CN109258468A - A kind of potato saline-alkali tolerant improvement breeding method - Google Patents
A kind of potato saline-alkali tolerant improvement breeding method Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a kind of potato saline-alkali tolerants to improve breeding method, and the breeding method is the following steps are included: a) choose Potato Cultivars and saline-alkali tolerant potato resource to be improved；B) Potato Cultivars and saline-alkali tolerant potato resource crossbreeding to be improved；C) improvement Potato Cultivars are treated and saline-alkali tolerant potato resource carries out anther Fiber differentiation respectively；D) identification of potato salt tolerance pressure；E) F1 generation cenospecies Anther Culture, salt tolerance；F) potato trains seedling strengthening seedling and rooting；G) colchicine processing flower training regeneration plant；H) further economical character, Saline alkali tolerance screen.Potato common cultivation kind is tetraploid, hereditary basis is complicated, the present invention goes out Dihaploid Potato using flower training technological guide, salt tolerance and improvement breeding are carried out on this basis, screening efficiency can be significantly improved, and highly shortened breeding time, accelerates breeding process, the improvement breeding of potato saline-alkali tolerant can be realized less than 3 years, the utilization of popularizing planting and saline alkali land resource to potato is all of great significance.
Description
Technical field
The present invention relates to a kind of potato saline-alkali tolerants to improve breeding method, belongs to crop planting and Cultivating techniques field.
Background technique
Potato (scientific name:Solanum tuberosum L.) category Solanaceae Solanum, annual herb plant, nickname potato,
Potato, taro, potato etc..Potato originates in South America andes region, and about Christian era can be traced in earliest artificial cultivation
Preceding 8000 to 5000 south of Peru area.Potato as a kind of wide adaptability, anti-disaster ability is strong, nutrition is comprehensive
High yield grain and vegetable crop are just widely applied plantation in the world, according to the system of FAO (Food and Agriculture Organization of the United Nation) (FAO)
Meter, the countries and regions of the potato of whole world plantation at present are up to 151, and wherein 41, Asia, 39 European, 37, Africa,
Middle and North America 18, South America 10,6, Oceania.Nowadays potato has become after rice, wheat, corn
The fourth-largest cereal crops in the world have high economic value and vast market prospect.
Potato has more than 400 years cultivation histories in China, with the propulsion of potato staple food grainization strategy, status
It will improve year by year, cultivated area will also expand therewith, this is significant for ensureing national food security.Come from nutritional point
It seeing, potato contains the ingredients such as much starch and vitamin abundant, minerals, amino acid, carotenoid, flavonoids, than
Cereal grain crop has more advantages, can supply a large amount of thermal energy of human body, moreover it is possible to provide in cereal grain and not had
Some vitamin As and vitamin C, and potato also has anti-oxidant, anti-aging, strengthening the spleen and stomach, reconciles dyspeptic function
Effect, can be described as " perfect food ".Coming from growth characteristics, potato yield is high, and it is stronger to the adaptability of environment, it plants
It is more easy, belongs to " four provinces " crop of " water-saving saves fertilizer, saves medicine, saves labour ".It is analyzed according to the Ministry of Agriculture, arrives the year two thousand twenty China
Grain demand increment is up to 100,000,000,000 jin or more, but constraint by cultivated land resource and planting benefit are influenced, wheat, rice etc.
The space that grain ration kind continues volume increase becomes smaller, difficulty increases, and therefore, potato staple food grain has become inevitable choice, it is contemplated that arrives
The year two thousand twenty, 50% or more potato will consume as staple food grain.
The salinization of soil is a global resource problem and ecological problem.According to statistics, there are various saline-alkali soil in the whole world about
9.5 hundred million hectares, the 10% of Global land area is accounted for, is distributed widely in a countries and regions more than 100.China is saline-alkali soil in the world
One of more country, for the salinized soil gross area up to 40,000 hectares, the 4.88% of land area can be utilized by accounting for the whole nation, mainly be divided
Cloth is in North China, northwest, northeast, areas formerly flooded by the Huanghe River and oceanfront, and with global warming, natural environment deterioration, saline-alkali soil area
To also constantly it expand, saline Land situation very severe.It, will if potato can be planted using the wetland with saline-alkaline of the length and breadth of land
Expand potato planting area as reasonable development and using saline alkali land resource and under the premise of not tying up three big staple food grains
Effective way.
Studies have shown that potato belongs to salt medium sensitivity crop, and when soil salt and alkali content is more than certain limit, potato
Growth and development will receive inhibition, will cause plant death when serious.Therefore to plant potato using wetland with saline-alkaline, just must
The strong Potato Cultivars of Saline alkali tolerance need to be selected.Conventional cross-breeding is the main means of current Potato Breeding, but horse
Bell potato is autotetraploid, and is height heterozygote, and Chromosome recombination frequency is high, no matter is selfed, is returned and can all generate separation.
And the Saline alkali tolerance of potato is by controlled by multiple genes and extremely complex, and raw by species and variety and genetype and internal physiological
Change the combined influence of reaction.Currently, breeding expert when carrying out the breeding of potato enduring alkali variety, need to cultivate hybridization big
It measures offspring and carries out Saline alkali tolerance identification, and identification method mostly uses greatly crop field multiple years identification method, i.e., these are miscellaneous for field planting
Offspring is handed over, and gives certain Saline Alkali Stress, then according to saline-alkali tolerant Traits change, then screens the Saline alkali tolerance of these offsprings.
Different breeders evaluates saline-alkali tolerant and the standard of non-enduring alkali variety/family is not quite similar, but is all according in the saline and alkaline side of body
Under compeling, what the Apparent characters such as potato morphological change, emergence rate, yield, commodity potato rate were divided.Since ring is identified in crop field
Border is not easy to control, the repetitive identified that these indexs generally require multiple years could obtain it is relatively believable as a result, therefore, it is conventional
The period of saline-alkali tolerant breeding is all very long, and generally all 7,8 years or even 10 years or more, and need to expend a large amount of manpower and material resources.
With the fast development of modern biotechnology, Potato Breeding technology will gradually integrate art methods and inhale
Newest research results of receiving form comprehensive breeding technology.Haploid breeding technology is to create the most effective hand of pure and mild genotype so far
Section, this technology are usually to be realized in a manner of Anther Culture.The common cultivation kind of potato is tetraploid, hereditary basis compared with
For complexity.The dihaploid that potato can be obtained by anther culture method, since its genome and chromosome quantitative subtract
Half, double haploid greatly improves the sensibility of stress from outside, is screened in this level, and sieve can be greatlyd improve
Select efficiency；Dihaploid Potato can obtain homozygous tetraploid, pass through different homozygous four times after artificial means double
Phase mutual cross between body can obtain unseparated first generation of hybrid true seed, become the utilization of Characters in Potato Hybrid advantage
May, this solves the allocation and transportation of the degeneration of kind and potato seed for being reserved seed for planting with true seed, and energy saving and cost has very
Important meaning.In addition, molecular labeling, transgeneic procedure, gene are fixed with the development of molecular biology and technique for gene engineering
Position, gene clone technology using increasingly extensive, flower training monoploid technology also will provide experimental material and research for the studies above
Basis.
Chinese scholar has been achieved for some research achievements, but tetraploid in terms of potato Anther Culture induces monoploid
Often ploidy mixes the regeneration plant that potato obtains by conventional Anther Culture approach, have dihaploid, tetraploid and
Mixoplod, and dihaploid proportion is not high, and this is for our the saline and alkaline sides of body of later use Dihaploid Potato plant pair
Compel more sensitive feature development Saline alkali tolerance screening operation to adversely affect, therefore we carry out potato Anther Culture process
Effective Regulation, improves the ratio of dihaploid in regeneration plant, to greatly improve screening efficiency, successfully develops one
The potato saline-alkali tolerant that kind breeding efficiency is high, breeding cycle is short improves breeding method, has important practical value.
Summary of the invention
The purpose of the present invention is the defects for conventional potato saline-alkali tolerant breeding, provide a kind of based on Anther Culture skill
The significant shortening potato saline-alkali tolerant breeding cycle of art, the potato saline-alkali tolerant for greatly speeding up potato saline-alkali tolerant breeding speed change
Good breeding method.
In order to achieve the above-mentioned object of the invention, technical solution provided by the invention is as follows:
A kind of potato saline-alkali tolerant improves breeding method, which is characterized in that the breeding method the following steps are included:
A) choose Potato Cultivars and saline-alkali tolerant potato resource to be improved: selection economical character is good but Saline alkali tolerance is insufficient
Potato cultivar is used as Potato Cultivars to be improved；It is evaluated according to Content Germplasm Resources in Potato Saline alkali tolerance, chooses saline-alkali tolerant
Germplasm materials kind of good performance is as saline-alkali tolerant potato resource；The field planting potato to be improved in a manner of potato seed sowing
Kind and saline-alkali tolerant potato resource；
B) Potato Cultivars and saline-alkali tolerant potato resource crossbreeding to be improved: with the Potato Cultivars to be improved of field planting
As female parent, saline-alkali tolerant potato resource harvests F1 generation cenospecies after solid as male parent, Post flowering artificial hybridization；
C) improvement Potato Cultivars are treated and saline-alkali tolerant potato resource carries out anther Fiber differentiation respectively: in potato squaring period
To flowering stage, acquires the bud of Potato Cultivars and saline-alkali tolerant potato resource to be improved and filter out at microspore development period
It in the bud of monokaryon middle and advanced stage, is transferred to after bud is pre-processed in superclean bench and carries out sterile working, first bud surface disappears
Then poison separates anther with scalpel and tweezers, be inoculated into progress anther Fiber differentiation in induced medium after cutting filigree,
Condition of culture is 24 ~ 26 DEG C, dark；
The ingredient of the induced medium are as follows: by without agar MS culture medium based on, then add altheine 4.7 ~
5.5mg/L, 23 ~ 27mg/L of lycopene, 13 ~ 19g/L of xylitol, 4.0 ~ 4.6mg/L of soybean lecithin, 30 ~ 36 μ of praseodymium sulfate
G/L, 0.15 ~ 0.20mg/L of triacontanol, chlorine is than 0.4 ~ 0.6mg/L of urea, 2,4-D 1.0 ~ 1.2mg/L, and potato starch 45 ~
55g/L, 9 ~ 15mg/L of hesperetin, 2.2 ~ 2.6mg/L of 5-bromouracil, pH value are 5.6 ~ 6.0；
D) NaCl and Na of gradient concentration the identification of potato salt tolerance pressure: is added in differentiation culture respectively2CO3Mixing
Solution causes Saline Alkali Stress condition, and Potato Cultivars and the saline-alkali tolerant potato resource to be improved to induce in step c) is cured
It when injured tissue grows to 2mm or so, is transferred on above-mentioned differential medium and carries out differentiation culture, selected according to callus differentiation rate
Suitable screening pressure level concentration is as potato salt tolerance pressure；
The ingredient of the differentiation culture are as follows: KNO32400 ~ 2600mg/L, NH4NO31020 ~ 1100mg/L, KH2PO4 350~
400mg/L, MgSO4·7H2O 340 ~ 380mg/L, CaCl2·2H2O 410 ~ 460mg/L, MnSO4·4H220 ~ 25mg/L of O,
Zinc methionine 8.5 ~ 9.5mg/L, H3BO311 ~ 14mg/L, KI 0.6 ~ 0.8mg/L, CuSO4·5H2O 0.023~0.027mg/
L, CoCl2·6H2O 0.023 ~ 0.027mg/L, Na2MoO4·2H2O 0.20 ~ 0.30mg/L, FeSO4·7H2O 27~28mg/
L, Na236 ~ 37mg/L of-EDTA, 0.13 ~ 0.17mg/L of nitroglycerin, 85 ~ 95mg/L of inositol, 4.4 ~ 5.0mg/L of glycylglycine,
0.5 ~ 0.6mg/L of thiamine hydrochloride, 0.5 ~ 0.6mg/L of puridoxine hydrochloride, 0.3 ~ 0.5mg/L of vitamin K, folic acid 0.5 ~
0.8mg/L, 3.0 ~ 4.0mg/L of gallic acid, 50 ~ 60mg/L of mashed potato, 1.6 ~ 2.0mg/L of epiphysin, increase production of amines 0.8 ~
1.2mg/L, 2,4- epi-brassinolide 0.5 ~ 0.7mg/L, 4- chlorine 0.4 ~ 0.6mg/L of this fluoroacetic acid, ethylene glycol contracting alditol 2.2 ~
2.4mg/L, oligofructose 7.5 ~ 8.1mg/L, 5'- monophosphate 11 ~ 15mg/L of cytidine, 33 ~ 37g/L of maltose, plant gel 5 ~
7g/L, pH value are 5.6 ~ 6.0；
E) F1 generation cenospecies Anther Culture, salt tolerance: will the sowing plantation of F1 generation cenospecies positive season in crop field, to seedling into
When entering squaring period to flowering stage, anther Fiber differentiation is carried out according to method described in step c), the callus induced is turned
It is connected on the differential medium (ingredient is identical as differential medium in step d)) for be added to potato salt tolerance pressure and carries out
Differentiation culture obtains potato and trains seedling；
F) potato trains seedling strengthening seedling and rooting: the potato training seedling by height in 3 ~ 5cm is transferred to strengthening seedling and rooting culture
In base, the vigorous flower training regeneration plant of robust growth, root system can get after cultivating 20-28d；
G) colchicine processing flower training regeneration plant: flower training regeneration plant is taken out from bottle, part root is cut off, stays root long degree
2 ~ 3cm, handles 16h with the mixed solution of 0.05% colchicine and 4% dimethyl sulfoxide at room temperature, and after treatment is by plant
The remaining medical fluid in root is cleaned, and the flower training regeneration plant that ploidy reverts to four times is obtained；
H) further economical character, Saline alkali tolerance screen: above-mentioned tetraploid flower training Transplantation of Regenerated Plantlets is carried out often into greenhouse
Cultivation management is advised, after entering and tying the potato phase, single plant harvests healthy potato seed, potato seed is further seeded into big field, to its salt tolerant
Alkalinity carries out evaluation identification, filter out Saline alkali tolerance improved, the good Potato Clones of economical character.
Potato Cultivars and the saline-alkali tolerant potato resource to be improved chosen in the step a) is that tetraploid is commonly planted
It cultivates, and the ability with sexual propagation.
The method that screening microspore development period is in the bud of monokaryon middle and advanced stage in the step c) are as follows: will be in bud
2 ~ 3 pieces of anther picking are pulverized, and release pollen, microscopy Observation of Microspore developmental stage, filters out list after being dyed with aceto-camine
The corresponding bud of core middle and advanced stage.
The pretreated method of bud in the step c) are as follows: be put into the mannitol solution of 40g/L after cleaning bud, room
Temperature is lower to pre-process 3d, then takes out bud and is placed on intensity to handle 30min in the uniform magnetic field of 400mT.
The mode of bud surface sterilization in the step c) are as follows: first bud is put into 75% ethyl alcohol and impregnates 60s, with sterilizing
Water rinses 2 ~ 3 times, then bud is put into 0.1% mercuric chloride solution and impregnates 15min, gently shakes when impregnating, then is sterilized with using
Water rinses 5 ~ 6 times.
NaCl and Na in the step d)2CO3In mixed solution, the mass ratio of the two is 9:5.
Suitable screening pressure level concentration refers in the step d): Potato Cultivars and saline-alkali tolerant potato to be improved
Resource callus differentiation rate is at 1.5% ~ 2.0%, NaCl and Na that differential medium adds respectively2CO3Mixed solution it is dense
Degree, and take the average value of the two.
The step d) and e) condition of middle differentiation culture are as follows: 25 ~ 27 DEG C of temperature, 2000 ~ 2400Lux of intensity of illumination, often
13 ~ 15h of its illumination.
The ingredient of strengthening seedling and rooting culture medium in the step f) are as follows: based on the 1/2MS culture medium without agar, then add
Add No. 5 0.1 ~ 0.2mg/L of root-inducing powder, 16 ~ 24mg/L of microcystin, 0.1 ~ 0.3g/L of active carbon, pH value is 5.6 ~ 6.0；It takes root
The condition of culture are as follows: 23 ~ 28 DEG C of temperature, intensity of illumination 2500 ~ 3000Lux, daily 15 ~ 17h of illumination.
Compared with prior art, the present invention there are following the utility model has the advantages that
1, the present invention problem not high for dihaploid ratio in conventional Tetraploid Potatoes Anther Culture regeneration plant, passes through
Regulate and control potato Anther Culture process, substantially increase the ratio of dihaploid in flower training regeneration plant, is double using potato
The haplobiont feature sensitive to Saline Alkali Stress carries out Saline alkali tolerance screening operation and provides material base.
2, the present invention improves and optimizes Tetraploid Potatoes anther-cultural system, improves callus induction rate, and
Find out a kind of differentiation culture process especially suitable for carrying out under conditions of Saline Alkali Stress.
3, breeding method of the invention combines haploid breeding technology, and F1 generation cenospecies is obtained double by Anther Culture
Monoploid Anther-culture, then use and contain NaCl and Na2CO3Differential medium simulation Saline Alkali Stress Saline alkali tolerance is carried out to it
Screening recycles chromosome doubling means to obtain homozygous tetraploid, sieves by further economical character, Saline alkali tolerance
Choosing, can be obtained potato Saline alkali tolerance new lines less than 3 years and greatly accelerates potato compared with conventional breeding methods
Saline-alkali tolerant improves the process of breeding, and the utilization of the popularizing planting and saline alkali land resource to potato is all of great significance.
Specific embodiment
Potato saline-alkali tolerant provided by the invention improvement breeding method is carried out below with reference to specific embodiment detailed
Illustrate, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Start within 2015, potato saline-alkali tolerant carried out using technical solution of the present invention and improves breeding work, the specific steps are as follows:
(1) choose Potato Cultivars and saline-alkali tolerant potato resource to be improved: potato 2 are used as potato product to be improved in selection
Kind, which is by the excellent variety of Chinese agricultural science institution vegetable or flower research institute breeding, which is early-maturing variety, yield
Height, stability are good, are in particular in that single plant knot potato is concentrated, and average every plant of 4-6 block, stem tuber is big and neat, 50 days after emergence
Commodity potato is harvested, for commodity potato rate 90% or more, the Tuber dormancy phase is short, is suitble to cultivate in two seasons, highly resistance floral leaf and leaf curl virus.
In production practice we have found that No. 2 Saline alkali tolerances of middle potato are poor, and main cultivation area Hebei, Henan, the Shandong, Shanxi of the kind
Etc. the salinization of soil problem on ground also get worse, therefore it is very necessary to carry out Saline alkali tolerance improvement to the kind.According to Ma Ling
The evaluation of potato germ plasm resource Saline alkali tolerance, the Saline alkali tolerance of Dongnong303 show as on, and the kind is also early-maturing variety, raw
It educates the phase 60 days or so, growing way is strong, and yield is high, and knot potato is concentrated, and quality is preferable, the ability with sexual propagation, therefore is elected to be salt tolerant
Alkali potato resource.Potato 2 and Dongnong303 in field planting in a manner of potato seed sowing in 2015.
B) Potato Cultivars and saline-alkali tolerant potato resource crossbreeding to be improved: the middle potato 2 with field planting in 2015
Number as maternal, Dongnong303 is used as male parent, and Post flowering artificial hybridization harvests F1 generation cenospecies after solid.
C) improvement Potato Cultivars are treated and saline-alkali tolerant potato resource carries out anther Fiber differentiation respectively: is existing in potato
Flower bud phase the bud of potato 2 and Dongnong303 and filters out microspore development period and is in monokaryon middle and advanced stage to flowering stage, in acquisition
Bud (screening technique are as follows: 2 ~ 3 pieces of anther picking in bud are pulverized, pollen is released, microscopy is seen after being dyed with aceto-camine
Microspore development period is examined, the corresponding bud of monokaryon middle and advanced stage is filtered out), the mannitol that 40g/L is put into after bud is cleaned is molten
In liquid, 3d is pre-processed at room temperature, and then bud is taken out and is placed on intensity to handle 30min in the uniform magnetic field of 400mT, will be located in advance
It is transferred to after bud after reason in superclean bench and carries out sterile working, first bud is put into 75% ethyl alcohol and impregnates 60s, with sterilizing
Water rinses 2 ~ 3 times, then bud is put into 0.1% mercuric chloride solution and impregnates 15min, gently shakes when impregnating, then is sterilized with using
Water rinses 5 ~ 6 times, then separates anther with scalpel and tweezers, cuts to be inoculated into induced medium after filigree and carries out
Anther Fiber differentiation, condition of culture is 25 DEG C, dark, the callus induction rate of potato 2 and Dongnong303 in statistics；
The ingredient of the induced medium of use are as follows: based on the MS culture medium without agar, then add altheine
5.1mg/L, lycopene 25mg/L, xylitol 16g/L, soybean lecithin 4.3mg/L, 33 μ g/L of praseodymium sulfate, triacontanol
0.18mg/L, chlorine is than urea 0.5mg/L, 2,4-D 1.1mg/L, potato starch 50g/L, hesperetin 12mg/L, 5-bromouracil
2.4mg/L, pH value 5.8.
D) identification of potato salt tolerance pressure: respectively differentiation culture in be added concentration be 0,0.1%, 0.2%,
0.3%, 0.4%, 0.5%, 0.6% NaCl and Na2CO3Mixed solution, wherein NaCl and Na2CO3Mass ratio be 9:5, cause salt
When the callus of alkaline stress condition, the middle potato when induce in step c) 2 and Dongnong303 grows to 2mm or so, transferred
Onto above-mentioned differential medium, differentiation training is carried out under conditions of 26 DEG C of temperature, intensity of illumination 2200Lux, daily illumination 14h
It supports, counts callus differentiation rate of the two kinds on each differential medium, and screen callus differentiation rate 1.5% ~ 2.0%
When, NaCl and Na that differential medium adds respectively2CO3The concentration of mixed solution takes the average value of the two resistance to as potato
Saline and alkaline screening pressure；Simultaneously to not adding NaCl and Na2CO3The potato seedling differentiated on the differential medium of mixed solution into
Row Ploidy Identification counts the ratio of Different Ploidy plant；
The ingredient of the differentiation culture of use are as follows: KNO32500mg/L, NH4NO31060mg/L, KH2PO4370mg/L, MgSO4·
7H2O 360mg/L, CaCl2·2H2O 435mg/L, MnSO4·4H2O 22.5mg/L, zinc methionine 9.0mg/L, H3BO3
12.5mg/L, KI 0.7mg/L, CuSO4·5H2O 0.025mg/L, CoCl2·6H2O 0.025mg/L, Na2MoO4·2H2O
0.25mg/L, FeSO4·7H2O 27.5mg/L, Na2- EDTA 36.5mg/L, nitroglycerin 0.14mg/L, inositol 90mg/L are double
Peptidylglycine 4.7mg/L, thiamine hydrochloride 0.55mg/L, puridoxine hydrochloride 0.55mg/L, vitamin K 0.4mg/L, folic acid
0.6mg/L, gallic acid 3.5mg/L, mashed potato 55mg/L, epiphysin 1.8mg/L, increase production of amines 1.0mg/L, 2,4- table rapes
The mono- phosphorus of plain lactone 0.6mg/L, 4- chlorine this fluoroacetic acid 0.5mg/L, ethylene glycol contracting alditol 2.3mg/L, oligofructose 7.8mg/L, 5'-
Sour cytidine 13mg/L, maltose 34g/L, plant gel 6g/L, pH value 5.8.
E) F1 generation cenospecies Anther Culture, salt tolerance: planting the positive season sowing of F1 generation cenospecies in crop field in 2016,
When seedling enters squaring period to flowering stage, anther Fiber differentiation is carried out according to method described in step c), by what is induced
Callus is transferred to differential medium (ingredient and the differential medium phase in step d) for being added to potato salt tolerance pressure
On together), differentiation culture is equally carried out under conditions of 26 DEG C of temperature, intensity of illumination 2200Lux, daily illumination 14h, obtains Ma Ling
Potato flower training seedling.
F) potato trains seedling strengthening seedling and rooting: the potato training seedling by height in 3 ~ 5cm is transferred to strengthening seedling and rooting
(the ingredient of the culture medium are as follows: based on the 1/2MS culture medium without agar, then add root-inducing powder 5 in culture medium
0.15mg/L, microcystin 20mg/L, active carbon 0.2g/L, pH value 5.8), at 26 DEG C of temperature, intensity of illumination 2750Lux, often
It can get the vigorous flower training regeneration plant of robust growth, root system after cultivating 20-28d under conditions of its illumination 16h.
G) colchicine processing flower training regeneration plant: flower training regeneration plant is taken out from bottle, part root is cut off, stays root
2 ~ 3cm of length, handles 16h with the mixed solution of 0.05% colchicine and 4% dimethyl sulfoxide at room temperature, and after treatment will
The remaining medical fluid in plant root is cleaned, and the flower training regeneration plant that ploidy reverts to four times is obtained.
H) further economical character, Saline alkali tolerance screening: by above-mentioned tetraploid flower training Transplantation of Regenerated Plantlets into greenhouse into
Row conventional cultivation management, after entering and tying the potato phase, single plant harvests healthy potato seed, potato seed is further seeded into crop field in 2017
In, evaluation identification is carried out to its Saline alkali tolerance, filter out Saline alkali tolerance improved, the good potato new product of economical character
System.
Comparative example 1
Compare the influence of different pretreatments method and anther induced medium to potato anther inducing effect: by embodiment 1
Preprocess method in step c) is changed to placing bud into 4 DEG C of refrigerator pretreatment 3d, and the induced medium of use is replaced with MS+
2,4-D 1.0mg/L+NAA 0.5mg/L+KT 1.0mg/L+ silver nitrate 30mg/L+ active carbon 3g/L+ sucrose 6%(W/V)+agar
0.6%(W/V), it is 107155874 A's of CN that the preprocess method and anther induced medium, which are quoted from application publication number,
Patent " a kind of method and its culture medium that Dihaploid Potato plant is obtained using Anther Culture ", potato 2 and east in statistics
The callus induction rate of agriculture 303.
Comparative example 2
Compare different differential mediums under conditions of Saline Alkali Stress, the influence to potato callus differentiation effect: will only implement
The differential medium used in 1 step d) of example replaces with MS+0.5mg/L gibberellin+2.0mg/L6-BA+3.0mg/L zeatin,
The differential medium is equally quoted from patent a kind of " method and its training for being obtained Dihaploid Potato plant using Anther Culture
Support base ", respectively in the differential medium be added concentration be 0,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% NaCl and
Na2CO3Mixed solution, potato 2 and callus differentiation rate of the Dongnong303 on each differential medium in statistics.
Comparative example 3
Compare influence of the different Anther Culture processes to potato Anther-culture ploidy: by the Anther Culture process in embodiment 1
Step c) and d) whole replace with patent a kind of " method and its training that Dihaploid Potato plant is obtained using Anther Culture
Support base " in the method that provides, centering potato 2 and Dongnong303 carry out Anther Culture, the specific steps are as follows:
1) select to budding takes length for the middle potato 2 of 5mm and the bud of Dongnong303 to flowering stage, places 4 DEG C of refrigerator pretreatments
3d.By the material handled well under superclean bench gnotobasis, bud is first placed in surface sterilizing in 75% alcoholic solution
30s aseptic water washing 1 time, then continues in 0.1% mercuric chloride solution the 8min that sterilizes, then with aseptic water washing 3 times, each 2min.
Sterilizing is placed on aseptic filter paper, is separated anther from bud with tweezers and transfer needle, and being inoculated in formula is MS+2,4-D
1.0mg/L+NAA 0.5mg/L+KT 1.0mg/L+ silver nitrate 30mg/L+ active carbon 3g/L+ sucrose 6%(W/V)+agar 0.6%
(W/V) in induced medium, the anther after inoculation is placed in dark culturing 72h in 35 DEG C of constant incubators, then continues at 24
DEG C, Fiber differentiation under dark condition；
2) white is formed at picking anther crack or faint yellow callus is transferred to and does not add NaCl and Na2CO3Differentiation culture
Break up culture in base, illumination condition is illumination 16h/ dark 8h, and cultivation temperature is 24 DEG C；Selective differentiation goes out the callus of green bud point
Tissue, which is transferred in subculture medium, cultivates 30d, and illumination condition is illumination 16h/ dark 8h, and cultivation temperature is 24 DEG C, differentiation culture
The formula of base and subculture medium is MS+0.5mg/L gibberellin+2.0mg/L6-BA+3.0mg/L zeatin, to the horse of acquisition
Bell potato seedling carries out Ploidy Identification, counts the ratio of Different Ploidy plant.
Interpretation of result
1, the influence of different pretreatments method and anther induced medium to potato anther inducing effect, by embodiment 1 with
Callus induction rate the result statistics such as the following table 1 of comparative example 1.
The callus induction rate of potato 2 and Dongnong303 respectively reaches in embodiment 1 it can be seen from upper table result
17.8% and 20.5%, it is significantly higher than the callus induction rate of comparative example 1.Preprocess method of the invention uses high temperature and fits
When the method that combines of magnetic field processing, especially magnetic field processing can change the physiological status of pollen, be more advantageous to starting arrhenokaryon
Development, to improve callus induction rate；Altheine, lycopene, wood are added in induced medium of the invention
Sugar alcohol, soybean lecithin, praseodymium sulfate, triacontanol 0.18mg/L, hesperetin, 5-bromouracil, and replaced with potato starch
Make coagulator for agar, all has the function of improving callus induction rate.
2, different differential mediums are under conditions of Saline Alkali Stress, and the influence to potato callus differentiation effect will be implemented
The phenylacetic acid result of example 1 and comparative example 2 statistics is such as the following table 2.
In embodiment 1 it can be seen from upper table result, middle potato 2 and Dongnong303 in addition various concentration NaCl and
Na2CO3Have certain callus differentiation rate on the differential medium of mixed solution, and No. 2 callus differentiation rates of middle potato 1.5% ~
When 2.0%, NaCl and Na that differential medium contains2CO3Mixed solution concentration is about 0.15%, and Dongnong303 callus differentiation rate exists
When 1.5% ~ 2.0%, NaCl and Na that differential medium contains2CO3Mixed solution concentration is about 0.4%, takes the average value of the two
Concentration 0.28% is used as salt tolerance pressure；In comparative example 2, middle potato 2 and Dongnong303 in addition various concentration NaCl and
Na2CO3Callus differentiation rate on the differential medium of mixed solution is extremely low or even undifferentiated, can not calculate salt tolerance pressure.
The data of comparing embodiment 1 and comparative example 2, hair there are currently no under conditions of Saline Alkali Stress, in two embodiments in
The callus differentiation rate difference of potato 2 and Dongnong303 is not significant, and under conditions of having Saline Alkali Stress, callus differentiation rate difference pole
Significantly, illustrate differential medium of the invention more suitable for the condition in environment stress, the potato anther of progress breaks up culture.
Differential medium of the invention improves the proportion of each element in minimal medium, and is added to nitroglycerin, glycylglycine, does not have
The objects such as gallate-based, epiphysin, increase production of amines, 2,4- epi-brassinolide, ethylene glycol contracting alditol, oligofructose, 5'- monophosphate cytidine
Matter can enhance the vitality of starting plant itself, improve physiological status, and stabilizing cell membrane enhances via plant immunity, improve
Anti-adversity.
Influence of the different Anther Culture processes to potato Anther-culture ploidy, by the potato of embodiment 1 and comparative example 3
Seedling ploidy identification result statistics such as the following table 3.
In embodiment 1 it can be seen from upper table result, the ratio of double haploid is up to 89.4% in potato seedling,
It is apparently higher than the ratio of double haploid in comparative example 3, is added to NaCl and Na in further Statistics Implementation example 12CO3Mixing
Double haploid ratio in the potato seedling differentiated in the differential medium of solution, both discoveries result have no significance difference
It is different, illustrate that potato Anther Culture process provided by the invention can greatly improve the ratio of double haploid in Anther-culture
Example, reduces the appearance of double haploid Natural double, provides material base for subsequent development Saline alkali tolerance screening operation.
In embodiment 1 obtain ploidy revert to four times flower training 127 plants of regeneration plant, further field planting,
Economical character, Saline alkali tolerance screening, finishing screen select Saline alkali tolerance improved, good 2 potatos of economical character it is new
Strain.
The above description is merely a specific embodiment, and the scope of protection of the present invention is not limited to this, any to be familiar with sheet
Those skilled in the art within the technical scope of the present disclosure, the simple change for the technical solution that can be become apparent to
Change or equivalence replacement is fallen within the protection scope of the present invention.
Claims (9)
1. a kind of potato saline-alkali tolerant improves breeding method, which is characterized in that the breeding method the following steps are included:
A) choose Potato Cultivars and saline-alkali tolerant potato resource to be improved: selection economical character is good but Saline alkali tolerance is insufficient
Potato cultivar is used as Potato Cultivars to be improved；It is evaluated according to Content Germplasm Resources in Potato Saline alkali tolerance, chooses saline-alkali tolerant
Germplasm materials kind of good performance is as saline-alkali tolerant potato resource；The field planting potato to be improved in a manner of potato seed sowing
Kind and saline-alkali tolerant potato resource；
B) Potato Cultivars and saline-alkali tolerant potato resource crossbreeding to be improved: with the Potato Cultivars to be improved of field planting
As female parent, saline-alkali tolerant potato resource harvests F1 generation cenospecies after solid as male parent, Post flowering artificial hybridization；
C) improvement Potato Cultivars are treated and saline-alkali tolerant potato resource carries out anther Fiber differentiation respectively: in potato squaring period
To flowering stage, acquires the bud of Potato Cultivars and saline-alkali tolerant potato resource to be improved and filter out at microspore development period
It in the bud of monokaryon middle and advanced stage, is transferred to after bud is pre-processed in superclean bench and carries out sterile working, first bud surface disappears
Then poison separates anther with scalpel and tweezers, be inoculated into progress anther Fiber differentiation in induced medium after cutting filigree,
Condition of culture is 24 ~ 26 DEG C, dark；
The ingredient of the induced medium are as follows: by without agar MS culture medium based on, then add altheine 4.7 ~
5.5mg/L, 23 ~ 27mg/L of lycopene, 13 ~ 19g/L of xylitol, 4.0 ~ 4.6mg/L of soybean lecithin, 30 ~ 36 μ of praseodymium sulfate
G/L, 0.15 ~ 0.20mg/L of triacontanol, chlorine is than 0.4 ~ 0.6mg/L of urea, 2,4-D 1.0 ~ 1.2mg/L, and potato starch 45 ~
55g/L, 9 ~ 15mg/L of hesperetin, 2.2 ~ 2.6mg/L of 5-bromouracil, pH value are 5.6 ~ 6.0；
D) NaCl and Na of gradient concentration the identification of potato salt tolerance pressure: is added in differentiation culture respectively2CO3Mixing
Solution causes Saline Alkali Stress condition, and Potato Cultivars and the saline-alkali tolerant potato resource to be improved to induce in step c) is cured
It when injured tissue grows to 2mm or so, is transferred on above-mentioned differential medium and carries out differentiation culture, selected according to callus differentiation rate
Suitable screening pressure level concentration is as potato salt tolerance pressure；
The ingredient of the differentiation culture are as follows: KNO32400 ~ 2600mg/L, NH4NO31020 ~ 1100mg/L, KH2PO4 350~
400mg/L, MgSO4·7H2O 340 ~ 380mg/L, CaCl2·2H2O 410 ~ 460mg/L, MnSO4·4H220 ~ 25mg/L of O,
Zinc methionine 8.5 ~ 9.5mg/L, H3BO311 ~ 14mg/L, KI 0.6 ~ 0.8mg/L, CuSO4·5H2O 0.023~0.027mg/
L, CoCl2·6H2O 0.023 ~ 0.027mg/L, Na2MoO4·2H2O 0.20 ~ 0.30mg/L, FeSO4·7H2O 27~28mg/
L, Na236 ~ 37mg/L of-EDTA, 0.13 ~ 0.17mg/L of nitroglycerin, 85 ~ 95mg/L of inositol, 4.4 ~ 5.0mg/L of glycylglycine,
0.5 ~ 0.6mg/L of thiamine hydrochloride, 0.5 ~ 0.6mg/L of puridoxine hydrochloride, 0.3 ~ 0.5mg/L of vitamin K, folic acid 0.5 ~
0.8mg/L, 3.0 ~ 4.0mg/L of gallic acid, 50 ~ 60mg/L of mashed potato, 1.6 ~ 2.0mg/L of epiphysin, increase production of amines 0.8 ~
1.2mg/L, 2,4- epi-brassinolide 0.5 ~ 0.7mg/L, 4- chlorine 0.4 ~ 0.6mg/L of this fluoroacetic acid, ethylene glycol contracting alditol 2.2 ~
2.4mg/L, oligofructose 7.5 ~ 8.1mg/L, 5'- monophosphate 11 ~ 15mg/L of cytidine, 33 ~ 37g/L of maltose, plant gel 5 ~
7g/L, pH value are 5.6 ~ 6.0；
E) F1 generation cenospecies Anther Culture, salt tolerance: will the sowing plantation of F1 generation cenospecies positive season in crop field, to seedling into
When entering squaring period to flowering stage, anther Fiber differentiation is carried out according to method described in step c), the callus induced is turned
It is connected on the differential medium (ingredient is identical as differential medium in step d)) for be added to potato salt tolerance pressure and carries out
Differentiation culture obtains potato and trains seedling；
F) potato trains seedling strengthening seedling and rooting: the potato training seedling by height in 3 ~ 5cm is transferred to strengthening seedling and rooting culture
In base, the vigorous flower training regeneration plant of robust growth, root system can get after cultivating 20-28d；
G) colchicine processing flower training regeneration plant: flower training regeneration plant is taken out from bottle, part root is cut off, stays root long degree
2 ~ 3cm, handles 16h with the mixed solution of 0.05% colchicine and 4% dimethyl sulfoxide at room temperature, and after treatment is by plant
The remaining medical fluid in root is cleaned, and the flower training regeneration plant that ploidy reverts to four times is obtained；
H) further economical character, Saline alkali tolerance screen: above-mentioned tetraploid flower training Transplantation of Regenerated Plantlets is carried out often into greenhouse
Cultivation management is advised, after entering and tying the potato phase, single plant harvests healthy potato seed, potato seed is further seeded into big field, to its salt tolerant
Alkalinity carries out evaluation identification, filter out Saline alkali tolerance improved, the good Potato Clones of economical character.
2. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step a)
Potato Cultivars and the saline-alkali tolerant potato resource to be improved chosen is tetraploid common cultivation kind, and has sexual propagation
Ability.
3. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step c)
The method that screening microspore development period is in the bud of monokaryon middle and advanced stage are as follows: 2 ~ 3 pieces of anther picking in bud are pulverized, are released
Pollen is released, microscopy Observation of Microspore developmental stage after being dyed with aceto-camine filters out the corresponding bud of monokaryon middle and advanced stage.
4. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step c)
The pretreated method of bud are as follows: be put into the mannitol solution of 40g/L after cleaning bud, pre-process 3d at room temperature, then will
Bud, which takes out to be placed in the uniform magnetic field that intensity is 400mT, handles 30min.
5. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step c)
The mode of bud surface sterilization are as follows: first bud is put into 75% ethyl alcohol and impregnates 60s, is rinsed 2 ~ 3 times with aqua sterilisa, then by bud
It is put into 0.1% mercuric chloride solution and impregnates 15min, gently shaken when impregnating, then rinsed 5 ~ 6 times with aqua sterilisa.
6. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step d)
NaCl and Na2CO3In mixed solution, the mass ratio of the two is 9:5.
7. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step d)
Suitable screening pressure level concentration refers to: Potato Cultivars and saline-alkali tolerant potato resource callus differentiation rate to be improved exists
When 1.5% ~ 2.0%, NaCl and Na that differential medium adds respectively2CO3The concentration of mixed solution, and take the average value of the two.
8. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that the step d) and
E) condition of differentiation culture in are as follows: 25 ~ 27 DEG C of temperature, intensity of illumination 2000 ~ 2400Lux, daily 13 ~ 15h of illumination.
9. a kind of potato saline-alkali tolerant according to claim 1 improves breeding method, which is characterized in that in the step f)
The ingredient of strengthening seedling and rooting culture medium are as follows: based on the 1/2MS culture medium without agar, then add root-inducing powder No. 5 0.1 ~
0.2mg/L, 16 ~ 24mg/L of microcystin, 0.1 ~ 0.3g/L of active carbon, pH value are 5.6 ~ 6.0；The condition of culture of rootage are as follows: temperature
23 ~ 28 DEG C of degree, intensity of illumination 2500 ~ 3000Lux, daily 15 ~ 17h of illumination.
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| CN110547191A (en) * | 2019-08-28 | 2019-12-10 | 铜仁万山九丰现代农业科技有限公司 | Seed cultivation method for improving salt tolerance of common head cabbage |
| CN112042543A (en) * | 2020-10-22 | 2020-12-08 | 郭爱英 | Method for obtaining haploid plant through in vitro culture of pear unfertilized ovule |
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| CN114532222A (en) * | 2022-03-03 | 2022-05-27 | 中国热带农业科学院热带作物品种资源研究所 | Efficient induction method for polyploidy of fagoya grandis |
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| CN113080065A (en) * | 2021-05-19 | 2021-07-09 | 南京农业大学 | Method for cultivating non-heading Chinese cabbage microspore plant |
| CN114532222A (en) * | 2022-03-03 | 2022-05-27 | 中国热带农业科学院热带作物品种资源研究所 | Efficient induction method for polyploidy of fagoya grandis |
| CN114532222B (en) * | 2022-03-03 | 2023-01-17 | 中国热带农业科学院热带作物品种资源研究所 | Efficient induction method for polyploidy of fagoya grandis |
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