CN112293248A - Chemical mutagenesis method for inducing and increasing mutation range of banana - Google Patents
Chemical mutagenesis method for inducing and increasing mutation range of banana Download PDFInfo
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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Abstract
The invention provides a chemical mutagenesis method for inducing and increasing mutation range of banana plantain, which comprises the following steps: the method comprises the following steps of preparation of a multiplication culture medium, mutation treatment of bananas, washing treatment and multiplication culture, wherein the lithium chloride solution used in the method is used for treating and inhibiting the growth of seedlings, the growth vigor of the seedlings has obvious difference, and single plants with special characters appear in the form; the mutation frequency is improved and the variation range is enlarged by treating with the mutagen, and the mutation frequency can be improved to 8.89% at most by treating with the chemical mutagen and is more than 1000 times higher than the natural mutation frequency; the mutation types are collectively expressed as leaf color mutation, leaf type mutation and plant type mutation, wherein 6.47% of plants express bright green leaves on leaves, 7.11% express yellow stripes, 1.96% express leaf edge curling, and 4.50% express short leaves, so that the artificial mutagenesis range is wide, and the mutation types are rich.
Description
Technical Field
The invention relates to the technical field of plant polyploid mutagenesis breeding and planting, in particular to a chemical mutagenesis method for inducing and increasing the mutation range of plantain.
Background
Bananas are a typical tropical and subtropical fruit tree and are widely distributed in tropical and southern subtropical regions. China is one of the major banana producing countries in the world, and planting areas are distributed in provinces such as Guangdong, Hainan, Guangxi, Fujian, Yunnan and Taiwan. However, at present, the development space of new varieties cultivated by bananas through a hybridization method is small, the stress resistance of the varieties is incomplete, the variation range is small, new gene mutants are difficult to appear through natural mutation, the mutagen tolerance to bananas is different at present, although a single mutagenesis method has high-efficiency mutation frequency on one aspect, the mutation rate of the overall characters is still low, the mutation types are few, and the mutagenesis breeding efficiency is low.
Disclosure of Invention
In view of the above, the present invention provides a chemical mutagenesis method for inducing and increasing mutation range of banana.
The technical scheme of the invention is realized as follows: a chemical mutagenesis method for inducing and increasing mutation range of banana: the method comprises the following steps:
s1, preparation of a proliferation medium: MS culture medium, 6-Benzylaminopurine (BAP) 1.0-5.0 mg/L, adenine 0.2-1.5 mg/L, agarose 2.0-4.0 g/L, sucrose 20-40 g/L;
s2, mutation treatment of bananas: cutting off single independent buds by using a scalpel, putting the buds into a triangular flask filled with a lithium chloride mutagen, performing shaking soaking culture for 4-24 h at 20-30 ℃ in a rotary shaking table at 138-200 rpm, then putting the buds into an illumination incubator, performing temperature change treatment for 1-3 h at an initial temperature of 15-20 ℃, adjusting the temperature to 22-30 ℃, and treating for 1-3 h; the lithium chloride mutagen is obtained by adding tween-80 and banana peel polysaccharide solution into lithium chloride solution in sterilized water, filtering and sterilizing;
s3, washing treatment: after the treatment of the steps, washing the stem tip in distilled water for 2-5 times;
s4, proliferation culture: transferring the treated adventitious bud into a propagation culture medium, performing 4-8 cycles, and transferring the bud into an MS basal culture medium containing 0.5-3.0 g/L of indolebutyric acid IBA, 2.0-4.0 g/L of agarose and 20-40 g/L of sucrose to obtain a mutagenized plant body.
Further, the pH value of the sucrose in the S1 is 4.0-6.0.
Further, the preparation of the propagation medium in S1: MS culture medium, 6-Benzylaminopurine (BAP)3.0mg/L, adenine 1.0mg/L, agarose 3.0g/L, sucrose 30 g/L.
Furthermore, the concentration of the mutagen in the S1 is 0.4-1.6%.
Further, in the step S2, the rotating speed of the rotary table is 150rpm, and the culture is performed by shaking and soaking at a constant temperature of 27 ℃.
Further, the light intensity in the step S2 is set to be 1000-3000 lx.
Further, the volume ratio of the lithium chloride solution, the sterilized water, the tween-80 and the banana peel polysaccharide solution in the step S2 is 1: 10-20: 0.3-2: 1-5.
Further, the proliferation culture medium of the S4 step is an MS basal culture medium of 1.0g/L of indolebutyric acid IBA, 3.0g/L of agarose and 30g/L of sucrose.
Compared with the prior art, the invention has the beneficial effects that:
the lithium chloride solution used in the invention is used for treating and inhibiting the growth of seedlings, the growth vigor has obvious difference, and single plants with special properties appear in the form; through the treatment of a mutagen, preparing a lithium chloride solution, sterilized water, Tween-80 and a banana peel polysaccharide solution into the mutagen according to a proportion, combining with an illumination incubator for culture, controlling illumination intensity and temperature change, obviously improving mutation frequency, expanding the variation range, and utilizing a chemical mutagen and a special treatment process to enable the mutation frequency to be improved to 8.89% at most and to be more than 1000 times higher than natural mutation frequency; the mutation types were collectively expressed as leaf color mutation, leaf type mutation and plant type mutation, with 6.47% of the plants expressing bright green leaves on the leaves and 7.11% expressing yellow streaks. 1.96% of the mutant shows leaf margin curling, and 4.50% shows short leaves, so that the artificial mutagenesis is wide in range and rich in variation types.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1
A chemical mutagenesis method for inducing and increasing mutation range of banana: the method comprises the following steps:
s1, preparation of a proliferation medium: MS culture medium, 6-Benzyl Aminopurine (BAP)1.0mg/L, adenine 0.2mg/L, agarose 2.0g/L, sucrose 20g/L, sucrose pH 4.0;
s2, mutation treatment of bananas: cutting off single independent buds by using a scalpel, putting the buds into a triangular flask filled with a lithium chloride mutagen, performing shaking soaking culture in a rotary shaking table at 138rpm and 20 ℃ for 4 hours, then putting the buds into an illumination incubator, setting the illumination intensity to be 1000lx, performing temperature change treatment, treating at an initial temperature of 15 ℃ for 1 hour, adjusting the temperature to be 22 ℃, and treating for 1 hour; the lithium chloride mutagen is obtained by adding tween-80 and the banana peel polysaccharide solution into a lithium chloride solution in sterilized water, and filtering and sterilizing, wherein the volume ratio of the lithium chloride solution to the sterilized water to the tween-80 to the banana peel polysaccharide solution is 1:10:0.3: 1;
s3, washing treatment: after the treatment of the steps, washing the stem tip in distilled water for 2 times;
s4, proliferation culture: transferring the treated adventitious bud into a propagation culture medium, performing 4 cycles, and transferring into an MS basal culture medium containing 0.5g/L of indolebutyric acid IBA, 2.0g/L of agarose and 20g/L of sucrose to obtain a mutagenized plant body.
Example 2
A chemical mutagenesis method for inducing and increasing mutation range of banana: the method comprises the following steps:
s1, preparation of a proliferation medium: MS culture medium, 6-Benzyl Aminopurine (BAP)5.0mg/L, adenine 1.5mg/L, agarose 4.0g/L, sucrose 40g/L, sucrose pH 6.0;
s2, mutation treatment of bananas: cutting off single independent buds by using a scalpel, putting the buds into a triangular flask filled with a lithium chloride mutagen, performing shaking immersion culture in a rotary shaking table at 200rpm and 30 ℃ for 24 hours, then putting the buds into an illumination incubator, setting the illumination intensity to be 3000lx, performing temperature change treatment, treating at the initial temperature of 20 ℃ for 3 hours, adjusting the temperature to be 30 ℃, and treating for 3 hours; the lithium chloride mutagen is obtained by adding tween-80 and the banana peel polysaccharide solution into a lithium chloride solution in sterilized water, and filtering and sterilizing, wherein the volume ratio of the lithium chloride solution to the sterilized water to the tween-80 to the banana peel polysaccharide solution is 1:20:2: 5;
s3, washing treatment: after the treatment of the steps, the stem tip is washed for 5 times in distilled water;
s4, proliferation culture: transferring the treated adventitious bud into a propagation culture medium, performing 8 cycles, and transferring into an MS basal culture medium containing 3.0g/L of indolebutyric acid IBA, 4.0g/L of agarose and 40g/L of sucrose to obtain a mutagenized plant body.
Example 3
A chemical mutagenesis method for inducing and increasing mutation range of banana: the method comprises the following steps:
s1, preparation of a proliferation medium: MS culture medium, 6-Benzyl Aminopurine (BAP)3.0mg/L, adenine 1.0mg/L, agarose 3.0g/L, sucrose 30 g/L;
s2, mutation treatment of bananas: cutting off single independent buds by using a scalpel, putting the buds into a triangular flask filled with a lithium chloride mutagen, performing shaking soaking culture in a rotary shaking table at the speed of 150rpm and the temperature of 27 ℃ for 14h, then putting the buds into an illumination incubator, setting the illumination intensity to be 2000lx, performing temperature change treatment, treating at the initial temperature of 18 ℃ for 2h, adjusting the temperature to be 26 ℃, and treating for 2 h; the lithium chloride mutagen is obtained by adding tween-80 and the banana peel polysaccharide solution into a lithium chloride solution in sterilized water, and filtering and sterilizing, wherein the volume ratio of the lithium chloride solution to the sterilized water to the tween-80 to the banana peel polysaccharide solution is 1:15:1.2: 3;
s3, washing treatment: after the treatment of the steps, washing the stem tip in distilled water for 3 times;
s4, proliferation culture: transferring the treated adventitious bud into a propagation culture medium, performing 6 cycles, and transferring into an MS basal culture medium containing 1.0g/L of indolebutyric acid IBA, 3.0g/L of agarose and 30g/L of sucrose to obtain a mutagenized plant body.
Example 4
This example differs from example 3 in that a chemical mutagenesis method to induce an increased mutation range of plantain: the volume ratio of the lithium chloride solution to the sterilized water to the tween-80 to the banana peel polysaccharide solution is 1:5:3: 7.
Example 5
This example differs from example 3 in that a chemical mutagenesis method to induce an increased mutation range of plantain: the light intensity is set to 4000lx in the step S2.
Example 6
This example differs from example 3 in that the concentration of the growth medium in the S1 step was 0.8%.
Comparative example 1
The difference between this comparative example and example 3 is that the bananas were not cultured in a light incubator during the mutagenesis treatment.
Comparative example 2
The comparative example differs from example 3 in that the temperature-swing treatment was not carried out in the light incubator for the mutagenesis treatment of bananas.
Comparative example 3
The difference between this comparative example and example 3 is that in the temperature-changing treatment, the initial temperature was 14 ℃ for 2 hours and then adjusted to 20 ℃.
Chemical mutagenesis induced mutation type and mutation rate
The induction methods of examples 1 to 6 and comparative examples 1 to 3 of the present invention were compared and each experiment was repeated 3 times to obtain the type of mutation induced by chemical mutagenesis and the rate of mutation (the following values are average values):
the variation rate calculation formula is as follows: different type variation rate (different phenotypic variation offspring/total number 100)
From the above table, the mutagenesis method of the present invention allows the banana mutations to be collectively expressed as leaf color mutation, leaf type mutation and plant type mutation. Of these, 6.47% showed bright green leaves on the leaves and 7.11% yellow streaks. 1.96% of the mutant shows leaf margin curling, and 4.50% shows short leaves, so that the artificial mutagenesis is wide in range and rich in variation types.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A chemical mutagenesis method for inducing and increasing mutation range of banana plantain is characterized in that: the method comprises the following steps:
s1, preparation of a proliferation medium: MS culture medium, 6-benzylaminopurine 1.0-5.0 mg/L, adenine 0.2-1.5 mg/L, agarose 2.0-4.0 g/L, sucrose 20-40 g/L;
s2, mutation treatment of bananas: cutting single independent buds, putting the buds into a lithium chloride mutagen, performing shaking soaking culture in a rotary table at the speed of 138-200 rpm and the temperature of 20-30 ℃ for 4-24 h, then putting the buds into an illumination incubator, performing temperature change treatment at the initial temperature of 15-20 ℃ for 1-3 h, adjusting the temperature to 22-30 ℃, and treating for 1-3 h; the lithium chloride mutagen is obtained by adding tween-80 and banana peel polysaccharide solution into lithium chloride solution in sterilized water, filtering and sterilizing;
s3, washing treatment: after the treatment of the steps, washing the stem tip in distilled water for 2-5 times;
s4, proliferation culture: transferring the treated adventitious bud into a propagation culture medium, performing 4-8 cycles, and transferring the bud into an MS basal culture medium containing 0.5-3.0 g/L of indolebutyric acid IBA, 2.0-4.0 g/L of agarose and 20-40 g/L of sucrose to obtain a mutagenized plant body.
2. A chemical mutagenesis method for inducing an increase in the mutation range of plantain as claimed in claim 1 wherein: the pH value of the sucrose in the S1 is 4.0-6.0.
3. A chemical mutagenesis method for inducing an increase in the mutation range of plantain as claimed in claim 1 wherein: preparation of the propagation medium in the S1: MS culture medium, 6-Benzylaminopurine (BAP)3.0mg/L, adenine 1.0mg/L, agarose 3.0g/L, sucrose 30 g/L.
4. A chemical mutagenesis method for inducing desirable mutation density in bananas according to claim 1, characterized by: the concentration of the proliferation culture medium in the S1 is 0.4-1.6%.
5. A chemical mutagenesis method for inducing desirable mutation density in bananas according to claim 1, characterized by: and in the step S2, the rotating speed of the rotary table is 150rpm, and the rotary table is subjected to shake soaking culture at the constant temperature of 27 ℃.
6. A chemical mutagenesis method for inducing desirable mutation density in bananas according to claim 1, characterized by: the illumination intensity in the step S2 is set to be 1000-3000 lx.
7. A chemical mutagenesis method for inducing desirable mutation density in bananas according to claim 1, characterized by: the volume ratio of the lithium chloride solution, the sterilized water, the tween-80 and the banana peel polysaccharide solution in the step S2 is 1: 10-20: 0.3-2: 1-5.
8. A chemical mutagenesis method for inducing desirable mutation density in bananas according to claim 1, characterized by: the proliferation culture medium of the S4 step is an MS basal culture medium of 1.0g/L of indolebutyric acid IBA, 3.0g/L of agarose and 30g/L of sucrose.
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| CN202011366771.4A CN112293248B (en) | 2020-11-30 | 2020-11-30 | Chemical mutagenesis method for inducing and increasing mutation range of banana |
| PCT/CN2021/109473 WO2022110864A1 (en) | 2020-11-30 | 2021-07-30 | Chemical mutagenesis method for increasing mutation range of dwarf bananas by means of induction |
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| WO2022110864A1 (en) * | 2020-11-30 | 2022-06-02 | 中国热带农业科学院海口实验站 | Chemical mutagenesis method for increasing mutation range of dwarf bananas by means of induction |
| CN117461558A (en) * | 2023-11-27 | 2024-01-30 | 中国热带农业科学院热带作物品种资源研究所 | Method for inducing diverse population of canna and identification |
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| WO2022110864A1 (en) | 2022-06-02 |
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