CN104186295B - Pocket orchid seed germination medium and cultural method - Google Patents
Pocket orchid seed germination medium and cultural method Download PDFInfo
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Abstract
The invention discloses a kind of pocket orchid seed germination medium and cultural method.Pocket orchid seed germination medium culture medium based on 1/4MS culture mediums of the present invention, and ascorbic acid, activated carbon, banana puree, gibberellin, agar powder and white granulated sugar is added with, the sprout time of pocket orchid seed can be shortened, seed germination rate is improved.
Description
Technical field
The present invention relates to Orchid Tissue culture technique field, more particularly to a kind of pocket orchid seed germination medium and culture
Method.
Background technology
Pocket is blue, also known as " slippers are blue ", " celestial shoe is blue ", is the general designation of orchid family Paphiopedilum (Paphiopedilum) plant.Pocket is blue
A most characteristic monoid in orchid, and most peculiar view and admire orchid, with its unique charm and many excellent spies
Property and enjoy doting on for world flower fan.
In recent years, the tissue culture technique of orchid and test tube seedling factorial praluction achieve swift and violent development, but as blue
The more original Paphiopedilum of section, its seed do not have endosperm, there are problems that germination rate is extremely low, sprout time.Existing pocket is blue to plant
Sub- germination medium generally on the basis of 1/4MS culture mediums, add the coconut milk of mass concentration 10%, the 6-BA of 0.5mg/L,
The activated carbon of the agar powder of NAA, 6g/L of 0.5mg/L, the sucrose of 30g/L and 2g/L.However, 6-BA and NAA is planted for broad spectrum activity
Thing growth regulator, is mainly used in taking root, sprouts and induce protocorm, poor to breaking seed dormancy effect.Therefore, using existing
Some pocket orchid seeds are sprouted culture and are cultivated, it is difficult to realize the blue quick breeding of pocket, it is difficult to meet the demand of large-scale planting.
Content of the invention
It is based on this, it is necessary to for the low problem of pocket orchid seed germination rate, there is provided one kind can improve pocket orchid seed germination rate
Culture medium.
Additionally, it is necessary to be directed to the pocket orchid low problem of seed germination rate, there is provided a kind of pocket orchid seed germination rate that can improve
Cultural method.
A kind of pocket orchid seed germination medium, its culture medium based on 1/4MS culture mediums, and be added with ascorbic acid,
Gibberellin, activated carbon, banana puree, agar powder and white granulated sugar.
Wherein in one embodiment, the concentration of ascorbic acid is 80～100mg/L, the concentration of gibberellin is 40～
60mg/L, the concentration of activated carbon is 1.8～2g/L, and the concentration of banana puree is 40～50g/L, and the concentration of agar powder is 6～7g/L,
The concentration of white granulated sugar is 25～30g/L.
Wherein in one embodiment, the concentration of ascorbic acid is 100mg/L, and the concentration of gibberellin is 50mg/L, active
The concentration of charcoal 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.
Wherein in one embodiment, the pH value of the pocket orchid seed germination medium is 6.0.
A kind of pocket orchid seed sprouts cultural method, comprises the following steps：
1) healthy growth, the pollination Fruit pod of 140～150 days are taken, with alcohol-pickled 25 that mass concentration is 70～75%～
30 seconds, then sterilized 18～25 minutes with the mercuric chloride solution that mass concentration is 0.10～0.12%, then clean with sterile water wash
Afterwards, Fruit pod is cut, takes out seed；
2) seed concentration is the KOH solution immersion treatment 8～10 minutes of 0.05～0.1mol/L, dry with sterile water wash
After net, reuse frequency is 50～60KHz, ultrasonication that power is 80～90W 25～30 minutes, is then seeded into the present invention
In described pocket orchid seed germination medium, cultivate under the conditions of light culture and sprout to seed, then in light dark period be 12h light
According to/12h is dark, intensity of illumination be to cultivate under conditions of 1800～2000lux to growing up to seedling.
Wherein in one embodiment, step 2) in, sterilizing filter paper is equipped with described pocket orchid seed germination medium,
Described sterilizing filter paper is through pocket of the present invention orchid seed germination medium immersion treatment.
In the pocket orchid seed germination medium of the present invention, using 1/4MS culture mediums, dense by the ion for reducing a great number of elements
Degree, is conducive to pocket orchid seed to sprout, improves germination rate；Additionally, by ascorbic acid, gibberellin, activated carbon and banana puree joint
Seed is acted on, seed sprouting can be effectively facilitated, seed germination rate is improved.Wherein, ascorbic acid is antioxidant, to seed without
Toxic and side effect, can be had an effect with the oxidation product quinones substance produced by browning so as to be reduced to aldehydes matter again,
So as to reduce browning degree, seed germination rate is improved；Gibberellin can adjust the physiological status of seed, by the transcriptive intermediate factor
The alpha-amylase gene in seed is affected, is promoted the formation of AMS mRNA and the synthesis of AMS, is improved AMS
Level, so as to promote α-amylasehydrolysis starch, increase the carbon source needed for seed is sprouted, break the dormancy of seed, promote kind
Son is sprouted, and significantly shortens sprout time；Activated carbon can phenols that effectively absorbed seed is produced, quinones substance, Browning control carries
High seed germination rate；Banana puree sprouts desired substance rich in multiple seeds such as amino acid, hormone and enzyme, can make up pocket orchid seed and not have
Albuminosus defect, provides nutriment for which, so as to improve germination rate, and makes protocorm robust growth；Agar is used as solid
Cultivation platform, provides nutrient, moisture and ventilative effectiveness for plant material, and using white granulated sugar as carbon source, in sprout time and sprouting
On the premise of rate is unaffected, the preparation cost of culture medium is reduced.
In the pocket orchid seed germination medium of the present invention, the concentration of ascorbic acid is preferably 80～100mg/L, if its consumption
Very few, then antioxidation is not obvious；The concentration of gibberellin is preferably 40～60mg/L, if its consumption is very few, it is difficult to promote to plant
Son is sprouted, if its consumption is excessive, explant can be caused to overgrow, and vitrifying is serious；The concentration of activated carbon is preferably 1.8～2g/
L, if its consumption is very few, it is impossible to which abundant absorbing phenolic and quinones substance, browning inhibition are poor, if its consumption is excessive, except
Outside absorbing phenolic and quinones substance, can also adsorb the nutriment in culture medium, affect seed to sprout and explant bulk-growth；Banana
The concentration of mud is preferably 40～50g/L, if its consumption is very few, is difficult to meet the nutrition needed for pocket orchid seed is sprouted.
The pocket orchid seed germination medium of the present invention, pH value is controlled 6.0, in weak acid environment, is conducive to the blue kind of pocket
The sprouting of son, so as to improve germination rate, shortens sprout time.
Pocket orchid seed germination medium of the present invention, by biochemical apparatus, adds ascorbic acid, resistance in the medium
Only aldehydes matter is oxidized to the quinones substance of brown；By physiology means, add gibberellin and banana puree in the medium, adjust
Seed physiology situation, breaks seed dormancy, promotes seed to sprout；And by physical means, add activated carbon in the medium, inhale
Phenols, quinones substance is received, tissue browning is reduced, so as to improve seed germination rate.The pocket orchid seed germination medium of the present invention, leads to
The synergy in terms of biochemical, physiology and physics three is crossed, the germination rate of pocket orchid seed can be significantly improved.
Pocket orchid seed of the present invention sprouts cultural method, employs the pocket orchid seed germination medium of the present invention, then
By chemistry, physiology etc. many and meanwhile effect, mutually promote, can significantly improve pocket orchid seed germination rate, seed sprout
Rate is up to 60%, shortens pocket orchid seed sprout time, and reduces explant browning rate.Seed is carried out after aseptic seed is obtained
KOH solution immersion and ultrasonication, to break seed dormancy, promote seed to sprout.Wherein, chemistry aspect, using KOH solution
Immersion, the material of energy dissolution inhibition seed sprouting, a corrosion kind skin make seed be easier sprouting, and raising germination rate is molten using KOH
Liquid rather than conventional NaOH solution, because the water imbibition of KOH is lower than NaOH, can preferably keep the moisture of seed；And physiology side
Face, using ultrasonication, then can make the activity increase of enzyme, abolish the dormancy of seed.Seed is soaked through KOH solution and ultrasonic wave
After process, being inoculated in pocket orchid seed germination medium of the present invention carries out light culture, can adjust seed physiology activity,
Break seed dormancy, promote seed to sprout.It is transferred to after germination in time under alternation of light and darkness environment and is cultivated, avoids original
Bulb growth is expanded, color bleaches, and can not turn green seedling.Additionally, sterilization treatment is carried out to Fruit pod using alcohol and mercury chloride,
Bacterium and fungi can be killed simultaneously, reach optimal sterilization effect.
The pocket orchid seed of the present invention is sprouted in cultural method, when making immersion treatment using KOH solution to seed, KOH solution
Concentration be preferably 0.05～0.1mol/L, soak time be preferably 8～10 minutes, if KOH solution excessive concentration or immersion when
Between long, pocket orchid seed can be damaged or even be allowed to inactivate, if KOH solution concentration is too low or soak time is too short, promote
Enter the DeGrain of seed sprouting；When carrying out ultrasonication to seed, frequency is preferably 50～60KHz, and power is preferably
80～90W, process time are preferably 25～30 minutes, if the frequency of ultrasonication, power are too high or process time is long, meeting
Cause seed physiology disorderly, destroy seed physiology structure, affect seed to sprout and even cause FCR de-vitalizing seed, if ultrasonication
Frequency, power are too low or process time is too short, then promote the DeGrain that seed is sprouted；Seed light culture is sprouted to seed,
If light culture overlong time, explant yellow can be caused, grow abnormal and explant vitrifying, if the light culture time is too short,
The DeGrain for then promoting seed to sprout.
Additionally, by laying the sterilizing filter paper that soaks through culture medium on seed germination medium, being prevented from seed and falling into
Enter in culture medium and suffocate and reduce seed germination rate.
Specific embodiment
Embodiment one：The preparation of pocket orchid seed germination medium of the present invention
Pocket orchid seed germination medium formula shown in 1/4MS culture medium prescriptions and table 2 according to table 1, prepares
Pocket orchid seed germination medium of the present invention.
By the amount for preparing 1100mL culture mediums, each composition and activated carbon, banana in 1/4MS culture mediums is weighed respectively
Mud, agar powder, white granulated sugar, are placed in triangular flask, add appropriate purified water, stirring and dissolving to be settled to 1000mL, and adjust pH value
To 6.0.Triangle bottle closure is placed in high-pressure sterilizing pot, in 121 DEG C of 20min that sterilize, be subsequently placed in superclean bench natural
Cooling, obtains sterilising medium mother liquor, standby.
By the amount for preparing 1100mL culture mediums, ascorbic acid and gibberellin is weighed respectively, add appropriate purifying water dissolves, mix
Even, 100mL is settled to, filtration sterilization in superclean bench is standby.
In superclean bench, after culture medium mother liquor subject to sterilization is cooled to 60 DEG C, the ascorbic acid and red after sterilizing is added
Mycin solution, mixes, and 1100mL pockets orchid seed germination medium is obtained.
Filter paper is taken, the size matched with tissue culture flasks is cut into, high-pressure sterilizing pot is placed in and is sterilized at 121 DEG C
20min, is put in superclean bench after sterilizing, and with obtained pocket orchid seed germination medium immersion, standby.
Obtained pocket orchid seed germination medium is taken, in dispensing to tissue culture flasks, is dried in the air cool, in its table after which solidifies
Face laying is through the aseptic filter paper after culture medium immersion, then by tissue cultures bottle closure, standby.
The composition and its consumption of 1 1/4MS culture mediums of table
Remarks：A great number of elements consumption in 1/4MS culture mediums for a great number of elements consumption of standard MS medium 1/4.
The composition and its consumption of 2 pocket of table orchid seed germination medium
Embodiment two：Pocket orchid seed of the present invention sprouts cultural method
Healthy growth, the pollination Fruit pod of 140 days is chosen, with alcohol-pickled 30 seconds that mass concentration is 75%, then quality is used
Concentration be 0.1% mercuric chloride solution sterilize 20 minutes, then with sterile water wash clean after, cut Fruit pod, take out seed, standby
With.
Tissue culture flasks prepared by Example one are standby, and pocket orchid seed of the tissue culture flasks equipped with solid-state sprouts culture
Base, the surface of culture medium are equipped with through the aseptic filter paper after culture medium immersion.
The aseptic seed obtained after disinfecting is taken, with the KOH solution immersion treatment that concentration is 0.1mol/L 10 minutes, is used
After sterile water wash is clean, it is the ultrasonication 30 minutes of 90W that reuse frequency is 60KHZ, power, is then seeded into embodiment
In one tissue culture flasks for preparing.Postvaccinal tissue culture flasks are placed in growth cabinet, are cultivated under the conditions of light culture
Sprout to seed within 12 days, the ambient parameter of growth cabinet is set to：26 ± 1 DEG C of temperature；Then in light dark period be 12h light
According to/12h is dark, intensity of illumination be 2000lux, temperature be to cultivate under conditions of 25 ± 1 DEG C to growing up to seedling.
Embodiment three：The test of pocket orchid seed germination medium of the present invention
Pocket orchid seed germination medium formula shown in 1/4MS culture medium prescriptions and table 3 according to table 1, reference
Culture medium preparation method described in embodiment one, prepares the pocket orchid seed germination medium of each test group, control group respectively.
3 pocket of table orchid seed germination medium
Healthy growth, the pollination Fruit pod of 140 days is chosen, with alcohol-pickled 30 seconds that mass concentration is 75%, then quality is used
Concentration be 0.1% mercuric chloride solution sterilize 20 minutes, then with sterile water wash clean after, cut Fruit pod, take out seed, in nothing
Under collarium border, be inoculated in the culture medium of each test group, control group shown in table 3 respectively, then in light dark period be 12h illumination/
12h is dark, intensity of illumination be 2000lux, temperature is to be cultivated under conditions of 25 ± 1 DEG C.Culture records seed after 30 days and sprouts
Rate, sprout time and melting brown rate is sent out, as a result as shown in table 4.
4 pocket of table orchid seed germination rate, sprout time, melting 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, using pocket of the present invention orchid seed germination medium, pocket can be significantly improved
Blue seed germination rate, shortens pocket orchid seed sprout time, and reduces its melting brown rate.By control group 2, control group 3 and test group 9
Comparative result is visible, there is collaboration facilitation, when both use simultaneously, further can carry between gibberellin and ascorbic acid
High pocket orchid seed germination rate, shortens seed sprout time.
Example IV：Pocket orchid seed of the present invention sprouts the test of cultural method
Healthy growth, the pollination Fruit pod of 140 days is chosen, with alcohol-pickled 30 seconds that mass concentration is 75%, then quality is used
Concentration be 0.1% mercuric chloride solution sterilize 20 minutes, then with sterile water wash clean after, cut Fruit pod, take out seed, standby
With.
Pocket orchid seed with reference to described in embodiment two sprouts cultural method, according to the processing method shown in table 5 and culture bar
Part, after the aseptic seed obtained after disinfecting is inoculated in pocket orchid seed germination medium, respectively to each test group, control group
Seed cultivated.Culture recorded seed germination rate and sprout time after 30 days, as a result as shown in table 5.
5 pocket of table orchid seed sprouts condition of culture and its result of the test
Remarks：1. "+" expression is processed；"-" is represented；
2. comprehensive grading y=a*0.7-b*0.3；
3. control group 4 and each test group are using the pocket orchid seed germination medium obtained by embodiment one, control group
5th, control group 6 using 1/4MS culture mediums and is added with the sucrose of the agar powder of 6g/L, 30g/L, and control group 7 is cultivated using 1/4MS
Base and be added with 10% coconut milk, the agar powder of NAA, 6g/L of 6-BA, 0.5mg/L of 0.5mg/L, the sucrose of 30g/L and
The activated carbon of 2g/L.
From the result of the test of table 5, on the basis of using pocket of the present invention orchid seed germination medium, then lead to
The interaction that light culture is processed after crossing KOH solution immersion treatment, ultrasonication and inoculation, and by laying through culture medium
The aseptic filter paper of immersion, further can significantly shorten the sprout time of pocket orchid seed, improve seed germination rate.Additionally, by right
Understand according to the result of the test for organizing 4,5,7, the culture effect of pocket orchid seed germination medium of the present invention, hence it is evident that better than basis training
Foster base and existing pocket orchid seed germination medium.The result of the test for having control group 5, control group 6 understands, consistent in culture medium
Under the conditions of, condition of culture is sprouted using pocket of the present invention orchid seed, pocket orchid seed germination rate can be also significantly improved, be shortened and plant
Sub- sprout time.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but simultaneously
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the guarantor of the present invention
Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (5)
1. a kind of pocket orchid seed germination medium, it is characterised in that：Culture medium based on 1/4MS culture mediums, and be added with anti-
Bad hematic acid, gibberellin, activated carbon, banana puree, agar powder and white granulated sugar；Wherein, the concentration of ascorbic acid is 80～100mg/L,
The concentration of gibberellin is 40～60mg/L, and the concentration of activated carbon is 1.8～2g/L, and the concentration of banana puree is 40～50g/L, agar
The concentration of powder is 6～7g/L, and the concentration of white granulated sugar is 25～30g/L.
2. pocket according to claim 1 orchid seed germination medium, it is characterised in that：The concentration of ascorbic acid is 100mg/
L, the concentration of gibberellin is 50mg/L, and the concentration of activated carbon is 2g/L, and the concentration of banana puree is 50g/L, and the concentration of agar powder is
6g/L, the concentration of white granulated sugar is 30g/L.
3. pocket according to claim 1 and 2 orchid seed germination medium, it is characterised in that：The pocket orchid seed sprouts training
The pH value of foster base is 6.0.
4. a kind of pocket orchid seed sprouts cultural method, comprises the following steps：
1) healthy growth, the pollination Fruit pod of 140～150 days are taken, with alcohol-pickled 25～30 seconds that mass concentration is 70～75%,
Sterilized 18～25 minutes with mercuric chloride solution that mass concentration is 0.10～0.12% again, then with sterile water wash clean after, cut
Fruit pod is opened, seed is taken out；
2) seed concentration is the KOH solution immersion treatment 8～10 minutes of 0.05～0.1mol/L, clean with sterile water wash
Afterwards, reuse frequency is 50～60KHz, ultrasonication that power is 80～90W 25～30 minutes, is then seeded into claim
Described in 1 pocket orchid seed germination medium in, cultivate under the conditions of light culture to seed sprout, then in light dark period be 12h
Illumination/12h is dark, intensity of illumination is to cultivate under conditions of 1800～2000lux to growing up to seedling.
5. pocket orchid seed according to claim 4 sprouts cultural method, it is characterised in that：Step 2) in, described pocket is blue
Sterilizing filter paper is equipped with seed germination medium, and described sterilizing filter paper sprouts training through the pocket orchid seed described in claim 1
Foster base 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 |
| CN110881478B (en) * | 2019-11-07 | 2021-04-13 | 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 |
| 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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Effective date of registration: 20191119 Address after: Room 607, block B, University Pioneer Park, No. 99, University Road, Tongshan District, Xuzhou hi tech Industrial Development Zone, Jiangsu Province Patentee after: Jiangsu Luzi Mali Food Co., Ltd Address before: 528313 Flower World Orchid Science Park, Chencun village, Shunde District, Foshan, Guangdong Patentee before: Foshan Shunde District Todayorchid Biological Technology Co., Ltd. |