CN114847159B - Tissue culture regeneration method for bulbus fritillariae cirrhosae - Google Patents
Tissue culture regeneration method for bulbus fritillariae cirrhosae Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a tissue culture and regeneration method of bulbus fritillariae cirrhosae. The tissue culture and regeneration method of the bulbus fritillariae cirrhosae comprises the following steps: the method comprises the following steps of S1 explant treatment, S2 embryogenic callus induction, S3 proliferation culture, S4 differentiation culture, S5 germination culture and S6 plant regeneration. The fritillaria cirrhosa bulb tissue culture regeneration method provided by the invention realizes mass proliferation in a short time by carrying out proliferation culture on fritillaria cirrhosa bulb calluses in a liquid environment, and improves the germination rate of somatic embryos by carrying out short-term low-temperature dark culture treatment on the somatic embryos in a germination culture link, thereby further improving the proliferation efficiency of plants.
Description
Technical Field
The invention relates to a plant tissue culture technology, in particular to a bulbus fritillariae cirrhosae bulb tissue culture regeneration method for quickly obtaining a large number of regeneration plants.
Background
Bulbus Fritillariae Cirrhosae (Fritillaria cirrhosa D. Don) is a perennial herb of the genus Fritillaria of the family Liliaceae. The medicine is prepared from the dry bulbs, is bitter in taste and cold in nature, has the effects of clearing heat, moistening lung, reducing phlegm, relieving cough, dissipating stagnation, eliminating carbuncle and the like, and is an important Chinese medicinal material for treating lung heat dry cough, dry cough with little phlegm, yin deficiency over-strained cough, bloody sputum and scrofula. The bulbus fritillariae cirrhosae is mainly distributed in places such as Sichuan, yunnan, tibet and the like, is mostly longer than wetland or rock cracks such as forests with the elevation of 2800-4700 meters, under bushes, grasslands, beaches, valleys and the like, is fond of cold and cool climatic conditions, and has the characteristics of cold resistance, moisture preference and shading preference. When the temperature is higher than 30 ℃ or the ground temperature is higher than 25 ℃, the plants will wither; therefore, it cannot survive in a region with a low altitude and a high temperature. The propagation modes of the fritillaria cirrhosa are mainly divided into seed propagation and bulb propagation. But due to the special habitat, the fritillaria cirrhosa seeds have long propagation dormancy period, low natural germination rate and poor seedling emergence rate in nature; when the artificial cultivation is carried out, the propagation coefficient of the bulbs is also lower; therefore, fritillaria cirrhosa is mainly harvested and dug wild resources all the time, but with the increasing demand of the market for fritillaria cirrhosa, the wild fritillaria cirrhosa is harvested excessively to cause resource shortage and is imminent to be extinct, and the wild fritillaria cirrhosa resources at present can not meet the market demand, so that how to actually and effectively solve the problem of fritillaria cirrhosa resource shortage becomes very urgent.
A large amount of unibract fritillary bulb regeneration plants can be obtained in a short time by utilizing the suspension culture technology, and the method is not limited by factors such as seasons, environment and the like. And the suspension culture also has the characteristics of less material consumption, high propagation efficiency and stable heredity, so that the industrial production of the bulbus fritillariae cirrhosae by selecting excellent single plants from wild bulbus fritillariae cirrhosae sources and then utilizing the suspension culture technology is one of the most effective methods for solving the problem of shortage of bulbus fritillariae cirrhosae resources. At present, although the research of obtaining the unibract fritillary bulb regeneration plant by using a suspension culture technology exists, the defects of long proliferation time, low somatic embryo germination rate and the like which influence the proliferation rate of the unibract fritillary bulb regeneration plant exist.
Disclosure of Invention
Based on the above, the present invention aims to provide a method for tissue culture and regeneration of fritillaria cirrhosa bulbs, which has the advantage of rapidly obtaining a large number of regenerated fritillaria cirrhosa plants.
A method for culturing and regenerating bulbus fritillariae cirrhosae bulb tissues comprises the following steps:
s1 explant treatment: selecting Bulbus Fritillariae Cirrhosae squama as explant, and sterilizing;
s2, embryogenic callus induction: inoculating the squamae treated by the S1 into an embryonic callus induction culture medium, and carrying out dark culture at 25 +/-2 ℃ until embryonic callus is formed;
s3, propagation culture: transferring the embryogenic callus obtained in S2 to a proliferation culture medium according to 40g/L, performing dark culture on a constant temperature shaking table at 25 ℃ for 120r/min, performing subculture for 1 time every 15 days, and performing continuous subculture for 4 times;
s4, differentiation culture: centrifuging the culture obtained in the step S3 to remove supernatant, transferring the remained cell mass into a differentiation culture medium, and carrying out dark culture on a constant-temperature shaking table at 25 ℃ for 120r/min, wherein the culture medium is replaced at least once during dark culture until somatic embryos with the diameter of 1-2 mm are differentiated;
s5, germination culture: inoculating the somatic embryo obtained in the step (S4) to a somatic embryo germination culture medium for germination, placing the somatic embryo at 10 ℃ for dark culture for 7-14 days, then transferring the somatic embryo to a culture condition with the temperature of 20-25 ℃, the photoperiod of 8/16h and the illumination intensity of 1000-1500 Lux for culture until the somatic embryo develops into milky-white bulblet with white and thick roots at the base;
s6, plant regeneration: separating the rooted bulblets obtained in the S5 from the callus, transferring the rooted bulblets to an SH basic culture medium, and continuously culturing until the bulblets are differentiated and sprout, wherein the fresh culture medium is replaced at least once in the period, the culture condition is 20-25 ℃, the photoperiod is 12/12h, and the illumination intensity is 2000Lux;
the embryogenic callus induction culture medium is calculated by per liter, and the preparation method comprises the following steps: adding 1-2 mg of 2,4-D, 0.5-1 mg of 6-BA, 0.5-2 mg of NAA and 30g of cane sugar into SH culture medium mother liquor, then using water to make volume constant to one liter, regulating pH value to 5.8, finally adding 2.5g of agar;
the proliferation culture medium is prepared by the following steps of: adding 1-2 mg of 2,4-D, 0.5-1 mg of NAA and 30g of cane sugar into SH culture medium mother liquor, then adding water to a constant volume of one liter, and adjusting the pH to 5.8;
the preparation method of the differentiation medium is as follows: adding 0.5-2 mg of 6-BA and 45g of cane sugar into SH culture medium mother liquor, then adding water to a constant volume of one liter, and adjusting the pH value to 5.8;
the germination culture medium is calculated by each liter, and the preparation method comprises the following steps: adding 0.5-2 mg of 6-BA and 30g of cane sugar into SH culture medium mother liquor, then adding water to a constant volume to one liter, adjusting the pH to 5.8, and then adding 0.5-2 g of active carbon and 2.5g of agar;
the SH basic culture medium is calculated by each liter, and the preparation method comprises the following steps: adding 30g of cane sugar into SH culture medium mother liquor, adding water to a constant volume to one liter, adjusting the pH value to 5.8, and adding 0.5-2 g of activated carbon and 2.5g of agar.
In the invention, the NAA is short for 1-naphthylacetic acid; the 6-BA is an abbreviation of 6-benzylaminopurine, and the 2,4-D is an abbreviation of 2,4-dichlorophenoxyacetic acid. In the invention, the SH culture medium is prepared from anhydrous calcium chloride (CaCl) 2) 151.02mg/L magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 400mg/L potassium nitrate (KNO) 3 ) 2500mg/L ammonium dihydrogen phosphate (NH) 4 H 2 PO 4 )300mg/L。
According to the bulbus fritillariae cirrhosae tissue culture regeneration method, massive proliferation is realized in a short time by performing proliferation culture on bulbus fritillariae cirrhosae callus in a liquid environment, and the germination rate of somatic embryos is improved by performing short-term low-temperature dark culture treatment on the somatic embryos in a germination culture link, so that the breeding rate of the bulbus fritillariae cirrhosae is further improved.
Further, in step S5 of the above method, the somatic embryo is cultured in the dark at 10 ℃ for 7 days. In this case, a better germination rate of the somatic embryo can be obtained.
Further, in step S3 of the above method, the embryogenic callus is a pale yellow, loose granular embryogenic callus selected from the embryogenic callus formed in S2.
Further, in step S3 of the above method, the culture was filtered with a 20-mesh stainless steel mesh every month, and the proliferation culture was continued with the cells having a diameter of less than 1mm being retained. In this case, cell lines having uniform size and good dispersibility can be obtained.
Further, in step S2 of the above method, the Bulbus Fritillariae Cirrhosae is cut into 0.5 cm-sized flakes 2 Inoculating the block into an embryogenic callus induction culture medium.
Further, in step S1 of the above method, the disinfection method is to wash the whole bulbs in running water to remove the rough skin and fibrous roots on the surface; then the scales are separated from inside to outside, and then dipped in the liquid detergent by a soft brush for rinsing, and finally the liquid detergent is rinsed under tap water. Putting the cleaned scales into a sterile bottle, transferring the sterile bottle into a super clean workbench, soaking the scales in 75% alcohol for 15-30 s, and then adding 0.1% HgCl 2 Soaking for 8-15 min, rinsing with sterile water for 5-8 times, and sucking surface water with sterile filter paper.
Further, in step S4 of the above method, the culture medium is replaced every 7 days; in step S6, the culture medium is replaced every 30 days.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a photograph of tissue culture regeneration of Bulbus Fritillariae Cirrhosae at various stages, wherein a is Bulbus Fritillariae Cirrhosae bulb; b is embryogenic callus induced from the explant; c is the liquid culture environment of the proliferation culture and differentiation culture stages; d is somatic embryo differentiated from embryonic cell suspension line; d is a regenerated bulblet with white and thick roots at the base part obtained by the germination of the somatic embryo; f is a differentiated, budding, regenerated bulblet.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A tissue culture regeneration method of Bulbus Fritillariae Cirrhosae bulb comprises the following steps:
s1 explant treatment: selecting fresh bulbus fritillariae cirrhosae bulbs with scales being closed tightly and without obvious wounds, mildewing and rotting on the outer surfaces as explants, firstly rinsing the whole bulbs under running water, and removing rough skins and fibrous roots on the surfaces; then the scales are separated from inside to outside, and then the soft brush is used for dipping the liquid detergent to wash the scales, and finally the liquid detergent is rinsed under tap water. Putting the cleaned scales into a sterile bottle, transferring the sterile bottle into a super clean workbench, soaking the scales in 75% alcohol for 15-30 s, and then adding 0.1% HgCl 2 Soaking for 8-15 min, rinsing with sterile water for 5-8 times, and sucking surface water with sterile filter paper.
S2, embryogenic callus induction: cutting the disinfected scale into 0.5cm pieces 2 Inoculating the left and right small blocks onto an embryogenic callus induction culture medium, culturing at 25 +/-2 ℃ in a dark environment, and beginning to form light yellow compact embryogenic callus after 30-45 days.
The embryogenic callus induction culture medium is calculated by per liter, and the preparation method comprises the following steps: 2mg of 2,4-D, 0.5mg of 6-BA, 0.5mg of NAA and 30g of sucrose are added to the SH medium mother liquor, then the volume is increased to one liter by water, the pH is adjusted to 5.8, and finally 2.5g of agar is added.
S3, propagation culture: and (3) selecting light yellow embryonic callus with loose structure and granular shape from the embryonic callus obtained in the step (S2), transferring the embryonic callus into a propagation culture medium according to the inoculation amount of 40g/L, putting the embryonic callus on a constant temperature shaking table at 25 ℃ for 120r/min, and performing propagation culture under the dark condition. The cells were subcultured 1 time every 15 days and 4 times consecutively to promote cell proliferation. In the culture process, original culture solution is removed by centrifugation at each subculture, and then equivalent fresh culture solution is supplemented; the culture was filtered with a 20 mesh stainless steel mesh every month, and cells having a diameter of less than 1mm were collected to continue proliferation culture.
The proliferation culture medium is prepared by the following steps of: adding 1mg of 2,4-D, 0.5mg of NAA and 30g of cane sugar into SH culture medium mother liquor, adding water to a constant volume of one liter, and adjusting the pH value to 5.8;
s4, differentiation culture: and (4) centrifuging the cell suspension obtained in the step (S3), removing the supernatant, transferring the remained cell clusters into a differentiation medium according to 1g/L, and performing embryoid induction culture on a constant-temperature shaking table at 25 ℃ for 120r/min under a dark condition. The culture medium is replaced every 7 days in the culture process, and the embryonic cell mass can be differentiated into somatic embryos with the diameter of 1-2 mm after about 35 days.
The preparation method of the differentiation culture medium comprises the following steps of: 1mg of 6-BA and 45g of sucrose are added into SH medium mother liquor, then the volume is increased to one liter by water, and the pH value is adjusted to 5.8.
S5, germination culture: and (4) transferring the somatic embryos with the diameter of 1-2 mm obtained in the step (S4) to a germination culture medium, wherein 20 somatic embryos are transferred into a dish, and 3 repeats are set. The somatic embryo is placed in a dark culture room at 10 ℃ for 7 days, then the culture room is turned to the culture temperature of 20 ℃, the photoperiod is 8/16h, the illumination intensity is 1000-1500 Lux, and the germination rate of the somatic embryo is counted after 20 days.
The germination culture medium is calculated by each liter, and the preparation method comprises the following steps: 1mg of 6-BA and 30g of sucrose are added into SH culture medium mother liquor, then the volume is increased to one liter by water, 0.75g of activated carbon and 2.5g of agar are added after the pH is adjusted to 5.8.
S6, plant regeneration: and (4) separating the regenerated bulblets obtained in the step (S5) from the callus, transferring the regenerated bulblets to an SH basic culture medium, and continuously culturing the regenerated bulblets on the SH basic culture medium under the conditions of 20 ℃, 12/12h of photoperiod and 2000Lux of illumination intensity until the regenerated bulblets form seedlings with white thick roots. During the culture period, the fresh medium was replaced every 30 days.
The SH basic culture medium is calculated by each liter, and the preparation method comprises the following steps: 30g of sucrose is added into SH culture medium mother liquor, then the volume is increased to one liter by water, 0.75g of activated carbon and 2.5g of agar are added after the pH is adjusted to 5.8.
Example 2
Step S5 of the first embodiment is changed into the step S4, somatic embryos with the diameter of 1-2 mm are transferred to a germination medium, 20 somatic embryos are placed in a dish, and 3 times of repetition are set. The culture medium is placed in a dark culture room at 10 ℃ for 14 days, and then is transferred to a culture room with the culture temperature of 20 ℃, the photoperiod of 8/16h and the illumination intensity of 1000-1500 Lux for culture. After 20 days, the germination rate of the somatic embryos is counted. The germination medium and the rest of the steps were the same as in example 1.
Comparative example
Step S5 of the first embodiment is changed to transfer the somatic embryos with the diameter of 1-2 mm obtained in step S4 to a germination medium, and 20 somatic embryos are placed in a dish for 3 times. And culturing in a culture room with the culture temperature of 20 ℃, the photoperiod of 8/16h and the illumination intensity of 1000-1500 Lux for 20 days, and then counting the germination rate of the somatic embryos. The germination medium and the remaining steps were as in example 1.
The germination rates of the somatic embryos of examples 1, 2 and comparative example 1 are shown in Table 1.
Wherein the germination rate of the somatic embryo (%) = (the number of the germinated somatic embryos/the total number of the inoculated somatic embryos) × 100, and the germinated somatic embryos are defined as somatic embryos which are expanded and developed into milky bulblets at the bases of the bulbs and have white root systems with smooth surfaces at the bases of the bulbs.
TABLE 1
| Experimental parameters | Comparative example | Example 1 | Example 2 |
| Low temperature dark culture treatment time (day) | 0 | 7 | 14 |
| Somatic embryo germination rate (%) | 35.0 | 75.0 | 65.0 |
Comparison of the comparative example and examples 1 and 2 in Table 1 shows that under otherwise identical conditions, embryos subjected to low-temperature dark culture for 7 days and 14 days, respectively, had higher germination rates than embryos not subjected to low-temperature dark culture. Wherein, when the low-temperature dark culture treatment time is 7 days, the germination rate of the somatic embryos is the highest and is 75.0 percent, which is more than twice of that of the comparative example. Therefore, the low-temperature dark culture treatment of 7-14 days on the somatic embryos can enable the somatic embryos to obtain higher germination rate, and the proliferation rate of the unibract fritillary bulb regeneration plants is improved. And when the low-temperature dark culture treatment time is 7 days, the highest germination rate of the somatic embryos can be obtained.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, to those skilled in the art, changes and modifications may be made without departing from the spirit of the present invention, and it is intended that the present invention encompass such changes and modifications.
Claims (7)
1. A method for culturing and regenerating bulbus fritillariae cirrhosae bulb tissue is characterized by comprising the following steps: the method comprises the following steps:
s1 explant treatment: selecting Bulbus Fritillariae Cirrhosae scales as explant, and sterilizing;
s2, embryogenic callus induction: inoculating the scales treated by the S1 into an embryonic callus induction culture medium, and performing dark culture at 25 +/-2 ℃ until embryonic callus is formed;
s3, propagation culture: transferring the embryogenic callus obtained in S2 to a proliferation culture medium according to 40g/L, performing dark culture on a constant temperature shaking table at 25 ℃ for 120r/min, performing subculture for 1 time every 15 days, and performing continuous subculture for 4 times;
s4, differentiation culture: centrifuging the culture obtained in the step S3 to remove supernatant, transferring the remained cell mass into a differentiation medium, and carrying out dark culture on a constant-temperature shaking table at 25 ℃ at 120r/min, wherein the medium is replaced at least once during dark culture until somatic embryos with the diameter of 1-2 mm are differentiated;
s5, germination culture: inoculating the somatic embryo obtained in the step S4 to a somatic embryo germination culture medium for germination, placing the somatic embryo at 10 ℃ for dark culture for 7-14 days, then transferring the somatic embryo to a culture condition with the temperature of 20-25 ℃, the photoperiod of 8/16h and the illumination intensity of 1000-1500 Lux for culture until the somatic embryo develops into milky-white bulblets with white and thick roots at the base;
s6, plant regeneration: separating the rooted bulblets obtained in the S5 from the callus, transferring the rooted bulblets to an SH basic culture medium, continuously culturing until the bulblets are differentiated and germinated, and replacing a fresh culture medium at least once in the period, wherein the culture condition is 20-25 ℃, the photoperiod is 12/12h, and the illumination intensity is 2000Lux;
the embryogenic callus induction culture medium is calculated by per liter, and the preparation method comprises the following steps: adding 1-2 mg of 2,4-D, 0.5-1 mg of 6-BA, 0.5-2 mg of NAA and 30g of cane sugar into SH culture medium mother liquor, then using water to make volume constant to one liter, regulating pH value to 5.8, finally adding 2.5g of agar;
the proliferation culture medium is calculated by each liter, and the preparation method comprises the following steps: adding 1-2 mg of 2,4-D, 0.5-1 mg of NAA and 30g of cane sugar into SH culture medium mother liquor, then adding water to a constant volume of one liter, and adjusting the pH to 5.8;
the preparation method of the differentiation culture medium comprises the following steps of: adding 0.5-2 mg of 6-BA and 45g of cane sugar into SH culture medium mother liquor, then adding water to a constant volume of one liter, and adjusting the pH value to 5.8;
the germination culture medium is calculated by each liter, and the preparation method comprises the following steps: adding 0.5-2 mg of 6-BA and 30g of cane sugar into SH culture medium mother liquor, then adding water to a constant volume to one liter, adjusting the pH to 5.8, and then adding 0.5-2 g of active carbon and 2.5g of agar;
the SH basic culture medium is calculated by each liter, and the preparation method comprises the following steps: adding 30g of sucrose into SH culture medium mother liquor, then adding water to a constant volume of one liter, adjusting the pH to 5.8, then adding 0.5-2 g of active carbon and 2.5g of agar, wherein the formula of the SH culture medium is anhydrous calcium chloride (CaCl) 2) 151.02mg/L magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 400mg/L, potassium nitrate (KNO) 3 ) 2500mg/L ammonium dihydrogen phosphate (NH) 4 H 2 PO 4 )300mg/L。
2. The tissue culture regeneration method of fritillaria cirrhosa bulbs according to claim 1, wherein the tissue culture regeneration method comprises the following steps: in step S5, the somatic embryo is placed at 10 ℃ for 7 days in dark culture.
3. The method for tissue culture regeneration of fritillaria cirrhosa bulbs according to claim 1, wherein the tissue culture regeneration method comprises the following steps: in step S3, the embryogenic callus is a pale yellow, loose granular embryogenic callus selected from the embryogenic callus formed in S2.
4. The method for tissue culture regeneration of fritillaria cirrhosa bulbs according to claim 3, wherein the method comprises the steps of: in step S3, the culture was filtered with a 20-mesh stainless steel mesh every month, and the proliferation culture was continued with the cells having a diameter of less than 1 mm.
5. The tissue culture regeneration method of fritillaria cirrhosa bulbs according to claim 1, wherein the tissue culture regeneration method comprises the following steps: in step S2Cutting Bulbus Fritillariae Cirrhosae into 0.5cm scale 2 Inoculating the block into an embryogenic callus induction culture medium.
6. The tissue culture regeneration method of fritillaria cirrhosa bulbs according to claim 1, wherein the tissue culture regeneration method comprises the following steps: in the step S1, the whole bulbs of the fritillaria cirrhosa are washed in running water to remove surface rough skin and fibrous roots; then separating the scales from inside to outside, dipping the scales in detergent by using a soft brush, rinsing the scales, and finally rinsing the detergent under tap water; putting the cleaned scales into a sterile bottle, transferring into a super clean bench, soaking in 75% alcohol for 15-30 s, and then 0.1% of HgCl 2 Soaking for 8-15 min, rinsing with sterile water for 5-8 times, and sucking surface water with sterile filter paper.
7. The method for tissue culture regeneration of fritillaria cirrhosa bulbs according to claim 1, wherein the tissue culture regeneration method comprises the following steps: in step S4, the culture medium is replaced every 7 days; in step S6, the culture medium is replaced every 30 days.
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