CN106818487A - A kind of screening technique of paddy rice high temperature resistant mutant strain - Google Patents
A kind of screening technique of paddy rice high temperature resistant mutant strain Download PDFInfo
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- CN106818487A CN106818487A CN201710104408.7A CN201710104408A CN106818487A CN 106818487 A CN106818487 A CN 106818487A CN 201710104408 A CN201710104408 A CN 201710104408A CN 106818487 A CN106818487 A CN 106818487A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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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
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Abstract
The invention discloses a kind of screening technique of paddy rice high temperature resistant mutant strain, step is as follows：1）The excellent japonica rice variety of selection/it is to screen source as high temperature resistant mutant strain；2）The flower pesticide for taking mutant strain screening source carries out Fiber differentiation, is heat-treated after obtaining antherderived callus；3）Select antherderived callus and be transferred to differential medium, carry out normal temperature differentiation culture~heat treatment cycle；4）Soaked during active callus is put into recovery, culture is broken up in continuation in being then transferred to new differential medium；5）After callus induction is emerged, strong sprout in subculture medium is transferred to, greenhouse is transplanted into after hardening；6）Bloom control individual plant sowing obtains flower training family, and further high temperature resistance screening obtains high temperature resistant mutant strain.The present invention screens pressure by selecting flower training callus heat treatment to be used as high temperature resistant mutant strain, and the survival of callus is ensured by cycle heat treatment, recovery treatment, can effectively obtain paddy rice high temperature resistant mutant strain, shortens the paddy rice high temperature resistance breeding cycle.
Description
Technical field
The present invention relates to agricultural biological technical field, specifically a kind of screening side of paddy rice high temperature resistant mutant strain
Method.
Background technology
China is eating rice production and consumption big country, and it is the first that rice growing area and yield occupy national cereal crops.Existing rank
Section China paddy year sown area breaks through 200,000,000 t, accounts for 40% and the world of national total output of grain in 30,000,000 more than hm, yield
30% or so of paddy total output.There is 60% or so population in the whole nation with rice as staple food, l.35 hundred million tons of annual consumption.According to the United Nations
Food and agricultural organization predicts that world population will rise to 9,100,000,000 people in the year two thousand fifty, and world food output need to increase by 70% and be possible to meet
The population demand for increasing rapidly.As can be seen here Rice Production as main food source be not only related to China's grain security but also
Directly influence world food safety.
Continued to develop as society is industrialized, substantial amounts of greenhouse gases are discharged into air so that global temperatures are not
Disconnected to rise, there is very common, even long-time high temperature sometimes in thermal extremes, and this makes the plant development of paddy rice be changed greatly by very
Become, global tropical area just has more than 4,000,000 hm2Paddy rice be heated evil stress, but also be continuously increased again.Paddy rice Climatic regionalization
It is also the Major Natural Disasters of Chinese rice work, occurs mainly on the south the Yangtze river basin, more serious area is big portion, the lake in Jiangxi
The east in south, the western part in Fujian, the west and south in Zhejiang, the northeast in the east in Sichuan and Guangdong.These regional late-seaon rices
Flowering and grouting phase, precocious semilate rice boot stage to full heading time, often in 7-8 month summer High Temperature seasons, so as to cause can not
Normal loose powder, fertilization, kernel grouting is not full, i.e., " high temperature is forced ripe ".
High temperature stress causes O in plant2 -Produced with a large amount of free radicals, Lipid peroxidation metabolism aggravation, the integrality of cell membrane
Destruction.Research finds, with high temperature stress time lengthening, O2 -Speed is produced to increase rapidly, generation and the removing of the free radical of oxygen
Balance is broken.High temperature declines crop plasma membrane degree of saturation, membrane permeability increase, the increase of Electrolyte Leakage amount, electrical conductivity liter
Height, the content increase of Lipid peroxidation metabolism product-MDA.High temperature mainly influences the physicochemical properties and structure group of thylakoid
Knit, cause the disintegration of cell membrane and the degraded of cellular component.Rice leaves under high nocturnal temperature chlorophyll-protein complexes
Conjugation and chlorophyll content decline, photosynthetic efficiency reduction, there is oxidative damage symptom.High Temperature during Grain Filling stress causes paddy rice
Sword-like leave chlorophyll content, the reduction of shuttle enzymatic activity, photosynthetic rate are remarkably decreased.Protein in high temperature stress ensuing crop body occurs
Degraded, free aminoacid content increases in crop body, when amino acid content is too high, plant can be made to produce ammonia poisoning.
Research finds that paddy rice generative growth phase is higher than vegetative growth phase to the sensitivity of high temperature stress.Paddy rice subtracts
Number division and heading flowering period period, extremely sensitive to temperature, optimum temperature is 25~30 DEG C, and more than 30 DEG C will produce
Adverse effect, when mean temperature is more than 30 DEG C, continuous more than 3d will result under the fertilization degree of pollen and the viability of flower pesticide
Drop, so that pollen is unable to normal development and ultimately results in pollen abortion, and then influences grain number per spike and setting percentage；Rice Heading is bloomed
Phase, to very temperature sensitive, is also to be shown that Rice Heading florescence continues by the research such as period the most serious, Satake is endangered
35 DEG C of high temperature above of 5d, will influence pollen tube growth and normal loose powder, cause to be fertilized and form empty, blighted grain；Grouting
Phase runs into high temperature can accelerate rice milking stage speed, shorten grouting time, and 35 DEG C of high temperature above may result in rice milking stage speed
It is decreased obviously, the circularity of seed is affected so that yield and grain weight are all significantly reduced.In terms of comprehensive, as yield forming
The decline of mostly important setting percentage is one of heat most sensitive index of evil in factor, can concentrated expression paddy rice floret opening,
Dusting and the comprehensive extent of injury of fertilization, the stability of setting percentage is the important indicator of heat resistance under high temperature.
The improved seeds for accelerating seed selection, popularization and application heat resistance strong are that the essence for defeating high temperature this natural calamity is arranged
Apply., it is necessary to give suitable high temperature stress in conventional rice high temperature resistance breeding work, so that different cultivars is expressed with family
Go out high temperature genetics of resistance property difference.Breeding man is identified using crop field multiple years mostly when heat resistant variety screening is carried out
Method, will identify that neoasozin is placed in the area that Climatic regionalization easily occurs and carries out, and when a large amount of offsprings are cultivated, often give one
Fixed high temperature stress, then according to Holstein Cattle difference, then the heat resistance for screening these offsprings.Different breeding men evaluate heat-resisting
Standard with nonrefractory kind/family is not quite similar, but be all according under high temperature stress, Plant Height of Rice, spike length, grain weight,
What the proterties such as setting percentage, production loss were divided.Because many factors are easily received in crop field identification（Apply fertilizer unequal cultivation condition
Difference, rice ear sprouting period difference etc.）Interference, these indexs generally require multiple years repetitive identified could obtain it is relative can
The result of letter, therefore, the cycle of conventional heat refractory breeding is all very long, typically all 7,8 years, or even more than 10 years, and needs consumption
Take substantial amounts of manpower and materials.
The success of traditional rice breeding depends on the excavation and utilization of a series of favorable genes, the excavation of mutant and
The new gene that functional genome finds will also greatly promote the seed selection of new rice variety.Mutant is that certain proterties occurs can be inherited
The material of variation, or the material that certain gene is undergone mutation.For a long time, rice breeding man tries to find and separate valuable
Natural mutation and mutative material.We find that Rice anther culture can produce abundant mutant in early stage is studied, by
Only it is often that single proterties there occurs heritable variation in mutant, therefore using the excellent only high temperature resistance deficiency of economical character
Breeding material directed screening paddy rice high temperature resistant mutants, are an effective shortcuts of paddy rice high temperature resistance breeding.
The content of the invention
The purpose of the present invention is directed to that the existing heat-resisting Japonica two line hybrid rice cycle is long, breeding high cost defect, there is provided a kind of
It is used as high temperature resistant mutant strain screening pressure by selecting flower training callus to be heat-treated, is protected by cycle heat treatment, recovery treatment
Hinder the survival of callus, can effectively obtain paddy rice high temperature resistant mutant strain, shorten the paddy rice in paddy rice high temperature resistance breeding cycle
The screening technique of high temperature resistant mutant strain.
The purpose of the present invention is solved by the following technical programs：
A kind of screening technique of paddy rice high temperature resistant mutant strain, it is characterised in that：The screening technique step is as follows：
1）The excellent japonica rice variety of selection/it is to screen source as high temperature resistant mutant strain：Choose yield high, meter Zhi Hao, resistance excellent
Good but heat labile japonica rice variety/it is as the screening source of high temperature resistant mutant strain；
2）The flower pesticide for taking mutant strain screening source carries out Fiber differentiation, is heat-treated after obtaining antherderived callus：Mutant strain is screened
The positive season plantation in source, choosing healthy and strong main Honoka medicine carries out flower pesticide Fiber differentiation, obtains anther callus, treats that anther callus are general
After 0.3~0.6cm to diameter long, regulation culture room temperature continues 5~7 days to 36~38 DEG C；
3）Select antherderived callus and be transferred to differential medium, carry out normal temperature differentiation culture~heat treatment cycle：Select a diameter of 0.4~
The anther callus of 0.7cm, are transferred to differential medium, carry out normal temperature differentiation culture, and temperature control is 25~27 DEG C, time control
It is made as 7~9 days, then is transformed to heat treatment, temperature control is 36~38 DEG C, and time control is 3~4 days, is then recovered to normal temperature
Differentiation culture, such normal temperature differentiation culture~heat treatment cycle 4~5 times to browning callus sectors reaches 90%；
4）Soaked during active callus is put into recovery, culture is broken up in continuation in being then transferred to new differential medium：After heat treatment
Callus overwhelming majority browning blackening, some callus albefaction loss of activity cut browning and albefaction part, will be left behind
, then be transferred to active callus in new differential medium and continue to break up culture by immersion treatment in active callus input recovery；
5）After callus induction is emerged, strong sprout in subculture medium is transferred to, greenhouse is transplanted into after hardening：Part callus warp
Survived after Overheating Treatment, recovery treatment, and differentiation and seedling emergence, treat that bloom control is long to transferring in subculture medium after 2cm
Strong sprout is carried out, it is long to opening sealed membrane hardening after 8cm after bloom control, it is transplanted into greenhouse after 10~15 days；
6）Bloom control individual plant sowing obtains flower training family, and further high temperature resistance screening obtains high temperature resistant mutant strain：In greenhouse
Flower training individual plant sowing, acquisition japonica rice training family is some, and the flower training family field planting that positive season will harvest, is right with the source of screening
According to the paddy rice high temperature resistant mutant strain that screening heat resistance is improved.
The step 2）In carry out flower pesticide Fiber differentiation detailed process it is as follows：
（2.1）Materials and pretreatment：Will choose mutant strain screening source Zheng Ji crop fields conventional cultivation, take pulvinus away from for 5~7cm,
The healthy and strong main fringe that fringe does not split in pistac, anther length with flower pesticide for 1/3~1/2 and booting of glume, retains 2~3
Blade, 75% alcohol surface sterilization is placed in Cold pretreatment 2~3 days in 4 DEG C of refrigerators after being wrapped up with wet gauze；
（2.2）Inoculation：After pretreatment, the rice bud containing main fringe is cut, surface disappears during the rice bud then is soaked in into 75% alcohol
3~4min of poison, the rice bud then being shifted, main fringe is peeled off in superclean bench, and it is of uniform size small further to strip small ear
Branch stalk, is next obstructed with the gauze wrapped ramuscule sterilized, then by the mercuric chloride sterilizing 10min that gauze input concentration is 0.1%,
Rinsed with sterile water 3~4 times, open gauze dry, finally cut off flower pesticide top with the scissors sterilized, tremble medicine method inoculation flower pesticide in
Inducing culture.
The step（2.2）In flower pesticide Fiber differentiation based formulas be：N6 a great number of elements+MS trace element+MS organic matters+
The plant gel of+5% sucrose of 2mg/L2,4-D+0.5mg/LNAA+0.5mg/L protein hydrolysate+0.8%, pH value is 5.8.
The step 2）The condition of culture of middle Fiber differentiation is controlled to：26~28 DEG C of temperature, light culture.
The step 3）Normal temperature differentiation culture is with the illumination condition of heat treatment：Intensity of illumination is 1000~1500Lux,
Photoperiod is that 14h light/10h is dark.
The step 3）With 4）Middle differential medium formula is：MS inorganic salts+MS organic matters+sucrose 3%+ agar 0.7%+
KT2.0mg/L+NAA0.5 mg/L+ protein hydrolysate 1g/L, pH value is 5.8.
The step 4）Middle active callus feature is：Short texture, surface tool spheric granules, of light color and transparent.
The step 4）Middle recovery composition is：0.3~0.4g/L of vitamin C, vitamin E 0.6~0.8g/L, Na2-
EDTA 0.8~1.0g/L, FeSO4∙7H2O 0.5~0.7g/L, ZnSeO4·5H2O1.2~1.5g/L, MgSO4·7H2O 0.6
0.02~0.03 μm of ol/L of~0.8g/L, plant vulcanization kinetin PSK- α.
The step 5）Middle squamous subculture based formulas are：Micro+MS the organic matters of a large amount of+MS of 1/2MS+sucrose 3%+ paclobutrazols
0.2~0.3mg/L+ agar 0.8%, pH value is 5.8.
The present invention has the following advantages compared to existing technology：
Rice material to be identified only has development to generative growth phase again by phenotype after heat treatment in conventional heat refractory Japonica two line hybrid rice
Difference identifies the heat resistance of rice material, because identification phenotype is easily disturbed by many factors, it is therefore desirable to multiple years
Repetition could obtain relatively believable result, so conventional heat refractory breeding cycle is all very long, typically all 7,8 years, or even 10 years
More than, and need to expend substantial amounts of manpower and materials.The screening technique of paddy rice high temperature resistant mutant strain of the invention is creative to be led to
Cross the training callus heat treatment of selection flower to carry out screening paddy rice high temperature resistant mutant strain, not only save substantial amounts of manpower and materials, and
And qualification result is more accurate, qualification cycle is shorter, so as to greatly reduce breeding cost.
Rice Callus be a short texture, surface tool spheric granules, parenchyma cell group of light color and transparent, it is non-
It is often tender and lovely, be usually placed under high temperature can gradually aging browning, lose growth activity.The present invention is broken up at culture~heat by normal temperature
The mode of circulation is managed, the purpose of heat treatment screening is both realized, callus damage is reduced again, and then soak by recovery
Process to improve the activity of callus, ensured the survival rate of heat-resisting callus.
Brief description of the drawings
Accompanying drawing 1 trains callus by activity recovery after recovery treatment for the flower of breeding trunk strain F199, is transferred to new differentiation culture
Base continues the photo of differential growth；
Accompanying drawing 2 is, using the technology of the present invention route, to differentiate high temperature resistance in the flower training callus of thermo-labile rice strain and dash forward
The photo of mutant.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
A kind of screening technique of paddy rice high temperature resistant mutant strain, it is characterised in that：The screening technique step is as follows：
1）The excellent japonica rice variety of selection/it is to screen source as high temperature resistant mutant strain：
Choose that yield is high, meter Zhi Hao, resistance be excellent but heat labile japonica rice variety/be screening as high temperature resistant mutant strain
Source；
2）The flower pesticide for taking mutant strain screening source carries out Fiber differentiation, is heat-treated after obtaining antherderived callus：
By the positive season plantation in mutant strain screening source, choosing healthy and strong main Honoka medicine carries out flower pesticide Fiber differentiation, the tool of flower pesticide Fiber differentiation
Body process is as follows：
（2.1）Materials and pretreatment：The positive season crop field conventional cultivation of BCF1 cenospecies that will be chosen, takes pulvinus away from being 5~7cm, fringe
The healthy and strong main fringe not split for 1/3~1/2 and booting of glume in pistac, anther length with flower pesticide, retains 2~3 leaves
Piece, 75% alcohol surface sterilization is placed in Cold pretreatment 2~3 days in 4 DEG C of refrigerators after being wrapped up with wet gauze；
（2.2）Inoculation：After pretreatment, the rice bud containing main fringe is cut, surface disappears during the rice bud then is soaked in into 75% alcohol
3~4min of poison, the rice bud then being shifted, main fringe is peeled off in superclean bench, and it is of uniform size small further to strip small ear
Branch stalk, is next obstructed with the gauze wrapped ramuscule sterilized, then by the mercuric chloride sterilizing 10min that gauze input concentration is 0.1%,
Rinsed with sterile water 3~4 times, open gauze dry, finally cut off flower pesticide top with the scissors sterilized, tremble medicine method inoculation flower pesticide in
Inducing culture（Flower pesticide Fiber differentiation based formulas are：N6 a great number of elements+MS trace element+MS organic matters+2mg/L2,4-D+
The plant gel of+5% sucrose of 0.5mg/LNAA+0.5mg/L protein hydrolysate+0.8%, pH value is 5.8）；
The condition of culture of flower pesticide Fiber differentiation is controlled to：25~27 DEG C of temperature, light culture；Flower pesticide Fiber differentiation obtains antherderived callus
Tissue, after callus is generally grown to 0.3~0.6cm of diameter, regulation culture room temperature continues 5~7 days to 35~36 DEG C；
3）Select antherderived callus and be transferred to differential medium, carry out normal temperature differentiation culture~heat treatment cycle：
The anther callus of a diameter of 0.4~0.7cm are selected, differential medium is transferred to（Differential medium formula is：MS is inorganic
Salt+MS organic matters+sucrose 3%+ agar 0.7%+KT2.0mg/L+NAA0.5 mg/L+ protein hydrolysate 1g/L, pH value is 5.8）, enter
The differentiation culture of row normal temperature, temperature control is 25~27 DEG C, and time control is 7~9 days, then is transformed to heat treatment, and temperature control is
35~36 DEG C, time control is 3~4 days, then recovers to normal temperature to break up to cultivate, and such normal temperature breaks up culture~heat treatment cycle
Exceed half to browning callus sectors 3~4 times, normal temperature differentiation culture is with the illumination condition of heat treatment：Intensity of illumination
It is 1000~1500Lux, the photoperiod is that 14h light/10h is dark；
4）Soaked during active callus is put into recovery, culture is broken up in continuation in being then transferred to new differential medium：
Callus sectors browning blackening after heat treatment, some callus albefaction loss of activity, cuts browning and albefaction part,
It is the active callus short texture that leaves, surface tool spheric granules, of light color and transparent, active callus input moist heat sterilization is crossed
Immersion treatment in recovery（Recovery composition is：Every liter of water containing 0.3~0.4g of vitamin C, 0.6~0.8g of vitamin E,
Na20.8~1.0g of-EDTA, FeSO4∙7H20.5~0.7g of O, ZnSeO4·5H2O1.2~1.5g, MgSO4·7H2O 0.6~
0.8g, 0.02~0.03 μm of ol of plant vulcanization kinetin PSK- α）, the time of immersion treatment is 6-8h；Then by active callus
Culture is broken up in continuation in being transferred to new differential medium；
5）After callus induction is emerged, strong sprout in subculture medium is transferred to, greenhouse is transplanted into after hardening：
Part callus is survived after Overheating Treatment, recovery treatment, and differentiation and seedling emergence, treat bloom control it is long to 2cm it
After transfer in subculture medium（Squamous subculture based formulas are：Micro+MS the organic matters of a large amount of+MS of 1/2MS+sucrose 3%+ paclobutrazols
0.2~0.3mg/L+ agar 0.8%, pH value is 5.8）Strong sprout is carried out, it is long to opening sealed membrane hardening after 8cm after bloom control, 10~
Greenhouse is transplanted into after 15 days；
6）Bloom control individual plant sowing obtains flower training family, and further high temperature resistance screening obtains high temperature resistant mutant strain：
Flower training individual plant sowing in greenhouse, acquisition japonica rice training family is some, the flower training family field planting that positive season will harvest, with sieve
It is control, the paddy rice high temperature resistant mutant strain that screening heat resistance is improved to select source.
Embodiment
First, selection F199 screens source as high temperature resistant mutant strain：
For many years breeding practice we filter out breeding trunk strain F199, F199 has yield high, good grain quality, resists more（Height is anti-
Stripe virus disease, false smut, rice blast, in anti-banded sclerotial blight, black streak dwarf）Advantage, in 151 days or so the time of infertility, be excellent
The breeding of ripe middle round-grained rice is key in good, but F199 high temperature resistance is not enough, and F199 kinds in 2013 are planted in Jiangsu Dong Xin farms, and then 7
~August has been subjected to Climatic regionalization, causes F199 high temperature to force ripe, and sterile grain rate reaches 31.2%, and mass of 1000 kernel have dropped 10.3%, high temperature
Resistance needs to be improved.
2nd, the flower pesticide for taking F199 carries out Fiber differentiation, is heat-treated after obtaining antherderived callus：
By the positive season plantations of F199, choosing healthy and strong main Honoka medicine carries out flower pesticide Fiber differentiation, the condition of culture control of flower pesticide Fiber differentiation
It is made as：26 DEG C of temperature, light culture；Flower pesticide Fiber differentiation obtains anther callus in 25 days or so, and callus is universal after 42 days
It is long to adjust culture room temperature to 35.5 DEG C to 0.3~0.6cm of diameter, continue 6 days.
3）Select antherderived callus and be transferred to differential medium, carry out normal temperature differentiation culture~heat treatment cycle：
After being once heat-treated, the anther callus of a diameter of 0.4~0.7cm are selected, be transferred to differential medium（Differential medium
It is formulated and is：MS inorganic salts+MS organic matters+sucrose 3%+ agar 0.7%+KT2.0mg/L+NAA0.5 mg/L+ protein hydrolysate 1g/L,
PH value is 5.8）, culturing room's culture is placed in, culturing room's illumination condition is：Intensity of illumination is 1300Lux, the photoperiod be 14h light/
10h is dark, first carries out normal temperature differentiation culture, and temperature control is 26 DEG C, and time control is 8 days, then is transformed to heat treatment, temperature control
It is 35.5 DEG C, time control is 3.5 days, then recovers to normal temperature to break up to cultivate, such normal temperature breaks up culture~heat treatment cycle,
Part callus color and luster starts dimmed, the brown line of callus edge appearance, part callus after 2 circulations after 1 circulation
Browning, browning position is spread rapidly from edge, and callus browning degree significantly, is calculated, about half callus by volume after 3 circulations
There is browning in tissue, after 4 circulations, callus browning degree is serious, is calculated by volume, about 90% callus occur browning or
Albefaction, now stops heat treatment.
4）Soaked during active callus is put into recovery, culture is broken up in continuation in being then transferred to new differential medium：
The callus that culturing room cultivates is cut into browning and albefaction part in superclean bench, the active callus structure for leaving
Loose, surface has spheric granules, of light color and transparent, immersion treatment in the recovery that active callus input moist heat sterilization is crossed
（Recovery composition is：Every liter of water 0.35g containing vitamin C, vitamin E 0.7g, Na2-EDTA 0.9g、FeSO4∙7H2O
0.6g、ZnSeO4·5H2O 1.35g、MgSO4·7H2O 0.7g, 0.025 μm of ol of plant vulcanization kinetin PSK- α）, at immersion
The time of reason is 7h；Then active callus is transferred in new differential medium and continues to break up culture.
Influence for inspection Different treatments to Rice Callus differentiation effect, the same period carries out contrast test, selects
The same or like callus of culturing room's culture is tested：First group carries out recovery immersion treatment experiment, during treatment
Between 7h；Second group uses aqua sterilisa immersion treatment, process time 7h；3rd group does not carry out any treatment.First group and second group
New differential medium is transferred to after treatment, the 3rd group is directly transferred to new differential medium.Callus seedling differentiation after 35 days（As said
Shown in bright book accompanying drawing 1 and Figure of description 2）, phenylacetic acid is counted, as a result such as table 1 below.
By the result of the test of table 1 it can be found that recovery immersion treatment is carried out to the callus after heat treatment can be notable
The activity of callus is improved, so as to substantially increase phenylacetic acid and green seedling ratio, a small amount of heat-resisting callus has been ensured
Survival, and the water process that sterilizes breaks up no remarkable result to callus.
5）After callus induction is emerged, strong sprout in subculture medium is transferred to, greenhouse is transplanted into after hardening：
A small number of callus are survived after Overheating Treatment, recovery treatment, and differentiation and seedling emergence, treat bloom control it is long to 2cm it
After transfer in subculture medium（Squamous subculture based formulas are：Micro+MS the organic matters of a large amount of+MS of 1/2MS+sucrose 3%+ paclobutrazols
0.2~0.3mg/L+ agar 0.8%, pH value is 5.8）Strong sprout is carried out, it is long to opening sealed membrane hardening, 13 days after 8cm after bloom control
After be transplanted into greenhouse.
6）Bloom control individual plant sowing obtains flower training family, and further economical character, heat-resisting screening obtain heat-resisting japonica rice new product
System：
In early August, 2015, flower training individual plant sowing in greenhouse obtains flower training family 23, the flower training that positive season in 2016 will harvest
Family field planting, is control with breeding backbone F199, and screening obtains the Keng rice lines that 2 heat resistances are improved（Remaining
21 is false high temperature resistant mutant strain）, into follow-up variety comparative test.
The present invention by select flower training callus be heat-treated be used as high temperature resistant mutant strain screen press, by cycle heat treatment,
Recovery processes to ensure the survival of callus, can effectively obtain paddy rice high temperature resistant mutant strain, shortens paddy rice high temperature resistance
Breeding cycle, is worth with larger Breeding Application.
Above example is only explanation technological thought of the invention, it is impossible to limit protection scope of the present invention with this, every
According to technological thought proposed by the present invention, any change done on the basis of technical scheme each falls within the scope of the present invention
Within；The technology that the present invention is not directed to can be realized by prior art.
Claims (9)
1. a kind of screening technique of paddy rice high temperature resistant mutant strain, it is characterised in that：The screening technique step is as follows：
1）The excellent japonica rice variety of selection/it is to screen source as high temperature resistant mutant strain：Choose yield high, meter Zhi Hao, resistance excellent
Good but heat labile japonica rice variety/it is as the screening source of high temperature resistant mutant strain；
2）The flower pesticide for taking mutant strain screening source carries out Fiber differentiation, is heat-treated after obtaining antherderived callus：Mutant strain is screened
The positive season plantation in source, choosing healthy and strong main Honoka medicine carries out flower pesticide Fiber differentiation, obtains anther callus, treats that anther callus are general
After 0.3~0.6cm to diameter long, regulation culture room temperature continues 5~7 days to 36~38 DEG C；
3）Select antherderived callus and be transferred to differential medium, carry out normal temperature differentiation culture~heat treatment cycle：Select a diameter of 0.4~
The anther callus of 0.7cm, are transferred to differential medium, carry out normal temperature differentiation culture, and temperature control is 25~27 DEG C, time control
It is made as 7~9 days, then is transformed to heat treatment, temperature control is 36~38 DEG C, and time control is 3~4 days, is then recovered to normal temperature
Differentiation culture, such normal temperature differentiation culture~heat treatment cycle 4~5 times to browning callus sectors reaches 90%；
4）Soaked during active callus is put into recovery, culture is broken up in continuation in being then transferred to new differential medium：After heat treatment
Callus overwhelming majority browning blackening, some callus albefaction loss of activity cut browning and albefaction part, will be left behind
, then be transferred to active callus in new differential medium and continue to break up culture by immersion treatment in active callus input recovery；
5）After callus induction is emerged, strong sprout in subculture medium is transferred to, greenhouse is transplanted into after hardening：Part callus warp
Survived after Overheating Treatment, recovery treatment, and differentiation and seedling emergence, treat that bloom control is long to transferring in subculture medium after 2cm
Strong sprout is carried out, it is long to opening sealed membrane hardening after 8cm after bloom control, it is transplanted into greenhouse after 10~15 days；
6）Bloom control individual plant sowing obtains flower training family, and further high temperature resistance screening obtains high temperature resistant mutant strain：In greenhouse
Flower training individual plant sowing, acquisition japonica rice training family is some, and the flower training family field planting that positive season will harvest, is right with the source of screening
According to the paddy rice high temperature resistant mutant strain that screening heat resistance is improved.
2. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 2）In
The detailed process for carrying out flower pesticide Fiber differentiation is as follows：
（2.1）Materials and pretreatment：Will choose mutant strain screening source Zheng Ji crop fields conventional cultivation, take pulvinus away from for 5~7cm,
The healthy and strong main fringe that fringe does not split in pistac, anther length with flower pesticide for 1/3~1/2 and booting of glume, retains 2~3
Blade, 75% alcohol surface sterilization is placed in Cold pretreatment 2~3 days in 4 DEG C of refrigerators after being wrapped up with wet gauze；
（2.2）Inoculation：After pretreatment, the rice bud containing main fringe is cut, surface disappears during the rice bud then is soaked in into 75% alcohol
3~4min of poison, the rice bud then being shifted, main fringe is peeled off in superclean bench, and it is of uniform size small further to strip small ear
Branch stalk, is next obstructed with the gauze wrapped ramuscule sterilized, then by the mercuric chloride sterilizing 10min that gauze input concentration is 0.1%,
Rinsed with sterile water 3~4 times, open gauze dry, finally cut off flower pesticide top with the scissors sterilized, tremble medicine method inoculation flower pesticide in
Inducing culture.
3. the screening technique of paddy rice high temperature resistant mutant strain according to claim 3, it is characterised in that：The step
（2.2）In flower pesticide Fiber differentiation based formulas be：N6 a great number of elements+MS trace element+MS organic matters+2mg/L2,4-D+
The plant gel of+5% sucrose of 0.5mg/LNAA+0.5mg/L protein hydrolysate+0.8%, pH value is 5.8.
4. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 2）In
The condition of culture of Fiber differentiation is controlled to：26~28 DEG C of temperature, light culture.
5. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 3）Often
Temperature differentiation culture is with the illumination condition of heat treatment：Intensity of illumination is 1000~1500Lux, and the photoperiod is that 14h light/10h is dark.
6. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 3）With
4）Middle differential medium formula is：MS inorganic salts+MS organic matters+sucrose 3%+ agar 0.7%+KT2.0mg/L+NAA0.5 mg/L+
Protein hydrolysate 1g/L, pH value is 5.8.
7. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 4）In
Active callus feature is：Short texture, surface tool spheric granules, of light color and transparent.
8. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 4）In
Recovery composition is：0.3~0.4g/L of vitamin C, vitamin E 0.6~0.8g/L, Na20.8~1.0g/L of-EDTA,
FeSO4∙7H2O 0.5~0.7g/L, ZnSeO4·5H2O1.2~1.5g/L, MgSO4·7H20.6~0.8g/L of O, plant vulcanization
0.02~0.03 μm of ol/L of kinetin PSK- α.
9. the screening technique of paddy rice high temperature resistant mutant strain according to claim 1, it is characterised in that：The step 5）In
Squamous subculture based formulas are：Micro+MS the organic matters of a large amount of+MS of 1/2MS+sucrose 3%+ paclobutrazol 0.2~0.3mg/L+ agar
0.8%, pH value is 5.8.
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| CN1968261A (en) * | 2005-11-14 | 2007-05-23 | 联想(北京)有限公司 | Method for resource sharing in WLAN |
| CN104429932A (en) * | 2015-01-04 | 2015-03-25 | 王开 | Novel indica-japonica hybrid gene introgression sheath blight resistance breeding method |
| CN104521744A (en) * | 2015-01-04 | 2015-04-22 | 黎建波 | New bacterial-blight-resisting japonica rice breeding method |
| CN105210864A (en) * | 2015-09-10 | 2016-01-06 | 宋立胜 | A kind of acquisition methods of dahlia detoxic seedling |
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| CN104429932A (en) * | 2015-01-04 | 2015-03-25 | 王开 | Novel indica-japonica hybrid gene introgression sheath blight resistance breeding method |
| CN104521744A (en) * | 2015-01-04 | 2015-04-22 | 黎建波 | New bacterial-blight-resisting japonica rice breeding method |
| CN105210864A (en) * | 2015-09-10 | 2016-01-06 | 宋立胜 | A kind of acquisition methods of dahlia detoxic seedling |
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