Method for establishing racomitrium japonicum gametophyte regeneration system

Abstract

The invention discloses a method for establishing a racomitrium japonicum gametophyte regeneration system. The method provided by the invention comprises the following steps of shearing racomitrium japonicum subjected to appropriate culture into small racomitrium japonicum segments from top to bottom, carrying out surface disinfection, inoculating a conventional MS culture medium with the sterilized small racomitrium japonicum segments, carrying out culture, carrying out screening to obtain aseptic explants, transferring the aseptic explants to a medium comprising a conventional MS medium and30g / L of cane sugar, inducing gametophytes to produce protonemata, transferring the protonemata to a propagation medium, carrying out culture for 50 days, transferring the protonemata obtained by theprevious step to a medium for producing gametophytes by induction, and carrying out culture for 70 days. The method for establishing a racomitrium japonicum gametophyte regeneration system is a novelmethod, has simple processes and a low cost, can realize propagation of a mass of racomitrium japonicum gametophytes in a short time, and guarantees the success of follow-up experiments.

Description

technical field

[0001] The invention relates to a method for establishing a gametophyte regeneration system of East Asian moss moss. Background technique

[0002] East Asian sand moss ( Racomitrium japonicum ) belongs to the genus Grimmiaceae (Grimmiaceae) Racomitrium ), born on rock face, rock face thin soil and sandy soil in low altitude areas. Widely distributed in China's Heilongjiang, Jilin, Liaoning, Henan, Jiangxi and other northern and southern provinces, Japan, North Korea, Russia (southeast of Siberia), Vietnam, Australia and other places are also distributed. In the long-term biological evolution process, most species in the family Aeruginaceae have developed the characteristics of adapting to the xerophytic environment in terms of community, stem, leaf and rhizoid structure and physiology. At present, in foreign countries, mosses in bryophytes have become ideal experimental materials for the study of plant molecular biology and model plants for studying gene ...

Examples

Embodiment 1

[0023] Take the East Asian sand moss that has been cultivated for a period of time, cut it into small pieces of 0.5-1cm from the top down, soak it in 2% detergent solution for 10 minutes, and then treat it with 0.02% mercury chloride for 45 seconds to carry out surface disinfection; after disinfection, inoculate Cultivate on regular MS + 0-40 g / L sucrose, improved Beneck + 0-40 g / L sucrose, and improved Knop + 0-40 g / L sucrose medium, respectively. The culture conditions are: light time 12h, light intensity 3000lux, culture temperature 20±2℃.

[0024] The cultivation results are as follows:

[0025] On the mediums of improved Beneck + 0-40 g / L sucrose and improved Knop + 0-40 g / L sucrose, the gametophyte infection rate is particularly high, mainly Penicillium and Rhizopus, and on this basis, increase disinfection The concentration of the solution and the disinfection time also obtained the same results. If the disinfection time and concentration were too high, the gametophyte...

Embodiment 2

[0027] The sterile explants screened in Example 1 were transferred to the culture medium for inducing gametophytes to produce protonema for about 20 days. The culture conditions were: light time 12 hours, light intensity 3000 lux, and culture temperature 20±2°C.

[0028] Prepare the culture medium for inducing gametophytes to produce protonema, and the formula consists of the following three types:

[0029] ① Conventional MS medium + 30 g / L sucrose;

[0030] ② Conventional MS medium + 30 g / L sucrose + 0.1-1.5mg / L 6-benzylaminopurine;

[0031] ③ Conventional MS medium + 30 g / L sucrose + 0.1-1.5mg / L 2,4-dichlorophenoxyacetic acid.

[0032] The composition of conventional MS medium is: KNO 3 1900mg / L, NH 4 NO 3 1650 mg / L, MgSO 4 ·7H 2 O 370mg / L, KH 2 PO 4 170 mg / L, CaCl 2 2H 2 O 440 mg / L, MnSO 4 4H 2 O 22.30 mg / L, ZnSO 4 ·7H 2O 8.6 mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 4 2H 2 O 0.25 mg / L, CuSO 4 ·5H 2 O 0.025 mg / L, CoCl 2 ·6H 2 O 0.025 mg / L, N...

Embodiment 3

[0039] The sterile explants screened in Example 1 were transferred to the conventional MS + 30 g / L sucrose medium for inducing gametocytes to produce protonema, and cultivated according to the following four culture conditions:

[0040] ① The culture temperature is 20±2℃, the light intensity is 3000 lux, and the light time is 10h;

[0041] ② The culture temperature is 20±2℃, the light intensity is 3000 lux, and the light time is 12h;

[0042] ③ The culture temperature is 20±2°C, the light intensity is 3000 lux, and the light time is 14 hours;

[0043] ④ The culture temperature is 20±2°C, the light intensity is 6000 lux, and the light time is 12 hours.

[0044] The result is as follows:

[0045] Under these four culture conditions, the protonema produced by sterile explants inoculated on conventional MS + 30 g / L sucrose medium had little difference in average diameter and average length, indicating that light intensity, light time It has little effect on the induction of gam...

technical field

[0001] The invention relates to a method for establishing a gametophyte regeneration system of East Asian moss moss. Background technique

[0002] East Asian sand moss ( Racomitrium japonicum ) belongs to the genus Grimmiaceae (Grimmiaceae) Racomitrium ), born on rock face, rock face thin soil and sandy soil in low altitude areas. Widely distributed in China's Heilongjiang, Jilin, Liaoning, Henan, Jiangxi and other northern and southern provinces, Japan, North Korea, Russia (southeast of Siberia), Vietnam, Australia and other places are also distributed. In the long-term biological evolution process, most species in the family Aeruginaceae have developed the characteristics of adapting to the xerophytic environment in terms of community, stem, leaf and rhizoid structure and physiology. At present, in foreign countries, mosses in bryophytes have become ideal experimental materials for the study of plant molecular biology and model plants for studying gene ...

Examples

Embodiment 1

[0023] Take the East Asian sand moss that has been cultivated for a period of time, cut it into small pieces of 0.5-1cm from the top down, soak it in 2% detergent solution for 10 minutes, and then treat it with 0.02% mercury chloride for 45 seconds to carry out surface disinfection; after disinfection, inoculate Cultivate on regular MS + 0-40 g / L sucrose, improved Beneck + 0-40 g / L sucrose, and improved Knop + 0-40 g / L sucrose medium, respectively. The culture conditions are: light time 12h, light intensity 3000lux, culture temperature 20±2℃.

[0024] The cultivation results are as follows:

[0025] On the mediums of improved Beneck + 0-40 g / L sucrose and improved Knop + 0-40 g / L sucrose, the gametophyte infection rate is particularly high, mainly Penicillium and Rhizopus, and on this basis, increase disinfection The concentration of the solution and the disinfection time also obtained the same results. If the disinfection time and concentration were too high, the gametophyte...

Embodiment 2

[0027] The sterile explants screened in Example 1 were transferred to the culture medium for inducing gametophytes to produce protonema for about 20 days. The culture conditions were: light time 12 hours, light intensity 3000 lux, and culture temperature 20±2°C.

[0028] Prepare the culture medium for inducing gametophytes to produce protonema, and the formula consists of the following three types:

[0029] ① Conventional MS medium + 30 g / L sucrose;

[0030] ② Conventional MS medium + 30 g / L sucrose + 0.1-1.5mg / L 6-benzylaminopurine;

[0031] ③ Conventional MS medium + 30 g / L sucrose + 0.1-1.5mg / L 2,4-dichlorophenoxyacetic acid.

[0032] The composition of conventional MS medium is: KNO 3 1900mg / L, NH 4 NO 3 1650 mg / L, MgSO 4 ·7H 2 O 370mg / L, KH 2 PO 4 170 mg / L, CaCl 2 2H 2 O 440 mg / L, MnSO 4 4H 2 O 22.30 mg / L, ZnSO 4 ·7H 2O 8.6 mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 4 2H 2 O 0.25 mg / L, CuSO 4 ·5H 2 O 0.025 mg / L, CoCl 2 ·6H 2 O 0.025 mg / L, N...

Embodiment 3

[0039] The sterile explants screened in Example 1 were transferred to the conventional MS + 30 g / L sucrose medium for inducing gametocytes to produce protonema, and cultivated according to the following four culture conditions:

[0040] ① The culture temperature is 20±2℃, the light intensity is 3000 lux, and the light time is 10h;

[0041] ② The culture temperature is 20±2℃, the light intensity is 3000 lux, and the light time is 12h;

[0042] ③ The culture temperature is 20±2°C, the light intensity is 3000 lux, and the light time is 14 hours;

[0043] ④ The culture temperature is 20±2°C, the light intensity is 6000 lux, and the light time is 12 hours.

[0044] The result is as follows:

[0045] Under these four culture conditions, the protonema produced by sterile explants inoculated on conventional MS + 30 g / L sucrose medium had little difference in average diameter and average length, indicating that light intensity, light time It has little effect on the induction of gam...