CN104186295A - Culture medium for paphiopedilum seed germination and culture method - Google Patents
Culture medium for paphiopedilum seed germination and culture method Download PDFInfo
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- CN104186295A CN104186295A CN201410514229.7A CN201410514229A CN104186295A CN 104186295 A CN104186295 A CN 104186295A CN 201410514229 A CN201410514229 A CN 201410514229A CN 104186295 A CN104186295 A CN 104186295A
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Abstract
The invention discloses a culture medium for paphiopedilum seed germination and a culture method. The culture medium for paphiopedilum seed germination, provided by the invention, takes 1/4 of culture medium as a basal culture medium, and ascorbic acid, activated carbon, banana puree, gibberellin, agar powder and white granulated sugar are added; therefore, the germination time of the paphiopedilum seed can be shortened, and the seed germination rate can be increased.
Description
Technical field
The present invention relates to Orchid Tissue culture technique field, particularly relate to the blue seed germination medium of a kind of pocket and cultural method.
Background technology
Pocket is blue, claims again " slippers are blue ", " celestial shoe is blue ", is the general designation of the orchid family Paphiopedilum (Paphiopedilum) plant.Pocket orchid is a monoid most characteristic in orchid, is also the most peculiar orchid of viewing and admiring, with its unique charm and many excellent specific properties and enjoy doting on of world flower fan.
In recent years, the tissue culture technique of orchid and test-tube plantlet batch production production have obtained swift and violent development, but as the more original Paphiopedilum of the orchid family, its seed does not have endosperm, have the problems such as germination rate is extremely low, sprout time is long.The blue seed germination medium of existing pocket conventionally on the basis of 1/4MS medium, add the coconut milk of mass concentration 10%, the NAA of the 6-BA of 0.5mg/L, 0.5mg/L, the sucrose of the agar powder of 6g/L, 30g/L and the active carbon of 2g/L.Yet 6-BA and NAA are broad spectrum activity plant growth regulator, be mainly used in taking root, sprout and induce protocorm, poor to breaking seed dormancy effect.Therefore, adopt the blue seed germination of existing pocket to cultivate, be difficult to realize the Fast-propagation of pocket orchid, be difficult to meet the demand of large-scale planting.
Summary of the invention
Based on this, being necessary, for the low problem of the blue seed germination rate of pocket, provides a kind of medium that can improve the blue seed germination rate of pocket.
In addition, be necessary, for the low problem of the blue seed germination rate of pocket, to provide a kind of cultural method that can improve the blue seed germination rate of pocket.
The blue seed germination medium of pocket, it take 1/4MS medium as basal medium, and is added with ascorbic acid, gibberellin, active carbon, banana puree, agar powder and white granulated sugar.
In an embodiment, the concentration of ascorbic acid is 80～100mg/L therein, and the concentration of gibberellin is 40～60mg/L, the concentration of active carbon is 1.8～2g/L, the concentration of banana puree is 40～50g/L, and the concentration of agar powder is 6～7g/L, and the concentration of white granulated sugar is 25～30g/L.
In an embodiment, the concentration of ascorbic acid is 100mg/L therein, and the concentration of gibberellin is 50mg/L, and the concentration of active carbon is 2g/L, and the concentration of banana puree is 50g/L, and the concentration of agar powder is 6g/L, and the concentration of white granulated sugar is 30g/L.
In an embodiment, the pH value of the blue seed germination medium of described pocket is 6.0 therein.
The blue seed germination cultural method of pocket, comprises the following steps:
1) get healthy growth, the pollination fruit pod of 140～150 days, by mass concentration, be 70～75% alcohol-pickled 25～30 seconds, then the mercuric chloride solution that is 0.10～0.12% by mass concentration sterilization 18～25 minutes, then after clean by sterile water wash, cut fruit pod, take out seed;
2) the KOH solution immersion treatment that seed is 0.05～0.1mol/L by concentration 8～10 minutes, after clean by sterile water wash, by frequency, be that the ultrasonic that 50～60KHz, power are 80～90W is processed 25～30 minutes again, then be inoculated in the blue seed germination medium of pocket of the present invention, under dark condition of culture, being cultured to seed germination, is to be then cultured to and to grow up to seedling under the condition that 12h illumination/12h is dark, intensity of illumination is 1800～2000lux in light dark period.
Therein in an embodiment, step 3) in, on the blue seed germination medium of described pocket, being equipped with sterilizing filter paper, described sterilizing filter paper is through the blue seed germination medium of pocket of the present invention immersion treatment.
In the blue seed germination medium of pocket of the present invention, adopt 1/4MS medium, by reducing the ion concentration of macroelement, be conducive to the blue seed germination of pocket, improve germination rate; In addition, by ascorbic acid, gibberellin, active carbon and banana puree, cooperate with seed, can effectively promote seed germination, improve seed germination rate.Wherein, ascorbic acid is antioxidant, and seed is had no side effect, can produce with brownization oxidation product---quinones substance is had an effect, make it again be reduced to aldehydes matter, thereby reduce brownization degree, improve seed germination rate; Gibberellin can regulate the physiological status of seed, by the alpha-amylase gene in transcriptive intermediate Effects of Factors seed, promote the formation of α-amylase mRNA and synthesizing of α-amylase, improve the level of α-amylase, thereby promote α-amylasehydrolysis starch, increase the required carbon source of seed germination, the dormancy of breaking seed, promote seed germination, significantly shorten sprout time; Active carbon phenols, quinones substance that effectively absorbed seed produces, Browning control, improves seed germination rate; Banana puree is rich in the multiple seed germination desired substances such as amino acid, hormone and enzyme, can make up the blue seed of pocket and not have albuminosus defect, for it provides nutriment, thereby improves germination rate, and makes protocorm robust growth; Agar is as solid culture platform, provides nutrient, moisture and ventilative effectiveness, and using white granulated sugar as carbon source for planting material, under sprout time and the impregnable prerequisite of germination rate, reduced the preparation cost of medium.
In the blue seed germination medium of pocket of the present invention, the concentration of ascorbic acid is preferably 80～100mg/L, if its consumption is very few, antioxidation is not obvious; The concentration of gibberellin is preferably 40～60mg/L, if its consumption is very few, is difficult to promote seed germination, if its consumption is too much, can cause explant overgrowing, and vitrifying is serious; The concentration of active carbon is preferably 1.8～2g/L, if its consumption is very few, and fully absorbing phenolic and quinones substance, brownization inhibition is poor, if its consumption is too much, except absorbing phenolic and quinones substance, also the nutriment in medium be can adsorb, seed germination and explant growth affected; The concentration of banana puree is preferably 40～50g/L, if its consumption is very few, is difficult to meet the required nutrition of the blue seed germination of pocket.
The blue seed germination medium of pocket of the present invention, is controlled at 6.0 by pH value, is weak acid environment, is conducive to the sprouting of the blue seed of pocket, thereby improves germination rate, shortens sprout time.
The blue seed germination medium of pocket of the present invention by biochemical means, adds ascorbic acid in medium, stops aldehydes matter to be oxidized to the quinones substance of brown; By physiology means, in medium, add gibberellin and banana puree, regulate seed physiology situation, break seed dormancy, promote seed germination; And by physical means, in medium, add active carbon, and absorb phenols, quinones substance, reduce and organize brownization, thereby improve seed germination rate.The blue seed germination medium of pocket of the present invention, by the synergy of biochemistry, physiology and physics three aspects:, can significantly improve the germination rate of the blue seed of pocket.
The blue seed germination cultural method of pocket of the present invention, adopted the blue seed germination medium of pocket of the present invention, again by many-sided effect simultaneously such as chemistry, physiology, mutually promote, can significantly improve the blue seed germination rate of pocket, seed germination rate, up to 60%, shortens the blue seed germination time of pocket, and reduces Brown rate.After obtaining aseptic seed, seed is carried out to the immersion of KOH solution and ultrasonic processing, to break seed dormancy, promote seed germination.Wherein, chemical aspect, adopts KOH solution to soak, the material of energy dissolution inhibition seed germination, corrosion kind of a skin, more easily sprout seed, improves germination rate, adopt KOH solution and non-common NaOH solution, because the water imbibition of KOH is lower than NaOH, can keep the moisture of seed better; And physiology aspect adopts ultrasonic to process, can make the activity of enzyme increase, the dormancy of abolishing seed.Seed, after the immersion of KOH solution and ultrasonic processing, is inoculated in the blue seed germination medium of pocket of the present invention and secretly cultivates, and can regulate seed physiology movable, breaks seed dormancy, promotes seed germination.After seed sprouting, transfer in time under alternation of light and darkness environment and cultivate, to avoid, protocorm growth is expanded, color bleaches, and can not turn green seedling.In addition, adopt alcohol and mercury chloride to carry out sterilization treatment to fruit pod, kill bacteria and fungi, reach best sterilization effect simultaneously.
In the blue seed germination cultural method of pocket of the present invention, while adopting KOH solution to make immersion treatment to seed, the concentration of KOH solution is preferably 0.05～0.1mol/L, soak time is preferably 8～10 minutes, if KOH solution concentration is too high or long soaking time, can damage and even make it inactivation the blue seed of pocket, if KOH solution concentration is too low or soak time is too short, promote the DeGrain of seed germination; When seed is carried out to ultrasonic processing, calibration is 50～60KHz, power is preferably 80～90W, processing time is preferably 25～30 minutes, if frequency, power that ultrasonic is processed are too high or the processing time is long, can cause seed physiology disorderly, destroy seed physiology structure, affect seed germination and even cause de-vitalizing seed, if frequency, power that ultrasonic is processed are too low or the processing time is too short, promote the DeGrain of seed germination; Seed is secretly cultured to seed germination, if dark incubation time is long, can cause explant yellow, and the undesired and explant vitrifying of growing, if dark incubation time is too short, promotes the DeGrain of seed germination.
In addition,, by lay the sterilizing filter paper soaking through medium on seed germination medium, can prevent that seed is absorbed in suffocates in medium and reduces seed germination rate.
Embodiment
Embodiment mono-: the preparation of the blue seed germination medium of pocket of the present invention
According to the blue seed germination culture medium prescription of the pocket shown in the 1/4MS culture medium prescription shown in table 1 and table 2, prepare the blue seed germination medium of pocket of the present invention.
By the amount of preparation 1100mL medium, take respectively each composition in 1/4MS medium and active carbon, banana puree, agar powder, white granulated sugar, be placed in triangular flask, add appropriate purified water, stirring and dissolving, is settled to 1000mL, and regulates pH value to 6.0.By triangular flask sealing, be placed in high-pressure sterilizing pot, in 121 ℃ of sterilizing 20min, be then placed in superclean bench naturally cooling, obtain sterilising medium mother liquor, standby.
Amount by preparation 1100mL medium, takes respectively ascorbic acid and gibberellin, adds appropriate purified water to dissolve, and mixes, and is settled to 100mL, and filtration sterilization in superclean bench is standby.
In superclean bench, medium mother liquor subject to sterilization is cooled to after 60 ℃, adds ascorbic acid and Gibberellins solution after sterilizing, mixes, and makes the blue seed germination medium of 1100mL pocket.
Get filter paper, be cut into the size matching with tissue culture flasks, be placed in high-pressure sterilizing pot sterilizing 20min at 121 ℃, after sterilizing, be put in superclean bench, and soak with the blue seed germination medium of prepared pocket, standby.
Get the blue seed germination medium of prepared pocket, divide and be filled in tissue culture flasks, dry in the air cool, after it solidifies, on its surface, lay the aseptic filter paper after medium soaks, then by tissue culture flasks sealing, standby.
Composition and the consumption thereof of table 1 1/4MS medium
Remarks: the macroelement consumption in 1/4MS medium be standard MS medium macroelement consumption 1/4.
Composition and the consumption thereof of the blue seed germination medium of table 2 pocket
| Composition | Final concentration |
| 1/4MS medium | - |
| Ascorbic acid | 100mg/L |
| Gibberellin | 50mg/L |
| Active carbon | 2g/L |
| Banana puree | 50g/L |
| Agar powder | 6g/L |
| White granulated sugar | 30g/L |
Embodiment bis-: the blue seed germination cultural method of pocket of the present invention
Choosing healthy growth, the pollination fruit pod of 140 days, is 75% with mass concentration alcohol-pickled 30 seconds, then the mercuric chloride solution that is 0.1% with mass concentration sterilization 20 minutes, then after clean by sterile water wash, cuts really pod, and taking-up seed is standby.
The tissue culture flasks of getting embodiment mono-preparation is standby, and this tissue culture flasks is equipped with the blue seed germination medium of solid-state pocket, and the surface of medium is equipped with the aseptic filter paper after medium soaks.
Get the aseptic seed obtaining after disinfecting, the KOH solution immersion treatment that is 0.1mol/L by concentration 10 minutes, after clean by sterile water wash, then be that the ultrasonic that 60KHZ, power are 90W is processed 30 minutes by frequency, be then inoculated in the tissue culture flasks of embodiment mono-preparation.Postvaccinal tissue culture flasks is placed in to climatic cabinate, cultivates 12 days to seed germination under dark condition of culture, the environmental parameter of climatic cabinate is set to: 26 ± 1 ℃ of temperature; Then in light dark period be that 12h illumination/12h is dark, intensity of illumination is that 2000lux, temperature are to be cultured to and to grow up to seedling under the condition of 25 ± 1 ℃.
Embodiment tri-: the test of the blue seed germination medium of pocket of the present invention
According to the blue seed germination culture medium prescription of the pocket shown in the 1/4MS culture medium prescription shown in table 1 and table 3, with reference to the medium preparation method described in embodiment mono-, prepare respectively the blue seed germination medium of pocket of each experimental group, control group.
The blue seed germination medium of table 3 pocket
Choose healthy growth, the pollination fruit pod of 140 days, by mass concentration, be 75% alcohol-pickled 30 seconds, the mercuric chloride solution that is 0.1% by mass concentration again sterilization 20 minutes, after clean by sterile water wash again, cut fruit pod, take out seed, under gnotobasis, be inoculated in respectively in the medium of each experimental group, control group shown in table 3, then in light dark period be that 12h illumination/12h is dark, intensity of illumination is that 2000lux, temperature are to cultivate under the condition of 25 ± 1 ℃.Cultivate after 30 days and record seed germination rate, sprout time and brown rate, result is as shown in table 4.
The blue seed germination rate of table 4 pocket, sprout time, brown rate test result
Remarks: comprehensive grading y=a*0.5-b*0.3-c*0.2.
From the result of the test of table 4, adopt the blue seed germination medium of pocket of the present invention, can significantly improve the blue seed germination rate of pocket, shorten the blue seed germination time of pocket, and reduce its brown rate.From control group 2, control group 3, contrasted with the result of experimental group 9, between gibberellin and ascorbic acid, have collaborative facilitation, when both use, can further improve the blue seed germination rate of pocket simultaneously, shorten the seed germination time.
Embodiment tetra-: the test of the blue seed germination cultural method of pocket of the present invention
Choosing healthy growth, the pollination fruit pod of 140 days, is 75% with mass concentration alcohol-pickled 30 seconds, then the mercuric chloride solution that is 0.1% with mass concentration sterilization 20 minutes, then after clean by sterile water wash, cuts really pod, and taking-up seed is standby.
With reference to the blue seed germination cultural method of the pocket described in embodiment bis-, according to the processing method shown in table 5 and condition of culture, the aseptic seed obtaining after disinfecting is inoculated in after the blue seed germination medium of pocket, respectively the seed of each experimental group, control group is cultivated.Cultivate and record seed germination rate and sprout time after 30 days, result is as shown in table 5.
The blue seed germination condition of culture of table 5 pocket and result of the test thereof
Remarks: 1. "+" represents to process; "-" represents not process;
2. comprehensive grading y=a*0.7-b*0.3;
3. control group 4 and each experimental group all adopt the blue seed germination medium of the prepared pocket of embodiment mono-, control group 5, control group 6 adopts 1/4MS medium to be also added with the agar powder of 6g/L, the sucrose of 30g/L, and control group 7 adopts 1/4MS medium and is added with 10% coconut milk, the 6-BA of 0.5mg/L, the agar powder of the NAA of 0.5mg/L, 6g/L, the active carbon of the sucrose of 30g/L and 2g/L.
Result of the test from table 5, adopting on the basis of the blue seed germination medium of pocket of the present invention, by KOH solution immersion treatment, ultrasonic, process again and the rear dark interaction of processing of cultivating of inoculation, and by laying the aseptic filter paper soaking through medium, can further significantly shorten the sprout time of the blue seed of pocket, improve seed germination rate.In addition,, from the result of the test of control group 4,5,7, the culture effect of the blue seed germination medium of pocket of the present invention, is obviously better than the blue seed germination medium of basal medium and existing pocket.There is the result of the test of control group 5, control group 6 known, under the consistent condition of medium, adopt the blue seed germination condition of culture of pocket of the present invention, also can significantly improve the blue seed germination rate of pocket, shorten the seed germination time.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. the blue seed germination medium of pocket, is characterized in that: take 1/4MS medium as basal medium, and be added with ascorbic acid, gibberellin, active carbon, banana puree, agar powder and white granulated sugar.
2. the blue seed germination medium of pocket according to claim 1, it is characterized in that: the concentration of ascorbic acid is 80～100mg/L, the concentration of gibberellin is 40～60mg/L, the concentration of active carbon is 1.8～2g/L, the concentration of banana puree is 40～50g/L, the concentration of agar powder is 6～7g/L, and the concentration of white granulated sugar is 25～30g/L.
3. the blue seed germination medium of pocket according to claim 2, is characterized in that: the concentration of ascorbic acid is 100mg/L, and the concentration of gibberellin is 50mg/L, the concentration of active carbon is 2g/L, the concentration of banana puree is 50g/L, and the concentration of agar powder is 6g/L, and the concentration of white granulated sugar is 30g/L.
4. according to the blue seed germination medium of the pocket described in one of them of claims 1 to 3, it is characterized in that: the pH value of the blue seed germination medium of described pocket is 6.0.
5. the blue seed germination cultural method of pocket, comprises the following steps:
1) get healthy growth, the pollination fruit pod of 140～150 days, by mass concentration, be 70～75% alcohol-pickled 25～30 seconds, then the mercuric chloride solution that is 0.10～0.12% by mass concentration sterilization 18～25 minutes, then after clean by sterile water wash, cut fruit pod, take out seed;
2) the KOH solution immersion treatment that seed is 0.05～0.1mol/L by concentration 8～10 minutes, after clean by sterile water wash, by frequency, be that the ultrasonic that 50～60KHz, power are 80～90W is processed 25～30 minutes again, then be inoculated in the blue seed germination medium of pocket claimed in claim 1, under dark condition of culture, being cultured to seed germination, is to be then cultured to and to grow up to seedling under the condition that 12h illumination/12h is dark, intensity of illumination is 1800～2000lux in light dark period.
6. the blue seed germination cultural method of pocket according to claim 5, it is characterized in that: step 3) in, on the blue seed germination medium of described pocket, be equipped with sterilizing filter paper, described sterilizing filter paper is through the blue seed germination medium of pocket claimed in claim 1 immersion treatment.
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| CN105325274A (en) * | 2015-10-30 | 2016-02-17 | 华南农业大学 | Paphiopedilum seed germination culture medium, culture method and application |
| CN106305113A (en) * | 2016-08-29 | 2017-01-11 | 宿州市埇桥区杨园种植专业合作社 | Planting method capable of improving onion germination rate |
| CN106717265A (en) * | 2016-12-02 | 2017-05-31 | 黑龙江省农业科学院草业研究所 | A kind of method that Leymus chinensis seeds are efficiently sprouted |
| CN107056414A (en) * | 2017-01-23 | 2017-08-18 | 中国林业科学研究院林业研究所 | A kind of broad spectrum activity hot bandwidth aseptic seeding culture medium two |
| CN107285933A (en) * | 2017-08-23 | 2017-10-24 | 太仓市新滨农场专业合作社 | A kind of crop seeds seedling culture medium |
| CN107493728A (en) * | 2017-09-12 | 2017-12-22 | 中国科学院华南植物园 | A kind of method for Helen's pocket orchid seed germination rate that cryopreservation is improved using ultrasonication |
| CN108157166A (en) * | 2018-01-24 | 2018-06-15 | 董春燕 | A kind of breed of variety of pocket Lanzhou and Xinjiang and tissue culture and rapid propagation method |
| CN110809935A (en) * | 2019-11-08 | 2020-02-21 | 广东省农业科学院果树研究所 | Method for improving germination rate of banana seeds |
| CN110881478A (en) * | 2019-11-07 | 2020-03-17 | 中国林业科学研究院林业研究所 | Method for promoting germination of seeds of denatolum and paphiopedilum by using ceriferous fungi |
| CN114507361A (en) * | 2022-02-28 | 2022-05-17 | 新疆农业大学 | Agar activated carbon hydrogel for soilless culture seeds and preparation method thereof |
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| CN105325274A (en) * | 2015-10-30 | 2016-02-17 | 华南农业大学 | Paphiopedilum seed germination culture medium, culture method and application |
| CN106305113A (en) * | 2016-08-29 | 2017-01-11 | 宿州市埇桥区杨园种植专业合作社 | Planting method capable of improving onion germination rate |
| CN106717265A (en) * | 2016-12-02 | 2017-05-31 | 黑龙江省农业科学院草业研究所 | A kind of method that Leymus chinensis seeds are efficiently sprouted |
| CN107056414A (en) * | 2017-01-23 | 2017-08-18 | 中国林业科学研究院林业研究所 | A kind of broad spectrum activity hot bandwidth aseptic seeding culture medium two |
| CN107285933A (en) * | 2017-08-23 | 2017-10-24 | 太仓市新滨农场专业合作社 | A kind of crop seeds seedling culture medium |
| CN107493728A (en) * | 2017-09-12 | 2017-12-22 | 中国科学院华南植物园 | A kind of method for Helen's pocket orchid seed germination rate that cryopreservation is improved using ultrasonication |
| CN108157166A (en) * | 2018-01-24 | 2018-06-15 | 董春燕 | A kind of breed of variety of pocket Lanzhou and Xinjiang and tissue culture and rapid propagation method |
| CN110881478A (en) * | 2019-11-07 | 2020-03-17 | 中国林业科学研究院林业研究所 | Method for promoting germination of seeds of denatolum and paphiopedilum by using ceriferous fungi |
| CN110809935A (en) * | 2019-11-08 | 2020-02-21 | 广东省农业科学院果树研究所 | Method for improving germination rate of banana seeds |
| CN114507361A (en) * | 2022-02-28 | 2022-05-17 | 新疆农业大学 | Agar activated carbon hydrogel for soilless culture seeds and preparation method thereof |
| CN114507361B (en) * | 2022-02-28 | 2024-01-19 | 新疆农业大学 | Agar activated carbon hydrogel for soilless culture seeds and preparation method thereof |
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