Agrobacterium-mediated genetic transformation method for sea barleygrass

Patent metadata

Field	Value
Publication number	US-12203079-B2
Application number	US-202217811764-A
Country	US
Kind code	B2
Filing date	Jul 11, 2022
Priority date	Oct 25, 2021
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

Abstract

An Agrobacterium -mediated genetic transformation method for sea barleygrass is provided. The method includes: S1, selecting immature embryo materials of sea barleygrass with immature embryos each having a length in a range of 0.5-1.0, sterilizing them with alcohol and sodium hypochlorite to obtain sterilized seeds; S2, separating the immature embryos, crosscutting the immature embryos, and inducing callus generation and proliferation; S3, adjusting pre-culture time and Agrobacterium infection time of calli based on the callus generation and Agrobacterium growth to thereby prevent excessive Agrobacterium liquid; and S4, performing adventitious bud induction culture and rooting induction culture under a shielding-formed low-light environment to obtain tissue culture plantlets. It relates to a tissue culture method for immature embryos of sea barleygrass with high green spot differentiation and plantlet formation rates, which is not limited by materials. A transformation and regeneration system has high genetic transformation and mutation efficiency.

First claim

What is claimed is:

1. An Agrobacterium -mediated genetic transformation method for sea barleygrass, comprising:
step S1, selection and sterilization of immature embryo materials of sea barleygrass, which comprises: selecting the immature embryo materials of sea barleygrass with immature embryos each having a length in a range of 0.5-1.0 millimeters (mm), and sterilizing selected immature embryo materials with alcohol and sodium hypochlorite to obtain sterilized seeds;
step S2, separation and callus induction of the immature embryos, which comprises: separating the immature embryos from the sterilized seeds, crosscutting the immature embryos, and inducing callus generation and proliferation, thereby obtaining calli;
step S3, Agrobacterium infection and selective culture, which comprises: adjusting pre-culture time and Agrobacterium infection time of the calli based on the callus generation and Agrobacterium growth to thereby prevent excessive Agrobacterium liquid; wherein a culture medium for the selective culture is added with a mixture of ticarcillin and clavulanic acid and hygromycin; and
step S4, callus differentiation, plantlet formation, and positive plantlet detection, which comprises: performing adventitious bud induction culture and rooting induction culture under a shielding-formed low-light environment to thereby obtain tissue culture plantlets, and then performing deoxyribonucleic acid (DNA) extraction and polymerase chain reaction (PCR) detection on obtained tissue culture plantlets to obtain PCR detection results, and determining whether target fragments are amplified based on the PCR detection results, to thereby determine whether the tissue culture plantlets are positive plantlets; wherein an Agrobacterium tumefaciens recombinant strain used for transformation carries a gene editing vector, further comprising performing PCR amplification on a sequence near a target of each of the positive plantlets, and detecting mutation sites by Sanger sequencing, and a hmsos1sg1F sequencing primer consisting of the nucleotide sequence comprising SEQ ID NO: 4.

2. The method according to claim 1 , wherein in the step S1, the selection and sterilization of immature embryo materials of sea barleygrass further comprises:
selecting the immature embryo materials of sea barleygrass with plump seeds, gelatinous endosperms, and translucent immature embryos each having the length in the range of 0.5-1.0 mm, retaining awns and keeping seed coats of the immature embryo materials to be intact, sterilizing the immature embryo materials of sea barleygrass with 70% alcohol, then soaking sterilized immature embryo materials in a prepared sodium hypochlorite solution, and subsequently rinsing soaked immature embryo materials repeatedly with sterilized water, to thereby obtain the sterilized seeds.

3. The method according to claim 1 , wherein in the step S2, the separation and callus induction of the immature embryos further comprises:
taking the sterilized seeds obtained from the step S1, and placing taken sterilized seeds on a petri dish covered with a double-layer sterile filter paper, wherein a back of each of the sterilized seeds faces up and a tail of the awn faces away from an operator;
using a clamping method with tips of double pairs of tweezers to pull off an entire back seed coat of each of the sterilized seeds to expose the immature embryo of each of the sterilized seeds, wherein tips of a pair of left-hand tweezers of the double pairs of tweezers clamp the sterilized seed, and tips of a pair of right-hand tweezers of the double pairs of tweezers tear the awn;
piercing ½ of an endosperm of each of the exposed immature embryos with the tips of the pair of left-hand tweezers and fixing the sterilized seed without the entire back seed coat, and obliquely pressing an upper of the endosperm of the immature embryo with the tips of the pair of right-hand tweezers to peel off the immature embryo, to thereby obtain peeled-off immature embryos;
placing the peeled-off immature embryos on a surface of a callus induction culture medium with fronts face upward, crosscutting middles of the peeled-off immature embryos one by one with the tips of one of the double pairs of tweezers; and
culturing crosscut immature embryos in dark at 22° C. for 7 days, removing minor seedling buds with the tips of one of the double pairs of tweezers, continuing culturing in dark for 7 days, and then removing seedling buds and non-embryonic calli; and performing callus culture in dark for 2-3 weeks after replacing a new callus induction culture medium, to thereby obtain the calli;
wherein a subsequent operation is performed according to the step S3 when Agrobacterium infection is required to obtain transgenic plants;
wherein when the calli are directly used for the callus induction, yellow and dense embryogenic calli are taken from the calli obtain from the step S2 and cultured in dark at 22° C. for 3-4 weeks, a callus induction culture medium without hygromycin and the mixture of ticarcillin and clavulanic acid is replaced every two weeks, and then a subsequent operation is performed according to the step S4.

4. The method according to claim 1 , wherein in the step S3, the Agrobacterium infection and selective culture further comprises:
taking yellow and dense embryogenic calli from the calli obtained from the step S2, and pre-culturing the taken yellow and dense embryogenic calli at 22° C. for 1 week, to thereby obtain pre-cultured callus blocks;
taking callus blocks each with a diameter of 2-3 mm from the pre-cultured callus blocks, using a 20 microliters (L) pipettor to suck Agrobacterium liquid, dropwise adding sucked Agrobacterium liquid in middles of taken callus blocks to wrap the taken callus blocks, then immediately sucking back excess Agrobacterium liquid, placing a petri dish in a tilted manner with the taken callus blocks on a clean bench in a tilted manner, and replacing a new callus induction culture medium after excess Agrobacterium liquid is dried, to thereby obtain infected callus blocks; and
co-culturing the infected callus blocks for 36-48 hours, reducing a number of co-cultured callus blocks to 20-30 per petri dish, replacing two rounds of callus induction screening culture medium, and culturing in dark for 3-4 weeks, to thereby obtain final co-cultured callus blocks.

5. The method according to claim 4 , wherein the Agrobacterium liquid employs a MG basic culture solution without antibiotic, and a concentration OD 600 (abbreviation for optical density at 600 nanometers) of the Agrobacterium liquid is in a range of 0.5-0.6.

6. The method according to claim 1 , wherein in the step S4, the callus differentiation, plantlet formation, and positive plantlet detection further comprises:
covering a petri dish cover and a culture tube cover with single-layer A4 sized papers throughout in stages of the callus differentiation and the rooting induction culture since the calli are exposed to light, to thereby create the shielding-formed low-light environment;
taking yellow and dense callus blocks obtained in the step S2 or obtained in the step S3, 15-20 numbers of individual callus blocks per petri dish, and then culturing taken callus blocks for 2 weeks in a differentiation culture medium or a differentiation screening culture medium under the shielding-formed low-light environment;
removing whitish parts of callus blocks, selecting callus blocks with differentiated green spots, transferring selected callus blocks with the differentiated green spots to a new differentiation culture medium or a new differentiation screening culture medium, and then culturing for 2-4 weeks under the shielding-formed low-light environment;
when the differentiated green spots grow to be plantlets each with a length i

Assignees

• Univ Zhejiang

Inventors

• Kuang Liuhui
• Li Qi
• Wu Dezhi
• Zhang Guoping

Classifications

• A01H4/008

  Methods for regeneration to complete plants · CPC title

• A01H1/04

  Processes of selection {involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection} · CPC title

• A01H6/46

  Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize · CPC title

• Y02P60/40

  Afforestation or reforestation · CPC title

• A01H6/4624

  Hordeum vulgarus [barley] · CPC title