CN114467757B - Method for reducing formation of basal callus in chimonanthus nitens stem tissue culture regeneration - Google Patents

Method for reducing formation of basal callus in chimonanthus nitens stem tissue culture regeneration Download PDF

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CN114467757B
CN114467757B CN202210225156.4A CN202210225156A CN114467757B CN 114467757 B CN114467757 B CN 114467757B CN 202210225156 A CN202210225156 A CN 202210225156A CN 114467757 B CN114467757 B CN 114467757B
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culture
stem
buds
callus
induction
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CN114467757A (en
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侯金艳
吴丽芳
王萍
苏鹏飞
王大成
丁双双
王军
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Hefei Institutes of Physical Science of CAS
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
    • A01G17/005—Cultivation methods
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
    • A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
    • A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/14—Measures for saving energy, e.g. in green houses

Abstract

The invention discloses a method for reducing formation of basal callus in chimonanthus nitens stem section tissue culture regeneration, which comprises the following steps: taking the current-year semi-lignified stem segment with axillary buds of a field perennial wintersweet excellent strain as an explant, cutting the explant into stem segments with axillary buds after surface disinfection, and inoculating the stem segments into an axillary bud induction culture medium for germination induction of the axillary buds; cutting the germinated axillary buds into sections, inoculating the sections into a specific culture medium, and performing induction and multiplication culture on adventitious buds; and separating the adventitious buds after propagation culture, and performing ex-vitro rooting culture to obtain a complete calyx canthus regeneration plant. The invention develops an efficient breeding method which can simplify the steps of tissue culture and breeding of the chimonanthus nitens, shorten the production period, reduce the production cost, reduce the formation of callus in the regeneration process and improve the transplanting survival rate of the chimonanthus nitens regenerated plants by combining the in-bottle adventitious bud induction with the in-bottle rooting technology.

Description

Method for reducing formation of basal callus in chimonanthus nitens stem tissue culture regeneration
Technical Field
The invention relates to the field of rapid propagation of plants, in particular to a method for reducing formation of basal callus in tissue culture regeneration of a chimonanthus nitens stem segment.
Background
The Chimonanthus praecox (Linn.) Link is perennial deciduous shrub of Chimonanthus family, has ornamental value of color, fragrance, shape and appearance, particularly has yellow flower like wax and pleasant flower fragrance, is open in winter, has fragrant flower fragrance, is unique precious flowers and trees in China, and is also a famous garden ornamental tree species in the world. Meanwhile, the essential oil extracted from the wintersweet flowers is rich in components, the price of the wintersweet aromatic oil in the international market is greatly higher than that of rose oil and jasmine aromatic oil, and the wintersweet flower essential oil has great development potential in the essential oil market. However, the lack of good varieties and effective large-scale rapid propagation technology of good varieties has become a bottleneck problem in the development of the wintersweet industry.
At present, the wintersweet is mainly propagated in a sowing mode, and due to heterozygosity of seeds, excellent agronomic characters of female parent plants are difficult to preserve; the excellent agronomic characters of the female parent plant of the chimonanthus nitens can be maintained by propagating the chimonanthus nitens through propagation modes such as cuttage, grafting, plant division and the like, but the problems of limited propagation materials, low propagation coefficient, complicated propagation process, season limitation on propagation and the like exist, and the demand of the market on the excellent seedlings of the chimonanthus nitens can not be met. Aiming at a plurality of problems in the breeding process of the Chimonanthus praecox, the inventor develops a method for efficient in-vitro regeneration of the Chimonanthus praecox plant (ZL 201710305050.4) in the early stage, and the tissue culture and rapid propagation of the Chimonanthus praecox plant are realized by taking a seed seedling as an explant source and utilizing a tissue culture technology. However, the inventor directly applies the technology to the rapid propagation of the field perennial wintersweet excellent strain to find that: the stem section obtained after axillary buds germinate can generate a large amount of callus at the base part in the regeneration process, and the absorption of nutrient substances is seriously influenced, so that the tissue culture regeneration bud is thin and small and yellowed, and the later growth of a regeneration plant is seriously influenced. Therefore, there is an urgent need to develop a method for reducing the formation of basal callus in the tissue culture regeneration of chimonanthus nitens stem segments.
Disclosure of Invention
The invention aims to solve the technical problems of how to reduce the formation of base callus in the regeneration process of tissue culture of the superior wintersweet strain stem section and improve the transplanting survival rate of the wintersweet regenerated plant.
The invention solves the technical problems through the following technical means:
a method for reducing the formation of basal callus in the tissue culture regeneration of a chimonanthus nitens stem segment comprises the following steps:
s1, taking an annual semi-lignified stem segment with an axillary bud of a field perennial wintersweet excellent strain as an explant;
s2, carrying out surface disinfection on the explant in the S1, cutting the explant into stem sections with axillary buds, and inoculating the stem sections with axillary buds to an axillary bud induction culture medium for germination induction of the axillary buds;
s3, cutting the axillary buds germinated in the step S2 into sections, inoculating the sections into a culture medium added with plant growth regulating substances, and performing induction and multiplication culture on adventitious buds; wherein the culture medium added with plant growth regulating substances is added with 0.2-0.5 mg/L6-BA, 0.2-1.0mg/L ZT and 5-20mg/L AgNO 3 20g/L sucrose and 8.0g/L agar in a 1/2DKW medium;
and S4, separating the adventitious buds after the propagation culture, and performing ex-vitro rooting culture to obtain complete wintersweet regenerated plants.
Taking axillary buds sprouting for a certain period of time in S2 as explants, cutting into stem segments with axillary buds with proper sizes, inoculating the stem segments in a culture medium added with plant growth regulating substances for adventitious bud induction, and then inoculating the stem segments in the culture medium added with the plant growth regulating substances for propagation culture, further realizing the expanded culture of superior wintersweet strains, and simultaneously avoiding the induction of callus at the base of the stem segments in the regeneration process.
Preferably, in S2, the method of surface disinfection of explants in S1 comprises the following steps: cutting the explant into 5-7cm sections, placing the sections in a plastic culture bottle, sealing the gauze, washing for 15-25min under running water, and adding 1-3ml of hand sanitizer during washing; then, wiping 1-3 times with 75% absolute ethyl alcohol, placing in a sterile operating platform, washing with sterile water for 2-4 times, then disinfecting with 75% absolute ethyl alcohol for 1-3 times, each time disinfecting for 25-35s, then washing with sterile water for 3-5 times, then disinfecting with 0.1% mercuric chloride solution for 1-2min, and finally washing with sterile water for 5-7 times.
Preferably, the method for surface disinfection of explants in S1 comprises the following steps: cutting the explant into 6 cm-sized sections, placing the sections into a 300ml plastic culture bottle, washing the gauze with running water for 20min after sealing the gauze, adding 2ml of a hand sanitizer during washing, disinfecting and wiping the gauze with 75% absolute ethyl alcohol for 1 time, placing the gauze in a sterile operating platform, washing the gauze with sterile water for 3 times, disinfecting the gauze with 75% absolute ethyl alcohol for 2 times, disinfecting the gauze for 30s each time, washing the gauze with sterile water for 4 times, disinfecting the gauze with 0.1% mercuric chloride solution for 2min, and finally washing the gauze with sterile water for 6 times; shaking continuously during the disinfection period, and absorbing the surface moisture after disinfection.
Preferably, in S2, the axillary bud induction medium includes: 1/2DKW, 0.05-0.2 mg/L6-BA, 0.05-0.5mg/L ZT, 20g/L sucrose, and 8.0g/L agar.
Preferably, in S3, the axillary buds germinated in S2 for 45-90 days are cut into suitable-size sections with axillary buds.
Preferably, in S3, the axillary buds germinated for 60 days in S2 are cut into suitable-size sections with axillary buds.
Preferably, the length of the section with the axillary buds is 1.0cm.
Preferably, in S3, the culture medium added with the plant growth regulating substance is added with 0.3 mg/L6-BA, 1.0mg/L ZT and 10mg/L AgNO 3 20g/L sucrose and 8.0g/L agar in a 1/2DKW medium.
Preferably, in S3, the induction and multiplication culture of adventitious buds is performed in a constant-temperature culture chamber; the temperature of the constant-temperature culture chamber is 20 +/-2 ℃, and the LED red light: blue light =3:1, the light period is 14/10h, and the illumination intensity is 2000lx.
Preferably, in S4, the rooting nutrient medium used for the ex-vitro rooting culture is a nutrient medium sprayed with 1/4 Hoagland nutrient solution added with 100-400mg/L K-IBA and 0.3% potassium permanganate.
Preferably, the nutrient medium is a mixture of nutrient soil, vermiculite and perlite, and the volume ratio of the nutrient soil to the vermiculite to the perlite is 2:2:1.
preferably, the culture conditions for germination induction of axillary buds in S2 and for ex vitro rooting culture in S4 are both: the temperature is 22 +/-2 ℃, the LED white light is emitted, the light intensity is 2000lx, and the illumination period is 14/10h.
Preferably, in S4, adventitious buds are separated when the adventitious buds to be propagation-cultured are 3 to 6cm long accompanied by 4 to 8 leaves.
Preferably, in S2, stem segments with axillary buds are cut to a length of 1.5 cm.
Preferably, the induction of germination of axillary buds in S2 and the induction and proliferation culture of adventitious buds in S3 are performed in culture flasks.
The percentages of 0.1% mercuric chloride solution, 0.3% potassium permanganate, 20% sodium hypochlorite solution and 10% hydrogen peroxide solution are all mass percentages.
The invention aims to overcome the defects of the prior art, provides a method for reducing the formation of basal callus in the tissue culture and regeneration of a chimonanthus nitens stem segment by utilizing a plant tissue culture technology, mainly takes the current-year semi-lignified stem segment of a perennial excellent chimonanthus nitens strain as an explant, and develops an efficient breeding technology which can simplify the tissue culture and breeding steps of chimonanthus nitens, shorten the production period, reduce the production cost, reduce the formation of callus in the regeneration process and improve the transplanting survival rate of chimonanthus nitens regenerated plants by combining the in-bottle adventitious bud induction with the out-bottle rooting technology;
the invention has the advantages that:
(1) The method takes the current-year semi-lignified stem segment of a field perennial wintersweet excellent strain as an explant, realizes the in vitro regeneration of the wintersweet plant by direct adventitious bud induction, and has the characteristics of convenient material acquisition, rich material and capability of maintaining the excellent properties of a wintersweet female parent strain;
(2) The regeneration method can effectively reduce the formation of callus in the stem regeneration process and effectively improve the quality of the regenerated plant;
(3) The method combines the rooting process and the domestication process of the wintersweet adventitious buds, namely, the wintersweet adventitious buds are directly planted in a nutrient medium after being separated and rooted and formed seedlings in a greenhouse; on one hand, the links of in-bottle rooting and out-bottle domestication and transplantation are simplified, the whole breeding process is simplified, and the seedling raising period is shortened; on the other hand, the transplanting survival rate and the production efficiency of the tissue culture seedlings are improved, the seedling breeding speed is accelerated, the production cost is saved, and a more reliable and more operational technical basis is provided for the industrial breeding of the wintersweet.
Drawings
FIG. 1 is a semi-lignified stem section diagram of a field superior wintersweet strain employed in the present invention;
FIG. 2 is a diagram of the induced germination of axillary buds of a stem segment of a field wintersweet good strain adopted by the invention;
FIG. 3 is the induction chart of adventitious buds of tissue cultured stem segments of Chimonanthus nitens in comparative example 1;
FIG. 4 is a graph showing the proliferation of adventitious buds of a stem section obtained by tissue culture of Chimonanthus nitens in comparative example 1;
FIG. 5 is an adventitious bud induction map of a tissue-cultured stem of Chimonanthus nitens in example 1 of the present invention;
FIG. 6 is a graph showing the proliferation of adventitious buds of tissue-cultured stem segments of Chimonanthus nitens in example 1 of the present invention;
FIG. 7 is a diagram of ex-vitro rooting and field planting of adventitious buds of tissue culture stem segments of Chimonanthus nitens in example 1 of the present invention;
FIG. 8 is a drawing showing the induction culture of extra-bottle rooting of wintersweet clumpy buds for 10 days in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Comparative example 1
A method for tissue culture regeneration of a superior wintersweet strain stem section comprises the following specific operations:
(1) Material taking: using the current year semi-lignified stem segment of the field perennial wintersweet excellent strain shown in figure 1 as an explant;
(2) Surface disinfection and axillary bud germination induction:
cutting the current-year semi-lignified stem segments of the superior wintersweet strain in the step (1) into 6 cm-sized cut segments, placing the cut segments into a 300ml plastic culture bottle, washing the cut segments for 20min under running water after the gauze is sealed, adding 2ml of hand sanitizer during washing, disinfecting and wiping the cut segments for 1 time by using 75% absolute ethyl alcohol, placing the cut segments into a sterile operating platform, and washing the cut segments for 3 times by using sterile water; sterilizing with 75% anhydrous ethanol for 2 times, each for 30s; then washing with sterile water for 4 times; then sterilizing for 2min by using 0.1% mercuric chloride solution, finally washing for 6 times by using sterile water, continuously shaking during the sterilization, absorbing surface moisture after the sterilization, cutting into 1.5cm long stem sections with axillary buds, inoculating the stem sections into a culture medium containing 1/2DKW, 0.05 mg/L6-BA, 0.2mg/L ZT, 20g/L sucrose and 7.5g/L agar, and performing germination induction culture on the axillary buds of the stem sections in a constant-temperature culture chamber with the temperature of 22 +/-2 ℃, LED white light, the illumination intensity of 2000lx and the light cycle of 14/10h, wherein after 20 days of culture, the stem section pollution rate is 2.0% as shown in figure 2; the germination rate of axillary buds is 89.8 percent.
(3) And (3) amplification culture:
taking the axillary buds germinated for 45 days in the step (2) as explant sources, cutting the axillary buds into cut segments with the length of 1.0cm and the axillary buds, and inoculating the cut segments into a wintersweet axillary bud germination induction culture medium (DKW, 0.1mg/L GA) related to a wintersweet axillary bud germination induction method (ZL 201710305050.4) developed by the inventor in earlier stage 3 1.0mg/L KT, 12.5mg/L VC, 30g/L sucrose, 7.0g/L agar) to induce adventitious buds; after 4 weeks of light culture, axillary buds of the stem segment germinated but the base of the stem segment formed a large amount of callus, as shown in FIG. 3; after removing the callus of the base part of the stem segment after the axillary buds germinate, transferring the stem segment after removing the callus of the base part of the axillary buds to a proliferation and elongation culture medium (DKW, 2.0mg/L KT, 0.1mg/L TDZ, 1.0mg/L IBA, 12.5mg/L VC, 30g/L sucrose and 7.0g/L agar) related to the invention patent (ZL 201710305050.4), carrying out proliferation culture in a constant-temperature culture room with the temperature of 22 +/-2 ℃, LED white light, the light intensity of 2000lx and the light period of 14/10h, after 4 weeks of illumination culture, proliferating the axillary buds, and averagely generating 3.1 adventitious buds per explant, but forming a large amount of callus at the base part of the stem segment, as shown in figure 4, leading the adventitious buds to yellowing and necrosis and seriously affecting the quality of the adventitious buds.
(4) Adventitious bud rooting outside the bottle:
separating cluster buds which are 3-6cm high and are accompanied with 4-8 leaves and formed in the step (3) from buds, cleaning the buds, and planting the buds in a nutrient medium sprayed with 1/4 Hoagland nutrient solution added with 100mg/L K-IBA and 0.3% potassium permanganate, wherein the nutrient medium is prepared by mixing the following components in percentage by volume: 2:1, performing induction culture of adventitious roots outside a bottle in a constant-temperature culture room with the temperature of 22 +/-2 ℃, LED white light, illumination intensity of 2000lx and light cycle of 14/10h, wherein after 2 weeks of culture, adventitious roots are formed at the base part of a stem section, and after 2 weeks of continuous culture, the adventitious root induction rate is 58.9%; after further cultivation for 2 months, the field transplanting seedling rate is 87.3 percent.
Example 1
A method for reducing the formation of basal callus in the tissue culture regeneration of a chimonanthus nitens stem segment comprises the following specific operations:
(1) Material taking: taking the current-year semi-lignified stem segment with axillary buds of a wild perennial wintersweet good strain as shown in figure 1 as an explant;
(2) Surface disinfection and axillary bud germination induction:
cutting the current-year stem of the superior wintersweet strain in the step (1) into 6 cm-sized cut segments, placing the cut segments into a 300ml plastic culture bottle, washing the cut segments with running water for 20min after the gauze is sealed, adding 2ml of hand sanitizer during washing, disinfecting and wiping the cut segments with 75% absolute ethyl alcohol for 1 time, and placing the cut segments into a sterile operating platform and washing the cut segments with sterile water for 3 times; sterilizing with 75% anhydrous ethanol for 2 times, each for 30s; then washing with sterile water for 4 times; then sterilizing with 0.1% mercuric chloride solution for 2min, and finally washing with sterile water for 6 times, and shaking continuously during sterilization; after disinfection, surface water is sucked dry, the cut pieces with axillary buds are cut into 1.5cm long sections, the sections are inoculated into a culture medium containing 1/2DKW, 0.2 mg/L6-BA, 0.05mg/L ZT, 20g/L sucrose and 8.0g/L agar, the germination induction culture of the axillary buds of the stem sections is carried out in a constant-temperature culture chamber with the temperature of 22 +/-2 ℃, LED white light, the illumination intensity of 2000lx and the light cycle of 14/10h, and the germination rate of the axillary buds is 100 percent after 20 days of culture.
(3) And (3) amplification culture:
taking the axillary buds germinated for 60 days in the step (2) as an explant source, and cutting the axillary buds into 1.0cm long sections with axillary budsInoculating to a culture medium containing 1/2DKW, 0.3 mg/L6-BA, 1.0mg/L ZT and 10mg/L AgNO 3 20g/L sucrose, 8.0g/L agar in an adventitious bud induction culture medium at a temperature of 20 +/-2 ℃, and an LED red light: blue light =3:1, performing induction culture of adventitious buds in a constant-temperature culture chamber with a photoperiod of 14/10h. After 4 weeks of illumination culture, the adventitious bud induction rate is as high as 92.4%, each explant averagely generates 3.3 adventitious buds, and only 10.4% of the stem segment base has a small amount of callus without formation, as shown in figure 5; then, the stem section from which the adventitious bud was induced was transferred to a fresh adventitious bud induction medium for subculture and multiplication culture of the adventitious bud. After 4 weeks of light culture, on average 6.5 adventitious shoots were produced per explant, with an average shoot length of 4.8cm, as shown in FIG. 6.
(4) Adventitious bud rooting outside the bottle:
separating cluster buds which are 3-6cm high and are accompanied with 4-8 leaves and formed in the step (3), cleaning, and planting the cluster buds in a nutrient medium sprayed with 1/4 Hoagland nutrient solution added with 300mg/L K-IBA and 0.3% potassium permanganate, wherein the nutrient medium contains 2 volume percent: 2:1, culturing adventitious roots outside a bottle in a constant-temperature culture chamber with the temperature of 22 +/-2 ℃, LED white light, illumination intensity of 2000lx and photoperiod of 14/10h, and performing induction culture on the adventitious roots outside the bottle, as shown in figure 7. After 10 days of culture, adventitious roots are formed at the base of the stem segment, and as shown in FIG. 8, the induction rate of the adventitious roots is 91.2%; after the cultivation is continued for 2 months, the field transplanting seedling rate is 95.6 percent.
Example 2
A method for reducing the formation of basal callus in the tissue culture regeneration of a chimonanthus nitens stem segment comprises the following steps:
(1) Material taking: taking the current-year semi-lignified stem section with axillary buds of a field perennial wintersweet excellent strain as an explant;
(2) Surface disinfection and axillary bud germination induction:
cutting the explants obtained in the step (1) into 5 cm-sized sections, placing the sections into a 300ml plastic culture bottle, washing the sections for 15min under running water after sealing gauze, adding 1ml of hand sanitizer during washing, disinfecting and wiping the sections for 3 times by using 75% absolute ethyl alcohol, and placing the sections into a sterile operating platform and washing the sections for 2 times by using sterile water; sterilizing with 75% anhydrous ethanol for 25s for 1 time; then washing with sterile water for 5 times; then sterilizing for 1min by using 0.1% mercuric chloride solution, finally washing for 7 times by using sterile water, cutting into 1.5cm long sections with axillary buds, inoculating the sections into a culture medium containing 1/2DKW, 0.05 mg/L6-BA, 0.5mg/L ZT, 20g/L sucrose and 8.0g/L agar, and performing germination induction culture on the axillary buds of the stem sections in a constant-temperature culture room with the temperature of 22 +/-2 ℃, LED white light, the illumination intensity of 2000lx and the light cycle of 14/10 h;
(3) And (3) amplification culture:
taking axillary buds germinated for 45 days in the step (2) as explant sources, cutting into sections with axillary buds and the lengths of 1.0cm, inoculating the sections with axillary buds to the section containing 1/2DKW, 0.2 mg/L6-BA, 0.2mg/L ZT and 5mg/LAgNO 3 20g/L sucrose and 8.0g/L agar, under the conditions that the temperature is 20 +/-2 ℃, and the temperature of an adventitious bud induction culture medium is as follows: blue light =3:1, carrying out induction culture of adventitious buds in a constant-temperature culture chamber with a photoperiod of 14/10h. After 4 weeks of illumination culture, transferring the stem segments from which the adventitious buds are induced to a fresh adventitious bud induction culture medium for subculture and multiplication culture of the adventitious buds;
(4) Adventitious bud rooting outside the bottle:
separating cluster buds which are 3-6cm high and are provided with 4-8 leaves and formed in the step (3), cleaning, and planting the cluster buds in a nutrient medium sprayed with 1/4 Hoagland nutrient solution added with 100mg/L K-IBA and 0.3% potassium permanganate, wherein the nutrient medium contains the components in a volume ratio of 2:2:1, culturing adventitious roots outside a bottle in a constant-temperature culture chamber with the temperature of 22 +/-2 ℃, LED white light, illumination intensity of 2000lx and photoperiod of 14/10h.
Example 3
A method for reducing the formation of basal callus in the tissue culture regeneration of a chimonanthus nitens stem segment comprises the following steps:
(1) Material taking: taking the current-year semi-lignified stem section with axillary buds of a field perennial wintersweet excellent strain as an explant;
(2) Surface disinfection and axillary bud germination induction:
cutting the explants obtained in the step (1) into 7 cm-sized sections, placing the sections into a 300ml plastic culture bottle, washing the sections with running water for 25min after sealing gauze, adding 3ml of a hand sanitizer during washing, disinfecting and wiping the sections with 75% absolute ethyl alcohol for 2 times, and placing the sections into a sterile operating platform and washing the sections with sterile water for 4 times; sterilizing with 75% anhydrous ethanol for 3 times, wherein the sterilizing time is 35s each time; then washing with sterile water for 3 times; then sterilizing for 2min by using 0.1% mercuric chloride solution, finally washing for 5 times by using sterile water, cutting into 1.5cm long sections with axillary buds, inoculating the sections into a culture medium containing 1/2DKW, 0.1 mg/L6-BA, 0.3mg/L ZT, 20g/L sucrose and 8.0g/L agar, and performing germination induction culture on the axillary buds of the stem sections in a constant-temperature culture room with the temperature of 22 +/-2 ℃, LED white light, the illumination intensity of 2000lx and the light cycle of 14/10 h;
(3) And (3) amplification culture:
taking the axillary bud germinated for 90 days in the step (2) as an explant source, cutting into 1.0cm long sections with axillary buds, inoculating the sections with 1/2DKW, 0.5 mg/L6-BA, 0.5mg/L ZT and 20mg/L AgNO 3 20g/L sucrose and 8.0g/L agar in an adventitious bud induction medium at a temperature of 20 + -2 ℃, LED red light: blue light =3:1, carrying out induction culture of adventitious buds in a constant-temperature culture chamber with a photoperiod of 14/10h. After 4 weeks of illumination culture, transferring the stem segments from which the adventitious buds are induced into a fresh adventitious bud induction culture medium for subculture and multiplication culture of the adventitious buds;
(4) Adventitious bud rooting outside the bottle:
separating cluster buds which are 3-6cm high and are accompanied with 4-8 leaves and formed in the step (3), cleaning, and planting the cluster buds in a nutrient medium sprayed with 1/4 Hoagland nutrient solution added with 400mg/L K-IBA and 0.3% potassium permanganate, wherein the nutrient medium contains 2 volume percent: 2:1, culturing adventitious roots outside a bottle in a constant-temperature culture chamber with the temperature of 22 +/-2 ℃, LED white light, illumination intensity of 2000lx and photoperiod of 14/10h.
The influence of different disinfection methods on the pollution rate of the wild wintersweet stems is tested. Cutting the current-year semi-lignified branch with axillary buds of a wild perennial wintersweet into 6 cm-sized sections, placing the sections into a 300ml plastic culture bottle, washing the sealed gauze for 20min under running water, adding 2ml of a hand sanitizer during washing, disinfecting and wiping the sections for 1 time by using 75% absolute ethyl alcohol, and placing the sections into a sterile operating platform and washing the sections for 3 times by using sterile water; sterilizing with 75% anhydrous ethanol for 2 times, each for 30s; then washing with sterile water for 4 times; then sterilizing with different kinds of sterilizing agents (20% sodium hypochlorite solution, 0.1% mercuric chloride solution and 10% hydrogen peroxide solution) for different time (1min, 2min,3min and 5 min), and washing with sterile water for 6 times while shaking. After sterilization, the surface water was blotted, and the stem section with axillary buds was cut into 1.5cm, and inoculated into the axillary bud induction medium of example 1 for axillary bud induction culture. The growth conditions were the same as in example 1. After 20d of culture, the average contamination rate of the stem segments and the average germination rate of axillary buds were counted, and the results are shown in table 1.
TABLE 1 Effect of different disinfection methods on the rate of wintersweet stem contamination and axillary bud germination
Figure BDA0003535394880000121
Figure BDA0003535394880000131
The results in table 1 show that of the three disinfectants tested, the 0.1% mercuric chloride solution has the best disinfection effect, the pollution rate is gradually reduced along with the prolonging of the disinfection time, the growth state of axillary buds of the stem segment is the best at the disinfection time of 2min, the average pollution rate of the stem segment is only 2.0%, and the average germination rate of the axillary buds is 100%.
To alleviate the induction of basal callus during regeneration of chimonanthus nitens sterile tissue culture stem sections, the inventors further tested various types and concentrations of plant regulators (6-BA, ZT and AgNO) 3 ) The content of macroelements, different light qualities and the explant age in the DKW culture medium, namely, the effect of axillary bud plants obtained from different days (30d, 45d,60d,75d and 90 d) of wild stem germination on adventitious bud induction and callus induction of the chimonanthus nitens stem segments. Taking axillary bud plants obtained after germination culture for 60d as explant materials, cutting into 1.0cm sections, inoculating to a culture medium added with different plant growth regulatorsThe stem axillary bud adventitious bud induction culture is carried out in a DKW culture medium with different macroelement contents in the agent in a constant-temperature culture chamber with the temperature of 20 +/-2 ℃, LED white light, illumination intensity of 2000lx and photoperiod of 14/10h, and the result is shown in Table 2. The study of Table 2 shows that the low-concentration macroelement and the low-concentration plant growth regulator in the culture medium are more beneficial to reducing the formation of the callus at the base of the stem segment, but the low-concentration plant growth regulator influences the induction rate of the adventitious bud, and the low-concentration macroelement also causes the weak growth of the adventitious bud and even causes the yellowing phenomenon. Therefore, based on the comprehensive consideration of adventitious bud inductivity, adventitious bud number, callus induction and adventitious bud growth, 0.3 mg/L6-BA, 1.0mg/L ZT and 10mg/L AgNO are added into the 1/2DKW culture medium 3 The induction effect of the adventitious bud of the chimonanthus nitens stem segment is optimal. After 30 days of illumination culture, the average adventitious bud induction rate is 89.2%, each explant averagely generates 3.1 adventitious buds, the average callus induction rate is 16.3%, and the average diameter of the callus is 0.6cm.
TABLE 2 Effect of different plant growth regulating substances and macroelements on induction of adventitious buds of Chimonanthus nitens stem segment
Figure BDA0003535394880000141
Meanwhile, the inventor tests the influence of the LED light quality on the growth vigor of the adventitious buds of the chimonanthus nitens stem segments, and researches find that the influence of the LED light quality on the length and the growth vigor of the adventitious buds of the stem segments is shown in table 3. Research finds that the LED light quality has an important influence on the growth vigor of the chimonanthus nitens adventitious bud, and in the tested light quality, the ratio of the LED red: blue is 3:1, it is most favorable for the elongation growth of the bud.
TABLE 3 influence of different LED light quality conditions on the height of adventitious bud of calyx canthus stem segment
Figure BDA0003535394880000142
Figure BDA0003535394880000151
On this basis, the inventors further tested the influence of the age of the explant on the callus and adventitious bud induction by inoculating the stem segments of axillary bud plants obtained by germinating the stem segments in the field on different days (30d, 45d,60d,75d and 90 d) in example 1 to the wild-type plants supplemented with 0.3mg/L of 6-BA, 1.0mg/L of ZT and 10mg/L of AgNO 3 In the 1/2DKW culture medium at a temperature of 20 +/-2 ℃, the LED emits red light: the stem adventitious bud induction culture was carried out in a thermostatical culture chamber with blue light (3. Researches show that the age of the explant has an important influence on the formation of adventitious buds and callus of the stem, the induction rate of the adventitious buds and the number of the adventitious buds generated by each explant averagely increase and then decrease with the increase of the age of the stem, meanwhile, the induction rate of the callus and the diameter of the callus tend to decrease gradually with the increase of the age of the stem, and the induction rate of the callus is 0% when the seedling age is 90 days. The induction conditions of adventitious buds and callus tissues are comprehensively considered, and when stem sections with the seedling age of 60 days are used as explants, the induction effect is the best.
TABLE 4 Effect of different explant ages on the induction of adventitious buds of Chimonanthus praecox
Figure BDA0003535394880000152
Figure BDA0003535394880000161
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (7)

1. A method for reducing the formation of basal callus in the tissue culture regeneration of a chimonanthus nitens stem segment is characterized by comprising the following steps:
s1, taking an annual semi-lignified stem segment with an axillary bud of a field perennial wintersweet excellent strain as an explant;
s2, carrying out surface disinfection on the explant in the S1, cutting the explant into stem sections with axillary buds, and inoculating the stem sections with axillary buds to an axillary bud induction culture medium for germination induction of the axillary buds; the axillary bud induction culture medium comprises the following components: 1/2DKW, 0.05-0.2 mg/L6-BA, 0.05-0.5mg/L ZT, 20g/L sucrose, 8.0g/L agar;
s3, cutting axillary buds sprouting in the S2 for 45-90 days into segments, inoculating the segments into a culture medium added with plant growth regulating substances, and performing induction and multiplication culture on adventitious buds; wherein the culture medium added with plant growth regulating substances is added with 0.3 mg/L6-BA, 1.0mg/L ZT and 10mg/L AgNO 3 20g/L sucrose and 8.0g/L agar in a 1/2DKW culture medium;
s4, separating the adventitious buds after the propagation culture, and then carrying out ex-vitro rooting culture to obtain complete wintersweet regenerated plants; the rooting nutrient medium used for the ex-vitro rooting culture is a nutrient medium sprayed with 1/4 Hoagland nutrient solution added with 100-400mg/L K-IBA and 0.3% potassium permanganate.
2. The method for reducing formation of basal callus in tissue culture regeneration of chimonanthus nitens stem segments according to claim 1, wherein in S2, the method for surface sterilization of explants in S1 comprises the following steps: cutting the explant into 5-7cm sections, placing in a plastic culture bottle, sealing with gauze, washing with running water for 15-25min, and adding 1-3ml of hand sanitizer during washing; then, wiping 1-3 times with 75% absolute ethyl alcohol, placing in a sterile operating platform, washing with sterile water for 2-4 times, sterilizing with 75% absolute ethyl alcohol for 1-3 times, each time for 25-35s, washing with sterile water for 3-5 times, sterilizing with 0.1% mercuric chloride solution for 1-2min, and washing with sterile water for 5-7 times.
3. The method for reducing formation of basal callus in tissue culture regeneration of chimonanthus nitens stem segments according to claim 1, wherein the length of the cut with axillary buds is 1.0cm.
4. The method for alleviating formation of callus on the basal part in tissue culture regeneration of a stem section of Chimonanthus praecox as claimed in claim 1, wherein in S3, induction and multiplication culture of adventitious bud is performed in a constant temperature culture chamber; the temperature of the constant-temperature culture chamber is 20 +/-2 ℃, and the LED red light: blue light =3:1, the photoperiod is 14/10h, and the illumination intensity is 2000lx.
5. The method for reducing the formation of basal callus in tissue culture regeneration of chimonanthus nitens stem segments according to claim 1, wherein the nutrient medium is a mixture of nutrient soil, vermiculite and perlite, and the volume ratio of the nutrient soil, the vermiculite and the perlite is 2:2:1.
6. the method for reducing the formation of basal callus in the tissue culture regeneration of chimonanthus nitens stem segments according to claim 1, wherein the conditions for the germination induction of axillary buds in S2 and the culture of ex vitro rooting in S4 are both: the temperature is 22 +/-2 ℃, the LED white light has the light intensity of 2000lx, and the illumination period is 14/10h.
7. The method for alleviating formation of basal callus in tissue culture regeneration of a stem section of Chimonanthus praecox as claimed in any one of claims 1 to 6, wherein adventitious shoots to be propagation-cultured are separated when they grow to 3 to 6cm and accompanied with 4 to 8 leaves in S4.
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CN1053345A (en) * 1990-06-15 1991-07-31 中国科学院武汉植物研究所 Isolated rapid reproduction method of wintersweet
CN102599060A (en) * 2012-03-29 2012-07-25 常熟市海虞茶叶有限公司 Calyx canthus tissue culture rapid propagation method
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CN112219721A (en) * 2020-11-12 2021-01-15 开远云之澳花卉有限公司 Breeding method of new variety of Australia wintersweet

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Publication number Priority date Publication date Assignee Title
CN1053345A (en) * 1990-06-15 1991-07-31 中国科学院武汉植物研究所 Isolated rapid reproduction method of wintersweet
CN102599060A (en) * 2012-03-29 2012-07-25 常熟市海虞茶叶有限公司 Calyx canthus tissue culture rapid propagation method
CN104285791A (en) * 2014-09-29 2015-01-21 中国计量学院 Method applied to tissue culture and rapid propagation of chimonanthus nitens
CN107006370A (en) * 2017-05-03 2017-08-04 中国科学院合肥物质科学研究院 A kind of wax plum plant high-efficiency in-vitro regeneration method
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