Culture medium and tissue culture method for reducing content of broussonetia papyrifera esterase

Abstract

The invention provides a culture medium for reducing the content of broussonetia papyrifera esterase and a tissue culture method, and belongs to the technical field of broussonetia papyrifera tissue culture. The culture medium for reducing the content of the broussonetia papyrifera esterase provided by the invention comprises an induction culture medium, a proliferation culture medium, a differentiation proliferation culture medium and a rooting culture medium, wherein the induction culture medium is composed of an MS culture medium, 6-BA and IAA; the proliferation culture medium is composed of a 1 / 2 MS culture medium, NAA, KT and IBA; the differentiation and proliferation culture medium consists of a B5 culture medium, folic acid, TDZ, 2, 4-D, sucrose and agar; the rooting culture medium consists of an N6 culture medium, gibberellin 3, ethylene, IAA, cane sugar and carrageenan. The culture medium and the tissue culture method provided by the invention can promote more than 98% of adventitious buds to root, ensure that the survival rate is more than 98% after acclimatization and transplantation, not only can effectively reduce the content of lignin in paper mulberry tissue culture seedlings, but also cannot influence the normal growth of the paper mulberry tissue culture seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of paper mulberry tissue culture, and in particular relates to a culture medium and a tissue culture method for reducing the wood content of paper mulberry. Background Art

[0002] Broussonetia papyrifera L., also known as shell tree, paper mulberry, and peach tree, belongs to the genus Broussonetia of the Moraceae family. It is a light-loving perennial deciduous tree. Broussonetia is an important economic forest tree. Its bark fiber is of excellent quality and has been an excellent raw material for papermaking since ancient times. Moreover, it has strong environmental adaptability and is an ideal tree species for rapidly greening barren mountains, wastelands, and saline-alkali land. Broussonetia has a high content of lignin. Although lignin plays an important role in maintaining the normal growth and development of plants and in human life, it has a very adverse effect on special industries such as papermaking and animal husbandry. Especially in the process of pulping and papermaking, the high and low content of lignin in trees and the different compositions directly affect the quality of papermaking materials. High content of lignin has become a limiting factor in the papermaking industry, increasing the cost and consumption of the pulping and papermaking process, and bringing increasingly serious environmental pollution. In animal husbandry, lignin in forage affects livestock's digestion and nutrient absorption of forage, and the level of lignin content is also one of the indicators of forage quality. Therefore, reducing the lignin content in the papermaking plant paper mulberry has become an important breeding goal. However, since the synthesis pathway of lignin in plants involves very complex physiological and biochemical processes, how to effectively reduce the lignin content from the beginning of seedling cultivation has always been a major difficulty in the field of paper mulberry cultivation. At present, there is no research on how to reduce the lignin content of paper mulberry tissue culture seedlings from the perspective of tissue culture. Summary of the invention

[0003] In view of this, one of the objects of the present invention is to provide a culture medium capable of reducing the lignin content of paper mulberry, and a second object of the present invention is to provide a tissue culture method capable of reducing the lignin content of paper mulberry. The culture medium and tissue culture method provided by the present invention can not only effectively reduce the lignin content in paper mulberry tissue culture seedlings, but also will not affect the normal growth of paper mulberry tissue culture seedlings.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a culture medium for reducing the content of lignin in paper mulberry, comprising an induction culture medium, a proliferation culture medium, a differentiation and proliferation culture medium and a rooting culture medium; the induction culture medium is an MS culture medium + 1.5-2.5 mg / L 6-BA + 0.5-1.5 mg / L IAA; the proliferation culture medium is 1 / 2MS culture medium + 0.8-1.5 mg / L NAA + 1.2-1.8 mg / L KT + 0.1-0.3 mg / L IBA; the differentiation and proliferation culture medium is a B5 culture medium + 0.3-0.8 mg / L folic acid + 1.1-1.5 mg / L TDZ + 0.9-1.3 mg / L 2,4-D+20-35g / L sucrose+8-10g agar; the rooting medium is N6 medium+0.6-1.1mg / L gibberellin 3+0.6-0.9mg / L ethylene+0.1-0.3mg / LIAA+18-25g / L sucrose+9-12g carrageenan.

[0006] Preferably, the pH of the induction medium is 5.5-6.5; the pH of the proliferation medium is 5.0-6.0; the pH of the differentiation and proliferation medium is 5.2-6.0; and the pH of the rooting medium is 6.0-6.5.

[0007] The invention also provides application of the culture medium in propagation of paper mulberry with low lignin content.

[0008] The present invention also provides a tissue culture method for reducing the content of wood fiber in mulberry, which includes the following steps based on the above-mentioned culture medium: taking the root tip of the mulberry seedling as an explant, inoculating it into an induction culture medium, inducing culture with blue light, and obtaining callus tissue; placing the callus tissue in a proliferation culture medium for white light proliferation culture, and then inoculating it into a differentiation proliferation culture medium for red light differentiation proliferation culture to obtain seedlings; and rooting the seedlings in a rooting culture medium under purple light to obtain mulberry regenerated seedlings.

[0009] Preferably, the amount of blue light is 70 to 80 μmol / (m 2 ·s), and the photoperiod was 3h / d.

[0010] Preferably, the amount of white light is 100 to 110 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0011] Preferably, the amount of red light is 60 to 70 μmol / (m 2 ·s), and the photoperiod was 13h / d.

[0012] Preferably, the amount of the purple light is 80 to 100 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0013] Preferably, the temperature of the induction culture is 23-25°C, and the time is 15-20 days; the temperature of the proliferation culture is 24-26°C, and the time is 10-15 days; the temperature of the differentiation and proliferation culture is 25-28°C, and the time is 30-35 days; the temperature of the rooting culture is 26-29°C, and the time is 20-23 days.

[0014] Preferably, the method for disinfecting the root apex is: after rinsing with water, pre-treating with 75% alcohol for 30 to 40 seconds, and disinfecting with a NaClO aqueous solution for 2 to 2.5 minutes, wherein the mass concentration of effective chlorine in the NaClO aqueous solution is 1 to 2%.

[0015] Beneficial effects of the present invention:

[0016] The induction culture medium provided by the present invention can better induce callus differentiation, improve the induction rate of callus, reduce the browning rate of callus, and improve the quality of callus; the proliferation culture medium provided by the present invention can promote rapid proliferation of callus; the differentiation and proliferation culture medium provided by the present invention can ensure rapid cultivation to obtain adventitious buds; the rooting culture medium provided by the present invention can promote more than 98% of adventitious buds to take root, and the promoted roots grow strong, ensuring a survival rate of more than 98% after hardening and transplanting.

[0017] The culture medium and tissue culture method provided by the invention can not only effectively reduce the content of lignin in the paper mulberry tissue culture seedlings, but also will not affect the normal growth of the paper mulberry tissue culture seedlings. DETAILED DESCRIPTION

[0018] The invention provides a culture medium for reducing the content of lignin in paper mulberry, comprising an induction culture medium, a proliferation culture medium, a differentiation and proliferation culture medium and a rooting culture medium; the induction culture medium is an MS culture medium + 1.5-2.5 mg / L 6-BA + 0.5-1.5 mg / L IAA; the proliferation culture medium is 1 / 2MS culture medium + 0.8-1.5 mg / L NAA + 1.2-1.8 mg / L KT + 0.1-0.3 mg / L IBA; the differentiation and proliferation culture medium is a B5 culture medium + 0.3-0.8 mg / L folic acid + 1.1-1.5 mg / L TTDZ + 0.9-1.3 mg / L 2,4-D + 20-35 g / L sucrose + 8-10 g agar; the rooting culture medium is an N6 culture medium + 0.6-1.1 mg / L gibberellin 3 + 0.6-0.9 mg / L ethylene + 0.1-0.3 mg / L IAA+18~25g / L sucrose+9~12g carrageenan.

[0019] The present invention has no particular limitation on the specific sources of the components in the induction medium, proliferation medium, differentiation and proliferation medium, and rooting medium. In the induction medium of the present invention, the concentration of 6-BA is preferably 1.8 to 2.2 mg / L, more preferably 2.0 mg / L, the concentration of IAA is preferably 0.8 to 1.2 mg / L, more preferably 1.0 mg / L, and the pH of the induction medium of the present invention is preferably 5.5 to 6.5, more preferably 5.8 to 6.2.

[0020] In the proliferation medium of the present invention, the concentration of NAA is preferably 1.0-1.4 mg / L, more preferably 1.2-1.3 mg / L; the concentration of KT is preferably 1.4-1.6 mg / L, more preferably 1.5 mg / L; the concentration of IBA is preferably 0.2 mg / L; the pH of the proliferation medium of the present invention is preferably 5.0-6.0, more preferably 5.2-5.6.

[0021] In the differentiation and proliferation medium of the present invention, the concentration of folic acid is preferably 0.4-0.7 mg / L, more preferably 0.5-0.6 mg / L; the concentration of TDZ is preferably 1.2-1.4 mg / L, more preferably 1.3 mg / L; the concentration of 2,4-D is preferably 1.0-1.2 mg / L, more preferably 1.1 mg / L; the concentration of sucrose is preferably 25-30 g / L, more preferably 26-28 g / L, the concentration of agar is preferably 8-10 g, more preferably 9 g; the pH of the differentiation and proliferation medium of the present invention is preferably 5.2-6.0, more preferably 5.4-5.8.

[0022] In the rooting medium of the present invention, the concentration of gibberellin 3 is preferably 0.8-1.0 mg / L, more preferably 0.9 mg / L; the concentration of ethylene is preferably 0.7-0.8 mg / L, more preferably 0.75 mg / L; the concentration of IAA is preferably 0.2 mg / L; the concentration of sucrose is preferably 20-24 g / L, more preferably 22-23 g / L; the concentration of carrageenan is preferably 10-11 g, more preferably 10.5 g; the pH of the rooting medium of the present invention is preferably 6.0-6.5, more preferably 6.2-6.4.

[0023] The MS culture medium, 1 / 2MS culture medium, B5 culture medium and N6 culture medium described in the present invention are all commercially available products in the art.

[0024] The invention also provides application of the culture medium in propagation of paper mulberry with low lignin content.

[0025] The present invention also provides a tissue culture method for reducing the content of wood fiber in mulberry, which includes the following steps based on the above-mentioned culture medium: taking the root tip of the mulberry seedling as an explant, inoculating it into an induction culture medium, inducing culture with blue light, and obtaining callus tissue; placing the callus tissue in a proliferation culture medium for white light proliferation culture, and then inoculating it into a differentiation proliferation culture medium for red light differentiation proliferation culture to obtain seedlings; and rooting the seedlings in a rooting culture medium under purple light to obtain mulberry regenerated seedlings.

[0026] In the present invention, the paper mulberry seedlings can be selected from sterile paper mulberry seedlings or paper mulberry seedlings cultivated in the field. When the paper mulberry seedlings are paper mulberry seedlings cultivated in the field, it is preferred to further include a disinfection and sterilization step, wherein the disinfection and sterilization method is preferably to rinse with water, pre-treat with 75% alcohol for 30 to 40 seconds, and disinfect with a NaClO aqueous solution for 2 to 2.5 minutes, wherein the mass concentration of effective chlorine in the NaClO aqueous solution is 1 to 2%. In the present invention, the length of the root tip is preferably 1 to 2 cm; and the water used for rinsing is preferably sterile water.

[0027] In the present invention, the temperature of the blue light induced culture is preferably 23-25°C, more preferably 24°C, the time of the blue light induced culture is preferably 15-20 days, more preferably 17-19 days, and the light intensity of the blue light is preferably 70-80 μmol / (m 2 ·s), more preferably 75 to 76 μmol / (m 2 ·s), and the illumination time is preferably 3h / d.

[0028] In the present invention, the temperature of the white light proliferation culture is preferably 24-26°C, more preferably 25°C, the time of the white light proliferation culture is preferably 10-15 days, more preferably 12-13 days, and the light intensity of the white light is preferably 100-110 μmol / (m 2 ·s), more preferably 105 to 108 μmol / (m 2 ·s), and the illumination time is preferably 12h / d.

[0029] In the present invention, the temperature of the red light differentiation and proliferation culture is preferably 25 to 28°C, more preferably 26 to 27°C, the time of the red light differentiation and proliferation culture is preferably 30 to 35 days, more preferably 32 to 34 days, and the light intensity of the red light is preferably 60 to 70 μmol / (m 2 ·s), more preferably 62 to 68 μmol / (m 2 ·s), and the illumination time is preferably 13h / d.

[0030] In the present invention, the temperature of the purple light rooting culture is preferably 26-29°C, more preferably 27-28°C, the time of the purple light rooting culture is preferably 20-23 days, more preferably 21-22 days, and the light intensity of the purple light is preferably 80-100 μmol / (m2 ·s), more preferably 85 to 95 μmol / (m 2 ·s), and the illumination time is preferably 12h / d.

[0031] The present invention combines light sources of different colors with culture media at each stage for timely irradiation culture, and ultimately achieves a browning rate of basically zero, a rooting rate and a transplant survival rate of more than 98%, and effectively reduces the content of lignin in paper mulberry tissue culture seedlings without affecting the normal growth of the paper mulberry tissue culture seedlings, achieving significant technical effects.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] A culture medium for reducing the content of lignin in paper mulberry, comprising an induction culture medium, a proliferation culture medium, a differentiation and proliferation culture medium and a rooting culture medium;

[0037] The induction medium is MS medium + 1.5 mg / L 6-BA + 1.5 mg / L IAA, with a pH of 6.0;

[0038] The proliferation medium is 1 / 2MS medium + 0.8mg / LNAA + 1.2mg / LKT + 0.3mg / L IBA, pH 5.5;

[0039] The differentiation and proliferation medium is B5 medium + 0.3 mg / L folic acid + 1.5 mg / L TDZ + 0.9 mg / L 2,4-D + 35 g / L sucrose + 8 g agar, pH 5.5;

[0040] The rooting medium is N6 medium + 0.6 mg / L gibberellin 3 + 0.9 mg / L ethylene + 0.1 mg / LIAA + 18 g / L sucrose + 12 g carrageenan, and the pH value is 6.2.

[0041] Example 2

[0042] A culture medium for reducing the content of lignin in paper mulberry, comprising an induction culture medium, a proliferation culture medium, a differentiation and proliferation culture medium and a rooting culture medium;

[0043] The induction medium is MS medium + 2.5 mg / L 6-BA + 0.5 mg / L IAA, with a pH of 5.5;

[0044] The proliferation medium is 1 / 2MS medium + 1.5mg / L NAA + 1.8mg / L KT + 0.1mg / L IBA, pH 5.0;

[0045] The differentiation and proliferation medium is B5 medium + 0.8 mg / L folic acid + 1.1 mg / L TDZ + 1.3 mg / L 2,4-D + 20 g / L sucrose + 10 g agar, with a pH of 5.2;

[0046] The rooting medium is N6 medium + 1.1 mg / L gibberellin 3 + 0.6 mg / L ethylene + 0.3 mg / LIAA + 25 g / L sucrose + 9 g carrageenan, and the pH value is 6.0.

[0047] Example 3

[0048] A culture medium for reducing the content of lignin in paper mulberry, comprising an induction culture medium, a proliferation culture medium, a differentiation and proliferation culture medium and a rooting culture medium;

[0049] The induction medium is MS medium + 2.0 mg / L 6-BA + 1.0 mg / L IAA, with a pH of 6.5;

[0050] The proliferation medium is 1 / 2MS medium + 1.2mg / LNAA + 1.5mg / LKT + 0.2mg / L IBA, pH 6.0;

[0051] The differentiation and proliferation medium is B5 medium + 0.5 mg / L folic acid + 1.3 mg / L TDZ + 1.1 mg / L 2,4-D + 25 g / L sucrose + 9 g agar, with a pH of 6.0;

[0052] The rooting medium is N6 medium + 0.8 mg / L gibberellin 3 + 0.7 mg / L ethylene + 0.2 mg / LIAA + 20 g / L sucrose + 10 g carrageenan, and the pH value is 6.5.

[0053] Example 4

[0054] A culture medium for reducing the content of lignin in paper mulberry, comprising an induction culture medium, a proliferation culture medium, a differentiation and proliferation culture medium and a rooting culture medium;

[0055] The induction medium is MS medium + 2.2 mg / L 6-BA + 1.2 mg / L IAA, with a pH of 6.2;

[0056] The proliferation medium is 1 / 2MS medium + 1.0 mg / L NAA + 1.6 mg / L KT + 0.2 mg / L IBA, pH 5.2;

[0057] The differentiation and proliferation medium is B5 medium + 0.6 mg / L folic acid + 1.4 mg / L TDZ + 1.2 mg / L 2,4-D + 30 g / L sucrose + 9 g agar, with a pH of 5.8;

[0058] The rooting medium is N6 medium + 1.0 mg / L gibberellin 3 + 0.8 mg / L ethylene + 0.2 mg / LIAA + 22 g / L sucrose + 10 g carrageenan, and the pH value is 6.1.

[0059] Example 5

[0060] A tissue culture method for reducing the wood fiber content of paper mulberry, comprising the following steps:

[0061] (1) The root tips of commercially available Broussonetia papyrifera seedlings were taken, rinsed with sterile water outside a clean bench, and then pretreated by immersion in 75% alcohol by volume for 40 seconds in a clean bench, and then disinfected by immersion in a NaClO aqueous solution with a mass concentration of 1% effective chlorine for 2 minutes. Then, 1 cm in length was cut with a blade as an explant, inoculated into the induction medium of Example 1, and induced and cultured at 25°C for 15 days under LED blue light to obtain callus tissue; the light intensity of the blue light was 70 μmol / (m 2 ·s), the illumination time is 3h / d;

[0062] (2) The callus tissue was placed in the proliferation medium of Example 1 and cultured for 15 days under LED white light at 24°C, and then inoculated into the differentiation and proliferation medium of Example 1 and cultured for 30 days under LED red light at 25°C to obtain sprouts; the light intensity of the white light was 100 μmol / (m 2 ·s), the illumination time is 12h / d; the light intensity of the red light is 60μmol / (m 2 ·s), the illumination time is 13h / d;

[0063] (3) The seedlings were placed in the rooting medium of Example 1 and cultured under LED purple light at 26° C. for 23 days to obtain paper mulberry regeneration seedlings; the light intensity of the purple light was 80 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0064] The root system of the regenerated paper mulberry seedlings obtained was thick and robust. According to statistics, the rooting rate was 99% and the browning rate was 0.

[0065] Example 6

[0066] A tissue culture method for reducing the wood fiber content of paper mulberry, comprising the following steps:

[0067] (1) The root tips of commercially available Broussonetia papyrifera seedlings were taken, rinsed with sterile water outside a clean bench, and then pretreated by immersion in 75% alcohol by volume for 30 seconds in a clean bench, and then disinfected by immersion in a NaClO aqueous solution with a mass concentration of 2% effective chlorine for 2.5 minutes. Then, 1.5 cm in length was cut with a blade as an explant, inoculated into the induction medium of Example 2, and induced and cultured at 23°C for 20 days under LED blue light to obtain callus tissue; the light intensity of the blue light was 80 μmol / (m 2 ·s), the illumination time is 3h / d;

[0068] (2) The callus tissue was placed in the proliferation medium of Example 2 and cultured for 10 days under LED white light at 26°C, and then inoculated into the differentiation and proliferation medium of Example 2 and cultured for 35 days under LED red light at 28°C to obtain sprouts; the light intensity of the white light was 110 μmol / (m 2 ·s), the illumination time is 12h / d; the light intensity of the red light is 70μmol / (m 2 ·s), the illumination time is 13h / d;

[0069] (3) The seedlings were placed in the rooting medium of Example 2 and cultured under LED purple light at 29° C. for 20 days to obtain paper mulberry regeneration seedlings; the light intensity of the purple light was 100 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0070] The root system of the regenerated paper mulberry seedlings obtained was strong. According to statistics, the rooting rate was 98% and the browning rate was 0.

[0071] Example 7

[0072] A tissue culture method for reducing the wood fiber content of paper mulberry, comprising the following steps:

[0073] (1) The root tips of commercially available Broussonetia papyrifera seedlings were taken, rinsed with sterile water outside a clean bench, and then pretreated by immersion in 75% alcohol by volume for 35 seconds in a clean bench, and then disinfected by immersion in a NaClO aqueous solution with a mass concentration of 2% effective chlorine for 2.2 minutes. Then, 2 cm in length was cut with a blade as an explant, inoculated into the induction medium of Example 3, and induced and cultured at 24°C for 19 days under LED blue light to obtain callus tissue; the light intensity of the blue light was 75 μmol / (m 2 ·s), the illumination time is 3h / d;

[0074] (2) The callus tissue was placed in the proliferation medium of Example 3 and cultured for 13 days under LED white light at 25°C, and then inoculated into the differentiation and proliferation medium of Example 3 and cultured for 32 days under LED red light at 26°C to obtain sprouts; the light intensity of the white light was 105 μmol / (m 2·s), the illumination time is 12h / d; the light intensity of the red light is 65μmol / (m 2 ·s), the illumination time is 13h / d;

[0075] (3) The seedlings were placed in the rooting medium of Example 3 and cultured under LED purple light at 27° C. for 22 days to obtain paper mulberry regeneration seedlings; the light intensity of the purple light was 90 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0076] The root system of the regenerated paper mulberry seedlings obtained was thick and robust. According to statistics, the rooting rate was 100% and the browning rate was 0.

[0077] Example 8

[0078] A tissue culture method for reducing the wood fiber content of paper mulberry, comprising the following steps:

[0079] (1) The root tips of commercially available Broussonetia papyrifera seedlings were taken, rinsed with sterile water outside a clean bench, and then pretreated by immersion in 75% alcohol by volume for 38 seconds in a clean bench, and then disinfected by immersion in a NaClO aqueous solution with an effective chlorine mass concentration of 1.5% for 2.4 minutes. Then, 1.5 cm in length was cut with a blade as an explant, inoculated into the induction medium of Example 4, and induced and cultured at 24°C for 18 days under LED blue light to obtain callus tissue; the light intensity of the blue light was 76 μmol / (m 2 ·s), the illumination time is 3h / d;

[0080] (2) The callus tissue was placed in the proliferation medium of Example 4 and cultured for 12 days under LED white light at 25°C, and then inoculated into the differentiation and proliferation medium of Example 4 and cultured for 34 days under LED red light at 27°C to obtain sprouts; the light intensity of the white light was 108 μmol / (m 2 ·s), the illumination time is 12h / d; the light intensity of the red light is 68μmol / (m 2 ·s), the illumination time is 13h / d;

[0081] (3) The seedlings were placed in the rooting medium of Example 4 and cultured under LED purple light at 28° C. for 21 days to obtain paper mulberry regeneration seedlings; the light intensity of the purple light was 95 μmol / (m 2 ·s), and the photoperiod was 12h / d.

[0082] The root system of the regenerated paper mulberry seedlings obtained was thick and robust. According to statistics, the rooting rate was 99% and the browning rate was 0.

[0083] Comparative Example 1

[0084] The difference from Example 7 is that the concentration of 6-BA in the induction medium is 1.0 mg / L, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is relatively strong, and the rooting rate is 82% and the browning rate is 40%.

[0085] Comparative Example 2

[0086] The difference from Example 7 is that the concentration of KT in the proliferation medium is 2 mg / L, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is relatively strong, and the rooting rate is 88% and the browning rate is 30%.

[0087] Comparative Example 3

[0088] The difference from Example 7 is that the differentiation and proliferation medium does not contain folic acid, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is relatively fine, and the rooting rate is 75% and the browning rate is 35%.

[0089] Comparative Example 4

[0090] The difference from Example 7 is that the concentration of ethylene in the rooting medium is 1.2 mg / L, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is fine and soft, and the rooting rate is 68% and the browning rate is 26%.

[0091] Comparative Example 5

[0092] The difference from Example 7 is that the light source for induction culture in step (1) is LED white light, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is strong, and the rooting rate is 72% and the browning rate is 45%.

[0093] Comparative Example 6

[0094] The difference from Example 7 is that the light source for differentiation and proliferation culture in step (2) is LED white light, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is strong, and the rooting rate is 88% and the browning rate is 18%.

[0095] Comparative Example 7

[0096] The difference from Example 7 is that the light source for rooting culture in step (3) is LED white light, and the rest is the same as Example 7. The root system of the obtained paper mulberry regeneration seedlings is strong, and the rooting rate is 65% and the browning rate is 32%.

[0097] Comparative Example 8

[0098] The difference from Example 7 is that the amount of blue light in step (1) is 90 μmol / (m 2 ·s), and the rest are the same as in Example 7. The root system of the obtained paper mulberry regeneration seedlings is thick, and the rooting rate is 89% and the browning rate is 14%.

[0099] Comparative Example 9

[0100] The difference from Example 7 is that the amount of purple light in step (3) is 70 μmol / (m 2 ·s), and the rest are the same as in Example 7. The root system of the obtained paper mulberry regeneration seedlings is thick, and the rooting rate is 93% and the browning rate is 28%.

[0101] Example 9

[0102] The paper mulberry regenerated seedlings obtained in Examples 5 to 8 and Comparative Examples 1 to 9 were first opened in an incubator for 2 days, then taken out of the culture bottle, the culture medium at the root was washed, and transplanted into a 8×8 cm nutrient pot filled with a cultivation medium (the cultivation medium was peat soil and vermiculite mixed in a volume ratio of 2:1, and the cultivation medium was disinfected with 0.1% carbendazim before potting) in a greenhouse. The ground film was covered for the first 4 days of transplanting, and the light intensity in the greenhouse was maintained at 200 μmol / (m 2 ·s), the temperature was 25-28°C, and the relative humidity was 70-80%. The experiment was repeated twice, with 100 plants in each repeat. The survival rate of seedlings in each group was calculated, where the survival rate of seedlings in the hardening was (the number of plants surviving in the nutrient pot / the number of transplanted test tube seedlings) × 100%.

[0103] After that, the mulch was removed and normal management such as watering and fertilization was carried out. After 2-3 weeks, new roots and new shoots appeared. When the seedlings reached 25 cm in height, they were transplanted to the outdoor field. The transplant survival rate of each group was calculated, where the transplant survival rate = (number of plants surviving in the field / total number of transplanted plants) × 100%.

[0104] After 6 months of growth after transplantation, the Klason method was used to determine the lignin content of different groups of paper mulberry. The commercial paper mulberry seedlings grown 6 months after transplantation were used as the control group. Five trees were randomly selected from each group, the bark was peeled off, and the stems were taken. The Klason method was used to determine lignin in accordance with the national standard (GB / T 2677.8-1994).

[0105] The above results are shown in Table 1. It can be seen that the culture medium and tissue culture method provided by the present invention can significantly reduce the content of lignin in tissue cultured B. emblica seedlings, and the survival rate of hardening and transplanting can be as high as 100%.

[0106] Table 1 Results of different groups

[0107] Group Hardening survival rate Transplant survival rate Lignin content Example 5 99％ 100％ 15.8％ Example 6 98％ 100％ 16.2％ Example 7 100％ 100％ 15.1％ Example 8 98％ 100％ 15.9％ Comparative Example 1 76％ 70％ 21.2％ Comparative Example 2 80％ 82％ 20.8％ Comparative Example 3 66％ 68％ 19.1％ Comparative Example 4 68％ 72％ 19.9％ Comparative Example 5 78％ 84％ 21.0％ Comparative Example 6 85％ 89％ 19.4％ Comparative Example 7 60％ 66％ 19.8％ Comparative Example 8 88％ 82％ 18.2％ Comparative Example 9 90％ 93％ 17.6％ Commercially available Broussonetia papyrifera seedlings - - 21.6％

[0108] Within two years after transplanting, the growth conditions of different groups of paper mulberry were counted, and the results showed that the paper mulberry of Examples 5 to 8 grew well in the field environment. Compared with the growth conditions of commercially available paper mulberry seedlings within two years after transplanting, the paper mulberry of Examples 5 to 8 groups were even better than those of commercially available paper mulberry seedlings in terms of plant height, tree circumference and leaf density.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A culture medium for reducing the content of lignin in paper mulberry, characterized in that: The invention comprises an induction medium, a proliferation medium, a differentiation and proliferation medium and a rooting medium; the induction medium is an MS medium + 1.5-2.5 mg / L 6-BA + 0.5-1.5 mg / L IAA; the proliferation medium is 1 / 2 MS medium + 0.8-1.5 mg / L NAA + 1.2-1.8 mg / L KT + 0.1-0.3 mg / L IBA; the differentiation and proliferation medium is a B5 medium + 0.3-0.8 mg / L folic acid + 1.1-1.5 mg / L TTDZ + 0.9-1.3 mg / L 2,4-D + 20-35 g / L sucrose + 8-10 g agar; and the rooting medium is an N6 medium + 0.6-1.1 mg / L gibberellin 3 + 0.6-0.9 mg / L ethylene + 0.1-0.3 mg / L IAA + 18-25 g / L sucrose + 9-12 g carrageenan.

2. The culture medium according to claim 1, characterized in that The pH of the induction medium is 5.5-6.5; the pH of the proliferation medium is 5.0-6.0; the pH of the differentiation and proliferation medium is 5.2-6.0; and the pH of the rooting medium is 6.0-6.

5.

3. Use of the culture medium according to claim 1 or 2 in the propagation of paper mulberry with low lignin content.

4. A tissue culture method for reducing the wood content of paper mulberry, characterized in that: The culture medium according to claim 1 or 2 comprises the following steps: taking the root tip of the paper mulberry seedling as an explant, inoculating it into the induction culture medium, inducing culture under blue light, and obtaining callus tissue; placing the callus tissue in the proliferation culture medium for white light proliferation culture, and then inoculating it into the differentiation proliferation culture medium for red light differentiation proliferation culture, and obtaining seedlings; subjecting the seedlings to purple light rooting culture in the rooting culture medium to obtain paper mulberry regenerated seedlings.

5. The tissue culture method according to claim 4, characterized in that The amount of blue light is 70 to 80 μmol / (m 2 ·s), and the photoperiod was 3h / d.

6. The tissue culture method according to claim 4, characterized in that The light intensity of the white light is 100-110 μmol / (m 2 ·s), and the photoperiod was 12h / d.

7. The tissue culture method according to claim 4, characterized in that: The amount of red light is 60 to 70 μmol / (m 2 ·s), and the photoperiod was 13h / d.

8. The tissue culture method according to claim 4, characterized in that The amount of the purple light is 80 to 100 μmol / (m 2 ·s), and the photoperiod was 12h / d.

9. The tissue culture method according to claim 4, characterized in that: The induction culture is carried out at a temperature of 23 to 25° C. for 15 to 20 days; the proliferation culture is carried out at a temperature of 24 to 26° C. for 10 to 15 days; the differentiation proliferation culture is carried out at a temperature of 25 to 28° C. for 30 to 35 days; and the rooting culture is carried out at a temperature of 26 to 29° C. for 20 to 23 days.

10. The tissue culture method according to claim 4, characterized in that: The method for disinfecting the root apex is as follows: after being rinsed with water, pre-treated with 75% alcohol for 30 to 40 seconds, and disinfected with a NaClO aqueous solution for 2 to 2.5 minutes, wherein the mass concentration of effective chlorine in the NaClO aqueous solution is 1 to 2%.