CN108522267A - A method of formulating the waterlogging mutant of wax gourd using EMS mutagens - Google Patents
A method of formulating the waterlogging mutant of wax gourd using EMS mutagens Download PDFInfo
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- CN108522267A CN108522267A CN201810269776.1A CN201810269776A CN108522267A CN 108522267 A CN108522267 A CN 108522267A CN 201810269776 A CN201810269776 A CN 201810269776A CN 108522267 A CN108522267 A CN 108522267A
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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
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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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
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Abstract
The present invention provides a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens, and this approach includes the following steps：(1) it uses 1 1.3%EMS phosphate buffers of volume ratio to handle 10 15h to gourd seed and obtains M1For seed；(2) the processed M of EMS mutagens will be used1It is placed in 25 30 DEG C of the CO that oxygen concentration is 10 15% for gourd seed2Vernalization in constant temperature germinating box；(3)M1It is sowed for seed, M is obtained after selfing2For seed；Then M2It is sowed for seed, after 15 25d of pollination self, 15 25cm of basal part of stem waterflooding is crossed using crop field and handles 3h 4h, selects waterlogging wax gourd strain, waterlogging strain, which is carried out single melon sowing, reserves seed for planting, and obtains waterlogging wax gourd mutant strain Mut.The present invention carries out mutagenic treatment using chemical mutagen EMS to gourd seed, obtains the waterlogging mutant of wax gourd, expands germ plasm resource source, formulate out new germ plasm resource.
Description
Technical field
The present invention relates to wax gourd germ plasm resources to formulate technical field, particularly, is related to a kind of using the EMS mutagens initiative winter
The method of the waterlogging mutant of melon.
Background technology
Wax gourd is China main vegetables crop, year about 4,800,000 mu of sown area, wax gourd product is for adjusting China vegetables week
Year supply plays an important role.Since wax gourd is mainly distributed on the countries in Southeast Asia such as China, India, Vietnam, Laos, however,
Relative to other vegetable crops, wax gourd material genetic background is relatively narrow, the serious initiative for limiting wax gourd new material and high-quality money
The expansion selection and breeding in source.Currently, wax gourd lacks waterlogging germ plasm resource, and the main planting area of wax gourd the Yangtze river basin and on the south
Area often meets with flood in planting process, causes huge economic loss, and passes through existing germ plasm resource at present
Crop field screening is difficult to obtain waterlogging-resistant wax gourd germ plasm resource, and the flood problem encountered in wax gourd production fails to solve very well.
Chemical mutagen EMS (ethyl methane sulfonate ethylmethane sulfonates) is a kind of single function alkylating agent,
One of most common efficient chemical mutagens in plant mutagenesis, due to its can be transformed into vivo electron deficient it is active in
Between product, be easy with base on N atoms and phosphate group occur nucleophilic substitution, so that DNA structure is changed, therefore wide
The general chemical mutagen as the initiative of high-quality crop new material.EMS chemical mutagenesis techniques are in muskmelon, wheat, rice, jade
Ideal Mutagenic Effect is obtained in the various crops such as rice, watermelon, cucumber, the crop of some genotype change is obtained, to crop
Functional genomics research and new varieties initiative and expansion selection and breeding play important impetus.
New germplasm materials can be provided for wax gourd breeding by creating mutant library screening mutant, to promote wax gourd germplasm
Innovation, but yet there are no the relevant mutant library construction work of wax gourd, therefore carry out wax gourd EMS mutant mutagenesis and waterlogging
Mutant screening is of great significance.
Invention content
Present invention aims at a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens is provided, to solve now
There is the technical issues of wax gourd lacks waterlogging germ plasm resource in technology.
To achieve the above object, the present invention provides it is a kind of using EMS mutagens formulate the waterlogging mutant of wax gourd method,
Include the following steps：
(1) gourd seed is handled using EMS mutagens, specially：The full gourd seed of health is selected, is used
After clear water rinses gourd seed, it is placed on the 25-40min that soaks seed in 50-60 DEG C of lukewarm water；Seed soaking is placed on 25-30 DEG C of perseverance
Incubator vernalization 30-40h；Then it uses volume ratio 1-1.3%EMS phosphate buffers to handle 10-15h, is terminated and reacted with terminate liquid；
After immersion, 10-15h is rinsed with clear water；
(2) seed of plantation processing：The processed gourd seed of EMS mutagens is placed in a concentration of 10- of oxygen volume
15% 25-30 DEG C of CO2Vernalization in constant temperature germinating box, the seed for selecting germination carry out seedling bed sowing；
(3) mutant obtains, specially：
First, M1The plantation in generation and seed collection：Seed Jing Guo mutagenic treatment is designated as M1, hole tray is educated after screening vernalization
Seedling, pollination self；By M1It is harvested respectively for each single plant, single melon is reserved seed for planting, and M is obtained2For seed；
Secondly, M2The plantation and selection in generation：By M2For seed a concentration of 10-15% of oxygen volume 25-30 DEG C of CO2It is permanent
Vernalization in warm germinating box is selected germination seed and is seeded in experimental plot；
Then, pollination self waits for that fruit setting carried out basal part of stem submerging treatment, selects waterlogging wax gourd strain, by waterlogging strain
It carries out single melon sowing to reserve seed for planting, obtains waterlogging wax gourd mutant seeds Mut.
Further, in step (1), the gourd seed, which needs to keep stirring during soaking, keeps seed heated equal
It is even.
Further, in step (1), gourd seed volume ratio 1.2%EMS phosphate buffers are handled into 12h.
Further, the EMS solution be configured so that PH be 7.4 0.2mol/L phosphate buffers configure volume it is dense
The EMS solution that degree is 1.2%.
Further, the terminator is the Na of mass concentration 5%2S2O3Solution.
Further, in step (3), the wax gourd M2After generation plantation, pollination self waits for that fruit setting 15-25d uses crop field
Basal part of stem waterflooding 15-25cm processing 3-4h are crossed, waterlogging wax gourd strain is picked out.
Further, the gourd seed is the full height of health for self-mating system gourd seed.
The invention has the advantages that：
The present invention provides a kind of methods for formulating the waterlogging mutant of wax gourd using EMS mutagens, use chemical mutagen EMS
Mutagenic treatment is carried out for self-mating system seed to wax gourd height, mutant library is obtained, expands germ plasm resource source, formulate out new
Germ plasm resource.Wherein, gourd seed 12h is handled using the EMS phosphate buffers of volume ratio 1.2%, is the half processing of wax gourd
Dosage can get maximum induced mutation rate, reach 54.67% germination percentage and 51.83% planting percent.This method is simple, mutagenesis
Significant effect.
The present invention provides a kind of methods for formulating the waterlogging mutant of wax gourd using EMS mutagens, using mutant seeds
Vernalization stage hypoxemia screens, i.e., gourd seed is placed in 25-30 DEG C of the CO of a concentration of 10-15% of oxygen volume2Constant temperature vernalization
Vernalization in case, can get waterlogging ability enhances mutant, passes through M2Waterlogging wax gourd mutating strain series Mut is filtered out for water-logging.
By to saltant type Mut and the adventitious root sprouting quantity under wild type WT wax gourd seedling stage difference Submergence times, length, net photosynthesis
Rate and survival rate carry out comparison and carry out catalase, peroxidase, malonaldehyde, alcohol dehydrogenase to its blade
And the variance analyses such as hydroxy radical removal ability, the results showed that use the method for the present invention mutagenesis and the wax gourd mutant filtered out
Gourd seed relative to wild type WT has significantly stronger waterlogging ability.It is provided by the invention a kind of using EMS mutagenesis
The method that the waterlogging mutant of wax gourd is formulated in agent, new germplasm materials is provided for wax gourd breeding, to promote wax gourd germplasm innovation.
Other than objects, features and advantages described above, the present invention also has other objects, features and advantages.
The present invention is further detailed explanation below.
Specific implementation mode
The embodiment of the present invention is described in detail below, but the present invention can be limited and be covered according to claim
Multitude of different ways implement.
The present invention provides a kind of methods for formulating the waterlogging mutant of wax gourd using EMS mutagens, include the following steps：
(1) EMS mutagenesis optimum condition is screened
Before carrying out the EMS mutagens initiative waterlogging mutant of wax gourd, first to EMS mutagenic treatments condition and mode into
It has gone contrast experiment, has selected the full DZ163 wax gourds height of health first for self-mating system seed, before mutagenesis, sprouted according to wax gourd
Rule (wax gourd vernalization needs 36h or so to start to show money or valuables one carries unintentionally, and 72h or so sprouts), after rinsing gourd seed with clear water, is placed on
Soak seed 30min in 55 DEG C of lukewarm water, and needing to keep stirring during seed soaking makes seed be heated evenly；Seed soaking is placed on 28 DEG C of perseverances
Incubator vernalization 36h.Then, different EMS concentration and time-triggered protocol are carried out.Each processing selected seed 100.The results detailed in Table
1。
The influence of 1 EMS concentration for the treatment of of table and processing time to wax gourd germination percentage and planting percent
As can be seen from Table 1：With the increase of EMS concentration for the treatment of and processing time, gourd seed germination percentage and seedling
Rate is on a declining curve, and wherein influence of influence of the concentration to gourd seed germination percentage and planting percent than processing time becomes apparent from.
According to the selection principle of median lethal rate, it is EMS concentration 1.2%, processing time 12h, at this to select best EMS treatment conditions
It can get 54.67% germination percentage and 51.83% planting percent under treatment conditions.
(2) gourd seed is handled using EMS mutagens, specially：Select the full DZ163 wax gourd kinds of health
Son after rinsing gourd seed with clear water, is placed on the 30min that soaks seed in 55 DEG C of lukewarm water, needs not stop to stir during seed soaking
Mixing makes seed be heated evenly, and seed soaking is placed on 28 DEG C of insulating box vernalization 36h；Matched with the PH 0.2mol/L phosphate buffers for being 7.4
The EMS solution that volume ratio is 1.2% is set, 1.2%EMS phosphate buffers selection processing 12h is then used, is 5% with mass concentration
Na2S2O3Solution terminates reaction；After immersion, 12h is rinsed with clear water.
(3) seed of plantation processing：It is a concentration of that the processed gourd seed of EMS mutagens is placed in oxygen volume
12% 28 DEG C of CO2Vernalization in constant temperature germinating box supplements sterile water in vernalization box in time, ensures that gourd seed is normally sprouted
Hair.
(4) mutant obtains, specially：
First, M1The plantation in generation and seed collection：Seed Jing Guo mutagenic treatment (is labeled as M1), hole plate seedling growth is fixed
It plants in solarium's greenhouse, pollination self；By M1It is harvested respectively for each single plant, single melon is reserved seed for planting, and M is obtained2For seed.
Secondly, M2The plantation and selection in generation：By M2M is pressed for seed1It is a concentration of in oxygen volume for same planting patterns
12% 28 DEG C of CO2Vernalization in constant temperature germinating box is selected germination seed and is seeded in experimental plot；
Then, pollination self waits for that fruit setting 20d crosses basal part of stem 20cm submerging treatments 2h using crop field, it is ensured that water level floods substantially
Do not have all wax gourd plant basal part of stem, select waterlogging wax gourd strain, waterlogging strain, which is carried out single melon sowing, reserves seed for planting, and obtains the waterlogging winter
Melon mutant seeds Mut.
(5) it will be seeded in nutritive cube after the waterlogging mutant strain Mut and wild type WT that screen respectively vernalization, wait for its length
When to 3 leaves wholeheartedly, 60 plants of the consistent seedling of growing way is chosen respectively carry out seedling stages and cross basal part of stem 10cm waterflooding experiment, it is ensured that water level
Substantially all seedling plants basal part of stem are flooded, the gourd seeds of the DZ163 using before mutagenic treatment compare as wild type WT, keep flooding
Water 5d, take respectively waterflooding 0h, for 24 hours, the Mut of 48h, 72h and 96h and WT carry out adventitious root quantity, Net Photosynthetic Rate, survival rate,
Malonaldehyde (MDA), catalase (CAT), proline (PRO), alcohol dehydrogenase (ADH) and hydroxy radical (OH) removal
The detections such as the anti-oxidant enzyme activity such as ability.This experiment carries out nursery, commercial seedling medium nursery (nursery base using 9*9cm nutritive cubes
Matter is bought in Shandong Academy of Agricultural Sciences's Horticultural Research Institute), 100 plants are sowed respectively.Proline (PRO) assay is using acid
Property ninhydrin method, malonaldehyde (MDA) content measurement using thiobarbituricacidα- method, soluble sugar content measure use anthrone
Method, peroxidase (POD), catalase (CAT), alcohol dehydrogenase (ADH), hydroxy radical (OH) are all made of colorimetric
Method (kit builds up Bioengineering Research Institute from Nanjing) measures, and photosynthesis rate, which measures, to be used using LI-6400XT just
Take formula photosynthesis measuring system measurement.Experimental result is shown in Table 2, table 3, table 4, table 5.
Dry weight and the difference of adventitious root under 2 waterlogging mutant strain Mut and WT seedling stage differences Submergence time of table
As can be seen from Table 2：In waterflooding 0-24h, the dry weight of waterlogging mutant strain Mut and WT, net photosynthesis, no
Determine radical amount, length and survival rate, difference is little.After waterflooding is more than 48h, waterlogging mutant strain Mut begins with certain amount
Adventitious root grow, and survival rate is 100%, and WT is almost without growing adventitious root, and begins with plant part death.
With the extension of Submergence time, waterlogging mutant strain Mut constantly have adventitious root grow with it is elongated, survival rate is still up to 80% after 96h
More than, and WT only has a small amount of adventitious root to grow, length is obviously shorter than waterlogging mutant strain Mut, and the later survival rates of 96h are significantly
It reduces, only 46.47%.It can be seen that by the method for the invention, mutagenesis and the wax gourd mutant filtered out have well
Waterlogging ability.
The difference of enzymatic activity under 3 waterlogging mutant strain Mut and WT seedling stage differences Submergence time of table
(CAT/nmol min-1g-1FW；POD/U g-1FW；ADH/μmol min-1g-1FW)
As antioxidase important in plant, activity is high for catalase (CAT) and peroxidase (POD)
Low energy reflects resilience of the plant to environment stress.Alcohol dehydrogenase (ADH) is a kind of important anaerobic respiration relevant enzyme,
Its activity height reflects the ability that plant carries out anaerobic respiration.As can be seen from Table 3：Before waterflooding, mutant and wild type
CAT, POD and ADH enzymatic activity no significant difference；After flooding stress, CAT, POD and ADH enzymatic activity rise rapidly；With the side of body
Compel the extension of time, the enzymatic activity of wild type is slowly fallen after rise, and the enzymatic activity of mutant can but maintain always higher level, and
It is significantly higher than wild type always.
MDA (nmol g under 4 Mut with WT seedling stage difference Submergence times of table-1) and the difference of (OH) Scavenging activity (%) FW
Malonaldehyde (MDA) as secondary metabolite important in plant, the height of content directly reflect plant by
The degree injured to adverse circumstance.Hydroxy radical (OH) is one kind of active oxygen in plant, and the higher content the easier to produce plant
Raw injury.Therefore, the height of oxygen scavenging activity ability can also reflect resilience of the plant to environment stress.It can be with from table 4
Find out：With the extension of Submergence time, MDA contents persistently rise wild type, until its content of 96h reaches 81.01nmol/g, it is 0h
8.57 times；And mutant waterflooding for 24 hours when its MDA content significantly increase, be 1.92 times of 0h, then with Submergence time
Extend its MDA content without apparent to rise, and the MDA contents of wild type are all remarkably higher than mutant during entire flooding stress.
With the extension of Submergence time, two material Hydroxyl radical-scavenging abilities constantly increase, but remove energy with mutant activity oxygen
Power is higher than wild type.
PRO (μ g g under 5 Mut with WT seedling stage difference Submergence times of table-1) and soluble sugar (mg g FW-1FW difference)
Proline (PRO) is not only osmotic adjustment, and be alternatively arranged as film and enzyme Protective substances and free radical it is clear
Except agent, to play a protective role to growth of the plant under osmotic stress.Under adverse environmental factor, proline in plant
Content dramatically increases, and proline content reflects the resistance of plant to a certain extent in plant.Soluble sugar is equally deposited
In similar effect.As can be seen from Table 5：The proline and soluble sugar of wild type and mutant have not after waterflooding
Incrementss with the increase of degree, but mutant are significantly higher than wild type.
By to saltant type Mut under wild type WT wax gourd seedling stage difference Submergence times adventitious root sprouting quantity, length,
Net Photosynthetic Rate and survival rate carry out comparison and carry out catalase, peroxidase, malonaldehyde, ethyl alcohol to its blade
The variance analyses such as dehydrogenase and hydroxy radical removal ability, the results showed that prominent using the method for the present invention mutagenesis and the wax gourd filtered out
Variant has significantly stronger waterlogging ability relative to the gourd seed of wild type WT.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens, which is characterized in that include the following steps：
(1) gourd seed is handled using EMS mutagens, specially：The full gourd seed of health is selected, is rushed with clear water
After washing gourd seed, it is placed on the 25-40min that soaks seed in 50-60 DEG C of lukewarm water；Seed soaking is placed on 25-30 DEG C of insulating box and urges
Bud 30-40h；Then it uses volume ratio 1-1.3%EMS phosphate buffers to handle 10-15h, is terminated and reacted with terminate liquid；Immersion terminates
Afterwards, 10-15h is rinsed with clear water；
(2) seed of plantation processing：The processed gourd seed of EMS mutagens is placed in a concentration of 10-15%'s of oxygen volume
25-30 DEG C of CO2Vernalization in constant temperature germinating box, the seed for selecting germination carry out seedling bed sowing；
(3) mutant obtains, specially：
First, M1The plantation in generation and seed collection：Seed Jing Guo mutagenic treatment is designated as M1, hole plate seedling growth after vernalization is screened, from
Hand over pollination；By M1It is harvested respectively for each single plant, single melon is reserved seed for planting, and M is obtained2For seed；
Secondly, M2The plantation and selection in generation：By M2For seed a concentration of 10-15% of oxygen volume 25-30 DEG C of CO2Constant temperature is urged
Vernalization in bud case is selected germination seed and is seeded in experimental plot；
Then, pollination self waits for that fruit setting carried out basal part of stem submerging treatment, selects waterlogging wax gourd strain, waterlogging strain is carried out
Single melon sowing is reserved seed for planting, and waterlogging wax gourd mutant seeds Mut is obtained.
2. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens according to claim 1, feature exist
In in step (1), the gourd seed needs to keep stirring during soaking that seed is made to be heated evenly.
3. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens according to claim 1, feature exist
In in step (1), by gourd seed volume ratio 1.2%EMS phosphate buffers processing 12h.
4. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens according to claim 3, feature exist
In the EMS solution is configured so that the 0.2mol/L phosphate buffers that PH is 7.4 configure the EMS that volumetric concentration is 1.2%
Solution.
5. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens according to claim 1, feature exist
In the terminator is the Na of mass concentration 5%2S2O3Solution.
6. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens according to claim 1, feature exist
In, in step (3), the wax gourd M2After generation plantation, pollination self waits for that fruit setting 15-25d crosses basal part of stem waterflooding 15- using crop field
25cm handles 3-4h, picks out waterlogging wax gourd strain.
7. a kind of method for formulating the waterlogging mutant of wax gourd using EMS mutagens according to claim 1, feature exist
In the gourd seed is the full height of health for self-mating system gourd seed.
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| CN112205294A (en) * | 2019-11-12 | 2021-01-12 | 广东石油化工学院 | Mutation breeding method for cyperus esculentus |
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