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

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.