CN111919748B - Culture medium for in-vitro induction and/or in-vitro preservation of germplasm of polygonatum cyrtonema potato blocks and application thereof - Google Patents
Culture medium for in-vitro induction and/or in-vitro preservation of germplasm of polygonatum cyrtonema potato blocks and application thereof Download PDFInfo
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Abstract
The invention provides a culture medium for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks and application thereof, belonging to the technical field of in vitro plant culture. The culture medium for in-vitro induction and/or in-vitro preservation of germplasm of polygonatum cyrtonema blocks provided by the invention takes an MS culture medium as a basic culture medium, and also comprises the following components in concentration: 0.2-0.8 mg/L of 5-nitroguaiacol sodium, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the culture medium is 5.6-6.0. The culture medium provided by the invention has three different purposes of tuber induction, rooting and in-vitro preservation, prolongs the single-generation preservation time of polygonatum cyrtonema germplasm resources, and reduces the cost of manpower and material resources caused by multiple preparation of culture medium and subculture inoculation; meanwhile, the method simplifies the operations of washing and planting the tissue culture seedlings, improves the survival rate, reduces the transplanting cost and solves the problem of high management difficulty of the conventional tissue culture rooted seedlings and the transplanted polygonatum cyrtonema.
Description
Technical Field
The invention belongs to the technical field of in-vitro plant culture, and particularly relates to a culture medium for in-vitro induction and/or in-vitro germ plasm preservation of polygonatum cyrtonema blocks and application thereof.
Background
Polygonatum cyrtonema (Polygonatum sibiricum Hua) is a perennial herb of Polygonatum of Liliaceae, and its rhizome contains starch, nicotinic acid, flavone, various amino acids, vitamins and various saponins, and has effects of invigorating qi, nourishing yin, strengthening tendons and bones, treating rheumatalgia, reducing blood lipid, lowering blood pressure, reducing blood glucose, resisting aging, and resisting tumor. With the development of the medicinal value of polygonatum cyrtonema, wild resources are artificially dug, so that the limited wild resources are gradually reduced, therefore, the production of polygonatum cyrtonema medicinal materials through artificial cultivation is necessary, and sufficient seedlings are an important basis for ensuring the success of artificial cultivation.
Polygonatum cyrtonema seeds can emerge only after two winter dormancy under natural conditions, and the problems of complicated propagation process, long seedling growing time, low seedling emergence rate, high seedling growing cost and the like exist in seedling growing by using the seeds. At present, the artificial planting of polygonatum cyrtonema is mainly based on tuber block breeding, so that the seed consumption is large, the breeding efficiency is low, the large-area popularization planting is seriously restricted, and meanwhile, because the tuber blocks are medicinal parts of the polygonatum cyrtonema, the breeding also influences the yield of polygonatum cyrtonema medicinal materials.
For over 10 years, research reports about polygonatum cyrtonema seedling tissue culture and rapid propagation are continuously reported, another good method and alternative way are provided for the high-efficiency and rapid propagation of polygonatum cyrtonema seedlings, although the polygonatum cyrtonema seedling tissue culture and rapid propagation technology has obvious advantages, a culture medium with multiple functions needs to be prepared to culture and store the polygonatum cyrtonema seedlings in the culture process of the tissue culture seedlings, the operation steps are complicated, and the culture and storage efficiency is greatly reduced; meanwhile, the tissue culture rooted seedlings are weak in leaves and tender plants, and the problems of high management difficulty in transplanting operation and after transplanting exist.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks and application thereof. The invention focuses on researching and improving the conventional rooting process in the polygonatum multiflorum tissue culture rapid propagation to obtain a three-in-one culture medium formula and culture conditions for tuber induction, rooting and germplasm in-vitro preservation, and has the advantages of simple operation, high tuber induction efficiency, long single-generation in-vitro preservation time and the like; and the potato blocks replace conventional rooted seedlings for transplanting, so that the method has the advantages of simple and extensive management after seedling emergence, seedling washing, planting and planting, high transplanting survival rate and the like.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a culture medium for in vitro culture and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks, which takes an MS culture medium as a basic culture medium and also comprises the following components in concentration: 0.2-0.8 mg/L of 5-nitroguaiacol sodium, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the culture medium is 5.6-6.0.
The invention provides an in vitro induction method of polygonatum cyrtonema potato blocks based on the culture medium of the technical scheme, which comprises the following steps:
inoculating the polygonatum cyrtonema cluster buds to the culture medium of the technical scheme, and carrying out low-light in-vitro culture to obtain polygonatum cyrtonema potato blocks with fibrous roots; the temperature of the low-light in-vitro culture is 23-33 ℃, the illumination time is 10-12h/d, and the illumination intensity is 5-15 mu mol.m-2·s-1Culturing for 30-50 days; the diameter of the polygonatum cyrtonema potato blocks with fibrous roots is 0.5-1.0 cm, and the number of the fibrous roots is 3-5.
Preferably, before the multiple yellow flower sperm cluster buds are inoculated, the cluster buds are cut and subjected to leaf removal treatment, and each treated cluster bud contains 1-2 buds.
Preferably, the method for obtaining the cluster buds comprises the following steps:
1) taking polygonatum cyrtonema potato blocks or seeds as explants, disinfecting, inoculating in a primary culture medium, and carrying out primary culture to form primary seedlings; when the explant is polygonatum cyrtonema potato blocks, culturing by illumination for 30-40 days; when the explant is a seed, culturing with weak light for 30-180 days;
2) inoculating the obtained primary seedlings into a subculture medium, and performing illumination culture to obtain subcultured cluster buds; the time of subculture is 25-35 d.
Preferably, the primary culture medium in step 1) takes an MS culture medium as a basal culture medium, and further comprises the following components in concentration: 6-BA 1.0-3.0 mg/L, IBA 0.1.1-0.3 mg/L, GA34.0-8.0 mg/L, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the primary culture medium is 5.6-6.0.
Preferably, the subculture medium in step 2) takes an MS medium as a basic medium, and further comprises the following components in concentration: 3.0-5.0 mg/L, IBA 0.3.3-0.5 mg/L of 6-BA, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the subculture medium is 5.6-6.0.
Preferably, the primary seedlings in the step 2) are cut and defoliated before being inoculated, and the treated primary seedlings contain 1-2 buds.
The invention provides a method for in-vitro preservation of polygonatum cyrtonema germplasm based on the culture medium of the technical scheme, which comprises the following steps: adopting the in-vitro induction method of polygonatum cyrtonema potato blocks in the technical scheme to obtain polygonatum cyrtonema potato blocks with fibrous roots; and continuously culturing the polygonatum cyrtonema potato blocks with fibrous roots in the culture medium of the technical scheme, and performing in-vitro preservation on polygonatum cyrtonema germplasm.
Preferably, the in-vitro preservation is carried out under the condition of weak light, the temperature of the in-vitro preservation is 23-33 ℃, the illumination time of the in-vitro preservation is 10-12h/d, and the illumination intensity of the weak light is 5-15 mu mol-m-2·s-1。
The invention provides the culture medium of the technical scheme, the polygonatum cyrtonema potato block in-vitro induction method of the technical scheme and the application of the polygonatum cyrtonema germplasm in-vitro preservation method of the technical scheme in the tissue culture and rapid propagation production of polygonatum cyrtonema seedlings.
The invention has the beneficial effects that:
the invention provides a culture medium for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks, which takes an MS culture medium as a basic culture medium and also comprises the following components in concentration: 0.2-0.8 mg/L of 5-nitroguaiacol sodium, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the culture medium is 5.6-6.0. The culture medium provided by the invention has three different functions of tuber induction, rooting and in-vitro preservation, the polygonatum multiflorum tissue culture intermediate is cultured under weak light, small tuber blocks with 3-5 fibrous roots can be induced and formed within 40 days, and because the buds on the small tuber blocks are in a semi-dormant state and the tuber blocks store rich water and nutrient substances, the single-generation preservation time of materials in tissue culture is greatly prolonged, the tuber blocks are favorable for replacing conventional rooted seedlings for transplanting, the transplanting operation difficulty is greatly reduced, and the management fineness after transplanting is greatly reduced.
Furthermore, the polygonatum cyrtonema tissue culture intermediate is preserved in vitro in a mode of inducing the small sweet potato blocks, the preservation time of a single generation is long (two years), the survival rate is high (73-100%), and the service life is more than 40 years; the service life of the tissue culture intermediate of polygonatum cyrtonema subjected to conventional subculture is only 2-3 years. In addition, in daily management, the polygonatum cyrtonema small potato blocks in an isolated preservation state are subcultured for 1 time every two years, namely, a culture medium is prepared for 1 time every two years and inoculated for 1 time; the tissue culture intermediate of polygonatum cyrtonema needs to be subcultured for at least 20 times within two years, namely, the culture medium is prepared for 20 times and inoculated for 20 times every two years. Therefore, the method can flexibly cope with the seedling market fluctuation while prolonging the effective service life of polygonatum cyrtonema germplasm resources and reducing the times of culture medium preparation and inoculation operation so as to reduce the germplasm preservation and culture cost, and is an important mode for storing and protecting polygonatum cyrtonema germplasm resources.
Furthermore, the invention provides a method for transplanting the polygonatum cyrtonema by inducing the polygonatum cyrtonema to generate small potato blocks through in vitro culture instead of traditional tissue culture rooted seedlings. The culture medium can directly induce the polygonatum cyrtonema subculture material to form small potato blocks with fibrous roots, and the small potato blocks can replace conventional tissue culture rooted seedlings to be transplanted, so that the polygonatum cyrtonema transplanting process is easier to operate, the production cost of seedlings is reduced, the transplanting survival rate is higher than that of the conventional rooted seedlings, and the transplanting survival rate can reach 100%; in addition, the small potato blocks store sufficient nutrition and moisture, have strong tolerance to temperature, moisture and illumination changes, have stronger stress tolerance and adaptability, are not limited in transplanting season, can be flexibly arranged in transplanting time, are more convenient and simpler to manage after transplanting, and obviously reduce the cost of transplanting labor and water and electricity. Fundamentally solves the problem that the conventional tissue culture rooting seedling transplanting operation and the management difficulty after transplanting are large.
Drawings
FIG. 1 is a schematic diagram of rhizoma Polygonati Odorati tuber induction, rooting and germplasm in vitro preservation, wherein A is a picture after just inoculation in tuber induction and/or germplasm in vitro preservation culture medium; b is a picture obtained by culturing for 30-50 days; c is a picture of A culture 760 d;
FIG. 2 example 1 is a subculture clump seedling obtained by culturing Polygonatum cyrtonema Hua potato blocks as explants;
FIG. 3 example 1 is a subculture clump seedling obtained by culturing Polygonatum cyrtonema seed as an explant;
FIG. 4 is the rhizome of Polygonatum cyrtonema with fibrous roots;
FIG. 5 example 1 Polygonatum cyrtonema germplasm material after two years of in vitro preservation;
FIG. 6 is a seedling grown by transplanting polygonatum cyrtonema rhizome blocks with fibrous roots into a seedbed 50d in example 1;
fig. 7 example 1 potato pieces formed by transplanting for 6 months and usable for field planting.
Detailed Description
The invention provides a culture medium for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks, which takes an MS culture medium as a basic culture medium and also comprises the following components in concentration: 0.2-0.8 mg/L of 5-nitroguaiacol sodium, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the culture medium is 5.6-6.0.
The method takes an MS culture medium as a basic culture medium, 0.2-0.8 mg/L of 5-nitroguaiacol sodium is added on the basis of the MS culture medium, and the concentration of the 5-nitroguaiacol sodium is further preferably 0.4 mg/L; the culture medium also comprises 25-35 g/L of sucrose, and the concentration of the sucrose is further preferably 30 g/L; the culture medium also comprises 3.3-3.7 g/L of agar, and the concentration of the agar is more preferably 3.5 g/L. The invention has no special requirements on the sources of all components in the culture medium, and can be prepared by adopting common commercial products.
The sodium 5-nitroguaiacol of the present invention has the effects of promoting root system development, promoting dry matter formation and transporting to storage organs such as tuberous roots, tubers or fruits. The invention realizes three different purposes of induction, rooting and in-vitro preservation of the potato tuber by using the 5-nitroguaiacol sodium under the same culture medium and the same culture condition, simplifies the preparation process of the culture medium, is simple and easy to operate, and reduces the cost of manpower and material resources.
The invention provides an in vitro induction method of polygonatum cyrtonema potato blocks based on the culture medium of the technical scheme, which comprises the following steps:
inoculating the polygonatum cyrtonema cluster buds to the culture medium in the technical scheme, and carrying out low-light in-vitro culture to obtain polygonatum cyrtonema potato blocks with fibrous roots; the temperature of the low-light in-vitro culture is 23-33 ℃, the illumination time is 10-12h/d, and the illumination intensity is 5-15 mu mol.m-2·s-1The culture time is 30-50 d; the diameter of the polygonatum cyrtonema potato blocks with fibrous roots is 0.5-1.0 cm, and the number of the fibrous roots is 3-5.
The method for obtaining the cluster buds preferably comprises the following steps:
1) taking polygonatum cyrtonema potato blocks or seeds as explants, disinfecting, inoculating in a primary culture medium, and carrying out primary culture to form primary seedlings; when the explant is polygonatum cyrtonema potato blocks, culturing by illumination for 30-40 days; when the explant is a seed, culturing with weak light for 30-180 d;
2) inoculating the obtained primary seedlings into a subculture medium, and performing illumination culture to obtain subcultured cluster buds; the time of subculture is 25-35 d.
The method comprises the steps of taking polygonatum cyrtonema blocks or seeds as explants, inoculating the sterilized polygonatum cyrtonema blocks or seeds into a primary culture medium, and carrying out primary culture to form primary seedlings.
In the present invention, the primary medium preferably uses an MS medium as a basic medium, and further preferably includes the following components in concentration: 6-BA 1.0-3.0 mg/L, IBA 0.1.1-0.3 mg/L, GA34.0-8.0 mg/L, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the primary culture medium is 5.6-6.0.
In the invention, when the initial culture is carried out by taking polygonatum cyrtonema potato blocks as explants, the illumination culture is carried out for 30-40 days; the preferable conditions of the illumination culture are that the temperature is 23-33 ℃, the illumination time is 10-12h/d, and the illumination intensity is 25-35 mu mol.m-2·s-1(ii) a More preferably, the temperature is 28 ℃, the light irradiation time is 11h/d, and the light irradiation intensity is 30 [ mu ] mol m-2·s-1。
In the present invention, the primary culture is performed with floretsWhen carrying out primary culture on polygonatum seeds serving as explants, carrying out weak light culture for 30-180 days; the preferable conditions of the low-light culture are that the temperature is 23-33 ℃, the illumination time is 10-12h/d, and the illumination intensity is 5-15 mu mol.m-2·s-1(ii) a More preferably, the temperature is 28 ℃, the light irradiation time is 11h/d, and the light irradiation intensity is 10 [ mu ] mol m-2·s-1。
After the primary seedlings are obtained, the primary seedlings are inoculated in a subculture medium and subjected to illumination culture to obtain subculture cluster buds.
In the present invention, the subculture medium preferably uses an MS medium as a basal medium, and further preferably includes the following components in concentrations: 3.0-5.0 mg/L, IBA 0.3.3-0.5 mg/L of 6-BA, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the subculture medium is 5.6-6.0. In the invention, before inoculation, the primary seedlings are preferably subjected to cutting and defoliation treatment, and the treated primary seedlings contain 1-2 buds.
In the invention, the temperature of the light irradiation culture is preferably 23-33 ℃, more preferably 28 ℃, the light irradiation time is preferably 10-12h/d, more preferably 11h/d, and the light irradiation intensity is preferably 25-35 mu mol.m-2·s-1More preferably 30. mu. mol. m-2·s-1。
The polygonatum cyrtonema clustered shoots are inoculated in the culture medium of the technical scheme and are subjected to low-light in-vitro culture to obtain polygonatum cyrtonema potato blocks with fibrous roots. In the invention, the polygonatum cyrtonema cluster buds are preferably subjected to cutting and defoliation treatment before inoculation, and the treated cluster buds preferably contain 1-2 buds. The method provided by the invention can be used for cutting cluster buds, so that the utilization rate of raw materials can be improved, and more polygonatum cyrtonema rhizome potato blocks with fibrous roots can be obtained and can be used for transplanting. The invention carries out defoliation treatment on cluster buds, reduces the consumption of the nutrition of the culture medium by leaves, ensures that the nutrient substances in the culture medium are intensively accumulated in the induced polygonatum multiflorum potato blocks without leaves and only with fibrous roots, and the buds on the small potato blocks formed by induction are in a semi-dormant state, thereby being convenient for long-time in-vitro preservation and subsequent field transplantation. Book (I)The temperature of the low-light isolated culture is 23-33 ℃, and the preferable temperature is 28 ℃; the illumination time of the low-light in-vitro culture is 10-12h/d, and the preferable illumination time is 11 h/d; the illumination intensity of the low-light in-vitro culture is 5-15 mu mol.m-2·s-1More preferably 10. mu. mol. m-2·s-1(ii) a The time of the weak light in-vitro culture is 30-50 d, and the preferable time is 40 d. The low-light culture condition is more beneficial to the induced growth of the potato blocks and the accumulation of nutrient substances such as starch in the potato blocks; in addition, the illumination cultivation in the normal plant tissue cultivation needs an illuminating lamp, the weak light cultivation does not need the illuminating lamp, and only scattered light of other illuminating light sources in the cultivation room is needed, so that electric energy is saved, and the cost is reduced.
After the low-light in-vitro culture, Polygonatum cyrtonema Hua tuber blocks with fibrous roots are obtained. In the invention, the diameter of the polygonatum cyrtonema potato blocks with fibrous roots is 0.5-1.0 cm; the number of the fibrous roots is 3-5.
After obtaining Polygonatum cyrtonema Hua potato blocks with fibrous roots, transplanting the Polygonatum cyrtonema Hua potato blocks with fibrous roots. The environment for transplanting is preferably a greenhouse, and the shading degree of the greenhouse is preferably 65-75%, and more preferably 70%; the planting density of polygonatum cyrtonema potato blocks is preferably 250-350 blocks/m2More preferably 300 blocks/m2。
The method for transplanting the polygonatum cyrtonema tuber blocks with fibrous roots can obviously improve the planting density which is 3 times of the transplanting density of the conventional leafy tissue culture rooted seedlings, the number of the produced seedlings in unit area is more, and the transplanting matrix and the greenhouse space are saved.
The method preferably broadcasts the polygonatum cyrtonema blocks with fibrous roots on a seedbed paved with a transplanting matrix, and then covers the transplanting matrix of 1.5-2.5 cm to keep the humidity of the seedbed.
In the invention, the transplanting matrix is preferably a mixed matrix of sand, yellow mud and humus soil, and the volume percentage of the components in the mixed matrix is preferably as follows: 20-40% of sand, 20-40% of yellow mud and 20-45% of humus soil; further preferably 30% of sand, 30% of yellow mud and 40% of humus. The invention has no special limit to the transplanting time, and can be transplanted all the year round; preferably transplanting in 4-10 months at relatively high temperature, culturing for 40 days after transplanting, and growing leaves to form plantlets; if the potatoes are transplanted from 11 months to 3 months in the next year, the transplanted small potato blocks firstly survive in a dormant state at low temperature, and the small potato blocks do not grow into leaves to form small seedlings until the temperature rises to more than 15 ℃ in spring. The invention preferably maintains the grown small seedlings according to a conventional field management method until the seedlings are poured in autumn and winter, and then the small potato blocks for field planting are obtained. The invention replaces the conventional tissue culture rooting seedling transplantation with the potato blocks with fibrous roots, so that the transplantation process is easier to operate, the management is easier after the transplantation, and the production cost of the seedlings is reduced.
The invention provides a method for in-vitro preservation of polygonatum cyrtonema germplasm based on the culture medium of the technical scheme, which comprises the following steps: adopting the in-vitro induction method of polygonatum cyrtonema potato blocks in the technical scheme to obtain polygonatum cyrtonema potato blocks with fibrous roots; continuously culturing the polygonatum cyrtonema potato blocks with fibrous roots in the culture medium of claim 1 under low light, and performing in-vitro preservation on polygonatum cyrtonema germplasm. The temperature for in-vitro preservation is 23-33 ℃, and the more preferable temperature is 28 ℃; the illumination time of the low-light culture is 10-12h/d, and the preferable illumination time is 11 h/d; the illumination intensity of the low-light culture is 5-15 mu mol.m-2·s-1More preferably 10. mu. mol. m-2·s-1。
The invention aims at inducing potato blocks, rooting and in vitro preservation of germplasm, and is completed in the same culture medium under the same culture condition. The method is used for inducing and rooting the potato tuber in the first 30-50 days of culture, and the storage period is from the later time to 760 days, and the two are combined to form the preservation period of the germplasm in vitro. The rhizoma polygonati multiflori can survive for a long time by virtue of the nutrition in the potato blocks after the nutrition in the culture medium is exhausted along with the prolonging of the culture time; and the buds on the small potato blocks are in a semi-dormant state and basically in a growth stagnation state, so that the consumed nutrition is little. The two are integrated together, and the in vitro preservation for a long time can be realized. FIG. 1 is a diagram of induction, rooting and germplasm in vitro preservation of polygonatum cyrtonema tuber potato pieces.
The invention provides the culture medium of the technical scheme, the polygonatum cyrtonema potato block in-vitro induction method of the technical scheme and the application of the polygonatum cyrtonema germplasm in-vitro preservation method of the technical scheme in rapid propagation of polygonatum cyrtonema seedlings.
To further illustrate the present invention, the culture medium and the application thereof for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema tubers are described in detail below with reference to the examples, which should not be construed as limiting the scope of the present invention.
Example 1
Culture medium formula for in-vitro induction and/or in-vitro preservation of germplasm of polygonatum cyrtonema potato blocks
MS culture medium + 5-nitroguaiacol sodium (5-NGS)0.4mg/L + sucrose 30g/L + agar 3.5g/L, pH 5.8.
In-vitro culture method of polygonatum cyrtonema potato blocks
1) Cleaning soil or seed coat on the surface of rhizoma Polygonati polygamae potato block or seed as explant, soaking in 2% detergent water solution for 4min, taking out, washing with running water, soaking in 75% ethanol for 60s, taking out, and placing in 0.1% HgCl2Soaking for 8min, taking out, and washing with sterile water for 5 times. Disinfection success rate and survival rate: 36% and 15% of potato blocks and 81% and 70% of seeds.
2) Inoculating the disinfected potato blocks or seeds into a primary culture medium, culturing the potato blocks in light for 30 days, and germinating and growing new buds of explants to form primary buds with the height of about 1 cm; and (5) culturing the seeds for 30-180 days in a weak light mode, and germinating and growing the seeds in sequence to form primary seedlings. The formula of the primary culture medium is as follows: MS +6-BA 2.0mg/L + IBA 0.2mg/L + GA36.0 mg/L + sucrose 30g/L + agar 3.5g/L (pH 5.8). The illumination culture conditions are as follows: the temperature is 28 +/-2 ℃, the illumination time is 11h/d, and the illumination intensity is 30 +/-2 mu mol.m-2·s-1(ii) a The low light culture conditions are as follows: the temperature is 28 +/-2 ℃, the illumination time is 11h/d, and the illumination intensity is 10 +/-2 mu mol.m-2·s-1. By using the method, the potato block explant is cultured for 40 days, and the induction rate of the primary bud seedling is 15%. By using the method, the seed explants are cultured for 30-180 days, the seeds germinate in succession, and the germination rate is 70%; the time span of seed germination is large, the time required for germinating the seeds is 30 days, and the time required for germinating the seeds is 180 days.
3) And cutting the obtained primary bud seedlings into single buds, reserving the base parts, cutting off the leaves, inoculating into a subculture multiplication medium, and carrying out illumination culture for 30d to obtain a subculture multiplication material. Wherein, the formula of the subculture multiplication medium is as follows: MS +6-BA4.0 mg/L + IBA 0.4mg/L + sucrose 30g/L + agar 3.5g/L (pH 5.8); the illumination culture conditions are as follows: the temperature is 28 + -2 deg.C, the illumination time is 10-12h/d, and the illumination intensity is about 30 + -2 μmol.m-2·s-1. Subculturing and proliferating by the method, the seedlings are strong, and the proliferation coefficient is 5.6/30 d. The polygonatum cyrtonema subculture seedling obtained by culturing the tuber as the explant is slower in growth and proliferation speed than the polygonatum cyrtonema subculture seedling obtained by culturing the seed as the explant 6 generations before subculture proliferation; after 6 generations of secondary proliferation, the growth and proliferation rates of the two tend to be consistent. FIG. 2 shows the subculture clumped seedlings obtained by culturing potato blocks, and FIG. 3 shows the subculture clumped seedlings obtained from seeds.
4) Cutting Polygonatum cyrtonema tissue culture subculture cluster bud seedlings as materials into single buds, reserving base parts, cutting off leaves, inoculating into Polygonatum cyrtonema tuber induction and/or germplasm in vitro preservation culture, and culturing for 40d under low light to obtain Polygonatum cyrtonema tuber blocks with fibrous roots, as shown in figure 4. The low light culture conditions are as follows: the temperature is 28 +/-2 ℃, the illumination time is 11h/d, and the illumination intensity is 10 +/-2 mu mol.m-2·s-1. The induction rate of the potato blocks cultured for 40 days is 100%, each potato block has 3-5 fibrous roots, and the average diameter of the small potato blocks is 1.0 cm.
The in vitro preservation method of the polygonatum cyrtonema tuber blocks with fibrous roots comprises the following steps: the polygonatum cyrtonema potato blocks with fibrous roots obtained by the culture method in the step 4) do not need to be transferred, the polygonatum cyrtonema potato blocks are continuously preserved for 720 days under the condition of normal-temperature weak light culture in polygonatum cyrtonema potato block induction and/or germplasm in-vitro preservation culture, the survival rate is 100 percent, and the regeneration and proliferation capacities of the preserved potato blocks are not influenced. The normal-temperature low-light culture conditions are as follows: temperature ofThe light intensity is 10 +/-2 mu mol.m at 28 +/-2 ℃ and the light time is 11h/d-2·s-1. The germplasm is preserved in vitro by the method, the survival rate is 100 percent, the polygonatum cyrtonema blocks with fibrous roots which are preserved in vitro for 760d (potato block induction for 40d + subsequent preservation for 720d) are shown in figure 5, and the material of figure 5 is subcultured again to have good proliferation and growth.
The transplanting method comprises the following steps: washing the obtained Polygonatum cyrtonema potato blocks with fibrous roots with clear water to remove the culture medium on the Polygonatum cyrtonema potato blocks, and transplanting. Transplanting environment: the shade degree of the greenhouse is 70%. The transplanting method comprises the following steps: according to the density of 300 small potato blocks per square meter, the potato blocks are sowed on a seedbed paved with a soft transplanting matrix, and then the transplanting matrix with the thickness of 2cm is covered, so that the humidity of the seedbed is kept; wherein, the transplanting substrate comprises the following components in percentage by weight (V/V): 30% of sand, 30% of yellow mud and 40% of humus soil. Transplanting is carried out in 5 months, after transplanting, the cultivation is carried out for about 40 days, leaves grow out to form small seedlings (figure 6), and the survival rate is 100%. And (3) curing the grown seedlings according to a conventional field management method to fall seedlings in autumn and winter to obtain sweet potato blocks for field planting (figure 7).
Example 2
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.2mg/L + sucrose 25g/L + agar 3.3g/L, pH 5.6;
the low light culture conditions are as follows: the temperature is 25 +/-2 ℃, the illumination time is 10h/d, and the illumination intensity is 7 +/-2 mu mol.m-2·s-1。
The transplanting matrix component content (V/V) is as follows: 40% of sand, 30% of yellow mud and 30% of humus soil.
As a result: the induction rate of the potato blocks is 100%, 3-5 fibrous roots are arranged on each potato block, and the average diameter of the small potato blocks is 0.8 cm; the induced small potato blocks are preserved in vitro at normal temperature for 760d without subculture of weak light, the survival rate is 100 percent, and the regeneration and proliferation capacities of the survival potato blocks are not influenced; the small potato blocks formed by induction are used for transplanting, and the survival rate is 100 percent.
Example 3
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.3mg/L + sucrose 35g/L + agar 3.7g/L, pH 6.0;
the low light culture conditions are as follows: the temperature is 30 +/-3 ℃, the illumination time is 12h/d, and the illumination intensity is 12 +/-3 mu mol.m-2·s-1。
The transplanting matrix component content (V/V) is as follows: 30% of sand, 40% of yellow mud and 30% of humus soil.
As a result: the induction rate of the potato blocks is 100%, 3-5 fibrous roots are arranged on each potato block, and the average diameter of the small potato blocks is 0.8 cm; the induced small potato blocks are preserved in vitro for 760d at normal temperature without subculture, the survival rate is 100 percent, and the regeneration and proliferation capacities of the survival potato blocks are not influenced; the small potato blocks formed by induction are used for transplanting, and the survival rate is 100 percent.
Example 4
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.5mg/L + sucrose 30g/L + agar 3.5g/L, pH 5.8;
as a result: the induction rate of the potato blocks is 100 percent, 3-5 fibrous roots are arranged on each potato block, and the average diameter of the small potato blocks is 0.8 cm; the induced small potato blocks are preserved in vitro for 760d at normal temperature without subculture, the survival rate is 100 percent, and the regeneration and proliferation capacities of the survival potato blocks are not influenced; the small potato blocks formed by induction are used for transplanting, and the survival rate is 100 percent.
Example 5
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.6mg/L + sucrose 30g/L + agar 3.3g/L, pH 5.8.
As a result: the induction rate of the potato blocks is 95%, 3-5 fibrous roots are arranged on each potato block, and the average diameter of the small potato blocks is 0.6 cm; the induced small potato blocks are preserved in vitro for 760d at normal temperature without subculture and with weak light, the survival rate is 89%, and the regeneration and proliferation capacities of the survival potato blocks are not affected; the small potato blocks formed by induction are used for transplanting, and the survival rate is 100 percent.
Example 6
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.7mg/L + sucrose 30g/L + agar 3.3g/L, pH 5.8.
As a result: the induction rate of the potato blocks is 90%, 3-5 fibrous roots are arranged on each potato block, and the average diameter of the small potato blocks is 0.6 cm; the induced small potato blocks are preserved in vitro for 760d at normal temperature without subculture, the survival rate is 84 percent, and the regeneration and proliferation capacity of the survival potato blocks are not influenced; the small potato blocks formed by induction are used for transplanting, and the survival rate is 100 percent.
Example 7
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.8mg/L + sucrose 30g/L + agar 3.3g/L, pH 5.8.
As a result: the inductivity of the potato blocks is 80%, 3-5 fibrous roots are arranged on each potato block, and the average diameter of the small potato blocks is 0.5 cm; the induced small potato blocks are preserved in vitro for 760d at normal temperature without subculture, the survival rate is 73 percent, and the regeneration and proliferation capacities of the survival potato blocks are not influenced; the small potato blocks formed by induction are used for transplanting, and the survival rate is 100 percent.
Comparative example 1
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 0.1mg/L + sucrose 30g/L + agar 3.3g/L, pH 5.8.
As a result: the potato block inductivity is low, and is only 66%; the formed potato pieces were also small, with an average diameter of 0.5 cm. The potato blocks formed by induction are preserved in vitro for 760 days at normal temperature and low light without subculture, and the survival rate is 42 percent; and (4) continuously preserving the rooted seedlings without transfer in vitro for 760d under normal temperature and weak light, wherein the survival rate is 0. Transplanting the small potato blocks formed by induction, wherein the transplanting survival rate is 98%.
Comparative example 2
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +5-NGS 1.5mg/L + sucrose 30g/L + agar 3.5g/L, pH 5.8.
As a result: the inductivity of the potato blocks is lower and is 56%, and fibrous roots on the potato blocks are reduced and shortened; the formed potato pieces are also small, with an average diameter of only 0.4 cm. The potato blocks formed by induction are preserved in vitro for 760 days at normal temperature and low light without subculture, and the survival rate is 25 percent; the rooted seedlings without the formed sweet potato blocks are not transferred and continuously stored in vitro at normal temperature and weak light, most of the seedlings can survive for only 90 days and die completely later. Transplanting the small potato blocks formed by induction, wherein the transplanting survival rate is 93%.
Comparative example 3
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS +2, 4-D0.4 mg/L + sucrose 30g/L + agar 3.5g/L, pH 5.8.
As a result: the inductivity of the potato blocks is 72 percent, and the induced small potato blocks are loose in texture, different in shape, size and color. The induced small potato blocks are preserved in vitro for 550 days at normal temperature and low light without subculture, and the survival rate is only 16 percent; the rooted seedlings without the formed sweet potato blocks are not transferred and continuously stored in vitro at normal temperature and weak light, most of the seedlings can survive for only 90 days and die completely later. Transplanting the small potato blocks formed by induction, wherein the transplanting survival rate is 92%.
Comparative example 4
Example 1 was repeated except that:
a basic culture medium MS culture medium of the polygonatum cyrtonema tuber in-vitro induction and/or germplasm in-vitro preservation culture medium is changed into 1/2MS culture medium.
As a result: the induced potato blocks are small, and the average diameter is less than 0.4 cm. The potato blocks formed by induction are not subjected to subculture, are preserved in vitro for 550 days under normal temperature and low light, and the survival rate is only 46 percent. Transplanting the small potato blocks formed by induction, wherein the transplanting survival rate is 96%.
Comparative example 5
Conventional rooting method with highest reported rooting rate
Example 1 was repeated except that:
the culture medium formula for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema potato blocks comprises:
MS, NAA 0.5mg/L, sucrose 30g/L, agar 3.5g/L and pH 5.8;
cutting the subculture cluster buds into single buds, and keeping leaves;
the culture condition is changed into illumination culture: the temperature is 28 +/-2 ℃, the illumination time is 10-12h/d, and the illumination intensity is 20 +/-2 mu mol.m-2·s-1；
Transplanting: hardening the rooted seedlings outdoors for 3-5 days, washing off the culture medium attached to the root systems by using clear water, transplanting the seedlings to a seedling bed paved with a transplanting matrix with the thickness of 5-7 cm in a greenhouse with the shading degree of 70% according to the plant-row spacing of about 7 multiplied by 12cm, wherein the component content (V/V) of the transplanting matrix is as follows: 30% of sand, 30% of yellow mud and 40% of humus soil, and the seedling washing and transplanting process avoids the damage of tender leaves to cause dead seedlings as much as possible; after the transplanting is finished, 500 times of liquid of 50 percent carbendazim wettable powder is used for sprinkling, so that the death caused by the contamination of bacteria to the wound in the transplanting process is prevented; watering and moisturizing according to weather conditions, and culturing for 40 d.
As a result: obtaining rooted seedlings, wherein the rooting rate is 98%, partial leaves are withered, and small potato blocks cannot be induced; the non-subculture survival time of the rooted seedlings in the culture medium is only about 90 days, the survival time is short, and the rooted seedlings cannot be used as an in-vitro preservation culture medium. The transplanting survival rate of the rooted seedlings is 90%, care needs to be taken to avoid dead seedlings caused by damage of tender leaves during seedling washing and transplanting, bactericides are used for preventing infection after transplanting, the moisture management requirement of a seedbed is more careful, only about 120 seedlings are planted in each square meter of seedbed, the transplanting time is limited in seasons with mild climate, and compared with the embodiment 1, the transplanting efficiency is low, and the management cost is high.
Comparative example 6
Conventional in vitro preservation
Example 1 was repeated except that:
the 5-NGS in the culture medium for in vitro induction and/or in vitro preservation of the germplasm of polygonatum cyrtonema blocks is changed into paclobutrazol which is a plant growth inhibitor commonly used in vitro preservation(PP333) The changed formula is as follows: MS + PP3331.0 mg/L + sucrose 30g/L + agar 3.5g/L, pH 5.8.
As a result: the potato blocks can not be induced, the potato blocks are cultured and stored for 200 days in the form of seedlings without subculture under normal temperature illumination, and the survival rate is only 60 percent. For 760d storage, transfer was required 4 times, i.e., medium was changed 4 times and inoculated 4 times.
The embodiment and the comparative example show that the culture medium provided by the invention has three different functions of tuber induction, rooting and in-vitro preservation, can achieve three different processes in one step by a three-in-one method, is simple and easy to operate in the whole process, has high tuber induction and rooting efficiency and long germplasm single generation preservation time, and reduces the cost of manpower and material resources in the aspects of germplasm preservation or tissue culture seedling transplantation. The problems of conventional tissue culture rooting seedling transplanting operation and large management difficulty after transplanting are solved, and meanwhile, the impact and influence of market fluctuation on seedling tissue culture production can be flexibly coped with.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A culture medium for in vitro induction and/or in vitro preservation of germplasm of polygonatum cyrtonema tuber blocks is characterized in that an MS culture medium is taken as a basic culture medium, and the culture medium further comprises the following components in concentration: 0.2-0.8 mg/L of 5-nitroguaiacol sodium, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the culture medium is 5.6-6.0.
2. An in vitro induction method of polygonatum cyrtonema potato pieces based on the culture medium of claim 1, comprising the following steps:
inoculating polygonatum cyrtonema cluster buds to the culture medium of claim 1, and carrying out low-light in-vitro culture to obtain polygonatum cyrtonema potato blocks with fibrous roots; the temperature of the low-light in-vitro culture is 23-33 ℃, the illumination time is 10-12h/d, and the illumination intensity is 5-15 mu mol·m-2·s-1The culture time is 30-50 d; the diameter of the polygonatum cyrtonema potato blocks with fibrous roots is 0.5-1.0 cm, and the number of the fibrous roots is 3-5.
3. The in vitro induction method according to claim 2, further comprising the step of cutting and defoliating the multiple shoots of polygonatum cyrtonema before inoculating the multiple shoots, wherein each processed multiple shoot contains 1-2 shoots.
4. The ex vivo induction method according to claim 2, wherein said multiple shoots are obtained by a method comprising:
1) taking polygonatum cyrtonema potato blocks or seeds as explants, disinfecting, inoculating in a primary culture medium, and carrying out primary culture to form primary seedlings; when the explant is polygonatum cyrtonema potato blocks, culturing by illumination for 30-40 days; when the explant is a seed, culturing with weak light for 30-180 days;
2) inoculating the obtained primary seedlings into a subculture medium, and performing illumination culture to obtain subcultured cluster buds; the time of subculture is 25-35 d.
5. The ex vivo induction method according to claim 4, wherein the primary medium in step 1) is MS medium as a basal medium, and further comprises the following components in concentration: 6-BA 1.0-3.0 mg/L, IBA 0.1.1-0.3 mg/L, GA34.0-8.0 mg/L, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the primary culture medium is 5.6-6.0.
6. The ex vivo induction method according to claim 4, wherein the subculture medium in step 2) is MS medium as a basal medium, and further comprises the following components in concentration: 3.0-5.0 mg/L, IBA 0.3.3-0.5 mg/L of 6-BA, 25-35 g/L of sucrose and 3.3-3.7 g/L of agar; the pH value of the subculture medium is 5.6-6.0.
7. The in vitro induction method according to claim 4, wherein the primary seedlings in step 2) are cut and defoliated before inoculation, and the treated primary seedlings contain 1-2 buds.
8. An in-vitro preservation method of polygonatum cyrtonema germplasm based on the culture medium of claim 1, comprising the following steps: obtaining polygonatum cyrtonema potato pieces with fibrous roots by adopting the polygonatum cyrtonema potato piece in-vitro induction method of claim 2; continuously culturing the polygonatum cyrtonema potato blocks with fibrous roots in the culture medium of claim 1, and performing in-vitro preservation on polygonatum cyrtonema germplasm.
9. The method for in-vitro preservation of polygonatum cyrtonema germplasm according to claim 8, wherein in-vitro preservation is performed under the condition of weak light, the temperature of in-vitro preservation is 23-33 ℃, the illumination time of in-vitro preservation is 10-12h/d, and the illumination intensity of weak light is 5-15 μmol-m-2·s-1。
10. The culture medium of claim 1, the polygonatum cyrtonema potato tuber in-vitro induction method of any one of claims 2 to 7 and the application of the polygonatum cyrtonema germplasm in-vitro preservation method of claim 8 or 9 in the polygonatum cyrtonema seedling tissue culture and rapid propagation production.
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