CN110839530A - Method for inducing flowering of Chinese rose in test tube - Google Patents
Method for inducing flowering of Chinese rose in test tube Download PDFInfo
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- CN110839530A CN110839530A CN201911166021.XA CN201911166021A CN110839530A CN 110839530 A CN110839530 A CN 110839530A CN 201911166021 A CN201911166021 A CN 201911166021A CN 110839530 A CN110839530 A CN 110839530A
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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
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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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention belongs to the technical field of plant tissue culture, and relates to a method for inducing Chinese rose to bloom in a test tube. The method comprises the following steps: sterilizing explants of Chinese roses to obtain sterile explants; inoculating the sterile explant to a first MS culture medium to induce callus; the first MS culture medium contains 0.1-10mg/L of 6-BA, 0.1-2mg/L of NAA, 4-10g/L of agar and 20-50g/L of sucrose; inoculating the callus to a second MS culture medium for differential culture to obtain a young stem; the second MS culture medium contains 0.01-2mg/L of 6-BA, 0.1-2mg/L of NAA, 4-10g/L of agar and 20-50g/L of sucrose; inoculating the young stem to a third MS culture medium, and alternately carrying out rooting and flowering culture in a photoperiod and a dark period; the third MS culture medium contains IBA 0.1-10mg/L, NAA 0.1-2mg/L, nano-iron particles 0.01-1mg/L, agar 4-10g/L and sucrose 20-50g/L, and has pH of 3-7. The method can make Chinese rose flower in test tube.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing Chinese rose to bloom in a test tube.
Background
The Chinese rose belongs to Rosaceae, Rosa and Chinese rose seed (Latin with the name of Rosa chinensis Jacq.). China rose is an important flower plant, is one of four cut flowers in the world, is native to China, and is widely distributed in the world. The Chinese rose can be used as ornamental plant or medicinal plant. Meanwhile, the Chinese rose is an important experimental material in the biological science research. The flowering of the Chinese rose in the test tube is realized by a tissue culture method, and a shortcut can be provided for the biological science research of the Chinese rose. Provides a theoretical basis for further exploring the principle mechanism of plant flowering.
The traditional Chinese rose tissue culture is mainly used for adjusting the differentiation and proliferation capacity of Chinese rose tissues so as to improve the propagation coefficient of Chinese rose test-tube plantlets, or for regulating and controlling the requirements of transgenic experiments, the Chinese rose test-tube plantlets are transferred into a rooting culture medium after being differentiated, and then are transplanted into soil after rooting. The method for culturing the tissues of the Chinese rose in the prior art is mainly used for the rapid propagation of good varieties and the basic operation of biotechnology in the Chinese rose transgenosis, but the research and the development of the method for culturing the tissues of the Chinese rose for flowering and fruiting in a test tube are not available, because the flowering and fruiting of the Chinese rose in the test tube are different from those of the Chinese rose in soil, the test tube is a relatively closed microenvironment controlled artificially, and the difficulty lies in the control of illumination and temperature and the proportion of nutrient components and hormones in a culture medium, so that the requirement that the Chinese rose can bloom in the test tube is met.
Disclosure of Invention
The invention aims to provide a method for inducing Chinese rose to bloom in a test tube so as to realize the flowering of the Chinese rose in the test tube.
In order to accomplish the above object, the present invention provides a method for inducing flowering of Chinese roses in a test tube, the method comprising the steps of:
s1, sterilizing explants of Chinese roses to obtain sterile explants;
s2, inoculating the sterile explant into a first MS culture medium to induce callus, so as to obtain callus; the first MS culture medium contains 0.1-10mg/L of 6-benzylamino adenine (6-BA), 0.1-2mg/L of naphthylacetic acid (NAA), 4-10g/L of agar and 20-50g/L of sucrose;
s3, inoculating the callus into a second MS culture medium for differential culture to obtain young stems; the second MS culture medium contains 0.01-2mg/L of 6-benzylamino adenine (6-BA), 0.1-2mg/L of naphthylacetic acid (NAA), 4-10g/L of agar and 20-50g/L of sucrose;
s4, inoculating the young stems into a third MS culture medium, and alternately performing rooting and flowering culture in a photoperiod and a dark period; the third MS culture medium contains 0.1-10mg/L of indolebutyric acid (IBA), 0.1-2mg/L of naphthylacetic acid (NAA), 0.01-1mg/L of nano-iron particles, 4-10g/L of agar and 20-50g/L of sucrose, and the pH value of the third MS culture medium is 3-7.
The explant can be sterilized by any known sterilization means by those skilled in the art. Preferably, in step S1, the sterilizing treatment of the explant of the Chinese rose comprises the following steps: firstly, washing the explant, then soaking the explant in 1% -10% of Tween-20 for 2-10 minutes, then rinsing the explant by using sterile water, and then disinfecting the explant by using 3% -5% of sodium hypochlorite for 15-30 minutes, or using 0.01% -1% of mercuric chloride (HgCl)2) Sterilizing the surface of the solution for 2-10 minutes, and finally rinsing with sterile water.
In a preferred embodiment of the present invention, in step S2, the first MS medium contains 0.8-2mg/L of 6-benzylamino adenine (6-BA), 0.5-1.5mg/L of naphthylacetic acid (NAA), 5-8g/L of agar, and 25-35g/L of sucrose. More preferably, the first MS medium contains 2mg/L of 6-benzylamino adenine (6-BA), 1mg/L of naphthylacetic acid (NAA), 6g/L of agar, and 30g/L of sucrose.
In a preferred embodiment of the present invention, in step S3, the second MS medium contains 1.5-2mg/L of 6-benzylamino adenine (6-BA), 0.5-1.5mg/L of naphthylacetic acid (NAA), 5-8g/L of agar, and 25-35g/L of sucrose. More preferably, the second MS medium contains 1mg/L of 6-benzylamino adenine (6-BA), 1mg/L of naphthylacetic acid (NAA), 6g/L of agar, and 30g/L of sucrose.
In a preferred embodiment of the present invention, in step S4, the third MS medium contains 0.8-1.5mg/L of indolebutyric acid (IBA), 1.5-2mg/L of naphthylacetic acid (NAA), 0.5-1mg/L of nano-iron particles, 5-8g/L of agar, and 15-20g/L of sucrose, and the pH of the third MS medium is 5.6-6.0. More preferably, the third MS medium contains 1mg/L of indolebutyric acid (IBA), 2mg/L of naphthylacetic acid (NAA), 0.5mg/L of nano-iron particles, 6g/L of agar, and 15g/L of sucrose, and the pH value of the third MS medium is 5.8.
In a preferred embodiment of the present invention, in step S4, the light cycle is 12 to 20 hours; the dark period is 12-4 hours.
In a preferred embodiment of the invention, during the rooting flowering culture, the young stems are irradiated with white fluorescence having a light intensity of 65-80 μ E/m2/s。
In a preferred embodiment of the present invention, the temperature of the rooting flowering culture is 23-25 ℃ in step S4.
In the present invention, except for the content of hormones, agar and sucrose, the first MS culture medium, the second MS culture medium, the third MS culture medium and the fourth MS culture medium may have the same major elements and trace elements, and the MS culture medium is widely used in plant tissue culture, and thus the present invention is not described herein again.
In the present invention, the time required for callus induction from sterile explants is 20-30 days; the time required for differentiation from the callus into young stems is 7-14 days; the time required for rooting and flowering of the young stems is 20-30 days.
The explant of a rose used in the present invention includes at least one of a stem tip, a lateral bud and a stem segment of a rose.
The method for inducing Chinese rose to bloom in the test tube realizes the flowering of Chinese rose in the test tube by selecting the culture medium used for inducing callus, the culture medium used for differentiation and the culture medium used for rooting and flowering.
The invention improves the types, concentrations and nutritional components of hormones in a culture medium, and the hormone regulation is mainly used for realizing the purpose of flowering by converting the vegetative growth into the reproductive growth in a test tube of the Chinese rose test-tube plantlet, so that the types, concentrations and nutritional components of the hormones are different from those of the traditional Chinese rose tissue culture. Because the flowering in the test tube is not limited by seasons and can be induced at any time, the tissue culture test tube has the advantages of high flowering rate, good repeatability, stable technology and the like, and the method can be widely applied to flowering research and test tube flower application.
The method for inducing Chinese rose to bloom in the test tube induces the Chinese rose to bloom in the test tube.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
Example 1
S1, using the stem tip of a miniature Chinese rose variety Hula Girl grown in a field as an explant, thoroughly washing the explant under tap water, then placing the explant in 5% Tween-20 for 5 minutes, and rinsing the explant with distilled water sterilized at high temperature and high pressure for 3-4 times, wherein 0.1% mercuric chloride (HgCl) is added2) Sterilizing the surface for 4 minutes, and rinsing with distilled water sterilized at high temperature and high pressure for 4-5 times to obtain a sterile explant.
S2, inoculating the sterile explant to a culture medium, and inducing callus: MS culture medium, 6-benzylamino adenine (6-BA)2mg/L, naphthylacetic acid (NAA)1mg/L, agar 6g/L, cane sugar 30g/L, and inducing callus for 25-30 days.
S3, transferring the callus to an MS basic culture medium, 1mg/L of 6-benzylamino adenine (6-BA), 1mg/L of naphthylacetic acid (NAA), 6g/L of agar and 30g/L of sucrose, wherein the pH value is 5.8, culturing for 7-10 days, and differentiating the callus into caulicles.
S4, inoculating the caulicles to an MS culture medium, 1mg/L of indolebutyric acid (IBA), 2mg/L of naphthylacetic acid (NAA), 0.5mg/L of nano-iron particles, 6g/L of agar and 15g/L of sucrose,the pH value is 5.8, the culture temperature of the tissue culture chamber is 23-25 ℃, and the white fluorescence intensity is 65 mu E/m2S, photoperiod of 16 hours, dark period of 8 hours. Rooting after 7 days, forming flower buds after 14 days, and flowering after 20 days.
Example 2
S1, using the stem tip of the Chinese rose Kori as an explant, thoroughly washing with tap water, placing in 5% Tween-20 for 5min, rinsing with distilled water sterilized at high temperature and high pressure for 3-4 times, and adding 0.1% mercuric chloride (HgCl)2) Sterilizing the surface for 4 minutes, and rinsing with distilled water sterilized at high temperature and high pressure for 4-5 times to obtain a sterile explant.
S2, inoculating the sterile explant to a culture medium, and inducing callus: MS culture medium, 6-benzylamino adenine (6-BA)2mg/L, naphthylacetic acid (NAA)1mg/L, agar 6g/L, cane sugar 30g/L, and inducing callus for 25-30 days.
S3, transferring the callus to an MS culture medium, 1mg/L of 6-benzylamino adenine (6-BA), 1mg/L of naphthylacetic acid (NAA), 6g/L of agar and 30g/L of sucrose, culturing for 7-10 days at the pH value of 5.8, and differentiating the callus into young stems.
S4, inoculating the caulicles to an MS culture medium, 1mg/L of indolebutyric acid (IBA), 2mg/L of naphthylacetic acid (NAA), 0.5mg/L of nano-iron particles, 6g/L of agar and 15g/L of sucrose, wherein the pH value is 5.8, the culture temperature of a tissue culture chamber is 23-25 ℃, and the white fluorescence light intensity is 65 mu E/m2S, photoperiod of 16 hours, dark period of 8 hours. Rooting after 7 days, forming flower buds after 20 days, and flowering after 30 days.
Comparative example 1
S1, using the stem tip of a miniature Chinese rose variety Hula Girl grown in a field as an explant, thoroughly washing the explant under tap water, then placing the explant in 5% Tween-20 for 5 minutes, and rinsing the explant with distilled water sterilized at high temperature and high pressure for 3-4 times, wherein 0.1% mercuric chloride (HgCl) is added2) Sterilizing the surface for 4 minutes, and rinsing with distilled water sterilized at high temperature and high pressure for 4-5 times to obtain a sterile explant.
S2, inoculating the sterile explant to a culture medium, and inducing callus: MS culture medium, agar 6g/L and cane sugar 30g/L, inducing callus for 25-30 days.
S3, transferring the callus to an MS basic culture medium, 0.5mg/L of 6-benzylamino adenine (6-BA), 6g/L of agar, 30g/L of cane sugar and a pH value of 5.8, culturing for 30 days, and differentiating the callus into a caulicle.
S4, inoculating the caulicles to an MS culture medium, 0.2mg/L of 6-benzylamino adenine (6-BA), 0.1mg/L of naphthylacetic acid (NAA), 6g/L of agar and 15g/L of cane sugar, wherein the pH value is 5.8, the culture temperature of a tissue culture chamber is 23-25 ℃, and the white fluorescent light intensity is 65 mu E/m2S, photoperiod of 16 hours, dark period of 8 hours. After 30 days, the roots were rooted and no flower buds were formed.
Comparative example 2
S1, using the stem tip of a miniature Chinese rose variety Hula Girl grown in a field as an explant, thoroughly washing the explant under tap water, then placing the explant in 5% Tween-20 for 5 minutes, and rinsing the explant with distilled water sterilized at high temperature and high pressure for 3-4 times, wherein 0.1% mercuric chloride (HgCl) is added2) Sterilizing the surface for 4 minutes, and rinsing with distilled water sterilized at high temperature and high pressure for 4-5 times to obtain a sterile explant.
S2, inoculating the sterile explant to a culture medium, and inducing callus: MS culture medium, 6-benzylamino adenine (6-BA)0.02mg/L, naphthylacetic acid (NAA)0.01mg/L, agar 6g/L and cane sugar 30g/L, and inducing callus for 25-30 days.
S3, transferring the callus to an MS basic culture medium, 0.01mg/L of 6-benzylamino adenine (6-BA), 0.01mg/L of naphthylacetic acid (NAA), 6g/L of agar and 30g/L of sucrose, culturing for 20 days, and differentiating the callus into young stems.
S4, inoculating the caulicles to an MS culture medium, 0.01mg/L of indolebutyric acid (IBA), 0.02mg/L of naphthylacetic acid (NAA), 0.005mg/L of nano-iron particles, 6g/L of agar and 15g/L of sucrose, wherein the pH value is 5.8, the culture temperature of a tissue culture chamber is 23-25 ℃, and the white fluorescence light intensity is 65 mu E/m2S, photoperiod of 16 hours, dark period of 8 hours. Rooting after 15 days, and no flower buds are formed.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Claims (9)
1. A method of inducing flowering of a rose in vitro, comprising the steps of:
s1, sterilizing explants of Chinese roses to obtain sterile explants;
s2, inoculating the sterile explant into a first MS culture medium to induce callus, so as to obtain callus; the first MS culture medium contains 0.1-10mg/L of 6-benzylamino adenine, 0.1-2mg/L of naphthylacetic acid, 4-10g/L of agar and 20-50g/L of cane sugar;
s3, inoculating the callus into a second MS culture medium for differential culture to obtain young stems; the second MS culture medium contains 0.01-2mg/L of 6-benzylamino adenine, 0.1-2mg/L of naphthylacetic acid, 4-10g/L of agar and 20-50g/L of cane sugar;
s4, inoculating the young stems into a third MS culture medium, and alternately performing rooting and flowering culture in a photoperiod and a dark period; the third MS culture medium contains 0.1-10mg/L of indolebutyric acid, 0.1-2mg/L of naphthylacetic acid, 0.01-1mg/L of nano-iron particles, 4-10g/L of agar and 20-50g/L of sucrose, and the pH value of the third MS culture medium is 3-7.
2. The method according to claim 1, wherein the step of sterilizing the explants of Chinese rose in step S1 comprises the steps of: firstly, washing the explant, then soaking the explant in 1% -10% of Tween-20 for 2-10 minutes, then rinsing the explant by using sterile water, then disinfecting the explant by using 3% -5% of sodium hypochlorite for 15-30 minutes, or sterilizing the surface of the explant by using 0.01% -1% of mercuric chloride solution for 2-10 minutes, and finally rinsing the explant by using sterile water.
3. The method of claim 1, wherein in step S2, the first MS medium comprises 0.8-2 mg/L6-benzylamino adenine, 0.5-1.5mg/L naphthylacetic acid, 5-8g/L agar, and 25-35g/L sucrose.
4. The method of claim 1, wherein in step S3, the second MS medium comprises 1.5-2 mg/L6-benzylamino adenine, 0.5-1.5mg/L naphthylacetic acid, 5-8g/L agar, and 25-35g/L sucrose.
5. The method of claim 1, wherein in step S4, the third MS medium comprises 0.8-1.5mg/L indolebutyric acid, 1.5-2mg/L naphthylacetic acid, 0.5-1mg/L nano-iron particles, 5-8g/L agar, and 15-20g/L sucrose, and the pH of the third MS medium is 5.6-6.0.
6. The method according to claim 1, wherein in step S4, the light period is 12-20 hours; the dark period is 12-4 hours.
7. The method according to claim 1, wherein during the rooting flowering culture, the young stems are irradiated with white fluorescence having an intensity of 65-80 μ E/m2/s。
8. The method according to claim 1, wherein the temperature of the rooting flowering culture is 23-25 ℃ in step S4.
9. The method of any one of claims 1-8, wherein the explant comprises: at least one of stem tip, lateral bud and stem segment of Chinese rose.
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Cited By (2)
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| CN113924977A (en) * | 2020-07-13 | 2022-01-14 | 中国科学院分子植物科学卓越创新中心 | Method for controlling flowering of Chinese rose test-tube plantlets |
| CN117770139A (en) * | 2024-02-26 | 2024-03-29 | 东北林业大学 | Culture medium for inducing China rose plant to regenerate in vitro and application thereof |
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Application publication date: 20200228 |