CN114902961A - Method for creating new ornamental pineapple variety by adopting EMS mutagenesis - Google Patents
Method for creating new ornamental pineapple variety by adopting EMS mutagenesis Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention belongs to the technical field of pineapple breeding, and particularly relates to a method for creating a new ornamental pineapple variety by EMS mutagenesis. The invention takes the tender lateral branch of Tainong 11 pineapple as an explant, establishes an aseptic tissue culture system, and adopts a method combining a tissue culture technology and EMS mutagenesis to obtain an EMS mutagenesis plant. The plant type of the EMS mutagenesis plant obtained by the method is beautiful and has ornamental value.
Description
Technical Field
The invention belongs to the technical field of pineapple breeding, and particularly relates to a method for creating a new ornamental pineapple variety by EMS mutagenesis.
Background
The ornamental pineapple is of the family of bromeliaceae (Bromeliaceae) The ornamental plant is originally produced in tropical and subtropical regions of America, and has unique plant type, beautiful leaf shape, rich and beautiful flower shape and color and long flowering phaseThe potted flower is a famous high-grade gift flower which has great development prospect and real name, has good flower and leaf watching performance and is mostly shady-resistant, is suitable for indoor long-term decoration and appreciation, is an extremely ideal indoor ornamental plant, is deeply loved by people all over the world and is a famous high-grade gift potted flower with great development prospect. With the introduction of ornamental pineapple in large quantities and the domestic cultivation, the related research is also gradually concerned.
The genetic material creation has very important function for the development of bioscience, induces the variation of plants, constructs a mutant library, screens target mutants from the mutant library, and can provide abundant basic materials for the research of plant gene functions. Ethyl Methane Sulfonate (EMS) is the most widely used chemical mutagen in the current germplasm resource innovation work, and has the characteristics of low mutagenesis cost, simple and convenient operation, high mutation rate, small damage to materials, difficult chromosome distortion, wide mutation spectrum and the like. According to the current research reports, the wheat EMS mutagenesis is generally carried out by using seeds, and the mutagenesis treatment is also partially carried out by using callus and the like, and the mutagenesis methods mainly adopted include a dropping method, a soaking method and a co-culture method. However, no report has been made on a method for constructing a novel ornamental pineapple variety by mutating a mutant of a pineapple plant with EMS.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for creating an induced pineapple mutant by EMS mutagenesis, and create a special mutant material, so as to enrich the genetic diversity of pineapple, improve the ornamental value of pineapple as an ornamental plant, and screen out an excellent germplasm resource of pineapple with ornamental property.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for creating a new ornamental pineapple variety by EMS mutagenesis is characterized by comprising the following steps: the method specifically comprises the following steps:
1) selection of test materials:
selecting tender lateral branches of more than one year of Tainong 11 pineapple plants to establish a tissue culture system, cutting the tender lateral branches into stem sections with the length of 2cm, and taking the prepared stem sections as explants for tissue culture;
2) preparing a sterile explant:
and (3) carrying out disinfection treatment on the explant: soaking 5% chlorothalonil overnight → washing with clear water for 10 minutes → washing with detergent diluent for 3 hours → washing with clear water until the surface of the explant is cleaned, washing with 75% alcohol for 5 minutes → washing with sterile distilled water for 3 times → sterilizing with 0.1% mercuric chloride for 10 minutes → washing with sterile distilled water for 3 times, and putting the sterilized explant on sterile filter paper for natural drying;
3) callus induction:
cutting the leaf of the dried explant into 2cm 2 Inoculating the explant into a callus induction culture medium, slightly pressing the explant to be attached to the culture medium, and placing the explant in a culture chamber to be cultured for 60 days to form callus; the culture conditions were: the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 The illumination time is 8 h/d;
4) EMS induction:
cutting the surviving callus into segments with length of 2cm, soaking in EMS-phosphate buffer solution with volume ratio concentration of 0.6%, shaking at 20 deg.C and 30 r/min for 6 hr, and adding Na with final concentration of 5% 2 S 2 O 3 Stopping reaction, cleaning callus with sterilized ultrapure water for 3 times, placing the cleaned callus on filter paper, and naturally drying;
5) adventitious bud induction and differentiation:
inoculating the callus subjected to EMS induction treatment into an adventitious bud induction differentiation culture medium, and placing the callus in a culture chamber for culturing for 45 days to form adventitious buds; the culture conditions were: the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 The illumination time is 8 h/d;
6) rooting induction:
inoculating the strong pineapple tissue culture seedlings grown by adventitious bud induction culture into a rooting induction culture medium, and placing the medium in a culture room for culture for 30 days to induce rooting; the culture conditions were: the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 The illumination time is 8 h/d;
7) and (3) promoting root and strengthening seedling culture:
transferring the pineapple tissue culture seedlings with the root length of more than 3 cm obtained by induced culture into a root promoting and seedling strengthening culture medium, and placing the medium in a culture chamber for culturing for 30 days to obtain complete pineapple tissue culture seedlings with developed and robust root systems;
8) hardening off and transplanting
After obtaining complete plants of the pineapple tissue culture seedlings with developed and robust roots, moving a culture bottle to an outdoor shade tent for strong light bottle closing and seedling exercising for 15 days, wherein the shade degree is preferably 50% -70%, then opening a bottle opening, continuously placing for 5 days, and then moving the tissue culture seedlings into nutrient soil for culture.
Further, the formula of the callus induction culture medium is as follows: MS powder 4.43g/L, sucrose 30g/L, plant gel 3g/L, BAP 4mg/L and NAA 0.2mg/L, and the pH value is 5.8.
Further, the EMS-phosphate buffer takes phosphate buffer as a solvent, and chemical mutagen ethyl methanesulfonate EMS is diluted to volume ratio concentration of 0.6%; wherein the molarity of the phosphate buffer solution is 0.1 mol/L, and the pH value is 5.8.
Further, the formula of the adventitious bud induction differentiation culture medium is 4.43g/L of MS powder, 30g/L of cane sugar, 3g/L of plant gel, 1mg/L of BAP and 0.2mg/L of NAA, and the pH value is 5.8.
Furthermore, the formula of the rooting induction culture medium is 4.43g/L of MS powder and 30g/L of sucrose, and the pH value is 5.8.
Furthermore, the formula of the root-promoting seedling-strengthening culture medium comprises 4.43g/L of MS powder, 30g/L of sucrose, 3g/L of plant gel and 0.2mg/L of NAA, and the pH value is 5.8.
Furthermore, the nutrient soil comprises retted coconut coir, organic fertilizer and grass peat.
The invention has the following remarkable advantages:
the invention provides a method for creating a new ornamental pineapple variety by EMS mutagenesis, which comprises the steps of taking the young side branch of Tainong 11 pineapple as an explant, establishing an aseptic tissue culture system, and obtaining an EMS mutagenesis plant by adopting a method combining a tissue culture technology and EMS mutagenesis. The method provides a basis for researching the genetic diversity of the pineapple, and is simple to operate and reliable in result.
Drawings
FIG. 1 shows photographs showing the proliferation and propagation of plant tissues of pineapple. A: young lateral branches of Tainong 11 pineapple plants. B: shearing into 2cm 2 The explant square of (1). C: inducing the formed callus. D: inducing the formed adventitious bud seedlings. E: and (4) rooting and inducing to obtain the pineapple tissue culture seedling. F-G: the pineapple tissue culture seedling with developed and robust root system is a complete plant.
The photograph shown in FIG. 2 is a table picture of appearance of wild type pineapple.
FIG. 3 is a photograph showing the phenotype of EMS-induced mutants.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
The embodiment provides a method for creating a new ornamental pineapple variety by EMS mutagenesis, which specifically comprises the following steps:
1) selection of test materials:
the test material is collected from Fujian Green Art landscape engineering Co., Ltd, young side branches of Tainong 11 pineapple plants for more than one year are selected for establishing a tissue culture system, the young side branches are cut into 2 cm-long stem sections, and the prepared stem sections are used as explants for tissue culture.
2) Preparing a sterile explant:
and (3) carrying out disinfection treatment on the explant: soaking 5% chlorothalonil overnight → washing with clear water for 10 minutes → washing with carving brand detergent diluent (adding 3 drops of detergent in 500ml of clear water) for 3 hours → washing with clear water until cleaning the surface foam of the explant → washing with 75% alcohol for 5 minutes → washing with sterile distilled water for 3 times → sterilizing with 0.1% mercuric chloride for 10 minutes → washing with sterile distilled water for 3 times, and placing the sterilized explant on sterile filter paper for naturally drying.
3) Callus induction:
cutting the dried explant into 2cm 2 Inoculating the square of the above-mentioned seed to a previously prepared callusIn a tissue induction culture medium, the formula of the callus induction culture medium is 4.43g/L of MS powder, 30g/L of cane sugar, 3g/L of plant gel, 4mg/L of BAP and 0.2mg/L, pH =5.8 of NAA; gently pressing to make the explant fit with the culture medium, inoculating, placing in a culture chamber, and culturing for 60 days under certain conditions to form callus, wherein the culture conditions comprise temperature of 32-35 deg.C, humidity of 80%, and illumination intensity of 40 μmol/m -2 ·s -1 And the illumination time is 8 h/d.
4) EMS induction:
cutting the viable callus into 3 cm segments, soaking in EMS-phosphate buffer solution (phosphate buffer solution with molar concentration of 0.1 mol/L and pH = 5.8) with volume concentration of 0.6%, shaking at 20 deg.C and 30 r/min for 6 hr, and adding Na with final concentration of 5% 2 S 2 O 3 To terminate the reaction, the callus was washed with sterilized ultrapure water for 3 times, and the washed callus was placed on filter paper and air-dried naturally.
5) Adventitious bud induction and differentiation:
inoculating callus subjected to EMS induction treatment to a prepared adventitious bud induction differentiation medium, wherein the adventitious bud induction differentiation medium comprises 4.43g/L of MS powder, 30g/L of cane sugar, 3g/L of plant gel, 1mg/L of BAP and 0.2mg/L, pH =5.8, and after inoculation, placing the inoculated callus in a culture chamber to be cultured for 45 days under certain conditions to form adventitious bud seedlings, wherein the culture conditions are that the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 And the illumination time is 8 h/d.
6) Rooting induction:
inoculating the strong pineapple tissue culture seedling cultured by adventitious bud induction culture into a rooting induction culture medium prepared in advance for inducing rooting, wherein the rooting induction culture medium is an MS liquid culture medium (MS powder is 4.43g/L + sucrose is 30g/L, pH = 5.8) without any exogenous hormone, the rooting induction time is determined according to the growth condition of the pineapple tissue culture seedling and is generally 30 days, the culture condition during the rooting induction period is that the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol. m -2 ·s -1 And the illumination time is 8 h/d.
7) And (3) promoting root and strengthening seedling culture:
the method comprises the steps of transferring pineapple tissue culture seedlings with the root length of more than 3 cm obtained through induction culture to a root and seedling promoting and strengthening culture medium prepared in advance for root and seedling promoting and strengthening culture, wherein the formula of the root and seedling promoting and strengthening culture medium comprises 4.43g/L of MS powder, 30g/L of cane sugar, 3g/L of plant gel and 0.2mg/L, pH =5.8, the culture conditions are that the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol. m -2 ·s -1 The illumination time is 8 h/d; and (5) promoting the root and strengthening the seedling for 30 d to obtain a complete plant of the pineapple tissue culture seedling with developed and robust root system.
8) Hardening off and transplanting
After obtaining complete plants of the pineapple tissue culture seedlings with developed and robust roots, moving a culture bottle to an outdoor shade tent for strong light bottle closing and seedling exercising for about 15 days, wherein the shade degree is preferably 50-70%, and then opening the bottle mouth and continuously standing for 5 days; then, gently taking out the tissue culture seedlings by using tweezers, putting the tissue culture seedlings into a clear water basin, carefully washing off root agar, then transplanting the tissue culture seedlings into nutrient soil, lightly covering and compacting the tissue culture seedlings by using the soil, watering the tissue culture seedlings by using a sprayer after the whole hole tray is fully planted, and finally putting the seedling culture tray into a greenhouse for normal management; the nutrient soil comprises the components of retted coconut chaff, organic fertilizer and grass peat.
FIG. 1 is a photograph showing the process of tissue proliferation and propagation of a pineapple plant of this example.
The photograph shown in FIG. 2 is a table picture of appearance of wild type pineapple. FIG. 3 is a photograph showing the phenotype of EMS-induced mutants. As can be seen from the figure, the wild pineapple has low ornamental value, the leaves are all green, and the color is single; the plant leaves and stems are clustered, and the leaves are long and narrow and are sword-shaped or strip-shaped. The pineapple after mutation induced by EMS has high ornamental value and beautiful plant type, and leaves are the main ornamental parts and can be appreciated for a long time; the center of the leather leaf is green, and the edge is similar to a gold edge; the middle of the tender leaves inside the pineapple is light green, and the leaf edge is light yellow; the middle of the outer old leaves is dark green, the edges of the leaves sometimes have a yellow-green gradual change color, and the pineapple leaves are clear in color gradation from inside to outside and are harmonious and uniform at the same time, so that the pineapple leaf tea has great ornamental value.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (6)
1. A method for creating a new ornamental pineapple variety by EMS mutagenesis is characterized by comprising the following steps: the method specifically comprises the following steps:
1) selection of test materials:
selecting tender lateral branches of more than one year of Tainong 11 pineapple plants to establish a tissue culture system, cutting the tender lateral branches into stem sections with the length of 2cm, and taking the prepared stem sections as explants for tissue culture;
2) preparing a sterile explant:
and (3) carrying out disinfection treatment on the explant: soaking 5% chlorothalonil overnight → washing with clear water for 10 minutes → washing with detergent diluent for 3 hours → washing with clear water until the foam on the surface of the explant is cleaned, washing with 75% alcohol for 5 minutes → washing with sterile distilled water for 3 times → sterilizing with 0.1% mercuric chloride for 10 minutes → washing with sterile distilled water for 3 times, and placing the sterilized explant on sterile filter paper for naturally airing;
3) callus induction:
cutting the leaf of the dried explant into 2cm 2 Inoculating the explant into a callus induction culture medium, slightly pressing the explant to be attached to the culture medium, and placing the explant in a culture chamber for culturing for 60 days to form callus; the culture conditions were: the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 The illumination time is 8 h/d;
4) EMS induction:
cutting the surviving callus into segments with length of 2cm, soaking in EMS-phosphate buffer solution with volume ratio concentration of 0.6%, shaking at 20 deg.C and 30 r/min for 6 hr, and adding Na with final concentration of 5% 2 S 2 O 3 Stopping reaction, cleaning the callus with sterilized ultrapure water for 3 times, placing the cleaned callus on filter paper, and naturally drying;
5) adventitious bud induction and differentiation:
inoculating the callus after EMS induction treatment to adventitious bud induction differentiation culture medium, and placing in a culture chamber for cultureCulturing for 45 days to form adventitious buds; the culture conditions were: the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 The illumination time is 8 h/d;
6) rooting induction:
inoculating the strong pineapple tissue culture seedlings grown by adventitious bud induction culture into a rooting induction culture medium, and placing the medium in a culture room for culture for 30 days to induce rooting; the culture conditions were: the temperature is 32-35 ℃, the humidity is 80%, and the illumination intensity is 40 mu mol.m -2 ·s -1 The illumination time is 8 h/d;
7) and (3) promoting root and strengthening seedling culture:
transferring the pineapple tissue culture seedlings with the root length of more than 3 cm obtained by induced culture into a root promoting and seedling strengthening culture medium, and placing the medium in a culture chamber for culturing for 30 days to obtain complete pineapple tissue culture seedlings with developed and robust root systems;
8) hardening off and transplanting
After obtaining complete plants of the pineapple tissue culture seedlings with developed and robust roots, moving a culture bottle to an outdoor shade tent for strong light bottle closing and seedling exercising for 15 days, wherein the shade degree is preferably 50% -70%, then opening a bottle opening, continuously placing for 5 days, and then moving the tissue culture seedlings into nutrient soil for culture.
2. The method for creating a new variety of ornamental pineapples by EMS mutagenesis according to claim 1, wherein the method comprises the following steps: the callus induction culture medium comprises the following components in percentage by weight: MS powder 4.43g/L, sucrose 30g/L, plant gel 3g/L, BAP 4mg/L and NAA 0.2mg/L, and the pH value is 5.8.
3. The method for creating a new variety of ornamental pineapple by EMS mutagenesis as claimed in claim 1, wherein: the EMS-phosphate buffer solution takes phosphate buffer solution as a solvent, and chemical mutagen ethyl methanesulfonate EMS is diluted to the volume ratio concentration of 0.6%; wherein the molarity of the phosphate buffer solution is 0.1 mol/L, and the pH value is 5.8.
4. The method for creating a new variety of ornamental pineapple by EMS mutagenesis as claimed in claim 1, wherein: the adventitious bud induction differentiation culture medium formula is 4.43g/L of MS powder, 30g/L of cane sugar, 3g/L of plant gel, 1mg/L of BAP and 0.2mg/L of NAA, and the pH value is 5.8.
5. The method for creating a new variety of ornamental pineapple by EMS mutagenesis as claimed in claim 1, wherein: the rooting induction culture medium comprises 4.43g/L of MS powder and 30g/L of sucrose, and the pH value is 5.8.
6. The method for creating a new variety of ornamental pineapple by EMS mutagenesis as claimed in claim 1, wherein: the formula of the root-promoting seedling-strengthening culture medium is 4.43g/L of MS powder, 30g/L of cane sugar, 3g/L of plant gel and 0.2mg/L of NAA, and the pH value is 5.8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115708480A (en) * | 2022-12-27 | 2023-02-24 | 中国热带农业科学院南亚热带作物研究所 | EMS in-vitro mutagenesis method for creating pineapple mutant |
| CN116138162A (en) * | 2023-04-19 | 2023-05-23 | 海南伯特生态休闲农业科技有限公司 | Method for mutagenesis of new germplasm by utilizing pineapple fruit crown buds |
| CN116210586A (en) * | 2023-04-23 | 2023-06-06 | 海南伯特生态休闲农业科技有限公司 | Method for breeding pineapple by synergistic mutagenesis of methyl carbamate and ultraviolet irradiation |
| CN116267596A (en) * | 2023-04-20 | 2023-06-23 | 海南伯特生态休闲农业科技有限公司 | Method for mutation breeding of pineapple stock plant primordial bud-sucking |
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| CN112931198A (en) * | 2019-11-26 | 2021-06-11 | 中国热带农业科学院海口实验站 | Preparation method of pineapple cold-resistant germplasm |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115708480A (en) * | 2022-12-27 | 2023-02-24 | 中国热带农业科学院南亚热带作物研究所 | EMS in-vitro mutagenesis method for creating pineapple mutant |
| CN116138162A (en) * | 2023-04-19 | 2023-05-23 | 海南伯特生态休闲农业科技有限公司 | Method for mutagenesis of new germplasm by utilizing pineapple fruit crown buds |
| CN116138162B (en) * | 2023-04-19 | 2023-06-30 | 海南伯特生态休闲农业科技有限公司 | Method for mutagenesis of new germplasm by utilizing pineapple fruit crown buds |
| CN116267596A (en) * | 2023-04-20 | 2023-06-23 | 海南伯特生态休闲农业科技有限公司 | Method for mutation breeding of pineapple stock plant primordial bud-sucking |
| CN116210586A (en) * | 2023-04-23 | 2023-06-06 | 海南伯特生态休闲农业科技有限公司 | Method for breeding pineapple by synergistic mutagenesis of methyl carbamate and ultraviolet irradiation |
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