WO2022110864A1 - 一种诱导增加粉蕉突变范围的化学诱变方法 - Google Patents
一种诱导增加粉蕉突变范围的化学诱变方法 Download PDFInfo
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- WO2022110864A1 WO2022110864A1 PCT/CN2021/109473 CN2021109473W WO2022110864A1 WO 2022110864 A1 WO2022110864 A1 WO 2022110864A1 CN 2021109473 W CN2021109473 W CN 2021109473W WO 2022110864 A1 WO2022110864 A1 WO 2022110864A1
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- mutation
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- bananas
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- 230000035772 mutation Effects 0.000 title claims abstract description 45
- 231100000350 mutagenesis Toxicity 0.000 title claims abstract description 39
- 238000002703 mutagenesis Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000000126 substance Substances 0.000 title claims abstract description 25
- 230000001965 increasing effect Effects 0.000 title claims abstract description 8
- 230000006698 induction Effects 0.000 title abstract description 3
- 244000291473 Musa acuminata Species 0.000 title abstract 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims abstract description 42
- 241000234295 Musa Species 0.000 claims abstract description 36
- 230000035755 proliferation Effects 0.000 claims abstract description 24
- 241000196324 Embryophyta Species 0.000 claims abstract description 14
- 239000003471 mutagenic agent Substances 0.000 claims abstract description 13
- 235000021015 bananas Nutrition 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 6
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 28
- 230000001939 inductive effect Effects 0.000 claims description 20
- 229930006000 Sucrose Natural products 0.000 claims description 18
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 18
- 239000005720 sucrose Substances 0.000 claims description 18
- 239000007640 basal medium Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 14
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 13
- 229920000936 Agarose Polymers 0.000 claims description 12
- 150000004676 glycans Chemical class 0.000 claims description 12
- 230000003505 mutagenic effect Effects 0.000 claims description 12
- 229920001282 polysaccharide Polymers 0.000 claims description 12
- 239000005017 polysaccharide Substances 0.000 claims description 12
- 231100000707 mutagenic chemical Toxicity 0.000 claims description 11
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 11
- 229920000053 polysorbate 80 Polymers 0.000 claims description 11
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 10
- 235000001484 Trigonella foenum graecum Nutrition 0.000 claims description 9
- 244000250129 Trigonella foenum graecum Species 0.000 claims description 9
- 235000001019 trigonella foenum-graecum Nutrition 0.000 claims description 9
- 229930024421 Adenine Natural products 0.000 claims description 7
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 7
- 229960000643 adenine Drugs 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims 1
- 238000007654 immersion Methods 0.000 claims 1
- 230000036438 mutation frequency Effects 0.000 abstract description 6
- 239000001963 growth medium Substances 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 8
- 241000318136 Musa velutina Species 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 208000020584 Polyploidy Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002962 chemical mutagen Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
Classifications
-
- 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
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
Definitions
- the present application relates to the planting field of plant polyploid mutation breeding technology, in particular to a chemical mutagenesis method for inducing and increasing the mutation range of fenugreek.
- Banana is a typical tropical and subtropical fruit tree, widely distributed in tropical and subtropical regions. my country is one of the main banana producing countries in the world, and the planting areas are distributed in Guangdong, Hainan, Guangxi, Fujian, Yunnan and other provinces.
- the development space of new banana varieties cultivated by hybridization is small, the variety's stress resistance is not comprehensive, and the variation range is small, it is difficult to generate new gene mutants through natural mutation, and the current resistance to bananas is mutagenic
- a single mutagenesis method has high mutation frequency in one aspect, the mutation rate of the overall trait is still low, resulting in less mutation types and low mutagenesis breeding efficiency.
- the present application proposes a chemical mutagenesis method for inducing an increase in the mutation range of fenugreek to solve the above problems.
- a chemical mutagenesis method for inducing an increase in the mutation range of the powder banana comprising the following steps:
- Mutagenesis treatment of banana use a scalpel to cut a single independent bud of banana, put it into a triangular flask containing lithium chloride mutagen, and shake and soak it in a rotary shaker at 138-200 rpm and 20-30 °C After 4 to 24 hours, it was placed in a light incubator for variable temperature treatment. The initial temperature was 15 to 20°C for 1 to 3 hours, and the temperature was adjusted to 22 to 30°C for 1 to 3 hours.
- the lithium chloride mutagen was Add Tween-80 and banana peel polysaccharide solution to the lithium chloride solution in sterilized water, and filter and sterilize to obtain;
- Proliferation culture The adventitious buds treated with S3 were transferred to the proliferation medium prepared by S1. After 4 to 8 passages of proliferation, they were transferred to IBA containing 0.5 to 3.0 g/L indole butyric acid, 2.0 to 4.0 The mutagenized plants were obtained in MS basal medium containing g/L agarose and 20-40 g/L sucrose, and the MS basal medium was the same as the MS basal medium in S1.
- the pH of the sucrose in the S1 is 4.0-6.0.
- the preparation of the proliferation medium in S1 MS basal medium, 6-benzylaminopurine (BAP) 3.0 mg/L, adenine 1.0 mg/L, agarose 3.0 g/L, sucrose 30 g/L .
- BAP 6-benzylaminopurine
- the mutagen concentration in the S1 is 0.4-1.6%.
- the rotating speed of the rotary shaker in S2 is 150 rpm, and the constant temperature is 27° C. for shaking and soaking culture.
- the light intensity in S2 is set to 1000-3000 lx.
- the volume ratio of lithium chloride solution, sterile water, Tween-80 and banana peel polysaccharide solution in S2 is 1:10-20:0.3-2:1-5.
- the MS basal medium in the step S4 is 1.0 g/L indole butyric acid IBA, 3.0 g/L agarose and 30 g/L sucrose.
- the lithium chloride solution used in this application inhibits the growth of seedlings, and there are obvious differences in growth, and individual plants with special characteristics appear in the form; -80 and banana peel polysaccharide solution are formulated into mutagen, combined with light incubator culture, control the light intensity and temperature changes, significantly increase the mutation frequency, expand the range of mutation, the use of chemical mutagens and special processing technology can make mutation The highest frequency increased to 8.89%, which was more than 1000 times higher than the natural mutation frequency.
- the mutation types concentrated in leaf color mutation, leaf type mutation and plant type mutation. Among them, 6.47% of plants showed bright green leaves on leaves, and 7.11% showed For yellow stripes. 1.96% showed curled leaf margins, and 4.50% showed short leaves, so it can be seen that artificial mutagenesis has a wide range and rich variation types.
- a chemical mutagenesis method for inducing an increase in the mutation range of fenugreek including the following steps:
- Mutagenesis treatment of banana use a scalpel to cut a single independent adventitious bud of banana, put it into a triangular flask containing a lithium chloride mutagen, and incubate it in a rotary shaker at 138 rpm and 20 °C for 4 hours. Then placed in a light incubator, the light intensity was set to 1000lx, and then the temperature was changed. The initial temperature was 15 °C for 1 hour, and the temperature was adjusted to 22 °C for 1 hour; the lithium chloride mutagen was a lithium chloride solution.
- Tween-80 and banana peel polysaccharide solution in sterilized water, filter and sterilize to obtain, the volume ratio of described lithium chloride solution, sterilized water, Tween-80 and banana peel polysaccharide solution is 1:10:0.3: 1;
- Proliferation culture The shoot tips of adventitious buds treated with S3 were transferred to the proliferation medium prepared by S1. Agarose and 20g/L sucrose MS basal medium, the mutagenized plants were obtained.
- a chemical mutagenesis method for inducing an increase in the mutation range of fenugreek including the following steps:
- Mutagenesis treatment of banana use a scalpel to cut a single independent adventitious bud of banana, put it into a triangular flask containing lithium chloride mutagen, and incubate it in a rotary shaker at 200 rpm and 30 °C for 24 hours. Then placed in a light incubator, the light intensity was set to 3000lx, and then the temperature was changed. The initial temperature was 20 °C for 3 hours, and the temperature was adjusted to 30 °C for 3 hours; the lithium chloride mutagen was a lithium chloride solution.
- Tween-80 and banana peel polysaccharide solution in sterilized water, filter and sterilize to obtain, the volume ratio of described lithium chloride solution, sterilized water, Tween-80 and banana peel polysaccharide solution is 1:20:2: 5;
- Proliferation culture The adventitious buds treated with S3 were transferred to the shoot tips and placed in the proliferation medium prepared by S1. After 8 times of proliferation, they were transferred to agar containing 3.0g/L indolebutyric acid IBA and 4.0g/L In MS basal medium of sugar and 40 g/L sucrose, mutagenized plants were obtained.
- a chemical mutagenesis method for inducing an increase in the mutation range of fenugreek including the following steps:
- Mutagenesis treatment of banana use a scalpel to cut a single independent adventitious bud of banana, put it into a triangular flask containing lithium chloride mutagen, and incubate it in a rotary shaker at 150rpm and 27°C for 14 hours. Then placed in a light incubator, the light intensity was set to 2000lx, and then the temperature was changed. The initial temperature was 18 °C for 2 hours, and the temperature was adjusted to 26 °C for 2 hours; the lithium chloride mutagen was a lithium chloride solution.
- Tween-80 and banana peel polysaccharide solution in sterilized water, filter and sterilize to obtain, the volume ratio of described lithium chloride solution, sterilized water, Tween-80 and banana peel polysaccharide solution is 1:15:1.2: 3;
- Proliferation culture The shoot tips of adventitious buds treated with S3 were transferred to the proliferation medium prepared by S1, and after 46 passages of proliferation, transferred to agar containing 1.0g/L indolebutyric acid IBA and 3.0g/L In MS basal medium of sugar and 30 g/L sucrose, mutagenized plants were obtained.
- Example 3 The difference between this example and Example 3 is that the chemical mutagenesis method for inducing an increase in the mutation range of fenugreek: the volume ratio of the lithium chloride solution, sterilized water, Tween-80 and banana peel polysaccharide solution is 1:5 :3:7.
- Example 3 lies in the chemical mutagenesis method for inducing an increase in the mutation range of the pink banana: the light intensity in the S2 is set to 4000lx.
- Example 3 lies in the chemical mutagenesis method for inducing an increase in the mutation range of the fenugreek: the concentration of the proliferation medium in the S1 is 0.8%.
- Example 3 The difference between this comparative example and Example 3 lies in the chemical mutagenesis method for inducing an increase in the mutation range of the pink banana: no light incubator is placed in the mutagenesis treatment of the S2 banana.
- Example 3 The difference between this comparative example and Example 3 is that the chemical mutagenesis method for inducing an increase in the mutation range of the pink banana: the S2 banana was not subjected to temperature change treatment in the light incubator for the mutagenesis treatment.
- Example 3 The difference between this comparative example and Example 3 lies in the chemical mutagenesis method for inducing an increase in the mutation range of the powder banana: in the temperature-changing treatment of S2, the initial temperature was 14°C for 2 hours and then adjusted to 20°C.
- the mutagenesis method of the present application makes the mutation types of pink banana concentrated in leaf color mutation, leaf type mutation and plant type mutation. Among them, 6.47% of the plants showed bright green leaves, 7.11% showed yellow stripes, 1.96% showed curled leaf margins, and 4.50% showed short leaves, so it can be seen that artificial mutagenesis has a wide range and rich variation types.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请提供一种诱导增加粉蕉突变范围的化学诱变方法，包括以下步骤：增殖培养基的配制、香蕉的诱变处理、洗涤处理、增殖培养，本申请使用的氯化锂溶液处理抑制幼苗生长，长势存在明显差异，在形态上出现了具有特殊性状的单株；通过诱变剂处理，提高了突变频率，扩大了变异范围，利用化学诱变剂处理可使突变频率最高提高到8.89％，比自然突变频率高1000倍以上；突变类型集中表现为叶色突变、叶型突变及株型突变，其中有6.47％的植株在叶片上表现为亮绿叶，7.11％表现为黄条纹，1.96％表现为叶缘卷曲，4.50％表现为短叶，从而可知人工诱变的范围广，变异类型丰富。
Description
本申请涉及植物多倍体诱变育技术种植领域，特别涉及一种诱导增加粉蕉突变范围的化学诱变方法。
香蕉是典型的热带亚热带果树，广泛分布于热带和南亚热带地区。我国是世界香蕉主产国之一，种植区域分布于广东、海南、广西、福建、云南等省区。但目前，香蕉通过杂交手段培育的新品种的发展空间小，品种的抗逆性不全面，且变异范围小，经自然突变难以出现新基因突变体，且目前对香蕉的诱变剂耐受能力不同，单一的诱变方法虽然在某一方面具有高效的突变频率，但总体性状的突变率仍较低，导致变异类型少，诱变育种效率低。
发明内容
鉴于此，本申请提出一种诱导增加粉蕉突变范围的化学诱变方法，解决上述问题。
本申请的技术方案是这样实现的：一种诱导增加粉蕉突变范围的化学诱变方法：包括以下步骤：
S1、增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤(BAP)1.0～5.0mg/L、腺嘌呤0.2～1.5mg/L、琼脂糖2.0～4.0g/L、蔗糖20～40g/L；
S2、香蕉的诱变处理：使用手术刀切割香蕉单个独立的芽，放入装有氯化锂诱变剂的三角瓶中，在旋转摇床中以138～200rpm、20～30℃振荡浸泡培养4～24h后，再放置于光照培养箱中，进行变温处理，初始温度为15～20℃处理1～3h，调整温度22～30℃，处理1～3h；所述氯化锂诱变剂是将氯化锂溶液在灭菌水中加吐温-80和香蕉皮多糖溶液，过滤灭菌得到；
S3、洗涤处理：上述步骤处理后，在蒸馏水中冲洗2～5次不定芽茎尖；
S4、增殖培养：将S3处理后的不定芽转接到S1配制的增殖培养基中，增殖继代4～8次后，转入含有0.5～3.0g/L吲哚丁酸IBA、2.0～4.0g/L琼脂糖和20～40g/L蔗糖的MS基础培养基中，获得诱变处理植物体，所述MS基础培养基与S1中的MS基础培养基相同。
优选地，所述S1中的蔗糖pH为4.0～6.0。
优选地，所述S1中增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤(BAP)3.0mg/L、腺嘌呤1.0mg/L、琼脂糖3.0g/L、蔗糖30g/L。
优选地，所述S1中诱变剂浓度0.4～1.6％。
优选地，所述S2中旋转摇床的转速为150rpm，恒温27℃荡浸泡培养。
优选地，所述S2中光照强度设定为1000～3000lx。
优选地，所述S2中氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液的体积比为1:10～20:0.3～2:1～5。
优选地，所述S4步骤的MS基础培养基为1.0g/L吲哚丁酸IBA、3.0g/L琼脂糖和30g/L蔗糖。
与现有技术相比，本申请的有益效果是：
本申请使用的氯化锂溶液处理抑制幼苗生长，长势存在明显差异，在形态上出现了具有特殊性状的单株；通过诱变剂处理，按比例将氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液配制成诱变剂，结合光照培养箱培养，控制光照强度和温度变化，明显提高了突变频率，扩大了变异范围，利用化学诱变剂和特殊的处理工艺可使突变频率最高提高到8.89％，比自然突变频率高1000倍以上；突变类型集中表现为叶色突变、叶型突变及株型突变，其中有6.47％的植株在叶片上表现为亮绿叶，7.11％表现为黄条纹。1.96％表现为叶缘卷曲，4.50％表现为短叶，从而可知人工诱变的范围广，变异类型丰富。
为了更好理解本申请技术内容，下面提供具体实施例，对本申请做优选地说明。
本申请实施例所用的实验方法如无特殊说明，均为常规方法。
本申请实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
诱导增加粉蕉突变范围的化学诱变方法：包括以下步骤：
S1、增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤(BAP)1.0mg/L、腺嘌呤0.2mg/L、琼脂糖2.0g/L、蔗糖20g/L，蔗糖pH为4.0；
S2、香蕉的诱变处理：使用手术刀切割香蕉单个独立的不定芽，放入装有氯化锂诱变剂的三角瓶中，在旋转摇床中以138rpm、20℃振荡浸泡培养4h后，再放置于光照培养箱中，光照强度设定为1000lx，再进行变温处理，初始温度为15℃处理1h，调整温度22℃，处理1h；所述氯化锂诱变剂是将氯化锂溶液在灭菌水中加吐温-80和香蕉皮多糖溶液，过滤灭菌得到，所述氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液的体积比为1:10:0.3:1；
S3、洗涤处理：上述步骤处理后，在蒸馏水中冲洗2次不定芽茎尖；
S4、增殖培养：将S3处理后的不定芽茎尖转接到S1配制的增殖培养基中，增殖继代4次后，转入含有0.5g/L吲哚丁酸IBA、2.0.0g/L琼脂糖和20g/L蔗糖的MS基础培养基中，获得诱变处理植物体。
实施例2
诱导增加粉蕉突变范围的化学诱变方法：包括以下步骤：
S1、增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤(BAP)5.0mg/L、腺嘌呤1.5mg/L、琼脂糖4.0g/L、蔗糖40g/L，蔗糖pH为6.0；
S2、香蕉的诱变处理：使用手术刀切割香蕉单个独立的不定芽，放入装有氯化锂诱变剂的三角瓶中，在旋转摇床中以200rpm、30℃振荡浸泡培养24h后，再放置于光照培养箱中，光照强度设定为3000lx，再进行变温处理，初始温度为20℃处理3h，调整温度30℃，处理3h；所述氯化锂诱变剂是将氯化锂溶液在灭菌水中加吐温-80和香蕉皮多糖溶液，过滤灭菌得到，所述氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液的体积比为1:20:2:5；
S3、洗涤处理：上述步骤处理后，在蒸馏水中冲洗5次不定芽茎尖；
S4、增殖培养：将S3处理后的不定芽转茎尖接到S1配制的增殖培养基中，增殖继代8次后，转入含有3.0g/L吲哚丁酸IBA、4.0g/L琼脂糖和40g/L蔗糖的MS基础培养基中，获得诱变处理植物体。
实施例3
诱导增加粉蕉突变范围的化学诱变方法：包括以下步骤：
S1、增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤(BAP)3.0mg/L、腺嘌呤1.0mg/L、琼脂糖3.0g/L、蔗糖30g/L；
S2、香蕉的诱变处理：使用手术刀切割香蕉单个独立的不定芽，放入装有氯化锂诱变剂的三角瓶中，在旋转摇床中以150rpm、27℃振荡浸泡培养14h后，再放置于光照培养箱中，光照强度设定为2000lx，再进行变温处理，初始温度为18℃处理2h，调整温度26℃，处理2h；所述氯化锂诱变剂是将氯化锂溶液在灭菌水中加吐温-80和香蕉皮多糖溶液，过滤灭菌得到，所述氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液的体积比为1:15:1.2:3；
S3、洗涤处理：上述步骤处理后，在蒸馏水中冲洗3次不定芽茎尖；
S4、增殖培养：将S3处理后的不定芽茎尖转接到S1配制的增殖培养基中，增殖继代46次后，转入含有1.0g/L吲哚丁酸IBA、3.0g/L琼脂糖和30g/L蔗糖的MS基础培养基中，获得诱变处理植物体。
实施例4
本实施例与实施例3的区别在于，诱导增加粉蕉突变范围的化学诱变方法：所述氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液的体积比为1:5:3:7。
实施例5
本实施例与实施例3的区别在于，诱导增加粉蕉突变范围的化学诱变方法：所述S2中光照强度设定为4000lx。
实施例6
本实施例和实施例3的区别在于，诱导增加粉蕉突变范围的化学诱变方法：所述S1中增殖培养基浓度0.8％。
对比例1
本对比例和实施例3的区别在于，诱导增加粉蕉突变范围的化学诱变方法：所述S2香蕉的诱变处理中未放置光照培养箱培养。
对比例2
本对比例和实施例3的区别在于，诱导增加粉蕉突变范围的化学诱变方法：所述S2香蕉的诱变处理的光照培养箱中未进行变温处理。
对比例3
本对比例和实施例3的区别在于，诱导增加粉蕉突变范围的化学诱变方法：所述S2的变温处理中，初始温度为14℃处理2h后再调整至20℃。
一、化学诱变诱导发生变异类型及发生的变异率
将本申请实施例1～6和对比例1～3的诱导方法进行对比实验，每次试验重复3次，得到化学诱变诱导发生变异类型及发生的变异率(以下数值为平均值)：
变异率计算公式：不同类型变异率＝不同表型变异后代/总数*100
从上表可知，本申请的诱变方法使得粉蕉突变类型集中表现为叶色突变、叶型突变及株型突变。其中有6.47％的植株在叶片上表现为亮绿叶，7.11％表现为黄条纹，1.96％表现为叶缘卷曲，4.50％表现为短叶，从而可知人工诱变的范围广，变异类型丰富。
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
Claims (8)
- 一种诱导增加粉蕉突变范围的化学诱变方法，其特征在于：包括以下步骤：S1、增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤1.0～5.0mg/L、腺嘌呤0.2～1.5mg/L、琼脂糖2.0～4.0g/L、蔗糖20～40g/L；S2、香蕉的诱变处理：切割香蕉单个独立的不定芽，放入氯化锂诱变剂中，在旋转摇床中以138～200rpm、20～30℃振荡浸泡培养4～24h后，再放置于光照培养箱中，进行变温处理，初始温度为15～20℃处理1～3h，调整温度22～30℃，处理1～3h；所述氯化锂诱变剂是将氯化锂溶液在灭菌水中加吐温-80和香蕉皮多糖溶液，过滤灭菌得到；S3、洗涤处理：上述步骤处理后，在蒸馏水中冲洗2～5次不定芽茎尖；S4、增殖培养：将S3处理后的不定芽茎尖转接到S1配制的增殖培养基中，增殖继代4～8次后，转入含有0.5～3.0g/L吲哚丁酸IBA、2.0～4.0g/L琼脂糖和20～40g/L蔗糖的MS基础培养基中，获得诱变处理植物体。
- 如权利要求1所述的一种诱导增加粉蕉突变范围的化学诱变方法，其特征在于：所述S1中的蔗糖pH为4.0～6.0。
- 如权利要求1所述的一种诱导增加粉蕉突变范围的化学诱变方法，其特征在于：所述S1中增殖培养基的配制：MS基础培养基、6-苄基氨基嘌呤(BAP)3.0mg/L、腺嘌呤1.0mg/L、琼脂糖3.0g/L、蔗糖30g/L。
- 如权利要求1所述的一种诱导香蕉理想突变密度的化学诱变方法，其特征在于：所述S1中增殖培养基浓度0.4～1.6％。
- 如权利要求1所述的一种诱导香蕉理想突变密度的化学诱变方法，其特征在于：所述S2中旋转摇床的转速为150rpm，恒温27℃荡浸泡培养。
- 如权利要求1所述的一种诱导香蕉理想突变密度的化学诱变方法，其特征在于：所述S2中光照强度设定为1000～3000lx。
- 如权利要求1所述的一种诱导香蕉理想突变密度的化学诱变方法，其特征在于：所述S2中氯化锂溶液、灭菌水、吐温-80和香蕉皮多糖溶液的体积比为1:10～20:0.3～2:1～5。
- 如权利要求1所述的一种诱导香蕉理想突变密度的化学诱变方法，其特征在于：所述S4的MS基础培养基为1.0g/L吲哚丁酸IBA、3.0g/L琼脂糖和30g/L蔗糖。
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