1. Manual De Produccion De Orquideas Pdf Para

Orchids require symbiotic fungi and/or specific conditions to germinate. Asymbiotic techniques have been shown successful for orchid germination. In Chile, Chloraea include many endemic, and potentially ornamental, terrestrial orchid species. In this study, individuals of Chloraea crispa, C. Gavilu and C. Virescens were manually autopollinated.

The resulting capsules were sterilized and seeds were aseptically obtained. We evaluated asymbiotic germination in: Agar Water (AW), Knudson C (KC), Banana Culture Media (CMB), Tomato Culture Media (CMT), Malmgren Modified (MM), Murashige and Skoog (MS), and MS modified (MS1/2). Seeds were incubated in the dark at 24±1 ºC for 2 weeks.

Then they were put in 16/8 h light/dark cycles for 14 weeks. We registered germination and embryo development in the different culture media.

After 8 weeks, all tested Chloraea species germinated in most culture media. After 16 weeks, embryos in MM showed an evident shoot.

In AW only the pre-germination stage was achieved. Gavilu germination was over 90% in MM, CMT, and CMB. In KC, a lower germination was obtained in all tested species compared to the other culture media, except for AW. Asymbiotic germination of endemic Chilean orchids could be a viable strategy for their conservation and propagation as ornamental species.KEYWORDS: Terrestrial orchids; endemic species; in vitro germination; culture media; orchid propagation; asymbiotic germination. ASYMBIOTIC GERMINATIONSeven culture media were used in the asymbiotic seed germination : agar water (AW), banana culture medium (CMB; ), tomato culture medium (CMT; ), ), Malmgren Modified Terrestrial Orchid Medium (MM; ), Murashige & Skoog medium (MS; ) and MS modified (MS1/2). All media were sterilized in an autoclave at 121 °C and 1 atm for 20 min and then poured into 40 mm-diameter Petri dishes in a laminar flow chamber.

Once all media were prepared, pH was adjusted to 5.8. Four plates (replicates) per culture media were used (four plates × seven culture media = 28 plates total).

Each plate contained at least 200 seeds. Petri dishes containing media and seeds were put in an incubator at 24 ± 1 °C in the dark. After two weeks, seed developmental stage was evaluated (see, adapted from ). Seeds were then put for four weeks in a growth chamber at 20 ± 1 °C with a photoperiod of 16/8-hours light/ dark (light: 70 μmol m-2s-1). Germination percentage was determined by counting the number of germinated seeds in all Petri dishes divided by total viable seeds. Seed viability was previously determined by the tetrazolium test (data not shown). Germination was achieved when the embryo emerged from the seed coat (stage 2 in ).

RESULTSFloral traits confirmed the identity of the studied species (A-C). After two weeks of the beginning of the experiment, seeds of some of the tested Chloraea species germinated in CMT, MM and CMB culture media (A). After eight weeks of incubation, embryos of all tested Chloraea species reached stage 3 (protocorm stage) in CMT and MM media (B). Also, only C. Gavilu seeds reached stage 3 in CMB. MS medium resulted in embryo development to stage 3 only in C.

At 16 weeks after the beginning of the experiment, embryos of all species in MM medium reached the highest development, evidencing a distinct shoot (Shoot stage, I, C), stage that is previous to true leaf initiation. Additionally, stage 4 (Rhizoid stage, H) was reached in all species in CMT and only in C.

Gavilu and C virescens in MS medium. Plants in AW showed poor development and only reaches stage 1 (pre-germination stage, C).The three tested Chloraea species showed differences in seed germination in the different culture media tested in our study (one-way ANOVA, p. FIGURE 3 Average seed germination in C. Crispa (A), C gavilu (B), and C. Virescens (C) after 16 weeks of incubation in different culture media. Different letters correspond to statistical differences (t-test, p. DISCUSSIONMany orchids are economically important (, ), mainly for their beautiful flowers which made them stand out among ornamental plants ( ).

Chloraea and Gavilea are the most represented orchid genus in Chile (, ). The Chilean orchids Chloraea crispa, C. Gavilu, and C. Virescens have high ornamental value and potential as cut flowers.

Thus, efficient propagation methods are required to propagate these species. In this study it was established that seeds of these orchid species can be asymbiotically germinated in laboratory conditions using different culture media. To our knowledge, asymbiotic (or symbiotic) germination for these species has not been previously reported.

Studies on terrestrial temperate orchids are relatively scarce (but see, ) compared to studies on tropical species. Moreover, studies on orchid biology and asymbiotic reproduction of Chilean species are even rarer and for many species non-existent (but see, ). This is particularly important considering that many of those species are considered critically endangered (, ). The shortage of ecological and functional studies on this group could be partially blamed on the lack of taxonomic knowledge in the country and the difficulties for the identification of the species in the field, especially individuals lacking reproductive structures. Currently, the only sources available for the identification of the species are out-of-date papers and divulgation books (, ).In nature, orchids reproduce by seeds and depend on direct association with specific (to some degree) mycorrhizal fungi to germinate (, ). As stated early, this dependency is due to the minimal amount, or even absence, of nutrients stored in the small seeds of orchids (, ).

The fungal partner supplies the required nutrients for embryo growth, nutrients that can also be supplied by specific culture media (, ). The appropriate choice of nutrient medium seems to be crucial for seed germination and also for embryo development (, ). Seed germination varied among the studied Chloraea species depending on the used culture media.

Crispa and C. Virescens the highest germination (over ∼75%) was achieved in CMT and MM. On the other hand, C. Gavilu germinated over 75% in all tested culture media except AW.

Previous studies on Bipinnula fimbriata reported a germination of 73 and 65.7% in MM and CMT, respectively ( ). Registered close to 89% asymbiotic seed germination of the terrestrial orchid Habenaria macroceratitis using Lindermann and Knudson C media after 7 weeks. However, in the same study MM medium was the only culture media that induced protocorm development to leaf-bearing after 16 weeks of incubation. More recent studies on the same orchid species reported asymbiotic germination percentages of 83.9% using MM medium, with embryos evidencing elongation of the first leaf after 21 weeks ( ).

In our study, Knudson C media resulted in relatively low germination (below 30%) in C. Crispa and C. However, the same media resulted in over 70% germination in C. These results reinforce the idea that interspecific differences in nutrient requirements exist, and specific protocols should be developed for each orchid species to be propagated. The ecological and physiological consequences of these differences should be addressed in future studies on temperate terrestrial orchids.In terms of embryo development MM media showed the best results, evidencing the more advanced stage (stage 5, shoot is differentiated from the protocorm) after 8 weeks compared to the other tested media. As mentioned above, the response of orchid seeds to different nutrient media varies between species (, ) and specific nutritional and environmental conditions are likely needed for in vitro germination of a determinate species (, ).

The asymbiotic media used in this study varied greatly in mineral salt, nitrogen organic additives and vitamin compositions. Gavilu, all tested media (except for AW) supported a high germination, suggesting that this species is able to utilize a broad range of carbon and nitrogen forms and concentration.

KC, MS and MS1/2 contain only inorganic sources of nitrogen (ammonium and nitrate). In contrast, MM (and probably CMB and CMT) contains only organic nitrogen sources, coinciding with the highest germination for C. Crispa and C.

Several researchers have reported that inorganic nitrogen may limit orchid germination, possibly due to low nitrate reductase activity during seed germination, and early protocorm development (, ). The high germination of C. Gavilu in media with inorganic nitrogen sources could be explained by an early activation of nitrate reductase during germination and/or because organic nitrogen does not play an important role in seed germination as other nutrients or environmental factors, such as in the terrestrial orchid Bletia purpurea (Lam.) DC. In the field, C. Gavilu shows a broad latitudinal distribution and can be found in diverse environments (, ).

In its wide geographical range it is likely that this species finds different nutrient, soil, and even biotic conditions, possibly explaining why is less demanding of specific nutrients and culture media in laboratory conditions. On the other hand, C. Crispa and C.

Manual De Produccion De Orquideas Pdf Para

Virescens have more narrow latitudinal distributions and more specific ecological conditions (, ). Following the same argument used above, this could explain the higher selectiveness for Nitrogen sources and culture media.In vitro germination of Chloraea species can shed light on possible in situ plant responses and their environmental requirements, and also further the knowledge of orchid development. This information can in turn be used to propagate plants for potential economic purposes and to establish better management policies for their in situ and ex situ conservation.

In vitro methods can achieve high germination in these species, but the reintroduction of adult individuals in the field could likely need previous inoculation with species fungal partners to increase plant survival. Further studies are needed to confirm these still open questions.