Search

CN-122004128-A - Method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway

CN122004128ACN 122004128 ACN122004128 ACN 122004128ACN-122004128-A

Abstract

The invention belongs to the field of plant biotechnology breeding, and particularly relates to a method for rapid tissue culture regeneration of alfalfa based on an organogenesis way, which comprises the following steps of sterilizing alfalfa seeds, placing the alfalfa seeds in a seed culture medium for dark culture and vernalization, and alternately culturing the vernalized seeds in an illumination environment and a dark environment to enable the seeds to germinate out cotyledons; cutting cotyledons, transferring the cotyledons into a bud induction culture medium, alternately culturing in an illumination environment and a dark environment to induce to obtain clustered buds, transferring the clustered buds into a bud elongation culture medium, alternately culturing in the illumination environment and the dark environment to enable the clustered buds to grow to obtain highly developed clustered buds, transferring the highly developed clustered buds into a rooting culture medium, alternately culturing in the illumination environment and the dark environment, culturing to root and obtaining complete plants. The regeneration material obtained by the invention can shorten the conventional tissue culture period from 6 months to 8 months to only 1 month to 2 months, thereby greatly shortening the tissue culture regeneration time.

Inventors

  • CONG LILI
  • CHAI MAOFENG
  • XIE HONGLI
  • LIU XIN
  • LIU XIANGJUN
  • XU RUI
  • FENG SIJIA
  • JIA SHIZHEN
  • BAI JIAHE
  • WANG ZENGYU
  • ZHANG KUN
  • DING WANG
  • YU AILING
  • Li Huici
  • GONG YAN

Assignees

  • 青岛农业大学
  • 歌拉斯(北京)科技发展有限公司

Dates

Publication Date
20260512
Application Date
20260109

Claims (10)

  1. 1. A method for rapid tissue culture regeneration of alfalfa based on an organogenesis pathway, comprising the steps of: (1) Sterilizing alfalfa seeds, placing the alfalfa seeds in a seed culture medium, dark culturing at 0-10 ℃ for vernalization for 1-2 days, and alternately culturing the vernalized seeds in 8-16 h illumination environment and 8-16 h dark environment for 1-3 days to enable the seeds to sprout cotyledons; (2) Cutting cotyledons, transferring the cotyledons into a bud induction culture medium, and alternately culturing for 14 days in an illumination environment of 8-16 hours and a dark environment of 8-16 hours to obtain clustered buds through induction, wherein the bud induction culture medium is an MSTB culture medium; (3) Transferring the obtained cluster buds into a bud elongation culture medium, and alternately culturing for 14 days in 8-16 h light environment and 8h dark environment to enable the cluster buds to grow so as to obtain highly developed cluster buds, wherein the bud elongation culture medium is MSZN culture medium; (4) And transferring the cluster buds which are highly developed into a rooting culture medium, and alternately culturing for 22-37 days in an illumination environment of 8-16 h and a dark environment of 8-16 h, so as to obtain a complete plant by culturing rooting, wherein the rooting culture medium is MSSG rooting culture medium.
  2. 2. The method of claim 1, wherein the temperature of the light environment is 20 ℃ to 26 ℃ and the temperature of the dark environment is 18 ℃ to 22 ℃.
  3. 3. The method of claim 1, wherein the illumination intensities are 4000lx to 4200lx.
  4. 4. The method of claim 1, wherein the excised cotyledons comprise cotyledon and hypocotyl junctions.
  5. 5. The method of claim 1, wherein the seed medium is 1/2MS medium.
  6. 6. The method according to claim 1, wherein the MSTB medium is prepared from 2.22-4.43 g of MS basal medium, 20-30 g of sucrose, 30 mu L of thidiazuron, 500 mu L of 6-benzyl amino purine, 3-3.2 g of plant gel, 1mL of cephalosporin and 1mL of timentin, and distilled water to 1L, wherein the concentration of the thidiazuron is 10mg/mL, the concentration of the 6-benzyl amino purine is 1mg/mL, the concentration of the cephalosporin is 200mg/mL, and the concentration of the timentin is 200mg/mL.
  7. 7. The method according to claim 1, wherein the MSZN culture medium comprises 2.22-4.43 g of MS basal medium, 20-30 g of sucrose, 0.05-0.5 mL of zeatin, 0.05-0.2 mL of naphthylacetic acid, 2.9-3.1 g of plant gel, 1mL of cephalosporin and 1mL of timentin, distilled water is supplemented to 1L, the concentration of the zeatin is 1mg/mL, the concentration of the naphthaleneacetic acid is 1mg/mL, the concentration of the cephalosporin is 200mg/mL, and the concentration of the timentin is 200mg/mL.
  8. 8. The method of claim 1, wherein the MSSG rooting medium comprises 1.11 g-4.43 g of MS basal medium, 10 g-20 g of sucrose, 0.1 mL-0.2 mL of indole-3-butyric acid, 3.2 g-3.4 g of plant gel, 1mL of cephalosporin, 1mL of timentin, and distilled water to 1L, wherein the concentration of indole-3-butyric acid is 1mg/mL, the concentration of cephalosporin is 200mg/mL, and the concentration of timentin is 200mg/mL.
  9. 9. The method of claim 1, wherein alfalfa seeds are sterilized using alcohol and sodium hypochlorite soaking.
  10. 10. The method according to any one of claims 1 to 9, wherein the alfalfa seeds are alfalfa No. 3 and alfalfa No. 1 seeds.

Description

Method for rapid tissue culture regeneration of alfalfa based on organogenesis pathway Technical Field The invention belongs to the field of plant biotechnology breeding, and particularly relates to a method for rapid tissue culture regeneration of alfalfa based on an organogenesis pathway. Background Alfalfa is used as perennial high-protein high-quality forage grass, and by virtue of the characteristics of high yield, high quality, good palatability and strong adaptability, the alfalfa enjoys the reputation of 'forage' and plays an important role in the development of animal husbandry. However, the current breeding quantity, speed and quality of alfalfa are difficult to meet the production requirements, so that alfalfa seeds are dependent on import for a long time, and industrial safety is challenging. The advent of CRISPR/Cas9 gene editing technology provides a revolutionary technological path for alfalfa breeding. However, alfalfa, as a cross-pollinated autotetraploid plant, its complex genetic background presents unique challenges to biological breeding. The technological development process is traced back, and since the callus induction path regeneration transformation system based on the true leaf explant is established in the 80 th 20 th century, though continuous optimization (including systematic improvement of culture medium components and hormone proportion) is carried out for nearly 40 years, the genetic transformation system based on the traditional callus path still faces three technical bottlenecks, namely 1) narrow receptor genotype range, suitability for few varieties, 2) long regeneration period, unstable transformation efficiency, 3) high operation cost and poor repeatability. The root of these technical bottlenecks is the inherent biological limitations of the traditional true leaf explant-callus regeneration system. In order to solve the difficult problem of biological breeding development of alfalfa, a technical path must be fundamentally innovated. Currently, the construction of a novel efficient and stable regeneration system has become a key point for promoting the breakthrough of genetic transformation technology. In summary, there is a need to establish a novel regeneration system based on direct organogenesis, which thoroughly avoids the traditional callus formation stage, so as to realize the shortening of the alfalfa cultivation period, high efficiency and wide variety. Disclosure of Invention In order to solve the problems, the invention provides a method for rapid tissue culture, culture and regeneration of alfalfa based on an organogenesis way. The invention is realized by the following technical scheme: a method for rapid tissue culture regeneration of alfalfa based on an organogenesis pathway, comprising the steps of: (1) Sterilizing alfalfa seeds, placing the alfalfa seeds in a seed culture medium, dark culturing at 0-10 ℃ for vernalization for 1-2 days, and alternately culturing the vernalized seeds in 8-16 h illumination environment and 8-16 h dark environment for 1-3 days to enable the seeds to sprout cotyledons; (2) Cutting cotyledons, transferring the cotyledons into a bud induction culture medium, and alternately culturing for 14 days in an illumination environment of 8-16 hours and a dark environment of 8-16 hours to obtain clustered buds through induction, wherein the bud induction culture medium is an MSTB culture medium; (3) Transferring the obtained cluster buds into a bud elongation culture medium, and alternately culturing for 14 days in 8-16 h light environment and 8h dark environment to enable the cluster buds to grow so as to obtain highly developed cluster buds, wherein the bud elongation culture medium is MSZN culture medium; (4) And transferring the cluster buds which are highly developed into a rooting culture medium, and alternately culturing for 22-37 days in an illumination environment of 8-16 h and a dark environment of 8-16 h, so as to obtain a complete plant by culturing rooting, wherein the rooting culture medium is MSSG rooting culture medium. Preferably, the temperature of the illumination environment is 20-26 ℃, and the temperature of the dark environment is 18-22 ℃. Preferably, the illumination intensity is 4000 lx-4200 lx. Preferably, the excised cotyledons include cotyledon and hypocotyl junctions. Preferably, the seed medium is a 1/2MS medium. Preferably, the MSTB culture medium comprises 2.22-4.43 g of MS basal culture medium, 20-30 g of sucrose, 30 mu L of thidiazuron, 500 mu L of 6-benzyl amino purine, 3-3.2 g of plant gel, 1mL of cephalosporin and 1mL of timentin, wherein distilled water is supplemented to 1L, the concentration of the thidiazuron is 10mg/mL, the concentration of the 6-benzyl amino purine is 1mg/mL, the concentration of the cephalosporin is 200mg/mL, and the concentration of the timentin is 200mg/mL. Preferably, the MSZN culture medium comprises 2.22-4.43 g of MS basic culture medium, 20-30 g of sucrose, 0.05-0.5 mL o