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CN-122004131-A - Rapid propagation method for tissue culture of large yellow crotalaria seed

CN122004131ACN 122004131 ACN122004131 ACN 122004131ACN-122004131-A

Abstract

The invention discloses a method for quickly breeding large yellow-flower crassifolia seeds by tissue culture, belonging to the field of plant seedling breeding. The tissue culture rapid propagation system comprises the steps of seed preparation and harvesting, explant disinfection, seed treatment, protocorm and adventitious bud, adventitious root induction, rooting and seedling strengthening culture, seedling transplanting and hardening and the like, the embryogenic rate of the seeds can be improved to be the average 86.76% according to the system, the germination rate of the seeds is improved to be 65.74%, the seeds can start to germinate to form protocorm in the fastest 60 days, adventitious buds grow out from the protocorm in the 100 days, and adventitious roots grow out in the 120 days. And then rooting and strengthening seedling culture and hardening seedling transplanting are carried out, so that high-quality seedlings can be produced and used for field normalization. The method is a high-efficiency, rapid and environment-friendly propagation technology system, can realize the large-scale production of the large-yellow-flower calanthe in a short period, and has important practical value for protecting and propagating the rare orchid.

Inventors

  • SHEN BAOMING
  • TAN ZHUMING
  • SHEN AIRONG
  • LIU LINA
  • TAN YUN
  • LI SAINAN

Assignees

  • 湖南省林业科学院

Dates

Publication Date
20260512
Application Date
20260226

Claims (8)

  1. 1. The method for rapidly propagating the large yellow croaker-orchid seed tissue culture is characterized by comprising the following steps: (1) Hybridization seed production is carried out on the large yellow shrimp ridge blue, and seeds are obtained under the aseptic condition after the non-cracked mature capsules are sterilized; (2) Treating seeds with sterile NaOH or KOH solution to destroy seed coats; (3) Inducing protocorms, adventitious buds and adventitious roots; (4) Rooting and strengthening seedlings; (5) Hardening off and transplanting seedlings; Step (3) under the aseptic condition, spreading the seeds obtained in the step (2) on the surface of a culture medium, wherein the culture medium comprises 1/2MS culture medium+1.0-5.0 mg/L6-BA+0.2-0.7 mg/L NAA+0.2-0.6 mg/L TDZ+1.0-4.0 g/L active carbon, and culturing for 60-80 d, wherein the protocorm is formed in a large amount, 100-120 d, adventitious buds are induced by the protocorm, 120-140 d, the adventitious buds are elongated, adventitious roots are produced at the base part, and seedlings are formed; Transferring the seedlings obtained in the step (3) into rooting and seedling strengthening culture media, wherein the culture media comprises 1/2MS culture media plus 0.2-0.7 mg/L NAA plus 1.0-5.0 g/L active carbon, culturing for 30-50 d, growing the seedlings to 2-4 cm high, 2-3 leaves and 2-5 roots of strong seedlings, and transferring the seedlings into a seedling hardening stage.
  2. 2. The method of claim 1, wherein in the step (1), in the season of 4 months, the daylily marjoram flowers are collected for hybridization pollination, and after pollination, the capsules naturally develop for 6-8 months of fruit maturity period until the pericarp is yellow-green, no disease spots exist on the surface, no worm damage exists and the healthy mature capsules are not cracked.
  3. 3. The method of claim 1, wherein the sterilization process of the capsule in the step (1) is carried out by washing the surface of the capsule with purified water, soaking the capsule in 70-75% alcohol for 30-90 s, preferably 40-60 s, washing the capsule with sterile water once, soaking the capsule in 6-10% NaClO for 8-12 min, preferably 7-8% NaClO for 9-10 min, washing the capsule with sterile water for three times, sucking the surface moisture of the capsule with sterile filter paper under sterile conditions, and then cutting to obtain seeds in the pericarp for later use.
  4. 4. The method according to claim 1, wherein the aseptic seeds obtained in step (1) are soaked in sterile NaOH or KOH solution with a concentration of 0.05-0.40 mol/L, preferably 0.20-0.30 mol/L, for 7-12 min, preferably 8-10 min, and rinsed three times with sterile water for later use.
  5. 5. The method of claim 1, wherein step (3) comprises adding 1mL of sterile water to the surface of the culture medium to uniformly spread the seeds on the surface of the culture medium.
  6. 6. The method of claim 1, wherein the sucrose concentration of the culture medium in the step (3) and the step (4) is 25-35 g/L, the agar powder concentration is 5-8 g/L, the pH is adjusted to 5.5-6.0, the culture temperature is 23+ -1 ℃, the humidity is 70-80%, the illumination time is 12-14 h/d, and the illumination intensity is 2000-3500lx.
  7. 7. The method of claim 1, wherein the step (5) is to transfer the strong seedlings obtained in the step (4) into a transplanting matrix after hardening the seedlings, and the transplanting matrix is formed by mixing peat soil, perlite and vermiculite according to a volume ratio of = (6-7): (1-2): (2-3).
  8. 8. The method of claim 7, wherein the tissue culture container with strong seedlings is placed under natural light and room temperature conditions, the tissue culture container is cultured for 3-5 days, then a bottle cap of the container is opened for half of the culture container for 12-24 hours, the seedlings are taken out of the container, the culture medium attached to roots is gently washed away in clean water at 20-30 ℃, the strong seedlings are transplanted into high-temperature sterilized transplanting matrixes, the tissue culture container is cultured in a natural light greenhouse for 2-4 times a day, the plant height is 8-10 cm until the maximum leaf length is 5-8 cm, the maximum leaf width is 2-4 cm, and the large yellow croaker seedlings are transplanted into a field suitable habitat.

Description

Rapid propagation method for tissue culture of large yellow crotalaria seed Technical Field The invention relates to a plant tissue culture technology, in particular to a tissue culture method of large yellow flower shrimp ridge orchid, which comprises the steps of hybridization seed production, disinfection of explants, seed treatment, protocorm and adventitious bud, adventitious root induction, rooting and seedling strengthening culture, seedling transplanting and hardening and the like. Background Orchids are one of the largest number of species among angiosperms. The large yellow-flower calanthe is a calanthe plant of the genus calanthe of the family Orchidaceae, has narrow distribution region and sparse population quantity. The number of the large yellow shrimp ridge is endangered. Protection points are established in the distribution areas of the Hunan Xinning lang mountain, yongzhou city Jindong forest farm and the like, and on-site conservation measures are carried out, but the ecological system is fragile, so that the field natural germination of seeds is extremely difficult, and the population is difficult to keep stable by only on-site protection and natural reproduction. Only the artificial breeding of the large yellow croaker orchid is realized, and the field normalization technology is combined to promote the population to be restored to a sufficient base number, so that the species can be ensured to reproduce the information sustainably in the primordium. Although a few reports exist on the breeding technology of the large-yellow-flower shrimp ridge orchid, the problems of long germination time, low germination rate and the like still limit the large-scale breeding process of the large-yellow-flower shrimp ridge orchid. In addition, the tissue culture methods of other orchid plants cannot be directly used as a reference, and have problems such as nutrient buds of an explant in a patent with a publication number of CN106258997A, and wounds are formed on tubers when the nutrient buds are obtained, so that pathogenic bacteria are easy to invade to cause death of the whole plant, the wild resource protection is not facilitated, and mercury lift disinfection with strong toxicity is also adopted. For another example, in the patent with publication number CN106386481a, a high-difficulty technical operation is required under a split microscope, so that a protocorm-like cell thin layer is obtained, which is not beneficial to popularization. Therefore, the invention discloses a method for improving the germination rate of the seed of the large yellow croaker and shortening the seedling period by using the seed of the large yellow croaker as a propagation material and controlling conditions such as environmental factors and biological factors such as nutrition conditions, temperature, humidity, illumination intensity and the like after the seed is treated, and the method also overcomes other defects in the prior art. Disclosure of Invention The invention aims to provide a simple, convenient and efficient method for sprouting and forming the large yellow croaker amaranth seeds, which aims at overcoming the defects of long sprouting time and low sprouting rate of the existing large yellow croaker amaranth seeds. The method utilizes the sterilizing method of HgCl 2 -free sterilization of the uncracked mature capsule of the large yellow crataegus pinnatifida to obtain a large number of embryogenic seeds as explants, and can rapidly induce protocorms, buds and roots, so that the germination time and germination rate reach excellent levels. The object of the present invention is achieved in the following manner. A method for rapidly propagating large yellow crotalaria seed tissue culture comprises the following steps: (1) Hybridization seed production is carried out on the large yellow shrimp ridge blue, and seeds are obtained under the aseptic condition after the non-cracked mature capsules are sterilized; (2) Treating seeds with sterile NaOH or KOH solution to destroy seed coats; (3) Inducing protocorms, adventitious buds and adventitious roots; (4) Rooting and strengthening seedlings; (5) Hardening off and transplanting seedlings; Step (3) under the aseptic condition, spreading the seeds obtained in the step (2) on the surface of a culture medium, wherein the culture medium comprises 1/2MS culture medium+1.0-5.0 mg/L6-BA+0.2-0.7 mg/L NAA+0.2-0.6 mg/L TDZ+1.0-4.0 g/L active carbon, and culturing for 60-80 d, wherein the protocorm is formed in a large amount, 100-120 d, adventitious buds are induced by the protocorm, 120-140 d, the adventitious buds are elongated, adventitious roots are produced at the base part, and seedlings are formed; Transferring the seedlings obtained in the step (3) into rooting and seedling strengthening culture media, wherein the culture media comprises 1/2MS culture media plus 0.2-0.7 mg/L NAA plus 1.0-5.0 g/L active carbon, culturing for 30-50 d, growing the seedlings to 2-4 cm hi