CN116114601A - Culture medium capable of widely promoting germination of orchid seeds and culture method thereof - Google Patents
Culture medium capable of widely promoting germination of orchid seeds and culture method thereof Download PDFInfo
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- CN116114601A CN116114601A CN202310080974.4A CN202310080974A CN116114601A CN 116114601 A CN116114601 A CN 116114601A CN 202310080974 A CN202310080974 A CN 202310080974A CN 116114601 A CN116114601 A CN 116114601A
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- 241000233855 Orchidaceae Species 0.000 title claims abstract description 59
- 230000035784 germination Effects 0.000 title claims abstract description 35
- 239000001963 growth medium Substances 0.000 title claims abstract description 32
- 230000001737 promoting effect Effects 0.000 title claims abstract description 16
- 238000012136 culture method Methods 0.000 title claims abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 15
- 240000004638 Dendrobium nobile Species 0.000 claims description 20
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 20
- 229930006000 Sucrose Natural products 0.000 claims description 20
- 239000005720 sucrose Substances 0.000 claims description 20
- 239000002609 medium Substances 0.000 claims description 17
- 241001076416 Dendrobium tosaense Species 0.000 claims description 16
- 241001523681 Dendrobium Species 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 241000196324 Embryophyta Species 0.000 claims description 10
- 238000005286 illumination Methods 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 241001517368 Coelogyne Species 0.000 claims description 6
- 241000605372 Fritillaria Species 0.000 claims description 4
- 230000007226 seed germination Effects 0.000 abstract description 7
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 230000008635 plant growth Effects 0.000 abstract description 2
- 241000026010 Dendrobium candidum Species 0.000 description 8
- 241000935235 Fritillaria meleagris Species 0.000 description 7
- 244000131844 Dendrobium aphyllum Species 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 238000000540 analysis of variance Methods 0.000 description 5
- 239000002775 capsule Substances 0.000 description 5
- 239000006870 ms-medium Substances 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 241001517354 Coelogyne cristata Species 0.000 description 2
- 241001678082 Dendrobium huoshanense Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000209510 Liliopsida Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
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- -1 /or Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 241000481604 Bolbostemma Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- 230000001261 florigenic effect Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000442 meristematic effect Effects 0.000 description 1
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- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 229940099259 vaseline Drugs 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention provides a culture medium for widely promoting germination of orchid seeds and a culture method thereof, belonging to the technical field of plant growth. The invention provides a culture medium for widely promoting seed germination of orchid, and a method for culturing orchid based on the culture medium has the characteristics of simple operation, low cost and easy culture. Therefore, the culture medium and the method for culturing orchid have important practicality and universality, are favorable for large-scale popularization and meet market demands.
Description
Technical Field
The invention belongs to the technical field of plant growth, and particularly relates to a culture medium for widely promoting germination of orchid seeds and a culture method thereof.
Background
Orchid (Orchidaceae) belongs to the class Lilium of monocotyledoneae, the phylum angiosperm, and is one of the largest florigenic plant groups worldwide. Orchids are a large variety, over 700 genera worldwide, 35000 species, and the number is next to that of asteraceae, the first major family in monocotyledons. Has important medical value, ornamental value, scientific research value and ecological value.
A typical feature of orchid is its extremely fine seed, several tens of thousands to millions of seeds in a capsule, consisting of tens of cells, the mature orchid seed has only undifferentiated primordia and no endosperm, and its natural germination rate is extremely low. Therefore, the orchid seeds are difficult to germinate by self nutrition only, and the nutrient substances needed by germination are needed to be provided from the outside. The problem can be solved by symbiotic germination or non-symbiotic germination technology, and the symbiotic germination needs proper germination-promoting fungi and has low germination rate. The non-symbiotic germination does not need the participation of fungi, is simple and convenient to operate and low in cost, can obtain a large number of young plants in a short period, obviously improves the germination rate of orchid seeds, increases the feasibility of large-scale culture of orchid plants, and meets the market demand.
However, the nutritional requirements of the seed of different orchids on germination are different, so that the seed of different orchids always needs to explore culture mediums with different formulas, and the culture mediums suitable for germination of various orchids are lacking in a broad spectrum.
Disclosure of Invention
In view of the above, the invention aims to provide a culture medium for widely promoting germination of orchid seeds, and provides a culture medium suitable for germination of orchid seeds by exploring key nutrients for germination of orchid seeds.
The invention provides a culture medium for widely promoting germination of orchid seeds, which takes water as a solvent and comprises the following components: 1800-2000 mg/L KNO 3 1600-1700 mg/L NH 4 NO 3 360-380 mg/L MgSO 4 .7H 2 KH of 160-180 mg/L and O 2 PO 4 430-450 mg/L CaCl 2 .2H 2 O, 28-32 g/L sucrose and 11-13 g/L agar.
Preferably, the water is used as a solvent and comprises the following components: KNO of 1900mg/L 3 1650mg/L NH 4 NO 3 370mg/L MgSO 4 .7H 2 KH of O, 170mg/L 2 PO 4 CaCl 440mg/L 2 .2H 2 O, 30g/L sucrose and 12g/L agar.
Preferably, the pH value of the culture medium is 5.6-6.0.
Preferably, the pH of the medium is 5.8.
The invention provides application of the culture medium in promoting seed germination of orchid.
Preferably, the orchid plant comprises a Dendrobium (Dendrobium) plant and/or a Coelogyne (Coelogyne) plant.
Preferably, the dendrobium plant comprises any one of the following: dendrobium officinale (Dendrobium officinale), dendrobium huoshanense (Dendrobium aphyllum) and dendrobium nobile (Dendrobium nobile);
the Fritillaria plant is Coelohde Thunbergii (Coelogyne cristata).
The invention provides a method for culturing orchid plants, which comprises the following steps:
seed of orchid is inoculated into the culture medium for culture.
Preferably, the culturing conditions are as follows: the day/night temperature is 28/25 ℃, and the relative humidity of the culture environment is 58% -62%;
the culture conditions also comprise illumination, the illumination intensity is 11000-13000 LX, and the illumination period is dark/bright for 12/12h.
Preferably, the time of the cultivation is 18 to 24 days.
The invention provides a culture medium for widely promoting germination of orchid seeds, which takes water as a solvent and comprises the following components: 1800-2000 mg/L KNO 3 1600-1700 mg/L NH 4 NO 3 360-380 mg/L MgSO 4 .7H 2 KH of 160-180 mg/L and O 2 PO 4 430-450 mg/L CaCl 2 .2H 2 O, 28-32 g/L sucrose and 11-13 g/L agar. The culture medium provided by the invention is based on an inorganic matter, organic matter and sucrose integrated culture system, key nutrient substances for germination of orchid seeds are further explored, and the culture medium for culturing orchid plants is provided, so that the germination rate of various orchid seeds can be improved. Experiments show that the culture medium is suitable for culturing various orchids and has universality.
Meanwhile, the orchid seeds are cultivated by the culture medium, and the germination time of the seeds can be shortened by only 3 weeks from the beginning of sowing to the formation of the original meristematic tissue of the seeds, namely the germination to form seedlings.
The invention also provides a culture method of the orchid, which has the advantages of simple culture operation method, low cost and easy culture. Therefore, the culture medium and the method for culturing orchid have important practicality and universality, are favorable for large-scale popularization and meet market demands.
Drawings
FIG. 1 is a diagram showing germination of the original corms of Dendrobium officinale seeds of Orchidaceae at different stages;
FIG. 2 is a graph showing germination of Dendrobium officinale seeds on different nutrient media for 40 days, wherein A: dendrobium aphyllum (sucrose+MS medium), seedlings; b: dendrobium nobile (sucrose+MS medium), seedling; c: dendrobium officinale (sucrose+MS medium), seedlings; d: unibract fritillary bulb (sucrose+ms medium), seedling; e: dendrobium aphyllum (sucrose+bulk medium), seedlings; f: dendrobium nobile (sucrose+bulk medium), seedling; g: dendrobium officinale (sucrose+bulk medium), seedlings; h: the seedling of the unibract fritillary bulb (sucrose+mass culture medium);
FIG. 3 shows the 40-day protocorm rate of Dendrobium officinale seeds cultured on different nutrient media, wherein Bar represents the standard deviation of three biological replicates, and different letters on Bar represent significant differences (P < 0.05); analyzing the data by adopting an ANOVA variance analysis method;
FIG. 4 shows the seedling rate of Dendrobium officinale seeds cultured on different nutrient media for 40 days, wherein Bar represents the standard deviation of three biological replicates, and different letters on Bar represent significant differences (P < 0.05); the data were analyzed using ANOVA analysis of variance.
Detailed Description
The invention provides a culture medium for widely promoting germination of orchid seeds, which takes water as a solvent and comprises the following components: 1800-2000 mg/L KNO 3 1600-1700 mg/L NH 4 NO 3 360-380 mg/L MgSO 4 .7H 2 KH of 160-180 mg/L and O 2 PO 4 430-450 mg/L CaCl 2 .2H 2 O, 28-32 g/L sucrose and 11-13 g/L agar.
In the present invention, the water as a solvent preferably comprises the following components: KNO of 1900mg/L 3 1650mg/L NH 4 NO 3 370mg/L MgSO 4 .7H 2 KH of O, 170mg/L 2 PO 4 CaCl 440mg/L 2 .2H 2 O, 30g/L sucrose and 12g/L agar. The pH of the medium is preferably 5.6 to 6.0, more preferably 5.8.
The method for preparing the medium is not particularly limited, and methods for preparing a medium known in the art may be employed.
The invention provides application of the culture medium in promoting seed germination of orchid.
In the present invention, the orchid preferably includes Dendrobium (Dendrobium) and/or Coelogyne (Coelogyne) plants. The dendrobium plant preferably comprises any one of the following: dendrobium officinale (Dendrobium officinale), dendrobium nobile (Dendrobium aphyllum) and dendrobium nobile (Dendrobium nobile), more preferably dendrobium officinale and dendrobium nobile, most preferably dendrobium nobile; the plant of the genus Fritillaria is preferably Fritillaria fusiforme (Coelogyne cristata).
The invention provides a method for culturing orchid plants, which comprises the following steps:
seed of orchid is inoculated into the culture medium for culture.
In the present invention, the conditions of the culture are preferably as follows: the day/night temperature is 28/25 ℃, and the relative humidity of the culture environment is 58% -62%; the culture conditions also comprise illumination, the illumination intensity is 11000-13000 LX, and the illumination period is dark/bright for 12/12h. The relative humidity of the culture environment was 60%. The illumination intensity was 12000LX. The time of the culture is preferably 18 to 24 days, more preferably 21 days. The cultivation is preferably carried out in a climatic incubator.
The invention provides a culture medium for widely promoting seed germination of orchid. The method for culturing orchid based on the culture medium has the characteristics of simple operation, low cost and easy culture. Therefore, the culture medium and the method for culturing orchid have important practicality and universality, are favorable for large-scale popularization and meet market demands.
The following is a detailed description of a culture medium and a method for culturing seeds of orchid, which are provided by the present invention, but are not to be construed as limiting the scope of the invention.
Example 1
Method for promoting germination of orchid seeds
1. Experimental raw materials
Seed: the experiment uses seeds of dendrobium candidum, dendrobium huoshima, dendrobium nobile, and unibract fritillary bulb as experimental materials, wherein dendrobium candidum capsules come from the biological technology limited company of holy products in Leqing city, and dendrobium huoshima, dendrobium nobile, and unibract fritillary bulb capsules come from the biological technology limited company of Murray Koenigii, yunnan province.
Culture medium: the composition of the medium is shown in Table 1.
TABLE 1 high efficient Medium for widely promoting germination of orchid
2. The method for culturing orchid seeds by using the culture medium comprises the following specific steps of
1. Disinfection of capsules and acquisition of sterile orchid seeds
Selecting a yellowing but not yet cracked mature orchid capsule, cleaning with sterile water for 1 time, sterilizing with 75% alcohol for 30 seconds in an ultra-clean workbench, cleaning with sterile water for 3 times, then sterilizing with 1% NaClO solution (effective chloride ion) for 8min, cleaning with sterile water for 3 times, placing on sterile filter paper for drying with sterile air, cutting the dendrobium candidum fruit with a sterile scalpel, taking out the seeds, wrapping with sterile filter paper, tying with rubber bands, marking the species name and date, finally wrapping the wrapped dendrobium candidum seed filter paper, placing in a sterile glass dryer with the bottom fully paved with anhydrous lithium chloride, coating a layer of vaseline on the sealing position, and sealing. After the seeds are dried, transferring the seeds into a sterile sealed freezing tube, labeling, placing the frozen tube into a closed container containing desiccant silica gel, and preserving the frozen tube in a refrigerator at the temperature of minus 20 ℃ to obtain sterilized seeds for later experiments.
2. Sowing and culturing
And (3) placing a proper amount of dendrobium candidum seeds and cooled sterile water into a 10ml centrifuge tube after sterilization in an ultra-clean workbench, and uniformly mixing to prepare the sterile seed suspension. 1ml of the suspension (containing about 1000 seeds) was then aspirated with a pipette and evenly sown on the surface of a medium of different nutritional composition (medium indicated in Table 1, denoted as high +sucrose, another medium denoted as MS, as control) with sterile filter paper. The culture dish is covered, the sealing film is sealed, and the mark is made. The above treatments were repeated for 10 dishes per group. Culturing in artificial climate incubator (12/12 h, light/dark) and temperature (25/22deg.C, day/night), observing every 5 days, covering every 20 days, grading the germination of Dendrobium officinale seeds according to table 2, and photographing each germination stage of Dendrobium officinale seed germination as shown in figure 1, recording the number of stages 1, 2, 3, 4 and 5, and calculating the ratio of protocorm and seedling according to formula I and formula II.
Protocorm ratio = (number of stage 2+number of stage 3+number of stage 4+number of stage 5)/total number of seeds×100% formula I
Seedling ratio = (number of stage 3+number of stage 4+number of stage 5)/total number of seeds×100% formula II
The standard for judging seed germination is that the seed swells due to water absorption, the embryo breaks through the seed coat, and the primary meristem is further formed, namely, the bud forms a seedling.
TABLE 2 description of seed germination stage of orchid
3. Data statistics and analysis
Data analysis was performed on the rates of ungerminated seeds, protocorms, and buds at each stage of orchid. For data which do not meet normal distribution, the non-parametric test method Kruskal-Wallis H test is adopted to carry out difference significance comparison (P < 0.05), then Mann-Whitney U test is adopted to carry out pairwise comparison (splsver.19.0, alpha=0.05), and for data which meet normal distribution, analysis of variance (ANOVA) and least significant difference method (LSD) multiple comparison are adopted.
The result shows that when the culture is continued for 40 days, the dendrobium huoshanense, dendrobium nobile, dendrobium candidum and unibract fritillary bulb of the orchid plants all have sprouting phenomenon, and the stage 3 is reached. Wherein Dendrobium aphyllum Rolfe is dark green, the buds are longer, dendrobium nobile and Dendrobium officinale are light green, the buds are shorter, and the unibract fritillary bulb is slower to develop, and only a small amount of protocorm forms a protogenic meristem (as shown in figure 2). Counting the number of seeds, protocorms and seedlings in stages, so as to calculate the seed, protocorm or seedling ratio of different stages.
The protocorm rate of the seed of the bract fritillary bulb and the seed of the dendrobium nobile on the sucrose and a large amount of culture mediums is obviously higher than that of the seed of the dendrobium nobile on the MS culture mediums, and the protocorm rates of the dendrobium nobile and the dendrobium candidum on the two culture mediums have no obvious difference (as shown in figure 3). The seedling rate of Dendrobium nobile is significantly higher than that of other orchid plants, reaching 26.91% + -2.63, and the seedling rates of Dendrobium nobile and Dendrobium aphyllum are not significantly different, respectively 8.90% + -1.47 and 9.80% + -1.94, and the seedling rate of Bolbostemma pentaphylla is lower, 4.27% + -2.58, but still further develops (as shown in figure 4). The sucrose and the macroelements not only promote the germination of the dendrobium candidum seeds, but also promote the germination of other orchid seeds, and the germination promotion effect of the sucrose and the macroelements is proved to have universality.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A culture medium for widely promoting germination of orchid seeds, which is characterized by comprising the following components by taking water as a solvent: 1800-2000 mg/L KNO 3 1600-1700 mg/L NH 4 NO 3 360-380 mg/L MgSO 4 .7H 2 KH of 160-180 mg/L and O 2 PO 4 430-450 mg/L CaCl 2 .2H 2 O, 28-32 g/L sucrose and 11-13 g/L agar.
2. The culture medium according to claim 1, wherein the water is used as a solvent and comprises the following components: KNO of 1900mg/L 3 1650mg/L NH 4 NO 3 370mg/L MgSO 4 .7H 2 KH of O, 170mg/L 2 PO 4 CaCl 440mg/L 2 .2H 2 O, 30g/L sucrose and 12g/L agar.
3. The medium according to claim 1 or 2, wherein the pH of the medium is 5.6-6.0.
4. A medium according to claim 3, characterized in that the pH value of the medium is 5.8.
5. Use of the medium according to any one of claims 1 to 4 for promoting germination of orchid seeds.
6. The use according to claim 5, wherein the orchid plants comprise Dendrobium (Dendrobium) plants and/or Coelogyne (Coelogyne) plants.
7. The use according to claim 6, wherein the dendrobe plant comprises any one of the following: dendrobium officinale (Dendrobium officinale), dendrobium huperzianum (Dendrobium aphycum), dendrobium nobile (Dendrobium nobile);
the plant of the genus Fritillaria is Coelohde (Coelogynecostata).
8. A method of culturing orchid plants, comprising the steps of:
inoculating orchid seeds into the culture medium according to any one of claims 1-4 for cultivation.
9. The culture method according to claim 8, wherein the culture conditions are as follows: the day/night temperature is 28/25 ℃, and the relative humidity of the culture environment is 58% -62%;
the culture conditions also comprise illumination, the illumination intensity is 11000-13000 LX, and the illumination period is dark/bright for 12/12h.
10. The method according to claim 8 or 9, wherein the time of the cultivation is 18 to 24 days.
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2023
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