Reproduction of rare tropical orchids in vitro

Authors: Antipina VA, Kolomeitseva G.L. (Main Botanical Garden named after NV Tsitsin of the Russian Academy of Sciences, Moscow, Russia).

The preservation of the biodiversity of the plant world is one of the most important tasks facing modern biological science. Among the families most affected by the anthropogenic factor is the Orchidaceae Juss. Family, whose representatives require detailed study and preservation.

The collection of tropical and subtropical orchids of the Main Botanical Garden of the Russian Academy of Sciences is a unique place to conduct this kind of research and create a center for studying plants of this family under the conditions of introduction.

The purpose of this work was seed reproduction in culture in vitro of rare and endemic orchid species, at different times introduced into the Stock Greenhouse conditions and grown under different temperature conditions: heat (+18 +23 ° С) and moderate (+14 +16 ° С) . Most of the studied species belong to the first group, species from the r. Cymbidium, as well as Masdevallia infracta. Of particular importance is the fact that among these species there are species from the “First CITES application” (Paphiopedilum appletonianum, P. haynaldianum, Phragmipedium longifolium) [6] and endemics of Vietnam (Coelogyne mooreana, Cymbidium erythrostylum) [8].

Under the conditions of introduction, there are particular difficulties associated with obtaining full-fledged seeds that are extremely small, lack a differentiated germ and nutrients. First of all, this is a limited number of clones represented in the collection, as well as instability and lack of flowering, caused by climatic and environmental factors of greenhouse cultivation. Seeds from these species were obtained as a result of artificial pollination, which was carried out in three different ways: autogamous (self-pollination), gaytonogamny (neighboring pollination) and xenogamic (cross-pollination) [4]. According to our data, the most effective was the last method, which requires the presence of several individuals of different origin in the collection [3].

Orchid fruit - dry box. Terms of fruit ripening are different and were:

2,5 мес. - Cymbidium crumenatum, Paphiopedilum longifolia; 3-3,5 мес. - studarettia macrocentra, Cymbidium albosanguineum, D.bullenianum, D.terminale; 5-6 мес. - Cymbidium brymerianum, D.dearei, Brassocattleya breaking Phalaenopsis haynaldianum, Robiquettia and compressed; 6,5-7 мес. - Listrostachys Magdalena Coelogyne tasseled Milton attractiveness; 8-8,5 мес. - Cymbidium loddigesii, Phalaenopsis appletonianum, Stanhopea serval, Vanda crested; 12 мес. и более - Coelogyne Mooreana, Cymbidium erythrostylum, C mastersii.

The obtained seeds were studied using a MICMED-5 light microscope, a Canon Power Shot A95 camera and the ImageJ program. The average length and width of the seed coat and the embryo (if it was visible) were calculated. They took from 50 to 200 seeds of each sample. Statistical data processing was performed using Excel.

The classification of orchid seeds presented in the literature is based on such indicators as the size and shape of the seed membrane, the morphological features of its cell walls, and the color of seeds. According to the R. Dressler system [7], the seeds of representatives of this family can be divided into 20 types, of which we found 6: Maxillaria, Disa, Dendrobium, Cymbidium, Gastrodia, Stanhopea, Vanda (Table 1). However, it should be noted that a strict distinction between the types of seeds is not always possible, especially with regard to differences in sculpturing and the size of the intercellular spaces in the seed coat (seeds of types Diuris and Disa), or density of contact between cell walls (seeds of types Vanda and Maxillaria). For this reason, the seeds of some taxa can be attributed simultaneously to two different types [2].

View Seed type
(Colour)
Size of this peel, micron Germ size, micron Early sprout day
length width length width
1 Aerangis macrocentra Maxillaria
(light brown)
327 ± 36 60 ± 9 168 ± 17 48 ± 6 60
2 Listrostachys magdalenae some
(dark brown)
259 ± 31 84 ± 11 - - 70
3 Coelogyne fimbriata Dendrobium
(white)
328 ± 25 117 ± 14 223 ± 20 192 ± 56 30
4 Coelogyne mooreana Dendrobium
(light green)
1470 ± 183 259 ± 34 493 ± 69 148 ± 18 30
5 Cymbidium erythrostylum Cymbidium
(cream)
1531 ± 138 240 ± 24 191 ± 22 122 ± 15 30
6 Cymbidium mastersii Cymbidium
(cream)
661 ± 113 188 ± 19 211 ± 30 124 ± 16 40
7 Dendrobium albosanguineum Dendrobium
(light yellow)
353 ± 44 95 ± 14 182 ± 22 70 ± 11 30
8 Dendrobium brymerianum Dendrobium
(yellow)
389 ± 34 102 ± 12 188 ± 17 102 ± 12 15
9 Dendrobium bullenianum Dendrobium
(yellow)
334 ± 35 70 ± 7 152 ± 12 70 ± 7 20
10 Dendrobium crumenatum Dendrobium
(yellow)
375 ± 51 70 ± 7 157 ± 19 61 ± 7 120
11 Dendrobium dearei Dendrobium
(yellow)
364 ± 34 71 ± 8 163 ± 15 59 ± 10 30
12 Dendrobium loddigesii Dendrobium
(yellow)
318 ± 29 64 ± 6 171 ± 14 55 ± 5 30
13 Dendrodium terminale Dendrobium
(yellow)
325 ± 31 71 ± 7 152 ± 10 71 ± 7 15
14 Brassocattleya peace Maxillaria
(yellowish)
209 ± 18 79 ± 8 132 ± 9 79 ± 8 60
15 Miltonia spectabilis Maxillaria
(yellowish)
189 ± 17 81 ± 8 - - 60
16 Paphiopedilum appletonianum Gastrodia (brown) 1516 ± 130 191 ± 16 210 ± 28 120 ± 12 60 / more
90
17 Paphiopedilum haynaldianum some
(dark brown)
416 ± 42 167 ± 21 - - 90
18 Phragmipedium longifolium some
(black and brown)
617 ± 66 135 ± 17 - - 15
19 Compressed Robiquettia Maxillaria
(yellowish)
314 ± 23 60 ± 5 168 ± 14 48 ± 5 30
20 Stanhopea tigrina Stanhopea
(white)
592 ± 62 356 ± 44 166 ± 19 124 ± 15 15
21 Vanda cristata Vanda
(brown)
259 ± 26 95 ± 10 - - 30

Table 1. Biometric characteristics and initial germination of seeds in the in vitro culture of some species of tropical orchids

Among the species studied by us, the maximum size of the seed coat was noted in Cymbidium erythrostylum (1531 ± 138 µm), and the minimum - in Miltonia spectabilis (189 ± 17 µm). The size of the embryo can be set if the seed coat is transparent or colorless. The seeds of some species were characterized by the presence of an opaque dark brown or black seed coat (Angraecum magdalenae, Miltonia spectabilis, Paphiopedilum haynaldianum, Phragmipedium longifolium, Vanda cristata), so that the size of the embryo could not be determined. The maximum germ sizes are characteristic for Coelogyne mooreana seeds (493 ± 69 µm), the minimum for Masdevallia infracta (132 ± 9 µm).

As a standard technology for germinating orchid seeds under asymbiotic conditions, sowing of mature seeds with sterilization in calcium hypochlorite solution (30 minutes) on standard Knudson-C medium was taken with subsequent seedling maintenance in climacameras with standard modes: temperature 22-24 ° С, humidity 50– 70%, illumination 2000 lux, 16-hour photoperiod. Sowing of seeds from an unopened fruit was also carried out. Observation of seed germination was carried out every 15 days.

The success of the germination of orchid seeds depends on many factors. First of all, on the degree of development of the embryo (the presence of phases of biological and ecological rest, seed germs, etc.). However, of no small importance is the structure of the seed coat, its density, the presence of tanning or other inhibiting substances in the cell walls [1].

The beginning of germination, according to the method proposed by J. Arditti [5], was considered the swelling of the developing embryo with subsequent rupture of the seed coat. Observations have shown that the difficulty of germination is characteristic, above all, for seeds with a thick seed coat and closely adjacent cells, as well as for seeds with painted cell walls. It should be noted that the presence of color in seeds is not always determined by the coloring of the cell walls of the seed coat. Seeds of some species have a transparent seed coat and a colored germ, the color and color intensity of which determines the overall color of the seeds. Thus, for example, among the majority of the species studied, the color of seeds determined the presence of a colored embryo. The exceptions were the seeds of such species as Aerangis macrocentra, Angraecum magdalenae, Paphiopedilum appletonianum, P.haynaldianum, Phragmipedium longifolium, Vanda cristata, which, as a rule, was characterized by slow germination. The longest germination we observed in seeds of Dendrobium crumenatum (120 days), which is probably due to the presence of biological or ecological dormancy in them. However, this issue requires more detailed study.

Of particular interest is the increase in the rate of germination of seeds after sterilization compared with seeds sown from an undisclosed fetus. Thus, the same degree of maturity of Paphiopedilum appletonianum seeds were sown in two given ways. Germination in the first case occurred more than 30 days earlier. This is probably due to the fact that during the sterilization process, leaching of substances inhibiting the germination of substances contained in the cells of the seed coat or its mechanical damage occurs.

Thus, the conducted studies allow us to conclude that there are some difficulties in obtaining viable seeds, determining their type, and identifying a correlation between the morphology of the seed coat and the susceptibility of seeds to germination. Thanks to modern methods of biotechnology, the problems of obtaining mass planting material of rare and endangered plant species are successfully solved, however, further study of the characteristics of orchid seed germination in asymbiotic culture is necessary, which is caused not only by theoretical significance (identification of phylogenetic relationships), but also by practical necessity.

Bibliography

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