light effects on seed germination of Calopogon tuberosus var. tuberosus, a native orchid with horticultural potential, were studied. Culture media included Knudson C, Malmgren modified terrestrial orchid, and PhytoTechnology orchid seed sowing. Effects of 8 weeks continual darkness, 8 weeks 16-h photoperiod, 2 weeks dark followed by 6 weeks 16-h photoperiod, 4 weeks dark followed by 4 weeks 16-h photoperiod, and 6 weeks dark followed by 2 weeks 16-h photoperiod were examined. Percent seed germination was highest on Knudson C after 8 weeks culture; however, seedling development was enhanced on PhytoTechnology seed sowing medium during 8 weeks culture under a 16-h photoperiod. This suggests that while KC and darkness promoted seed germination, P723 and light enhanced further seedling development. Seedlings of C. tuberosus readily acclimated to greenhouse conditions.     

DNA synthesis and mRNA accumulation during germination of embryos of the orchidSpathoglottis plicata

Summary Germination of embryos of the orchid,Spathoglottis plicata Blume involves the formation of a protocorm in which DNA synthesis and cell divisions are confined to the proximal end whereas cells at the distal end undergo enlargement. Although³H-thymidine was incorporated into the distal cells of the embryo during the early period of germination, DNA synthesis was not followed by mitosis and cytokinesis. Poly(A)-RNA detected by in situ hybridization of sections to³H-poly-(U) was present uniformly in all cells of the embryo of the dry seed. However, coincident with the formation of the apical meristem, there was an increase in the density of auto-radiographic silver grains in the cells of the embryo, with a concentration of silver grains in the proximal part. The results indicate that regulatory events in the embryo prior to seed maturity determine the fate of its proximal and distal parts during germination.Dedicated to the memory of Professor John G. Torrey     

Mycorrhizal compatibility and symbiotic reproduction of Gavilea australis,an endangered terrestrial orchid from south Patagonia

Gavilea australis is a terrestrial orchid endemic from insular south Argentina and Chile. Meeting aspects of mycorrhizal fungi identity and compatibility in this orchid species is essential for propagation and conservation purposes. These knowledge represent also a first approach to elucidate the mycorrhizal specificity of this species. In order to evaluate both the mycorrhizal compatibility and the symbiotic seed germination of G. australis, we isolated and identified its root endophytic fungal strains as well as those from two sympatric species: Gavilea lutea and Codonorchis lessonii. In addition, we tested two other strains isolated from allopatric terrestrial orchid species from central Argentina. All fungal strains formed coilings and pelotons inside protocorms and promoted, at varying degrees, seed germination, and protocorm development until seedlings had two to three leaves. These results suggest a low mycorrhizal specificity of G. australis and contribute to a better knowledge of the biology of this orchid as well as of other sympatric Patagonian orchid species, all of them currently under serious risk of extinction.     

Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

The rapid loss of native orchid habitat throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate plans for the propagation and reintroduction of many native orchid species. Ideally, symbiotic orchid seed germination methods are utilized in the production of orchid seedlings to be used in plant reintroduction programs. In the current study we (1) describe an efficient symbiotic seed germination protocol to germinate seeds of the rare sub-tropical terrestrial orchid Habenaria macroceratitis; (2) discuss the in vitro fungal specificity demonstrated by this species; and (3) describe the effects of three photoperiods (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic seed germination of H. macroceratitis. Six fungal mycobionts were isolated from both vegetative and flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds were cultured with fungal mycobiont Hmac-310. Seeds of H. macroceratitis demonstrated a degree of specificity toward fungal mycobionts isolated from plants originating from the same site where seed was collected. Continual darkness (0/24 h L/D) inhibited initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2; 53.5%). These findings will aid in developing an efficient symbiotic seed germination protocol for the conservation of this rare Florida terrestrial orchid, and may prove useful in the conservation of other sub-tropical terrestrial orchid species.     

Studies of Mycorrhizal Fungi of Chinese Orchids and Their Role in Orchid Conservation in China—A Review

China has over 1,200 species of native orchids in nearly 173 genera. About one fourth of native species are of horticultural merit. Some species are of Chinese medicinal value. In fact, the demand on orchid species with high Chinese medicinal values such as Gastrodia elata, Dendrobium offcinale, along with demands on species of cultural importance, such as those in the genus of Cymbidium, is a major factor causing wild populations to diminish and in some cases, drive wild populations to the brink of extinction. These market demands have also driven studies on the role of mycorrhizal fungi in orchid seed germination, seedling and adult growth, and reproduction. Most of these mycorrhizal studies of Chinese orchids, however, are published in Chinese, some in medical journals, and thus overlooked by the mainstream orchid mycorrhizal publications. Yet some of these studies contained interesting discoveries on the nature of the mycorrhizal relationships between orchids and fungi. We present a review of some of these neglected publications. The most important discovery comes from the mycorrhizal studies on G. elata, in which the researchers concluded that those fungi species required to stimulate seed germination are different from those that facilitate the growth of G. elata beyond seedling stages. In addition, presence of the mycorrhizal fungi associated with vegetative growth of post-seedling G. elata hindered the germination of seeds. These phenomena were unreported prior to these studies. Furthermore, orchid mycorrhizal studies in China differ from the mainstream orchid studies in that many epiphytic species (in the genus of Dendrobium, as medicinal herbs) were investigated as well as terrestrial orchids (mostly in the genus Cymbidium, as traditional horticultural species). The different responses between epiphytic and terrestrial orchid seeds to fungi derived from roots suggest that epiphytic orchids may have a more general mycorrhizal relationship with fungi than do terrestrial orchid species during the seed germination stage. To date, orchid mycorrhizal research in China has had a strongly commercial purpose. We suggest that this continuing research on orchid mycorrhizal relationships are a solid foundation for further research that includes more rare and endangered taxa, and more in-situ studies to assist conservation and restoration of the endangered orchids. Knowledge on the identities and roles of mycorrhizal fungi of orchids holds one of the keys to successful restoration and sustainable use of Chinese orchids.     

Noël Bernard (1874–1911): orchids to symbiosis in a dozen years,one century ago

Noël Bernard, the famous discoverer of the symbiotic germination of orchid seeds, died a one hundred years ago in 1911, at the age of 37. Here we remember his life, personality, training and contributions to science. He studied at the Ecole Normale Supérieure and took courses on microbiology at the Pasteur institute. He was also an admirer of Darwin. We briefly summarize some of his inspiring research in botany, plant physiology and symbiosis, especially that on orchid seed germination and tuber formation, published over a period of only 12 years. We briefly consider his legacy and the research that has recently been published in Symbiosis, which extends the various fields of research that Noël Bernard pioneered.     

美花石斛菌根真菌接菌方式与接种效应初步研究

自然条件下,兰科菌根真菌的共生对于兰科植物种子萌芽和植株生长是必不可少的。为探讨有益共生真菌对兰科植物生长的促进作用,特别是在实验室环境下接菌方式的改变与接种效应直接的联系,本研究从野生美花石斛(Dendrobium loddigesii Rolfe.)新鲜营养根中分离、筛选出3种菌根真菌（M1、M2和M3）,采用单一接菌和混合接菌的接种方式,分析3个菌株及其不同接种方式对美花石斛生长的影响。研究得到优势菌株M1和M3,并证实混合接菌对美花石斛的生物量增长具有较好的正效应,两两混合接种方式M1-M2、M2-M3及3个混合接种方式M1-M2-M3均能较好的促进美花石斛生物量的积累。充分发挥混合接菌对兰科植物生长发育所产生的效能,提高生产效率,具有较强的现实意义。     

Influence of growth environment on tolerance to UV-B radiation, germination speed, and morphology of Metarhizium anisopliae var. acridum conidia

We previously reported wide variability in UV-B tolerance among different Metarhizium anisopliae isolates [Braga, G.U.L., Flint, S.D., Miller, C.D., Anderson, A.J., Roberts, D.W., 2001a. Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S. J. Invertebr. Pathol. 78, 98-108] as well as wide phenotypic variability in some of these isolates in response to alterations in their growth environments [Rangel, D.E.N., Braga, G.U.L., Flint, S.D., Anderson, A.J., Roberts, D.W., 2004. Variations in UV-B tolerance and germination speed of M. anisopliae conidia produced on artificial and natural substrates. J. Invertebr. Pathol. 87, 77-83]. Studies on other biological systems have demonstrated that strong selective pressure for tolerance to a stress factor may reduce the phenotypic variability of this trait. In the present study, conidia of the isolate most tolerant to radiation and heat, ARSEF 324, presented very little phenotypic variability in UV-B tolerance in response to production on either artificial culture medium or infected insects. The phenotypic plasticities in two other traits (conidial morphology and germination speed), however, were considerably higher.     

Erratum to: Ozpolat B,Akar U,Mehta K,Lopez-Berenstein G. PKCδ and tissue transglutaminase are novel inhibitors of autophagy in pancreatic cancer cells. Autophagy 2007; 3:480-3

Ozpolat B, Akar U, Mehta K, Lopez-Berenstein G. PKCδ and tissue transglutaminase are novel inhibitors of autophagy in pancreatic cancer cells. Autophagy 2007; 3:480-3. This addendum included figures from a previously published paper but omitted the proper attribution to Akar U, Ozpolat B, Mehta K, Fok J, Kondo Y, Lopez-Berestein G. Tissue transglutaminase inhibits autophagy in pancreatic cancer cells. Mol Cancer Res 2007; 5:241-9. Fig. 1A and B were modified from Fig. 1A and B, Fig. 1C was taken from Fig. 4D, and Fig. 1D was from Fig. 3D. Fig. 2A and B were from Fig. 4A and Fig. 6, respectively. Finally, part of Fig. 3A was from Fig. 7A.     

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.     

Mechanical Constraints in the Endosperm and Endocarp are Major Causes of Dormancy in Sinojackia xylocarpa Hu (Styracaceae) Seeds

Sinojackia xylocarpa Hu, a member of the first new genus described and published by the Chinese botanist Hu, is an endangered plant endemic to China. We analysed mechanisms of seed dormancy in S. xylocarpa at the cytological level. Cell composition, structure, morphology, and hydration in fruit and seeds were observed using paraffin section detection, transmission electron microscopy, scanning electron microscopy, and magnetic resonance imaging. The results indicate that mechanical constraints in the endosperm and endocarp are important causes of dormancy. Germination tests indicated that embryos are non-dormant; rather, the endosperm imposes significant mechanical constraints on germination, particularly around the radicle.

     

In vitro symbiotic germination of myco-heterotrophic Gastrodia elata by Mycena species

Symbiotic germination was analyzed in the myco-heterotrophic orchid Gastrodia elata. Seeds were placed on water agar medium containing a leaf-disc of Quercus accutissima that had been previously inoculated with one of six different Mycena species. Among the six fungi, KFRI1212 (HQ662845) and KFRI2121 (HQ662846) germinated 60.1 and 47.0 % seeds, respectively, while others germinated less than 3.5 %. Although KFRI1212 induced a significantly higher germination rate than KFRI2121, initiation of protocorm development was induced much faster by KFRI2121 than by KFRI1212. Molecular identification and phylogenetic analysis showed that the two fungi belonged to one clade that includes Mycena chlorophos, M. amicta and M. tenerrima, suggesting that seed germination of G. elata depends on a narrow taxonomic range of fungi. In conclusion, this study not only showed fungal preference of G. elata for seed germination but also confirmed molecular identities of mycorrhizal fungi for the first time, which will allow us to better understand the process of symbiotic events at the germination stage of G. elata.     

Enhanced symbiotic seed germination of Cypripedium macranthos var. rebunense following inoculation after cold treatment

Cypripedium macranthos var. rebunense is a well-known wild orchid in Japan, and is considered to be a symbol for rare plant conservation. A fungus isolated from roots of C. macranthos var. rebunense induced symbiotic germination of the species in vitro. Cold treatment of the seeds at 4°C prior to fungal inoculation was required for the symbiotic germination. Changing the timing of inoculation of the fungus to the seeds greatly improved germination frequency. Maximum germination was attained after seeds were inoculated just after the cold treatment for 12 weeks, and approximately 20% of the seeds developed into protocorms more than 1 mm long. These results suggest that fungal inoculation takes place at the beginning of spring in nature, and the tough impervious seed coat may preserve the seed from the infection during autumn and winter seasons. The lengthy culture period of more than 16 weeks at 20°C on the same medium with the fungus caused gradual browning and rot of the protocorms. By elimination of the fungus with a fungicide and by transfer to a nutrient rich medium, approximately 20% of the protocorms developed into healthy plantlets. The methods obtained here appear to be applicable to symbiotic germination of many other threatened Cypripedium spp.     

Receptors for B-melanocyte-stimulating hormone exhibit positive cooperativity in synchronized melanoma cells

Cloudman S91 mouse melanoma cells respond in culture to B-melanocyte-stimulating hormone (B-MSH) with changes in morphology, growth rates, and melanin production. The effects of MSH appear to be mediated through a stimulation of the cyclic AMP system. It was reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle [Wong, G., Pawelek, J., Sansone, M., & Morowitz, J. (1974) Nature (London) 248, 351-354] and that the apparent reason for this cell cycle restriction is that receptors for MSH are most active in the G2 phase [Varga, J. M., DiPasquale, A., Pawelek, J., McGuire, J., & Lerner, A. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1590-1593]. In this report, we found that by two separate methods of obtaining populations of cells in the G2 phase of their cycle--centrifugal elutriation or synchronization with thymidine--we observed increased binding of MSH by cells in the G2 and possibly late S phases of their cycle. However, cultures of cells passing through their cycle in synchrony were quite different from nonsynchronized (random) cultures. Both synchronized and random cultures expressed receptors for MSH in the G2 and possibly late S phases of their cycle, but synchronized cultures bound severalfold more MSH per cell than random cultures. This increased binding of MSH by synchronized cells was accompanied by an increase in tyrosinase activity and pigment production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.     

Ecological specialization in mycorrhizal symbiosis leads to rarity in an endangered orchid

Terrestrial orchid germination, growth and development are closely linked to the establishment and maintenance of a relationship with a mycorrhizal fungus. Mycorrhizal dependency and specificity varies considerably between orchid taxa but the degree to which this underpins rarity in orchids is unknown. In the context of examining orchid rarity, large scale in vitro and in situ germination trials complemented by DNA sequencing were used to investigate ecological specialization in the mycorrhizal interaction of the rare terrestrial orchid Caladenia huegelii. Common and widespread sympatric orchid congeners were used for comparative purposes. Germination trials revealed an absolute requirement for mycorrhisation with compatibility barriers to germination limiting C. huegelii to a highly specific and range limited, efficacious mycorrhizal fungus. DNA sequencing confirmed fidelity between orchid and fungus across the distribution range of C. huegelii and at key life history stages within its life cycle. It was also revealed that common congeners could swap or share fungal partners including the fungus associated with the rare orchid but not vice versa. Data from this study provides evidence for orchid rarity as a cause and consequence of high mycorrhizal specialization. This interaction must be taken into account in efforts to mitigate the significant extinction risk for this species from anthropogenically induced habitat change and illustrates the importance of understanding fungal specificity in orchid ecology and conservation.     

Fruit harvesting time and corresponding morphological changes of seed integuments influence in vitro seed germination of <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl.

In vitro asymbiotic seed germination of Dendrobium nobile varied significantly with fruit harvesting time and growth medium used for culturing seeds. Seeds harvested 129 days after pollination (DAP) possessing globular shaped embryos and a discontinuous cuticle layer showed a substantially greater germination on P668 medium. Alternatively, immature seeds harvested 96 and 116 DAP displayed a significantly lower germination response on various growth media. Most of the ovules at 96 DAP are in archesporial and megaspore mother cell stages, whereas the majority of ovules are mature and fertilized at 116 DAP. Mature seeds harvested 158 DAP also germinated at a higher frequency at Stage 5 (emergence of the first leaf) after 8 weeks of culture on different growth media indicating the absence of testa imposed dormancy in this endangered epiphytic orchid.