CN102217537A - Low-temperature conservation method of banana tissue culture plantlet provenances - Google Patents

Abstract

The invention discloses a low-temperature preservation method for the seed source of banana tissue culture seedlings. It is to inoculate the clustered shoots of bananas obtained through tissue culture into low-temperature preservation medium after cutting, and cultivate them under suitable conditions, and then cut the provenance of low-temperature preservation and inoculate them into recovery growth medium under suitable conditions. Proliferation and rapid propagation to obtain clustered buds, and the clustered shoots were rooted and cultured to obtain test-tube plantlets. The present invention adopts culture conditions such as unique culture medium, suitable culture temperature and culture light to carry out low-temperature preservation treatment on the cluster buds (provenance), so that the cluster buds are changed from the original subculture period of 30 to 40 days to low-temperature-preserved successors. The generation cycle is 3 to 4 months. Due to the prolongation of the subculture cycle, the preservation time of the clustered buds is prolonged. When test-tube seedlings are needed, the clustered shoots preserved at low temperature can be restored to growth through provenance, subcultured and multiplied to obtain clustered buds. A large number of test-tube seedlings can be obtained in a short period of time after the clustering buds are cultured by rooting, which provides an effective way for timely production of banana seedlings.

Description

A kind of cryopreservation method of seed source of banana tissue culture seedling

Technical field:

The invention relates to a method for plant tissue culture, in particular to a method for low-temperature preservation of the seed source of banana tissue culture seedlings.

Background technique:

my country is one of the main banana-producing countries and one of the countries with the longest history of cultivating bananas in the world. Our country is rich in varieties of bananas and has made great contributions to the world's banana industry. my country has a large population, and the consumption of bananas is huge, but at present, the average annual consumption of bananas per person is less than 4kg. With the improvement of transportation, banana fresh-keeping technology and the improvement of people's living standards, the demand for bananas will still increase greatly. The scale of production will inevitably continue to expand. In order to improve the competitiveness of my country's banana industry, it is urgent and necessary to develop its related high-tech biotechnology and to promote and apply it.

The successful promotion of banana tissue culture seedlings and the application of annual cultivation technology have made banana planting more profitable than rice and other crops, achieved obvious economic and social benefits, and achieved the purpose of high yield, high quality and high efficiency of bananas. my country's banana production has basically realized the improved variety of planting varieties and the industrialization of seedling production. At present, the coverage rate of good varieties of banana planting in China has reached more than 90%. Through the banana group cultivation technology, a large number of high-quality, virus-free and neatly grown banana test-tube seedlings have been cultivated to replace the traditional sucking buds, thereby greatly improving the quality of bananas in my country. production level. Further planned introduction of foreign famous new varieties with good quality, high yield and strong stress resistance, and large-scale production of seedlings through tissue culture to achieve regional and standardized production can promote the rapid development of my country's banana industry.

However, there are some problems in the production of banana tissue culture seedlings in our country, such as the identification and control of mutant strains, the preservation of tissue culture seedling source, the control of production cost, the stability of tissue culture seedling quality and so on. In particular, there are very few reports on the preservation of the seed source of tissue culture seedlings. Due to the seasonality and instability of the demand for banana seedlings, sometimes there are many seed sources, and it will cause a lot of waste when there is no need for seedlings on the market, and when there is a sudden need for a large number of seedlings in the market, there are not enough seed sources available Therefore, the preservation of the source of tissue culture seedlings sometimes plays a key role in the large-scale production of bananas. At present, there is no report on the preservation of the provenance of banana tissue cultured seedlings at home and abroad, and there is no patent application for the preservation of the provenance of banana tissue cultured seedlings. The invention adopts unique culture medium, suitable culture conditions such as culture temperature and culture light to effectively preserve the seed source of banana tissue culture seedlings.

The banana materials used in this experiment include Emperor Banana, Brazil Banana, Fen Banana and so on. Emperor banana (Musa paradisiaca AA), native to Indonesia, is a unique edible diploid (AA type) banana. It is widely cultivated in Southeast Asia. In ancient times, Thailand and Vietnam paid tribute to the emperor of our country, and it was also called tribute banana. In Myanmar, it is also called sweet banana because of its high sweetness. Emperor Banana fruit is small, about 10 cm long, seedless, thin skin, golden-yellow skin after ripening, bright color, beautiful appearance, orange-yellow flesh, sweet and fragrant, sweet and delicious, it is very popular in my country as soon as it is launched. Emperor banana fruit is rich in nutrition. It is reported that every 100 grams of edible part contains 20 grams of carbohydrates, 1.2 grams of protein, 0.6 grams of fat, and also contains multiple vitamins. It is one of the world's recognized high-grade banana varieties. Brazilian banana (Musa AAA Giant Cavendish cv. Baxi) is currently the largest banana variety in my country, with a genotype of AAA, high yield and good fruit quality. The pink banana (Musa ABB) is a hybrid cultivar of the genus Musa, also known as Saigon banana, Annan banana, and milk banana. Its genotype is ABB, with strong wind resistance, cold resistance, and drought resistance. Thin, milky white or light orange pulp, tender and sweet taste, slightly fragrant, high edible rate, the planting area in my country has expanded rapidly in recent years.

Invention content:

The purpose of the present invention is to provide a kind of preservation cost is low, simple to operate, good preservation effect, can effectively preserve the method for the seed source of banana tissue culture seedling, utilize this method to be able to preserve earlier the banana tissue culture seed source, When large-scale growth of banana seedlings is required, a large number of test-tube seedlings can be obtained in a short period of time after the seedlings of banana tissue culture seedlings preserved in low temperature are restored to growth, subcultured and rooted. Timely production of seedlings provides an effective way.

The cryopreservation method of banana tissue culture seedling seed source of the present invention comprises the following steps:

(1) Low-temperature preservation of the provenance: cut the clustered buds obtained from bananas through tissue culture and inoculate them into the low-temperature preservation medium. The cultivation temperature is 14-18° C., the cultivation light is 500-800 lx, and the light time is 6-10 hours/ The cryopreservation medium contains 0.1-0.5 mg of 6-benzylaminoadenine, 60-90 mg of diethylhydrazide, 10-20 g of sucrose, 6-7 g of agar, and 1/2 MS per liter of medium. The pH is 5.5-5.8, thereby preserving the provenance of banana tissue culture seedlings; the subculture period is 3-4 months, sometimes a small amount of roots will be produced during the preservation process, and the multiplication coefficient of each cycle is 1.8-2.2.

(2) Restoration of growth of provenance: cut the seedlings of tissue culture seedlings preserved in low temperature and inoculate them into the recovery growth medium. The seedlings can recover to normal growth in 8 to 10 days, and the seedlings of normal growth will resume in 30 to 35 days. The source is inoculated into the new recovery growth medium for rapid multiplication to obtain clustered buds, and the multiplication factor can reach 2.5-3 times. Culture conditions: culture temperature 25-30 ℃, light intensity 1500-2000lx, light time 12-16 hours/day , the recovery growth medium contains 3-5 mg of 6-benzylaminoadenine, 0.2-0.5 mg of indolebutyric acid, 20-30 g of sucrose, 6-7 g of agar, and the balance of MS per liter of medium, pH 5.5 ～5.8; The test-tube seedlings were obtained by conventional rooting culture of clustered shoots.

The test-tube seedlings are routinely transplanted and hardened to obtain cultivated seedlings, which can be used for production and cultivation.

In the described step (1), the clustered buds obtained by bananas through tissue culture are preferably obtained by the following method, the selection and cultivation of explants: choose the 40～50cm high sucking buds of healthy and disease-free banana plants As an explant, wash it with water, cut off the upper outer pseudostem, leave a 4-8cm high stem and the lower outer pseudostem, and peel off the leaf sheath wrapped around the stem layer by layer under sterile conditions, each layer Wipe once with an alcohol solution with a volume fraction of 70-75%. When the explant is 2cm×2cm×2cm, soak it in the alcohol solution with a volume fraction of 70-75% for 10-15 seconds, and then use the mass volume percentage Concentration of 0.05% ~ 0.2% mercury solution disinfection 1 ~ 2 times, 3 ~ 6 minutes each time, rinse with sterile water 4 ~ 5 times, cut off the brown part of the base, and then peel the explants to the growth point, Inoculate it into the axillary bud initiation medium and cultivate it. When the axillary bud grows, it will be cut off and proliferated on the new axillary bud initiation medium;

Culture conditions: culture temperature 25-30°C, illuminance 1500-2000lx, light time 12-16 hours/day, the axillary bud initiation medium contains 4-6 mg of 6-benzylaminoadenine and indoledin per liter of medium Acid 0.1 ~ 0.5mg, sucrose 20 ~ 30g, agar 6 ~ 7g, the balance is MS, pH 5.5 ~ 5.8.

The subculture cycle of cluster bud proliferation is 30 to 40 days, more preferably when the 3rd to 4th generations are multiplied, each explant has 20 to 30 buds, and 2 to 4 of them are sampled for virus immunity of "Panama" disease Biochemical testing to test pathogen-free strains for provenance preservation and subsequent growth.

The preferred step of rooting culture described in the step (2) is: after carrying out multiplication and rapid propagation in the new recovery growth medium to obtain clustered buds and cutting them into single buds, then transfer to the rooting medium for rooting culture, and the culture temperature is 25-25℃. 30°C, illuminance 1500-2000lx, light time 12-16 hours/day, until it grows into test-tube plantlets, the rooting medium contains 1.0-2.0mg of indolebutyric acid and 0.2-0.8mg of naphthaleneacetic acid per liter of medium , 20-30g of sucrose, 6-7g of agar, the balance is MS, pH 5.5-5.8. After rooting culture, the rooting rate of buds can reach 100%.

Described MS is the culture medium commonly used in the world, and its composition and preparation method refer to literature (Edited by Tan Wencheng and Dai Cegang. Ornamental Plant Tissue Culture Technology. Beijing: China Forestry Press, 1991); Described 1/2MS is the A culture medium in which the concentration of macroelements is halved while the concentrations of other components remain unchanged.

The present invention adopts culture conditions such as unique culture medium, suitable culture temperature and culture light to carry out low-temperature preservation treatment on the cluster buds (provenance), so that the cluster buds are changed from the original subculture period of 30 to 40 days to low-temperature-preserved successors. The generation cycle is 3 to 4 months. Due to the prolongation of the subculture cycle, the preservation time of the clustered buds is prolonged. When test-tube seedlings are needed, the clustered shoots preserved at low temperature can be restored to growth through provenance, subcultured and multiplied to obtain clustered buds. A large number of test-tube seedlings can be obtained in a short period of time after the clustering buds are cultured by rooting, which provides an effective way for timely production of banana seedlings.

The invention has the advantages of low preservation cost, simple operation and good preservation effect. When large-scale growth of seedlings is required, the provenance preserved at low temperature can be restored to growth and subcultured to obtain clustered buds in a short period of time. After rooting and culturing, a large number of test-tube seedlings can be obtained in a short period of time, which provides an effective way for timely production of banana seedlings.

Detailed ways:

The following is a further description of the present invention, rather than a limitation of the present invention.

Embodiment 1: Low temperature preservation of the seed source of the emperor banana tissue culture seedling

(1) Selection and cultivation of explants:

In the emperor banana planting base without disease, select the 40-50cm high suction buds of healthy and disease-free plants in the growing season as explants, after washing the surface soil under tap water, remove the upper outer pseudostem, Leave the stem 4-8cm high and the lower outer pseudostem. Under the aseptic condition of the ultra-clean workbench, the leaf sheath wrapped around the stem was peeled off layer by layer, and each peeled layer was wiped once with a 70% alcohol solution by volume fraction. When the explant was 2cm×2cm×2cm in size, Soak in 70% alcohol aqueous solution for 10 seconds, then sterilize twice with mercuric chloride solution with a concentration of 0.05% by mass volume, 6 minutes each time, rinse with sterile water 4 times, cut off the browned part of the base, Then the explants are stripped to a growth point of about 0.5 cm high, inoculated into the axillary bud initiation medium and cultivated. The axillary bud initiation medium contains 4 mg of 6-benzylaminoadenine (6-BA) and indoletin per liter of medium. Acid (IBA) 0.1mg, sucrose 20g, agar 6g, the rest is MS, pH 5.5, culture temperature 25°C, light intensity 1500lx, light time 12 hours/day, when the axillary bud grows, cut it off and start the new axillary bud Proliferate clustered buds on the culture medium, the culture temperature is 25°C, the light intensity is 1500lx, and the light time is 12 hours/day. The subculture cycle is 40 days. When the 4th generation is multiplied, each explant has 20 buds, and 2 of them are taken for virus immunological detection of "Panama" disease. Only the strains without pathogenic bacteria can be used for provenance preservation and subsequent production.

(2) Low temperature preservation of provenance:

The clustered buds (provenance) obtained by the subculture in the previous step were inoculated into the cryopreservation medium after being cut and inoculated into the cryopreservation medium, which contained 6-benzylamino gland per liter of medium Purine (6-BA) 0.1mg, butyrhydrazide (bijiu, B9) 60mg, sucrose 10g, agar 6g, balance 1/2MS, pH 5.5, culture temperature 14°C, culture light 500lx, light time 10 hours/day, and the subculture cycle is 4 months. During the preservation process, sometimes a small amount of roots are produced, and the multiplication coefficient per cycle is 1.8, thereby preserving the provenance of the tissue culture seedlings.

(3) Restoration of provenance:

Inoculate in recovery growth medium after the tissue culture plant seed source cutting of cryopreservation in last step, this recovery growth medium is that every liter contains 6-benzylaminoadenine (6-BA) 3mg, indolebutyric acid ( IBA) 0.2mg, sucrose 20g, agar 6g, the rest is MS, pH 5.5, culture temperature 25°C, illuminance 1500lx, light time 16 hours/day, normal growth can be restored in 10 days, and then transferred to a new recovery growth culture in 35 days Proliferation and rapid propagation on the base, the multiplication factor can reach 2.5 times.

(4) rooting culture:

The clustered buds obtained by the previous step multiplication are cut into single buds and then transferred to the rooting medium, which contains 1.0 mg of indolebutyric acid (IBA) and 0.2 mg of naphthaleneacetic acid (NAA) per liter. Sucrose 20g, agar 6g, the rest is MS, pH 5.5, culture temperature 25°C, light intensity 1500lx, light time 16 hours/day, after rooting culture, the rooting rate can reach 100%, and then grow into test-tube plantlets.

Embodiment 2: the cryopreservation of the seed source of the banana tissue culture seedling

(1) Selection and cultivation of explants:

In the non-disease-free Brazilian banana planting base, select the 40-50cm high suction buds of healthy and disease-free plants as explants during the growing season. After washing the soil on the surface under tap water, cut off the upper outer pseudostem , leaving 4-8cm high stems and lower outer pseudostems. Under the aseptic condition of the ultra-clean workbench, the leaf sheath wrapped around the stem was peeled off layer by layer, and each peeled layer was wiped once with a 75% alcohol aqueous solution. When the explant was 2cm×2cm×2cm in size, Soak in 75% alcohol aqueous solution for 15 seconds, then sterilize once with 0.2% mercuric chloride solution by mass volume percentage, 3 minutes each time, rinse with sterile water 5 times, cut off the browned part of the base, and then The explants are stripped to a growth point of about 0.5 cm high, and inoculated into the axillary bud initiation medium, which contains 6-benzylaminoadenine (6-BA) 6 mg, indolebutyric acid (IBA) per liter of medium. ) 0.5mg, sucrose 30g, agar 7g, and the rest are MS and pH 5.8. When the axillary buds grow, they are cut off and proliferated on a new axillary bud initiation medium. The above-mentioned culture conditions are: culture temperature 30°C, The illuminance is 2000lx, and the light time is 16 hours/day. The subculture cycle is 30 days. When the multiplication reaches 3 generations, each explant has 30 buds, and 2 of them are taken for virus immunological detection of "Panama" disease. Only strains without pathogenic bacteria can be used for provenance preservation and Subsequent production.

(2) Low temperature preservation of provenance:

The clustered shoots of the strains obtained by subculture and tested to be free of pathogenic bacteria were cut as the provenance and inoculated into the cryopreservation medium, which contained 6-benzylaminoadenine (6-BA) per liter 0.5mg, butyrhydrazide (Bijiu, B9) 90mg, sucrose 20g, agar 7g, the rest is 1/2MS, pH 5.8, the culture temperature is 18°C, the culture light is 800lx, and the light time is 6 hours/day. The subculture cycle is 3 months, and sometimes a small amount of roots are produced during the preservation process, and the multiplication coefficient of each cycle is 2.2, thereby preserving the provenance of the tissue culture seedlings.

(3) Provenance recovery growth:

Inoculate in recovery growth medium after the tissue culture plant seed source of cryopreservation of last step is cut, and every liter of culture medium of this recovery growth medium contains 6-benzylaminoadenine (6-BA) 5mg, indolebutyric acid ( IBA) 0.5mg, sucrose 30g, agar 7g, the rest is MS, pH 5.8, culture temperature 30°C, illuminance 2000lx, light time 12 hours/day, normal growth can be resumed in 8 days, and then transferred to a new recovery growth culture in 30 days Proliferation and rapid propagation are carried out on the base, the culture temperature is 30°C, the light intensity is 2000lx, and the light time is 12 hours/day, and the multiplication factor can reach 3.0 times.

(4) rooting culture:

The clustered shoots obtained by the previous step multiplication were cut into single buds and then transferred to the rooting medium. The rooting medium contained 2.0 mg of indolebutyric acid (IBA) and 0.8 mg of naphthaleneacetic acid (NAA) per liter of medium. mg, sucrose 30g, agar 7g, the rest is MS, pH 5.8, culture temperature 30°C, light intensity 2000lx, light time 12 hours/day, after rooting culture, the rooting rate can reach 100%, and then grow into test-tube plantlets.

Embodiment 3: the cryopreservation of the seed source of the tissue cultured seedling of the banana

(1) Selection and cultivation of explants:

In the non-disease-free banana planting base, select the 40-50cm high suction buds of healthy and disease-free plants as explants during the growing season, rinse the surface soil under tap water, and cut off the upper outer pseudostem , leaving 4-8cm high stems and lower outer pseudostems. Peel off the leaf sheath covering the stem layer by layer under the sterile condition of the ultra-clean workbench, and wipe each layer with a volume fraction of 72% alcohol solution once, when the explant is 2cm×2cm×2cm in size , soaked in 72% alcohol aqueous solution for 12 seconds, then sterilized twice with mercuric chloride solution with a concentration of 0.1% by mass volume percent, 5 minutes each time, rinsed with sterile water 4 times, and cut off the browned part of the base , then strip the explants to a growth point of about 0.5 cm high, and inoculate them into the axillary bud initiation medium, which contains 6-benzylaminoadenine (6-BA) 5 mg, indole butyric acid (IBA) per liter. ) 0.3mg, sucrose 25g, agar 6.5g, the rest is MS, pH 5.6, culture temperature 28°C, light intensity 1800lx, light time 14 hours/day. When the axillary bud grows, it can be cut off and carried out clustered bud proliferation in a new axillary bud starting medium, 28° C. of culture temperature, 1800 lx of illumination, and 14 hours/day of illumination time. The subculture cycle is 35 days. When the 4th generation is multiplied, each explant has 25 buds, and 4 of them are taken for virus immunological detection of "Panama" disease. Only strains free of pathogenic bacteria can be used for provenance preservation and subsequent production.

(2) Low temperature preservation of provenance:

Cut the clustered buds of the strains obtained by subculture in the previous step and detect no pathogenic bacteria and inoculate them in the cryopreservation medium. The cryopreservation medium contains 6-benzylaminoadenine (6-BA) per liter. 0.2mg, butyrhydrazide (bijiu, B ₉ ) 80mg, sucrose 15g, agar 6.5g, the rest was 1/2MS, pH 5.6, the culture temperature was 16°C, the culture light was 700lx, and the light time was 8 hours/day. The generation cycle is 100 days. During the preservation process, sometimes a small amount of roots were produced, and the multiplication coefficient per cycle was 2.0, thereby preserving the provenance of the tissue culture seedlings.

(3) Provenance recovery growth:

Inoculate in recovery growth medium after cutting the seed source of the tissue culture plantlet that last step cryopreservation, every liter of this recovery growth medium contains 6-benzylaminoadenine (6-BA) 4mg, indole butyric acid (IBA) ) 0.4mg, sucrose 25g, agar 6.5g, the rest is MS, pH 5.6, culture temperature 28°C, illuminance 1800lx, light time 14 hours/day, normal growth can be resumed in 9 days, and then transferred to a new recovery growth culture in 35 days Proliferation and rapid propagation are carried out on the base, the culture temperature is 28°C, the light intensity is 1800lx, and the light time is 14 hours/day, and the multiplication factor can reach 2.8 times.

(4) rooting culture:

Cut the clustered shoots obtained from the multiplication in the previous step into single shoots and then transfer them to the rooting medium, which contains 1.5 mg of indole butyric acid (IBA), 0.4 mg of naphthaleneacetic acid (NAA), sucrose, and 25g, agar 6.5g, and the rest are MS, pH 5.6, culture temperature 28°C, illuminance 1800lx, light time 14 hours/day, after rooting culture, the rooting rate can reach 100%, and then grow into test-tube plantlets.

Claims (4)

1. the low temperature store method of a tissue culture seedlings of bananas provenance is characterized in that, may further comprise the steps:

(1) low temperature of provenance is preserved: preserve in the medium being inoculated into low temperature after the bud cutting of growing thickly of banana by the tissue culture acquisition, cultivation temperature is 14～18 ℃, cultivation illumination is 500～800lx, light application time 6～10 hours/day, it is that every liter of medium contains 6-benzyl aminoadenine 0.1～0.5mg, daminozide 60～90mg that described low temperature is preserved medium, sucrose 10～20g, agar 6～7g, surplus are 1/2MS, and pH 5.5～5.8, have preserved the provenance of tissue culture seedlings of bananas therefrom;

(2) recovery of provenance growth: be inoculated into after the tissue cultivating seedling provenance cutting with the low temperature preservation and recover in the growth medium, in the time of 30～35 days, the provenance that restore normal growth is inoculated into breeds fast numerous acquisition bud of growing thickly in the new recovery growth medium, condition of culture: 25～30 ℃ of cultivation temperature, illuminance 1500～2000lx, light application time 12～16 hours/day, described recovery growth medium be every liter of medium to contain 6-benzyl aminoadenine 3～5mg, indolebutyric acid 0.2～0.5mg, sucrose 20～30g, agar 6～7g, surplus be MS, pH 5.5～5.8; The bud of growing thickly obtains test-tube plantlet through conventional culture of rootage.

2. the low temperature store method of tissue culture seedlings of bananas provenance according to claim 1, it is characterized in that, in the described step (1) is to obtain by the following method banana by the bud of growing thickly that tissue culture obtains, the selection of explant and cultivation: choose robust growth, the high suction bud of 40～50cm of disease-free banana plant is as explant, wash, the outer false stem on excision top, stay false stem outside high stem of 4～8cm and the bottom, under the aseptic condition, successively peel off and wrap in the outer leaf sheath of stem, every stripping one deck is wiped 1 time with the alcohol water blend of volume fraction 70～75%, when explant is 2cm * 2cm * 2cm, be immersed in volume fraction and be in 70～75% the alcohol water blend 10～15 seconds, be 0.05%～0.2% mercuric chloride solution sterilization 1～2 time with the quality concentration expressed in percentage by volume again, each 3～6 minutes, aseptic water washing 4～5 times, cut the part of base portion browning, again explant is shelled to growing point, be inoculated in the axillalry bud startup medium and cultivate, when axillalry bud grows, its cutting-out is carried out adventitious buds proliferation on new axillalry bud startup medium;

Condition of culture: 25～30 ℃ of cultivation temperature, illuminance 1500～2000lx, light application time 12～16 hours/day, described axillalry bud start medium be every liter of medium to contain 6-benzyl aminoadenine 4～6mg, indolebutyric acid 0.1～0.5mg, sucrose 20～30g, agar 6～7g, surplus be MS, pH 5.5～5.8.

3. the low temperature store method of tissue culture seedlings of bananas provenance according to claim 2, it is characterized in that, describedly start at new axillalry bud that to carry out adventitious buds proliferation on the medium be when adventitious buds proliferation to the during 3～4 generations, each explant has 20～30 buds, take a sample 2～4 and carry out " Panama " sick virological immunology detection, detecting aseptic strain is to be used for provenance low temperature to preserve.

4. the low temperature store method of tissue culture seedlings of bananas provenance according to claim 1, it is characterized in that, culture of rootage step described in the step (2) is: will breed fast numerous acquisition and grow thickly after bud is cut into simple bud in new recovery growth medium, change culture of rootage in the root media again over to, 25～30 ℃ of cultivation temperature, illuminance 1500～2000lx, light application time 12～16 hours/day, until growing up to test-tube plantlet, described root media is that every liter of medium contains indolebutyric acid 1.0～2.0mg, methyl 0.2～0.8mg, sucrose 20～30g, agar 6～7g, surplus is MS, and pH 5.5～5.8.