Hormone-free culture method for citrus shoot tip

A hormone-free culture method for a citrus shoot tip, comprising the following steps:

• (1) selecting an adult citrus branch, cutting off a leave, washing a dust on a surface of the adult citrus branch, and then cutting into a stem segment with a length of 6-7 cm;
• (2) disinfecting the stem segment first with alcohol for 20-30 s and then with a sodium hypochlorite solution for 15-25 min, then washing with sterile water, and finally, removing surface water with a sterile filter paper to obtain a sterilized stem segment;
• (3) inserting the sterilized stem segment into a Murashige and Skoog (MS) solid medium, and then placing in an incubator for a branch culture for 10-15 days to allow the sterilized stem segment to grow a new bud;
• (4) preparing a Murashge and Tucker (MT) liquid medium solution, and separately packaging after sterilization, wherein a mass concentration of sucrose in the MT liquid medium solution is 7-8%;
• adding the MT liquid medium solution into a culture dish to prepare a liquid medium, and sealing the liquid medium with a sealing membrane for later use;
• (5) removing an outer leave of the new bud, and cutting a shoot tip with a length of 0.15-0.25 mm from a resulting bud by a sterile blade;
• inoculating the shoot tip in the liquid medium and then placing in an incubator for a primary culture for 25-40 days;
• (6) selecting a seed of a trifoliate orange or a citrange and peeling off a testa, disinfecting first with the alcohol for 20-30 s and then with the sodium hypochlorite solution for 15-25 min, and then washing with the sterile water to obtain a sterilized rootstock seed;
• (7) inoculating the sterilized rootstock seed into a test tube containing the MS solid medium and culturing in dark for 13-15 days to obtain an etiolated seedling with a height of 6-9 cm;
• (8) selecting an etiolated seedling with a diameter of a root neck of greater than or equal to 2 mm and placing in a sterile culture dish;
• cutting off an upper part of a stem of the etiolated seedling and cutting a T-shaped incision at a position 3-5 mm from an upper end of a resulting stem;
• placing the shoot tip after the primary culture in a middle of the T-shaped incision to obtain a grafted seedling;
• and
• (9) placing the grafted seedling in an incubator for a grafting culture, and then conducting a secondary grafting in a greenhouse when a scion bud grows to 0.5-1 cm.