阅读说明：本技术 一种组织培养快速繁育花叶八角金盘的方法 (method for rapidly breeding floral leaf illicium verum discs through tissue culture ) 是由 李秋静 林丹 卿霞 周天宇 唐忠炳 张文昊 庞玉华 秦祥 于 2019-09-20 设计创作，主要内容包括：本发明涉及一种组织培养快速繁育花叶八角金盘的方法,包括：(1)无菌材料的获得；(2)芽的分化和增殖；(3)生根培养；(4)炼苗与移栽。与现有技术相比,本发明采用组培快速繁育的方法,可以在短时间内获得大量株型整齐,性状优良的植株,以满足市场的大量需求。(The invention relates to a method for quickly breeding a floral leaf illicium verum disc by tissue culture, which comprises the following steps: (1) obtaining a sterile material; (2) differentiation and proliferation of shoots; (3) rooting culture; (4) hardening and transplanting the seedlings. Compared with the prior art, the invention adopts a tissue culture rapid breeding method, can obtain a large number of plants with regular plant types and excellent properties in a short time, so as to meet a large number of demands of the market.)

1. a method for tissue culture and rapid propagation of Fatsia japonica Makino is characterized by comprising the following steps:

(1) obtaining of sterile Material

Collecting tender stem segments with terminal buds of the female parent of the floral leaf illicium verum in spring for 3 months, washing, sequentially soaking in 75% alcohol and 1 ‰ mercuric chloride, washing with sterile water, cutting terminal buds into small segments, and inoculating on terminal bud induction culture medium;

(2) Differentiation and proliferation of shoots

After the buds are inoculated on the apical bud induction culture medium for 15 days, the base parts of the buds begin to expand, obvious callus can be seen in 25 days, the culture is carried out for 30 days, and the callus with the cut buds is put into an adventitious bud multiplication culture medium for culture;

(3) Rooting culture

Taking seedlings in an adventitious bud multiplication culture medium, cutting robust plantlets, transferring the plantlets into a rooting culture medium to induce rooting, differentiating a plurality of white root primordia at the seedling base part after 25 days, growing to 2-3.5cm after 35 days, and ensuring that the rooting rate is 95%;

(4) Hardening and transplanting seedlings

when the root system grows to 2-3cm, selecting aseptic seedlings with developed and strong root systems, placing the aseptic seedlings in a room with the temperature of 15 ℃, natural illumination and humidity for a week, taking out the seedlings, cleaning root agar, transplanting the seedlings onto a seedbed, watering the planted seedlings thoroughly, opening a greenhouse every day, keeping enough ventilation, properly watering when the soil is dry, and ensuring that the transplanting survival rate can reach 95%.

2. the method for tissue culture to rapidly propagate Fatsia japonica Linn according to claim 1, wherein in step (1), good Fatsia japonica Linn plants with pure variety, stout plant, good growth vigor and no plant diseases and insect pests are selected as the matrix.

3. The method for tissue culture for rapid propagation of Fatsia japonica Lindl according to claim 1, wherein in step (1), the washing method comprises: flushing the collected explants under the running water to clean dust on the surfaces of the explants; then, washing with soapy water, and shaking the bottle during washing to ensure that the explants fully contact with the soapy water; and washing with running water after washing.

4. The method for tissue culture of rapid propagation of Fatsia japonica Makino in claim 1, wherein in step (1), the apical bud induction medium contains macroelements, ferric salt, inositol, organic substances, trace elements, 6-BA, NAA,

the macroelement content is 0.4423g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, the trace element content is 0.2708g/1000L, and the 6-BA content is 3.0mg/L, NAA and 0.1 mg/L;

The macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

The pH value of the terminal bud induction culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the terminal bud induction culture medium, the culture temperature of the terminal bud induction culture medium is 21-23 ℃, and the illumination is 1800-2200 Lx.

5. The method for tissue culture of rapid propagation of Fatsia japonica Makino in claim 1, wherein in step (2), the adventitious bud propagation medium contains macroelements, ferric salt, inositol, organic substances, trace elements, 6-BA, NAA,

the macroelement content is 0.4423g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, the trace element content is 0.2708g/1000L, the 6-BA content is 1.5mg/L, NAA and 0.1mg/L,

The macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

The pH value of the adventitious bud propagation culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the adventitious bud propagation culture medium, the culture temperature of the adventitious bud propagation culture medium is 21-23 ℃, and the illumination is 1800-2200 Lx.

6. The method for tissue culture of rapid propagation of Fatsia japonica Makino in claim 1, wherein in step (3), the rooting medium contains macroelements, iron salt, inositol, organic substances, trace elements, IBA and NAA,

the macroelement content is 0.2212g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, and the trace element content is 0.2708g/1000L, IBA, the trace element content is 3.0mg/L, NAA, and the trace element content is 0.1 mg/L;

the macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

the pH value of the rooting culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the rooting culture medium, the culture temperature of the rooting culture medium is 21-23 ℃, and the illumination is 1800-.

7. The method for tissue culture of rapid propagation of Fatsia japonica Lindl according to claim 1, wherein in step (4), the substrate on the seedbed is a mixture of turf and perlite in a volume ratio of 1:1,

Uniformly stirring the matrix, spreading the matrix on a seedbed, wherein the spreading thickness is 20cm, and spraying 500 times of sodium diurethane liquid medicine on the spread matrix for disinfection.

8. the method for tissue culture to rapidly propagate Fatsia japonica Linn according to claim 1, wherein in step (4), the transplanting is carried out by inserting a tool into the hole, then transplanting the plantlets after the root system is spread by tweezers, and the planting depth is preferably that the matrix covers the base of the plantlets and the root is not exposed.

9. The method for tissue culture to rapidly propagate Fatsia japonica Linn according to claim 1, wherein in step (4), the liquid medicine is applied once a week, and carbendazim and Yinong-Dasheng M45 are used alternately to prevent drug resistance and reduce the times in sunny days.

Technical Field

The invention relates to a plant tissue culture breeding method, in particular to a method for rapidly breeding a floral leaf illicium verum by tissue culture.

Background

the floral leaf Dysosma versipellis (the scientific name: Fatsia japonica 'Variegata') is an evergreen shrub or a small arbor plant of the Araliaceae Dysosma genus, and the plant height can reach 5 m. Smooth and non-thorn. The length of the petiole is 10-30 cm; big leaf, leathery, nearly circular, yellow or white spot on leaf surface, 12-30 cm diameter, 7-9 deep cleft palm, oval lobe, short tip and heart shape at base. Growing the top of the panicle by 20-40 cm; the diameter of the umbrella-shaped inflorescence is 3-5 cm, and the inflorescence axis is covered by brown villus; calyx is close to the whole margin and has no hair; petal 5, oval triangle, length 2.5-3 mm, yellow white, without hair; stamen 5, filament and petal are equal in length; the lower part of the ovary, 5 chambers, each chamber has 1 blastocyst; separating the flower column 5; the flower disc is convex and semicircular. The fruit is approximately spherical, 5 mm in diameter and black when cooked. The flowering period is 10-11 months, and the fruit ripening period is 4 months next year.

the Fatsia japonica is a special variety of Fatsia japonica, and is evergreen shrub or small arbor of Fatsia japonica of Araliaceae, and is a popular indoor and outdoor foliage plant with evergreen and beautiful leaf shape. The floral leaf illicium verum dish has strong shade resistance and bright color, is widely applied to the aspects of landscape configuration, indoor potted plant appreciation and the like, and has large market demand. The floral leaf illicium verum discs adopt propagation modes such as cuttage and the like, and have low propagation coefficient and survival rate. By adopting the tissue culture rapid breeding method, a large number of plants with regular plant types and excellent properties can be obtained in a short time, so as to meet a large number of demands of the market.

Chinese patent CN102239805A discloses a tissue culture rapid propagation method of oriole tail, comprising: the method comprises the following steps: preparing a culture medium, including a basic culture medium; a bud induction medium; subculture multiplication medium; strong seedling culture medium; rooting culture medium; step two: culturing the virus-free tissue culture plantlets of the warburg tail, including selection and sterilization of explants; carrying out bud induction culture; bud multiplication culture; strong seedling culture; rooting culture; step three: hardening and transplanting the tissue culture seedlings, including hardening the seedlings; and (5) transplanting and culturing. The method remarkably reduces the contamination rate of oriole explants by adjusting the types and the content of culture medium hormones and adding antibiotics, so that the inoculation survival rate reaches more than 90 percent, the induced differentiation rate of the explants is also obviously improved, the induced differentiation rate is increased from 70-80 percent to 90 percent in the prior art, and the strong seedling rate of oriole tissue culture is improved.

chinese patent CN109588318A relates to a method for tissue culture and rapid breeding of plantain, which comprises (1) obtaining sterile materials; (2) differentiation and proliferation of shoots; (3) rooting culture; (4) hardening and transplanting the seedlings. The method can obtain a large number of plants with regular plant types and excellent properties in a short time so as to meet a large number of demands of the market. The plant stem tip is selected according to the material category, and the culture conditions are as follows: shoot tip induction medium: macroelement, iron salt, inositol, organic, microelement, 6-BA2.0 and NAA 0.2; adventitious bud propagation medium: macroelement, iron salt, inositol, organic, microelement, 6-BA0.2 and NAA 0.1; rooting culture medium: macroelement + iron salt + inositol + organic + microelement + NAA 0.1.

An article "tissue culture research of illicium verum dish" in the fifth stage of 2009 from Xinjiang agricultural science (author extra, total of the year of Yiba extraction, Qi Yun, xu Hui, Song peak, Cao Ye Fei, from Yan Shao scenic spot management station in Wuluqi city, Xinjiang Yu engineering design and consultation Limited) introduced a tissue culture and rapid propagation method of illicium verum dish, wherein the culture conditions used MS as a basic culture medium. (1) A bud induction culture medium is MS + TDZ 1 mg.L- (-1) (the same below the unit) + NAA 0.1; (2) subculture multiplication medium MS +6-BA1.5+ NAA 0.1; (3) rooting culture medium MS + NAA0.2+ IBA 0.3. The culture temperature is (25 +/-1) ° C, the illumination intensity is 2000-.

disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for tissue culture and rapid propagation of Fatsia japonica.

The purpose of the invention can be realized by the following technical scheme:

a method for tissue culture and rapid breeding of Fatsia japonica Makino comprises the following steps:

(1) Obtaining of sterile Material

collecting tender stem segments with terminal buds of the female parent of the floral leaf illicium verum in spring for 3 months, washing, sequentially soaking in 75% alcohol and 1 ‰ mercuric chloride, washing with sterile water, cutting terminal buds into small segments, and inoculating on terminal bud induction culture medium;

(2) differentiation and proliferation of shoots

after the buds are inoculated on the apical bud induction culture medium for 15 days, the base parts of the buds begin to expand, obvious callus can be seen in 25 days, the culture is carried out for 30 days, and the callus with the cut buds is put into an adventitious bud multiplication culture medium for culture;

(3) Rooting culture

Taking seedlings in an adventitious bud multiplication culture medium, cutting robust plantlets, transferring the plantlets into a rooting culture medium to induce rooting, differentiating a plurality of white root primordia at the seedling base part after 25 days, growing to 2-3.5cm after 35 days, and ensuring that the rooting rate is 95%;

(4) Hardening and transplanting seedlings

When the root system grows to 2-3cm, selecting aseptic seedlings with developed and strong root systems, placing the aseptic seedlings in a room with the temperature of 15 ℃, natural illumination and humidity for a week, taking out the seedlings, cleaning root agar, transplanting the seedlings onto a seedbed, watering the planted seedlings thoroughly, opening a greenhouse every day, keeping enough ventilation, properly watering when the soil is dry, and ensuring that the transplanting survival rate can reach 95%.

in the step (1), a good-quality Fatsia japonica plant with pure variety, stout plant, good growth vigor and no plant diseases and insect pests is selected as a matrix.

In the step (1), the washing method comprises the following steps: washing the collected explants under fluid water, and cleaning dust on the surfaces of the explants for 10 min; washing with soapy water for 15min, and shaking the bottle during washing to make the explant fully contact with the soapy water; and washing with running water after washing.

In the step (1), the terminal bud induction culture medium contains macroelements, ferric salt, inositol, organic substances, trace elements, 6-BA and NAA,

The macroelement content is 0.4423g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, the trace element content is 0.2708g/1000L, and the 6-BA content is 3.0mg/L, NAA and 0.1 mg/L.

The macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

The pH value of the terminal bud induction culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the terminal bud induction culture medium, the culture temperature of the terminal bud induction culture medium is 21-23 ℃, and the illumination is 1800-2200, preferably 2000 Lx.

in the step (2), the adventitious bud propagation culture medium contains macroelements, ferric salt, inositol, organic substances, trace elements, 6-BA and NAA,

The macroelement content is 0.4423g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, the trace element content is 0.2708g/1000L, the 6-BA content is 1.5mg/L, NAA and 0.1mg/L,

The macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

the pH value of the adventitious bud propagation culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the adventitious bud propagation culture medium, the culture temperature of the adventitious bud induction culture medium is 21-23 ℃, and the illumination is 1800-2200, preferably 2000 Lx.

In the step (3), the rooting culture medium contains macroelements, ferric salt, inositol, organic substances, trace elements, IBA and NAA,

The macroelement content is 0.2212g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, and the trace element content is 0.2708g/1000L, IBA, the trace element content is 3.0mg/L, NAA, and the trace element content is 0.3 mg/L;

The macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

The pH value of the rooting culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the rooting culture medium, the culture temperature of the rooting induction culture medium is 21-23 ℃, the illumination is 1800-.

in the step (4), the substrate on the seedbed is a mixture of turf and perlite in a volume ratio of 1:1,

Uniformly stirring the substrate, spreading the substrate on a seedbed, wherein the spreading thickness is about 20cm, and spraying 500 times of sodium diurethane liquid medicine on the spread substrate for disinfection.

in the step (4), a tool is used for inserting holes during transplanting, then the seedlings are planted after the roots of the seedlings are spread by forceps, and the planting depth is that the base parts of the seedlings are covered by the matrix to prevent the roots from exposing.

in the step (4), because the soil is moist in rainy days and the seedlings are easy to mildew and die, the liquid medicine needs to be applied once every week, and the carbendazim and the Dow Yinong-Dasheng M45 are alternately used to prevent the occurrence of drug resistance and reduce the times in sunny days.

The method for rapid breeding by plant tissue culture is particularly suitable for breeding floral leaf illicium verum varieties.

Because the bred plants are different, different plant cultures are different in tissue culture conditions, and different in requirements on culture media, the method for quickly breeding the plantain lily by tissue culture, which is related to Chinese patent CN109588318A, is not suitable for the floral leaf illicium verum disk of the application, and the application changes the culture conditions, especially researches the element proportion in the culture media, and finds out the culture medium composition most suitable for the floral leaf illicium verum disk culture.

compared with the tissue culture research of the illicium verum in the background technology, the proliferation culture medium and the rooting culture medium are different, and the using amount is more accurate; the environmental conditions are different, the pH value is accurate to 5.8, 30g/L of cane sugar and 3g/L of agar are added into the rooting culture medium, the temperature range is reduced to 21-23 ℃, and the illumination condition is about 2000 Lx. The cultivated seedlings of the floral leaf illicium verum have strong seedlings and the transplanting survival rate is 95 percent.

the invention adopts a tissue culture rapid breeding method, can obtain a large number of plants with regular plant types and excellent properties in a short time, so as to meet a large number of demands of the market.

Detailed Description

The present invention will be described in detail with reference to specific examples.

A method for tissue culture and rapid breeding of Fatsia japonica Makino comprises the following steps:

(1) Obtaining of sterile Material

Collecting tender stem segments with terminal buds of the female parent of the floral leaf illicium verum in spring for 3 months, washing, sequentially soaking in 75% alcohol and 1 ‰ mercuric chloride, washing with sterile water, cutting terminal buds into small segments, and inoculating on terminal bud induction culture medium;

(2) Differentiation and proliferation of shoots

After the buds are inoculated on the apical bud induction culture medium for 15 days, the base parts of the buds begin to expand, obvious callus can be seen in 25 days, the culture is carried out for 30 days, and the callus with the cut buds is put into an adventitious bud multiplication culture medium for culture;

(3) Rooting culture

Taking seedlings in an adventitious bud multiplication culture medium, cutting robust plantlets, transferring the plantlets into a rooting culture medium to induce rooting, differentiating a plurality of white root primordia at the seedling base part after 25 days, growing to 2-3.5cm after 35 days, and ensuring that the rooting rate is 95%;

(4) hardening and transplanting seedlings

when the root system grows to 2-3cm, selecting aseptic seedlings with developed and strong root systems, placing the aseptic seedlings in a room with the temperature of 15 ℃, natural illumination and humidity for a week, taking out the seedlings, cleaning root agar, transplanting the seedlings onto a seedbed, watering the planted seedlings thoroughly, opening a greenhouse every day, keeping enough ventilation, properly watering when the soil is dry, and ensuring that the transplanting survival rate can reach 95%.

In the step (1), good plants with pure varieties, stout plants, good growth vigor and no plant diseases and insect pests are selected as the mother body of the floral octagonal Japan Fatsia.

in the step (1), the washing method comprises the following steps: washing the collected explants under fluid water, and cleaning dust on the surfaces of the explants for 10 min; washing with soapy water for 15min, and shaking the bottle during washing to make the explant fully contact with the soapy water; and washing with running water after washing.

in the step (1), the terminal bud induction culture medium contains macroelements, ferric salt, inositol, organic substances, trace elements, 6-BA and NAA,

the macroelement content is 0.4423g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, the trace element content is 0.2708g/1000L, and the 6-BA content is 3.0mg/L, NAA and 0.1 mg/L.

the macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

the pH value of the terminal bud induction culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the terminal bud induction culture medium, the culture temperature of the terminal bud induction culture medium is 21-23 ℃, and the illumination is 2000 Lx.

In the step (2), the adventitious bud propagation culture medium contains macroelements, ferric salt, inositol, organic substances, trace elements, 6-BA and NAA,

the macroelement content is 0.4423g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, the trace element content is 0.2708g/1000L, the 6-BA content is 1.5mg/L, NAA and 0.1mg/L,

The macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

the pH value of the adventitious bud propagation culture medium is 5.8, 30g/L of cane sugar and 3g/L of agar are added into the adventitious bud propagation culture medium, the culture temperature of the adventitious bud induction culture medium is 21-23 ℃, and the illumination is 2000 Lx.

In the step (3), the rooting culture medium contains macroelements, ferric salt, inositol, organic substances, trace elements, IBA and NAA,

the macroelement content is 0.2212g/L, the iron salt content is 0.0651g/L, the inositol content is 0.01g/L, the organic matter content is 0.031g/L, and the trace element content is 0.2708g/1000L, IBA, the trace element content is 3.0mg/L, NAA, and the trace element content is 0.3 mg/L;

the macroelements comprise one or more of the following substances: KNO3, NH4NO3, MgSO4, CaCl2, KH2PO 4; the iron salt is FeSO4 & 7H 2O; the organic substance comprises one or more of the following substances: pyridoxine hydrochloride, thiamine hydrochloride, glycine, nicotinic acid; the trace elements comprise one or more of the following substances: MnSO4 & 4H2O, ZnSO4 & 7H2O, H3BO3, KI, Na2MoO4 & 2H2O, CuSO4 & 5H2O, CoCl2 & 5H 2O;

The pH value of the rooting culture medium is 5.8, 30g/L of sucrose and 3g/L of agar are added into the rooting culture medium, the culture temperature of the rooting induction culture medium is 21-23 ℃, and the illumination is 2000 Lx.

in the step (4), the substrate on the seedbed is a mixture of turf and perlite in a volume ratio of 1:1,

uniformly stirring the substrate, spreading the substrate on a seedbed, wherein the spreading thickness is about 20cm, and spraying 500 times of sodium diurethane liquid medicine on the spread substrate for disinfection.

In the step (4), a tool is used for inserting holes during transplanting, then the seedlings are planted after the roots of the seedlings are spread by forceps, and the planting depth is that the base parts of the seedlings are covered by the matrix to prevent the roots from exposing.

In the step (4), because the soil is moist in rainy days and the seedlings are easy to mildew and die, the liquid medicine needs to be applied once every week, and the carbendazim and the Dow Yinong-Dasheng M45 are alternately used to prevent the occurrence of drug resistance and reduce the times in sunny days.

The method for rapid breeding by plant tissue culture is particularly suitable for breeding floral leaf illicium verum varieties.

the embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.