Method for obtaining regenerated seedlings of Brassica campestris L.ssp.chinensis from embryonic tip tissue

Patent metadata

Field	Value
Publication number	US-12201076-B2
Application number	US-202418651494-A
Country	US
Kind code	B2
Filing date	Apr 30, 2024
Priority date	Jun 8, 2023
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

Abstract

A method for obtaining regenerated seedlings of Brassica campestris L. ssp. chinensis from embryonic tip tissues, including the following steps. A seed is inoculated to a germination medium for dark culture for 60 h. The testa, root tip, two cotyledons and middle growing point of the resultant germinated seed are removed, and an embryonic tip with a length of 3-5 mm is retained as an explant. The explant is sequentially subjected to low-temperature pre-culture in a pre-culture medium for 36 h, room-temperature shaking culture in a liquid bud induction culture medium for 10 min, and bud induction culture in a bud induction culture medium for 20 d. A regenerated plant with 5-6 leaves is transferred to a rooting medium for rooting culture for about 2 weeks, and a well-rooted plant is collected, and subjected to hardening and transplantation into a filed.

First claim

What is claimed is:

1. A method for obtaining regenerated seedlings of Brassica campestris L. ssp. chinensis from an embryonic tip tissue, comprising:
(S1) obtaining an aseptic seed of Brassica campestris L. ssp. chinensis;
(S2) inoculating the aseptic seed obtained in step (S1) into a germination culture medium for dark culture for 60 hours, wherein the germination culture medium is a Murashige and Skoog (MS) culture medium containing 3 wt. % of sucrose, 1.0 mg/L of 6-benzylaminopurine (6-BA) and 0.8 wt. % agar, and has a pH of 5.8, and the dark culture is performed at a day temperature of 24° C., a night temperature of 18° C. and a humidity of 80%;
(S3) collecting a germinated seed, removing testa, root tip, two cotyledons and middle growing point from the germinated seed, retaining an embryonic tip with a length of 3-5 mm as an explant;
(S4) subjecting the explant to pre-culture in a pre-culture medium for 36 hours to obtain a primarily-cultured explant, wherein the pre-culture is performed at a day temperature of 18° C., a night temperature of 12° C., and a humidity of 80%, and wherein the pre-culture medium is an MS culture medium containing 3 wt. % of sucrose, 2.0 mg/L of 6-BA, 0.4 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 wt. % of agar, and has a pH of 5.8;
(S5) subjecting the primarily-cultured explant to shaking culture in a first bud induction culture medium to obtain a secondarily-cultured explant, wherein the shaking culture is performed at room temperature under a rotating speed of 180 rpm for 10 min, and wherein the first bud induction culture medium is an MS culture medium containing 3 wt. % of sucrose, 0.8 mg/L of 6-BA, 0.2 mg/L of naphthaleneacetic acid (NAA) and 2 mg/L of isopentenyladenine, and has a pH of 5.8;
(S6) transferring the secondarily-cultured explant to a second bud induction culture medium for bud induction, wherein the second bud induction culture medium is an MS culture medium containing 0.8 mg/L of 6-BA, 0.2 mg/L of NAA, 0.8 wt. % of agar and 3 wt. % of sucrose, and has a pH of 5.8, and wherein culture conditions of the bud induction are listed as follows: culture time: 20 days; day temperature: 24° C.; night temperature: 18° C.; day light-exposure length: 16 hours; night light-exposure length: 8 hours; and humidity: 80%;
(S7) collecting a regenerated plant with 5-6 leaves, and transferring the regenerated plant to a rooting culture medium for rooting culture, wherein the rooting culture medium is an MS culture medium containing 3 wt. % of sucrose, 1.0 mg/L of indole butyric acid (IBA) and 0.8 wt. % of agar, and has a pH of 5.8, and wherein culture conditions of the rooting culture are listed as follows: culture time: 2 weeks; day temperature: 24° C.; night temperature: 18° C.; day light-exposure length: 16 hours; night light-exposure length: 8 hours; and humidity: 80%; and
(S8) collecting a rooted plant, and subjecting the rooted plant to hardening followed by transplantation into a field.

Assignees

• Univ Anhui Agricultural
• Yingshang Shili Ecological Agriculture Tech Co Ltd

Inventors

• Chen Guohu
• Yin Qian
• Li Ting
• Wang Chenggang
• Tang Xiaoyan
• Wang Ying
• Liu Xueqing
• Wen Hongwei
• Wu Siwen

Classifications

• A01H4/002 Primary

  Culture media for tissue culture · CPC title

• A01H4/005

  Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques · CPC title

• Y02P60/40

  Afforestation or reforestation · CPC title

• A01H4/008 Primary

  Methods for regeneration to complete plants · CPC title