Search

CN-121970685-A - Culture method for directly inducing cluster buds by utilizing gingko aseptic stems

CN121970685ACN 121970685 ACN121970685 ACN 121970685ACN-121970685-A

Abstract

The invention discloses a culture method for directly inducing cluster buds by utilizing aseptic stems of ginkgo, and relates to the technical field of ginkgo tissue culture. The culture method comprises the following steps of removing the outer seed coats of ginkgo fruits, washing, airing, storing, sterilizing, washing with sterile water, taking out ginkgo embryos, inoculating the ginkgo embryos into an initial culture medium for culture, cutting the cultured sterile seedlings into stem segments, inoculating into a proliferation culture medium for culture, and directly inducing cluster buds to germinate. The invention shortens the culture period, improves the induction rate and proliferation coefficient of the cluster buds, avoids the risk of genetic variation, and effectively solves the problems of low proliferation efficiency, long culture period, high risk of genetic variation and the like in the prior art.

Inventors

  • DENG HUIHONG
  • XU XUEJIAO
  • WANG JIANBING
  • Xiong Shengguo
  • LI LINRUI
  • FENG SHAOBO

Assignees

  • 开江县林业科研所

Dates

Publication Date
20260505
Application Date
20260403

Claims (10)

  1. 1. The culture method for directly inducing the cluster buds by utilizing the aseptic stems of the ginkgo is characterized by comprising the following steps of: S1, removing the exocarp of ginkgo fruits, washing, airing and storing, washing with sterile water after disinfection treatment, taking out ginkgo embryos, and inoculating the ginkgo embryos into an initial culture medium for culture; S2, cutting the cultured sterile seedlings, inoculating the cut sterile seedlings into a proliferation culture medium for culturing, and directly inducing the cluster buds to germinate, wherein the proliferation culture medium is an MS culture medium containing 0.5-0.7 mg/L cytokinin, 0.1-0.2 mg/L auxin, 15-25 mL/L gingko sterile embryo milk, 5-15 mL/L gingko sterile cotyledon juice, 25-35 g/L sucrose and 6-8 g/L agar, and the pH value is 5.6-5.8.
  2. 2. The method for directly inducing multiple shoots using aseptic stems of ginkgo biloba according to claim 1, wherein in step S1, the storage temperature is 20-25 ℃ and the relative humidity is 40-50%.
  3. 3. The method according to claim 1, wherein in step S1, the ginkgo seed is sterilized by treating 25-35% S in 74-76 vt% ethanol solution, and then treating 10-15 min in sodium hypochlorite solution containing 0.02-0.04. 0.04 wt% tween-20 at a concentration of 0.4-0.6-wt%.
  4. 4. The method for directly inducing multiple shoots using aseptic stems of ginkgo according to claim 1, wherein in step S1, the initial medium is an MS medium containing 0.4-0.6 mg/L6-benzylaminopurine, 0.1-0.3 mg/L naphthylacetic acid, 25-35g/L sucrose and 6-8g/L agar, and the pH is 5.6-5.8.
  5. 5. The method for directly inducing multiple shoots by using aseptic stems of ginkgo according to claim 1, wherein in step S1, the cultivation temperature is 24-26 ℃, the illumination intensity is 2800-3000 lx, the photoperiod is 16 h illumination/8 h darkness, and the cultivation is 20-30 d.
  6. 6. The method for directly inducing multiple shoots using aseptic stems of ginkgo according to claim 1, wherein in step S2, the cytokinin is 6-benzylaminopurine or kinetin, and the auxin is indolebutyric acid or indoleacetic acid.
  7. 7. The method for culturing ginkgo sterile embryo milk by directly inducing cluster buds according to claim 1, wherein in the step S2, ginkgo sterile embryo milk is prepared by taking ginkgo nut endosperm, washing, cutting, adding ultrapure water, grinding to obtain endosperm homogenate, heating the endosperm homogenate, cooling, filtering, adjusting conductivity value, and sterilizing to obtain ginkgo sterile embryo milk.
  8. 8. The method for culturing ginkgo biloba sterile cotyledon juice by directly inducing cluster buds according to claim 1, wherein in the step S2, ginkgo biloba sterile cotyledon juice is prepared by cutting off cotyledons from aseptic seedlings, washing, adding ultrapure water, grinding to obtain cotyledon homogenate, heating the cotyledon homogenate, cooling, filtering, adjusting conductivity value, and sterilizing to obtain ginkgo biloba sterile cotyledon juice.
  9. 9. The method for directly inducing multiple shoots using aseptic stems of ginkgo biloba according to claim 1, wherein in step S2, the conductivity value of the aseptic embryo milk of ginkgo biloba is 500-800 μs/cm, and the conductivity value of the aseptic cotyledon juice of ginkgo biloba is 300-500 μs/cm.
  10. 10. The method for directly inducing cluster buds according to claim 1, wherein in step S2, the culturing temperature is 24-26 ℃, the illumination intensity is 3000-3500 lx, the photoperiod is 16h illumination/8 h darkness, and the culturing is 25-30 d.

Description

Culture method for directly inducing cluster buds by utilizing gingko aseptic stems Technical Field The invention relates to the technical field of ginkgo tissue culture, in particular to a culture method for directly inducing cluster buds by utilizing aseptic stems of ginkgo. Background Gingko (Ginkgo biloba L.) is used as the wig plant in the fourth glacier period of the new generation, is a rare and precious tree species with unique leaves and fruits and excellent properties in China, is called as "activated stone" of gymnosperm, and has extremely high medical, edible, ornamental, economic and ecological values. The seeds contain various nutritional components and effective components such as protein, fat, calcium, phosphorus, iron, carotene, lactone and the like, and have good curative effect on cardiovascular and cerebrovascular diseases. The active ingredients such as flavone and terpene lactone in ginkgo leaf have remarkable effects in eliminating free radicals, resisting tumors, resisting oxidization and protecting nervous system. Meanwhile, gingko cultivation and management are convenient, the service life is long, the ornamental performance is good, and the gingko cultivation and management method becomes a main tree species for urban landscaping. Along with the continuous deep development and utilization of the ginkgo, the market demand for high-quality ginkgo seedlings, especially good ginkgo seedlings, is gradually increased. Conventional ginkgo breeding modes comprise sowing, grafting, cutting and the like, but have the problems of low breeding efficiency, long period, limited breeding coefficient and the like, and are difficult to meet the requirement of large-scale production. The plant tissue culture technology is used as an effective means for rapid propagation of high-quality seedlings, can realize high-efficiency propagation of improved varieties of ginkgo, and has important significance for promoting clone breeding of ginkgo and industrialized production of ginkgo secondary metabolites. However, the tissue culture process of ginkgo leaves not only has the technical problems of high pollution rate, difficult rooting, low proliferation coefficient, long culture period and the like, but also has the key hidden trouble that most of the prior art relies on a callus induction differentiation path to obtain cluster buds or adventitious buds, cells in the callus formation process can undergo dedifferentiation and redifferentiation, genetic variation is easily generated due to recombination or mutation of genetic materials, so that regenerated plant characters are inconsistent with female parents, the stability of fine variety characters is seriously affected, and the method is a core problem to be solved in large-scale ginkgo leaf cultivation which needs to keep excellent genetic characteristics. Therefore, the ginkgo tissue culture technical scheme is optimized, a sterile stem is constructed to directly induce a callus-free path of the cluster buds, the risk of genetic variation is avoided, meanwhile, the induction rate and proliferation coefficient of the cluster buds are improved, the culture period is shortened, the pollution risk is reduced, and the method becomes a key technical requirement in the current ginkgo tissue culture field. In the prior art, a method for culturing the gingko aseptic seedlings by taking gingko seed kernels and gingko embryo as inoculating materials and adding gingko endosperm juice with different concentrations is provided, but the method is only focused on the primary culture of the aseptic seedlings, does not relate to related schemes of cluster bud induction and multiplication culture, cannot realize the large-scale rapid propagation of the seedlings, and the preferred culture medium is suitable for the initial growth of the aseptic seedlings, but does not optimize the nutritional requirements of cluster bud induction, has a longer culture period and does not avoid the risk of genetic variation. The conventional ginkgo tissue culture propagation method has the problems of complex sterile treatment process, complex culture medium components and hormone combination, limited explant selection, limited proliferation efficiency, long proliferation period and genetic variation risk. Disclosure of Invention Aiming at the defects in the prior art, the invention provides a culture method for directly inducing arbuscular buds by utilizing gingko sterile stems, which effectively solves the problems of low proliferation efficiency, long culture period, high genetic variation risk and the like in the prior art. In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is that a culture method for directly inducing cluster buds by utilizing aseptic stems of ginkgo is provided, which comprises the following steps: S1, removing the exocarp of ginkgo fruits, washing, airing and storing, washing with sterile water af