METHOD FOR DISINFECTING EXPLANTS OF KADSURA COCCINEA STEMS WITH BUDS AND METHOD FOR DIRECTLY INDUCING RAPID PROLIFERATION OF STERILE BUDS BY USING EXPLANTS OF KADSURA COCCINEA STEMS WITH BUDS

Abstract

A method for disinfecting explants of Kadsura coccinea stems with buds and a method for directly inducing rapid proliferation of sterile buds by using the explants of Kadsura coccinea stems with buds involve processes such as selection, treatment and disinfection of explants, primary culture, subculture proliferation culture. The problem of difficulty in tissue culture and primary culture of Kadsura coccinea stems is solved, and the advantages include low contamination rate of explants, high propagation rate and robust proliferation of axillary buds. This allows obtaining sterile axillary buds of Kadsura coccinea through tissue culture, and provides support for tissue culture, rapid propagation and factory seedling of Kadsura coccinea in the future.

Claims

1. A method for disinfecting an explant of a Kadsura coccinea stem with buds, sequentially comprising the following steps: (1) obtaining the explant: transplanting Kadsura coccinea cultivated in a field indoors, cutting off stems and leaves of overground parts, and using g germinated semi-lignified stem with buds as the explant after 3-5 months; (2) treating the explant: cutting the germinated semi-lignified stem with the buds into 3-4 cm long stems, wherein each of the 3-4 cm long stems is a semi-lignified Kadsura coccinea stem containing axillary bud, washing the semi-lignified Kadsura coccinea stem with a detergent 1-2 times, and then rinsing the semi-lignified Kadsura coccinea stem with running water for at least 1 hour; and (3) disinfecting the explant: soaking the semi-lignified Kadsura coccinea stem after rinsing with running water in 75% alcohol for 1 minute and in another 75% alcohol for 1 minute, rinsing the semi-lignified Kadsura coccinea stem with sterile water 3-4 times, disinfecting the semi-lignified Kadsura coccinea stem with 0.1% mercuric chloride for 45 minutes, and finally rinsing the semi-lignified Kadsura coccinea stem with sterile water 5-6 times to obtain a disinfected explant of the Kadsura coccinea stem with the buds, wherein the 0.1% mercuric chloride is changed every 15 minutes, 3 times in total.

2. The method according to claim 1, wherein step (1) comprises transplanting biennial to triennial Kadsura coccinea cultivated in the field into pots indoors from January to April, cutting off the stems and the leaves of the overground parts, and using the germinated semi-lignified stem with the buds as test materials after 3-5 months.

3. The method according to claim 2, wherein the pots in step (1) each have a height of 25-30 cm and a diameter of 20-25 cm.

4. A method for directly inducing rapid proliferation of buds in a disinfected explant of a Kadsura coccinea stem containing the buds, comprising 1) inducing axillary buds: inoculating the disinfected explant of the Kadsura coccinea stem containing the buds into a first culture medium of ½ MS+2.0-3.0 mg/L 6-BA+0.2 mg/L IAA to induce the axillary buds, culturing the disinfected explant of the Kadsura coccinea stem in dark for 2 days, and then transferring the disinfected explant of the Kadsura coccinea stem to culture under light conditions; and 2) performing subculture proliferation: cutting and inoculating the axillary buds obtained after a stem differentiation in step 1 into a second culture medium of ½ MS+2.0-3.0 mg/L 6-BA+0.1-0.5 mg/L IBA for proliferation culture of the axillary buds.

5. The method according to claim 4, wherein during step 1, the disinfected explant of the Kadsura coccinea stem is cultured in light dark for 2 days and then transferred to culture under the light conditions for 30-35 days.

6. The according to claim 4, wherein for the light conditions, a culture temperature is 26±1° C., a light culture intensity is 2100-2200 lx, and a light culture time 12-14 h/d.

7. The method according to claim 4, wherein the first culture medium for inducing the axillary buds is ½ MS+2.0 mg/L 6-BA+0.2 mg/L IAA.

8. The method according to claim 4, wherein the second culture medium for the subculture proliferation is ½ MS+3.0 mg/L 6-BA+0.1-0.5 mg/L IBA.

9. The method according to claim 4, wherein the first culture medium and the second culture medium are each additionally provided with 30 g/L sucrose and 7 g/L agar, and a pH adjusted to 5.5-5.8.

10. The method according to claim 4, wherein the disinfected explant of the Kadsura coccinea stem containing the buds is disinfected using a method comprising the following steps: (1) obtaining an explant: transplanting Kadsura coccinea cultivated in a field indoors, cutting off stems and leaves of overground parts, and using a germinated semi-lignified stem with buds as the explant after 3-5 months; (2) treating the explant: cutting the germinated semi-lignified stem with the buds into 3-4 cm long stems, wherein each of the 3-4 cm long stems is a semi-lignified Kadsura coccinea stem containing one axillary bud, washing the semi-lignified Kadsura coccinea stem with a detergent 1-2 times, and then rinsing the semi-lignified Kadsura coccinea stem with running water for at least 1 hour; and (3) disinfecting the explant: soaking the semi-lignified Kadsura coccinea stem after rinsing with running water in 75% alcohol for 1 minute and in another 75% alcohol for 1 minute, rinsing the semi-lignified Kadsura coccinea stem with sterile water 3-4 times, disinfecting the semi-lignified Kadsura coccinea stem with 0.1% mercuric chloride for 45 minutes, and finally rinsing the semi-lignified Kadsura coccinea stem with sterile water 5-6 times to obtain the disinfected explant of the Kadsura coccinea stem containing the buds, wherein the 0.1% mercuric chloride is changed every 15 minutes, 3 times in total.

11. The method according to claim 10, wherein step (1) comprises transplanting biennial to triennial Kadsura coccinea cultivated in the field into pots indoors from January to April, cutting off the stems and the leaves of the overground parts, and using the germinated semi-lignified stem with the buds as test materials after 3-5 months.

12. The method according to claim 11, wherein the pots in step (1) each have a height of 25-30 cm and a diameter of 20-25 cm.

13. The method according to claim 5, wherein the disinfected explant of the Kadsura coccinea stem containing the buds is disinfected using a method comprising the following steps: (1) obtaining an explant: transplanting Kadsura coccinea cultivated in a field indoors, cutting off stems and leaves of overground parts, and using a germinated semi-lignified stem with buds as the explant after 3-5 months; (2) treating the explant: cutting the germinated semi-lignified stem with the buds into 3-4 cm long stems, wherein each of the 3-4 cm long stems is a semi-lignified Kadsura coccinea stem containing one axillary bud, washing the semi-lignified Kadsura coccinea stem with a detergent 1-2 times, and then rinsing the semi-lignified Kadsura coccinea stem with running water for at least 1 hour; and (3) disinfecting the explant: soaking the semi-lignified Kadsura coccinea stem after rinsing with running water in 75% alcohol for 1 minute and in another 75% alcohol for 1 minute, rinsing the semi-lignified Kadsura coccinea stem with sterile water 3-4 times, disinfecting the semi-lignified Kadsura coccinea stem with 0.1% mercuric chloride for 45 minutes, and finally rinsing the semi-lignified Kadsura coccinea stem with sterile water 5-6 times to obtain the disinfected explant of the Kadsura coccinea stem containing the buds, wherein the 0.1% mercuric chloride is changed every 15 minutes, 3 times in total.

14. The method according to claim 13, wherein step (1) comprises transplanting biennial to triennial Kadsura coccinea cultivated in the field into pots indoors from January to April, cutting off the stems and the leaves of the overground parts, and using the germinated semi-lignified stem with the buds as test materials after 3-5 months.

15. The method according to claim 14, wherein the pots in step (1) each have a height of 25-30 cm and a diameter of 20-25 cm.

16. The method according to claim 6, wherein the disinfected explant of the Kadsura coccinea stem containing the buds is disinfected using a method comprising the following steps: (1) obtaining an explant: transplanting Kadsura coccinea cultivated in a field indoors, cutting off stems and leaves of overground parts, and using a germinated semi-lignified stem with buds as the explant after 3-5 months; (2) treating the explant: cutting the germinated semi-lignified stem with the buds into 3-4 cm long stems, wherein each of the 3-4 cm long stems is a semi-lignified Kadsura coccinea stem containing one axillary bud, washing the semi-lignified Kadsura coccinea stem with a detergent 1-2 times, and then rinsing the semi-lignified Kadsura coccinea stem with running water for at least 1 hour; and (3) disinfecting the explant: soaking the semi-lignified Kadsura coccinea stem after rinsing with running water in 75% alcohol for 1 minute and in another 75% alcohol for 1 minute, rinsing the semi-lignified Kadsura coccinea stem with sterile water 3-4 times, disinfecting the semi-lignified Kadsura coccinea stem with 0.1% mercuric chloride for 45 minutes, and finally rinsing the semi-lignified Kadsura coccinea stem with sterile water 5-6 times to obtain the disinfected explant of the Kadsura coccinea stem containing the buds, wherein the 0.1% mercuric chloride is changed every 15 minutes, 3 times in total.

17. The method according to claim 16, wherein step (1) comprises transplanting biennial to triennial Kadsura coccinea cultivated in the field into pots indoors from January to April, cutting off the stems and the leaves of the overground parts, and using the germinated semi-lignified stem with the buds as test materials after 3-5 months.

18. The method according to claim 17, wherein the pots in step (1) each have a height of 25-30 cm and a diameter of 20-25 cm.

19. The method according to claim 7, wherein the disinfected explant of the Kadsura coccinea stem containing the buds is disinfected using a method comprising the following steps; (1) obtaining an explant: transplanting Kadsura coccinea cultivated in a field indoors, cutting off stems and leaves of overground parts, and using a germinated semi-lignified stem with buds as the explant after 3-5 months; (2) treating the explant; cutting the germinated semi-lignified stem with the buds into 3-4 cm long stems, wherein each of the 3-4 cm long stems is a semi-lignified Kadsura coccinea stem containing one axillary bud, washing the semi-lignified Kadsura coccinea stem with a detergent 1-2 times, and then rinsing the semi-lignified Kadsura coccinea stem with running water for at least 1 hour; and (3) disinfecting the explant: soaking the semi-lignified Kadsura coccinea stem after rinsing with running water in 75% alcohol for 1 minute and in another 75% alcohol for 1 minute, rinsing the semi-lignified Kadsura coccinea stem with sterile water 3-4 times, disinfecting the semi-lignified Kadsura coccinea stem with 0.1% mercuric chloride for 45 minutes, and finally rinsing the semi-lignified Kadsura coccinea stem with sterile water 5-6 times to obtain the disinfected explant of the Kadsura coccinea stem containing the buds, wherein the 0.1% mercuric chloride is changed every 15 minutes, 3 times in total.

20. The method according to claim 19, wherein step (1) comprises transplanting biennial to triennial Kadsura coccinea cultivated in the field into pots indoors from January to April, cutting off the stems and the leaves of the overground parts, and using the germinated semi-lignified stem with the buds as test materials after 3-5 months.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 is a photograph showing contamination of a stem with a bud of Kadsura coccinea cultivated in a field in an early stage of inoculation according to the present invention;

[0027] FIG. 2 is a photograph showing browning of a stem with a bud of Kadsura coccinea cultivated in a field when the disinfection time is long according to the present invention;

[0028] FIG. 3 is a photograph showing contamination of a stem with a bud of Kadsura coccinea cultivated in a field when culture is performed for 1 month according to the present invention;

[0029] FIG. 4 is a photograph showing germinated new shoots when seedlings of Kadsura coccinea cultivated in a field are transplanted into a laboratory pot according to the present invention;

[0030] FIG. 5 is a photograph showing potted seedlings of Kadsura coccinea when primary culture is performed for 10 days according to the present invention;

[0031] FIG. 6 and FIG. 7 are photographs showing potted seedlings of Kadsura coccinea when primary culture is performed for 20 days according to the present invention;

[0032] FIG. 8 and FIG. 9 are photographs showing potted seedlings of Kadsura coccinea when primary culture is performed for 30 days according to the present invention;

[0033] FIG. 10, FIG. 11, and FIG. 12 are photographs showing potted seedlings of Kadsura coccinea when subculture proliferation culture is performed for 10 days according to the present invention; and

[0034] FIG. 13 is a photograph showing whole potted seedlings of Kadsura coccinea during primary culture according to the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The following embodiment is used to further illustrate the present invention, but not to limit the present invention.

Embodiment 1

[0036] Following operations are performed in sequence:

[0037] 1. Transplanting of field materials: Biennial to triennial Kadsura coccinea cultivated in a field were transplanted into plastic pots with a height of 30 cm and a diameter of 25 cm from January to April each year, with stems and leaves of the overgrounds cut off, watered and brought into a greenhouse on the roof of a laboratory of a tree building of Central South University of Forestry and Technology for normal water and fertilizer management for 3-5 months, and germinated semi-lignified stems with buds were used as test materials in the present invention.

[0038] 2. Treatment of explants: The semi-lignified stems with buds were cut into 3-4 cm long stems, each containing one axillary bud, and the stems were washed with a detergent 1-2 times in a breaker and then rinsed with running water for at least 1 hour to rinse out mucus secreted by the stems for use.

[0039] 3. Disinfection treatment of the explants: The semi-lignified Kadsura coccinea stems were selected in a sunny morning from July to October in 2018 to 2019 and brought to the laboratory, after the leaves were removed, the collected stems were rinsed with running water 1-2 times, rinsed with an appropriate amount of a detergent solution 1-2 times, repeatedly rinsed with running water for 60 minutes to rinse out the mucus secreted by the stems, placed on an ultra-clean workbench and disinfected with 75% alcohol, a 0.1% mercuric chloride solution and a 5% sodium hypochlorite solution. Different time gradients were set, the materials collected directly from the field were shown in Table 1, and the materials collected on the roof were shown in Table 2. The stems were stirred constantly with tweezers during disinfection and rinsed with sterile water 5-6 times, water on surfaces of the stems is absorbed with sterile filter paper, browned parts at both ends are cut off with a sterile knife, each stem with one bud was inoculated into a culture medium, and the contamination rate and the browning rate are counted.

TABLE-US-00001 TABLE 1 Disinfection effects of different disinfection treatment on stems of Kadsura coccinea cultivated in field 0.1% Induced 5% sodium mercuric Contamination Browning survival rate 75% hypochlorite chloride rate rate of axillary buds Number alcohol(s) (min) (min) (%) (%) (%) A.sub.1 1.5 0 30 100 0 0 A.sub.2 1.5 0 35 90.47 9.5 0 A.sub.3 1.5 0 40 82.61 17.19 0 A.sub.4 2 0 35 88.24 11.76 0 A.sub.5 2 0 40 74.07 25.93 0 A.sub.6 2 0 45 63.98 36.02 0 A.sub.7 2 0 50 45.62 54.38 0 A.sub.8 9 23 0 100 0 0 A.sub.9 2 25 0 100 0 0 A.sub.10 3 0 35 53.85 46.15 0 A.sub.11 3 0 40 43.52 56.58 0

TABLE-US-00002 TABLE 2 Disinfection effects of different disinfection treatment on potted Kadsura coccinea stems 0.1% 5% sodium mercuric Contamination Browning Induction rate 75% hypochlorite chloride rate rate of axillary buds Number alcohol(s) (mm) (min) (%) (%) (%) A.sub.1 1.5 0 30 100 0 0 A.sub.2 1.5 0 35 65.4 16.6 18.0 A.sub.3 1.5 0 40 53.1 14.3 32.6 A.sub.4 2 0 30 72.6 7.2 20.2 A.sub.5 2 0 35 58.2 16.8 25.0 A.sub.6 2 0 40 43.0 20.4 36.6 A.sub.7 2 0 45 18.5 21.7 59.8 A.sub.8 2 0 50 10.5 33.7 55.8 A.sub.9 2 25 0 84.1 15.9 0 A.sub.10 3 0 40 38.6 28.3 33.1 A.sub.11 3 0 45 15.1 30.5 54.4

[0040] It is finally confirmed that a preferred disinfection method of the present invention includes the following steps:

[0041] (1) obtaining materials; transplanting Kadsura coccinea cultivated in a field indoors, and using germinated semi-lignified stems with buds as test materials;

[0042] (2) treating explants, cutting the semi-lignified stems with buds into 3-4 cm long stems, each containing one axillary bud, washing the stems with a detergent 2 times, and then rinsing the stems with running water for 1 hour; and

[0043] (3) disinfecting the explants: soaking the semi-lignified Kadsura coccinea stems with buds after rinsing with running water in 75% alcohol for 1 minute and in another 75% alcohol for 1 minute, rinsing the stems with sterile water 4 times, disinfecting the stems with 0.1% mercuric chloride for 45 minutes, and finally rinsing the stems with sterile water 5-6 times, where mercuric chloride is changed every 15 minutes, 3 times in total.

[0044] The disinfected explants are inoculated into a ½MS culture medium according to a hormone formula in Table 3 for induction of axillary buds, cultured in dark for 2 days and then cultured under light conditions for 30-35 days, and the highest induction rate can reach 46.15%.

[0045] 30 g/L sucrose and 7 g/L agar are additionally provided, and the pH is 5.5-5.8. The culture temperature is 26±1° C., the light intensity is 2100-2200 lx, and the light time is 12-14 h/d; (see FIG. 5), and the proliferation coefficient after primary culture can reach 8 or above.

TABLE-US-00003 TABLE 3 Effects of different plant growth regulators on primary culture of Kadsura coccinea stems Induction Number of 6-BA IAA GA.sub.3 rate of axillary concentration/ concentration/ concentration/ axillary buds Number mg/L mg/L mg/L buds/% (pieces) Growth conditions B.sub.1 0 0 0 0 0 No axillary buds grow B.sub.2 0 0.1 0 5.53 1.0 Axillary buds are weak, thin and yellow B.sub.3 0 0.5 0 10.67 1.0 Axillary buds are slender and yellow B.sub.4 1.0 0.1 0 35.39 5.0 Axillary buds are short, and callus tissues grow in stem base parts at the upper end of the culture medium B.sub.5 1.0 1.0 0 31.07 3.0 Axillary buds are tender and slender B.sub.6 2.0 0.2 0 46.15 8.2 Axillary buds are robust and grow well with many cluster buds B.sub.7 2.0 0.5 0 37.5 7.0 Callus tissues grow in stem base parts, and axillary buds are green B.sub.8 2.0 1.0 0 32.14 6.0 Axillary buds are short, and leaves are small and a little curly with red front parts B.sub.9 3.0 0.2 0 53.12 7.6 Stem base parts are big with a few of callus tissues, and many axillary buds grow B.sub.10 3.0 0.5 0 48.50 4.0 Axillary buds are tight, and stem base parts are big and green B.sub.11 3.0 1.0 0 32.14 3.5 A large number of callus tissues grow in stem base parts B.sub.12 0 0 1.0 10.35 1.0 Axillary buds are short and brittle with red front parts B.sub.13 0 0 2.0 27.14 1.0 Callus tissues grow in base parts, and buds grow poorly

[0046] The axillary buds are subjected to subculture proliferation culture. The axillary buds P obtained after primary culture are cut and inoculated into a ½MS culture medium according to a hormone formula in Table 4 for proliferation culture under light conditions for 30-35 days, and the highest proliferation coefficient is 8.5. 30 g/L sucrose and 7 g/L agar are additionally provided, and the pH is 5.5-5.8. The culture temperature is 26±1° C., the light intensity is 2100-2200 lx, and the light time is 12-14 h/d (see FIG. 10 to FIG. 12).

TABLE-US-00004 TABLE 4 Effects of different plant growth regulators on subculture proliferation culture of potted Kadsura coccinea ZT IAA 2ip IBA 6-BA concentration/ concentration/ concentration/ concentration/ concentration/ Proliferation Number mg/L mg/L mg/L mg/L mg/L coefficient C.sub.1 1 0.1 1 0 0 1.0 C.sub.2 2 0.1 1 0 0 2.0 C.sub.3 2 0.2 2 0 0 4.0 C.sub.4 0 0 0 0.1 1 3.2