Method of hormone-free shoot tip culture for citrus

Abstract

A hormone-free culture method for a citrus shoot tip is provided, including: (1) selecting a citrus branch to make an explant; (2) washing with sterile water after disinfection; (3) inserting the explant into a Murashige and Skoog (MS) solid medium and culturing to produce a branch with new buds; (4) preparing a Murashge and Tucker (MT) liquid medium solution; (5) taking a shoot tip, and inoculating in a liquid medium for primary culture; (6) selecting a seed of a trifoliate orange or a citrange and peeling off a testa; washing with the sterile water after disinfection; (7) inoculating a resulting seed into a test tube containing the MS solid medium to obtain an etiolated seedling; (8) cutting a T-shaped incision and placing the shoot tip in a middle of the T-shaped incision to obtain a grafted seedling; and (9) conducting a secondary grafting.

Claims

1. A hormone-free culture method for a citrus shoot tip, comprising the following steps: (1) selecting an adult citrus branch, cutting off a leave from the adult citrus branch, washing a dust on a surface of a resulting adult citrus branch, and then cutting a washed adult citrus branch into a plurality of stem segments each with a length of 6-7 cm; (2) disinfecting each of the plurality of stem segments first with alcohol for 20-30 s and then with a sodium hypochlorite solution for 15-25 min to obtain a disinfected stem segment, then washing the disinfected stem segment with sterile water, and finally, removing surface water from the disinfected stem segment 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 the MS solid medium containing the sterilized stem segment in a first incubator for a branch culture for 10-15 days to allow the sterilized stem segment to grow a bud; (4) preparing a Murashge and Tucker (MT) liquid medium solution, and separately packaging after a sterilization to obtain a sterilized MT liquid medium solution, wherein a mass concentration of sucrose in the MT liquid medium solution is 7-8%; adding the sterilized 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 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 the liquid medium containing the shoot tip in a second 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 from the seed, disinfecting a resulting seed first with the alcohol for 20-30 s and then with the sodium hypochlorite solution for 15-25 min, and then washing a disinfected seed 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 the sterilized rootstock seed 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 the etiolated seedling with the diameter of the root neck of greater than or equal to 2 mm in a sterile culture dish; cutting off an upper part of a stem of the etiolated seedling with the diameter of the root neck of greater than or equal to 2 mm 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 the first incubator for a grafting culture, and then conducting a secondary grafting of the grafted seedling in a greenhouse when a scion bud grows to 0.5-1 cm.

2. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (2) and step (6), a mass concentration of the alcohol is 70-80%.

3. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (2) and step (6), a mass concentration of the sodium hypochlorite solution is 1-1.5%.

4. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (2), the disinfected stem segment is washed with the sterile water 3-5 times, and in step (6), the disinfected seed is washed with the sterile water 3-5 times.

5. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (1), each of the plurality of stem segments has 3-4 buds.

6. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (3) and step (9), a culture condition of the first incubator is 1600-2200 Lux of a light intensity, and 16 h of light at 25±0.5° C. and 8 h of darkness at 25±0.5° C. per 24 h.

7. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (5), the outer leave of the bud is removed and the resulting bud is cut into the shoot tip with the help of a stereoscope in a super-clean worktable.

8. The hormone-free culture method for the citrus shoot tip according to claim 1, wherein in step (5), a culture condition of the second incubator is 1600-2200 Lux of a light intensity, and 16 h of light at 40±0.5° C. and 8 h of darkness at 30±0.5° C. per 24 h.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a photo showing an appearance of branches growing new buds in embodiment 1 of the present invention;

[0046] FIGS. 2A-2B are photos showing an appearance of a shoot tip after a primary culture in embodiment 1 of the present invention, where FIG. 2A is cultured for 3 days, and FIG. 2B is cultured for 4 weeks;

[0047] FIG. 3 is a photo showing an appearance of etiolated seedlings in embodiment 1 of the present invention; and

[0048] FIGS. 4A-4B are photos showing an appearance of a micro-bud grafting and scion buds growing to 0.5 cm after the grafting in embodiment 1 of the present invention. In FIGS. 4A-4B, FIG. 4A is a grafted seedling that completed the micro-bud grafting, and FIG. 4B is the grafted seedlings with the scion buds growing to 0.5 cm.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] Citrus varieties such as Ehime Kashi No. 28 28, Navvelina, Citrus reticulata ‘Chun Jian’, and Newhall oranges are adopted in the embodiments of the present invention.

[0050] The survival rate of culturing is at least 95% in the embodiments of the present invention.

[0051] The adult citrus branch grows well and has no obvious disease spot on the appearance in the embodiments of the present invention.

[0052] A full and healthy seed of the trifoliate orange or the citrange is selected in the embodiments of the present invention.

[0053] The condition of the culturing in the dark is culturing at 25±0.5° C. for 24 h without light in the embodiments of the present invention.

Embodiment 1

[0054] (1) An adult citrus branch is selected, leaves are cut off, the dust on the surface of the adult citrus branch are wahsed, and then the adult citrus branch is cut into 7 cm long stem segments. Each stem segment has 4 buds. [0055] (2) First the stem segment is disinfected with alcohol for 30 s, then disinfected with the sodium hypochlorite solution for 25 min, then washed with sterile water, and finally, surface water of the stem segment is removed with the sterile filter paper to obtain a sterilized stem segment. The mass concentration of the alcohol is 70%, the mass concentration of the sodium hypochlorite solution is 1%, and the stem segment is washed with sterile water 5 times. [0056] (3) The sterilized stem segment is inserted into the MS solid medium, and then placed in an incubator for the branch culture for 15 days to allow the stem segment to grow a new bud. The culture condition of the incubator is 1600 Lux of the light intensity, and 16 h of light at 25±0.5° C. and 8 h of darkness at 25±0.5° C. per 24 h.

[0057] The appearance of the stem segment with new buds is shown in FIG. 1. [0058] (4) The MT liquid medium solution is prepared and separately packaged after sterilization where the mass concentration of sucrose is 8%. The liquid medium solution is added into a culture dish to prepare a liquid medium, and the liquid medium is sealed with a sealing membrane for later use. The MT liquid medium solution is separately packaged by 100 ml/bottle after sterilization. The culture dish is a glass culture dish with a diameter of 60 mm after sterilization. The liquid medium solution is added into several culture dishes on the super-clean worktable. An amount of the liquid medium solution added to each culture dish is 3 ml. [0059] (5) The new bud is taken and the outer leave of the bud is removed, and a shoot tip with a length of 0.25 mm is cut by a sterile blade. The shoot tip is inoculated in the liquid medium and then placed in an incubator for a primary culture for 40 days. The outer leave of the new bud is removed and the resulting bud is cut into the shoot tip with the help of a stereoscope in a super-clean worktable. The culture condition of the incubator is 1600 Lux of the light intensity, and 16 h of light at 40±0.5° C. and 8 h of darkness at 30±0.5° C. per 24 h.

[0060] The shoot tip cultured 3 days and 4 weeks of the primary culture is shown in FIGS. 2A and 2B, respectively. [0061] (6) A seed of a trifoliate orange is selected and peeled off a testa, first the resulting seed is disinfected with the alcohol for 30 s, then disinfected with the sodium hypochlorite solution for 25 min, and then washed with sterile water to obtain a sterilized rootstock seed. The mass concentration of the alcohol is 70%. The mass concentration of the sodium hypochlorite solution is 1%. Washed with sterile water 5 times. Disinfecting and washing are carried out on the super-clean worktable. [0062] (7) The sterilized rootstock seed is inoculated into a test tube containing the MS solid medium and cultured in the dark for 14 days to obtain an etiolated seedling with a height of 7-8 cm.

[0063] The appearance of the etiolated seedling cultured in the dark for 4, 8, 12, and 14 days is shown in FIG. 3. [0064] (8) The etiolated seedling with a diameter of a root neck of greater than or equal to 2 mm is selected and placed on a sterile culture dish. An upper part of the stem of the etiolated seedling is cut off and a T-shaped incision at the position of 3-5 mm from the upper end of the resulting stem is cut. The shoot tip after the primary culture is placed in the middle of the T-shaped incision to obtain a grafted seedling. Cutting is carried out on the super-clean worktable. [0065] (9) The grafted seedling is placed in an incubator for a grafting culture on the super-clean worktable, and then a secondary grafting is conducted in the greenhouse when the scion bud grows to 0.8 cm. The culture condition of the incubator is 1600 Lux of the light intensity, and 16 h of light at 25±0.5° C. and 8 h of darkness at 25±0.5° C. per 24 h.

[0066] The appearance of the micro-bud grafting and scion buds growing to 0.5 cm after the grafting is shown in FIGS. 4A-4B.

Embodiment 2

[0067] This embodiment differs from Embodiment 1 in the following: [0068] (1) 6 cm long stem segments are cut and each stem segment has 3 buds. [0069] (2) Disinfect with alcohol for 25 s, then disinfect with sodium hypochlorite solution for 20 min. The mass concentration of the alcohol is 75%. The mass concentration of the sodium hypochlorite solution is 1.3%. Washed with sterile water 4 times. [0070] (3) The stem segment is cultured for 13 days, and the light intensity of the incubator is 2000 Lux. [0071] (4) The mass concentration of sucrose is 8% in the MT liquid medium solution. [0072] (5) The new bud is taken and the outer leave of the bud is removed, and a shoot tip with a length of 0.2 mm is cut by a sterile blade. The primary culture lasts for 30 days. The light intensity of the incubator is 2000 Lux. [0073] (6) A seed of the citrange is selected, disinfected with alcohol for 25 s, then disinfected with the sodium hypochlorite solution for 20 min. The mass concentration of the alcohol is 75%. The mass concentration of the sodium hypochlorite solution is 1.3%. Washed with sterile water 4 times. [0074] (7) Cultured in the dark for 13 days to obtain an etiolated seedling with a height of 6-7 cm. [0075] (8) A T-shaped incision at the position of 3 mm from the upper end of the stem is cut. [0076] (9) A secondary grafting is conducted when the scion bud grows to 0.5 cm. The light intensity of the incubator is 2000 Lux.

Embodiment 3

[0077] This embodiment differs from Embodiment 1 in the following: [0078] (1) 6.5 cm long stem segments are cut, and each stem segment has 3 buds. [0079] (2) Disinfect with alcohol for 20 s, then disinfect with sodium hypochlorite solution for 15 min. The mass concentration of the alcohol is 80%. The mass concentration of the sodium hypochlorite solution is 1.5%. Washed with sterile water 3 times. [0080] (3) The stem segment is cultured for 10 days, and the light intensity of the incubator is 2200 Lux. [0081] (4) The mass concentration of sucrose is 7% in the MT liquid medium solution. [0082] (5) The new bud is taken and the outer leave of the bud is removed, and a growing point of the shoot tip with a length of 0.15 mm is cut by a sterile blade. The primary culture lasts for 25 days. The light intensity of the incubator is 2200 Lux. [0083] (6) A seed of the citrange is selected, disinfected with alcohol for 20 s, then disinfected with the sodium hypochlorite solution for 15 min. The mass concentration of the alcohol is 80%. The mass concentration of the sodium hypochlorite solution is 1.5%. Washed with sterile water 3 times. [0084] (7) Cultured in the dark for 15 days to obtain an etiolated seedling with a height of 8-9 cm. [0085] (8) A T-shaped incision at the position of 5 mm from the upper end of the stem is cut. [0086] (9) A secondary grafting is conducted when the scion bud grows to 1 cm. The light intensity of the incubator is 2200 Lux.