CN107889742A - The method that haloduric corn kind is cultivated using corn allopolyploid - Google Patents
The method that haloduric corn kind is cultivated using corn allopolyploid Download PDFInfo
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- CN107889742A CN107889742A CN201711189085.2A CN201711189085A CN107889742A CN 107889742 A CN107889742 A CN 107889742A CN 201711189085 A CN201711189085 A CN 201711189085A CN 107889742 A CN107889742 A CN 107889742A
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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
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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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
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Abstract
The invention discloses the method that haloduric corn kind is cultivated using corn allopolyploid, belong to breeding method field.The present invention is with the corn allohexaploid MTF 1 of Sichuan Agricultural University's original creation for bridge material, pass through the method for backcrossing, and backcross progeny is selected under the adverse environmental factor of salt stress, salt-tolerance character from corn Wild related germplasm category in MTF 1 is imported into the excellent corn inbred line of economical character, obtains the corn inbred line of salt tolerant.The inventive method, for bridge material, is overcome the difficulty of distant hybridization between corn and its Related species, has widened the Germplasm Bases of corn salt tolerant breeding with MTF 1；The corn variety good salt tolerance that the inventive method is cultivated, suitable for being planted in varieties in saline-alkali areas, expands corn planting scope；Using the characteristics of vermiculite Water-saving effect is good, gas permeability is strong, true and stable salt environment also is provided for the Salt-Tolerance Identification of the present invention by the present invention；Breeding method of the present invention is simple, efficient, cost is low.
Description
Technical field
The invention belongs to corn breeding method field, and in particular to cultivate haloduric corn kind using corn allopolyploid
Method.
Background technology
Salinization of soil is one of main abiotic stress for limiting crop yield, and the quickening of global salinization of soil process aggravates
The saline and alkaline threat to the mankind.10% or so of the whole world land gross area is salt-soda soil, about 9.54 hundred million hectares, and it is raw to agricultural
Loss is only second to arid caused by production.It is the most economical effective of crop yield on raising salt-soda soil to cultivate salt tolerant variety of crops
Method, but due in existing germ plasm resource salt tolerant base material it is few, and it is quantitative character by controlled by multiple genes that salt tolerance etc. is mostly
Deng the successful case of Salt And Alkali Tolerance breeding at present is less.In long-term evolutionary process, some wild materials in saline and alkaline aerial
With merits such as salt tolerances, but due to reasons such as reproduction isolation, it is difficult to be applied in breeding.
MTF-1 (Tripsazea creammaize are T.2n=76) is that Sichuan Agricultural University is beautiful with the perennial class of tetraploid
Intergeneric hybrid F between rice and the tetraploid corn (corn doubles) introduced and finger-like friction standing grain1Generation (2n=56) hybridization obtains
Ternary hybrid, it be one containing corn (Zea mays L.), tetraperennial teosinte (zea perennis, 2n=
And a full set of genomic allohexaploid (the expensive tetra- of Su Yue of friction standing grain (Tripsacum dactyloides are L.2n=72) 40)
River agriculture university Master's thesis, 2009), its Classification And Nomenclature is that beautiful broomcorn millet standing grain belongs to Yu Qi leaching grass seeds by Sichuan Agricultural University.It is therein
Finger-like friction standing grain (also known as gama grass, Tripsacum dactyloides are L.2n=72), it is and corn affiliation
Nearest category, America is grown in, anti-multiple diseases, survival ability is strong, there is very strong anti-adversity ability, is suitable for acid, saline alkali
And wetland environmental condition (the .Proceedings of the NationalAcademy such as Jack R of
Sciences, 1977,74 (8)：3494-3497).Tetraperennial teosinte (Zeaperennis, 2n=40) is mainly distributed
In height above sea level 1500-2000 meter bands, there is perennial characteristic, can be survived for many years under the unfavorable conditions such as cold wet.Corn-rub
Wipe standing grain filial generation the NaCl resistances coerced and winter resistance are shown heterobeltiosis (Branson.J.Econ.Entomol,
1972,65：195-196), therefore by both remote edge materials and corn the MTF-1 being polymerized, carries good degeneration-resistant base
Cause, can be as the fine germplasm resources of genetic improvement.Conventional friction standing grain or tetraperennial teosinte and corn hybridization, by
In its offspring height infertility, therefore all it is merely resting on F1Generation, and ternary hybrid MTF-1 and corn, tetraploid class corn or rub
Fertile offspring can be produced by wiping standing grain hybridization, therefore using MTF-1 as bridge material, by being returned with corn, what can be carried is near
The excellent adversity gene of the wild kind of edge imports corn, so as to cultivate degeneration-resistant corn variety.
Through retrieval, do not find to cultivate the report of haloduric corn kind using corn allopolyploid.
The content of the invention
Problem is lost to caused by Maize Production for salt damage, present invention aims at provide corn allopolyploid training
Educate the purposes in haloduric corn kind.
Another object of the present invention is to provide the method for cultivating haloduric corn kind using corn allopolyploid.
3rd purpose of the invention is to provide a kind of breeding method of haloduric corn self-mating system.
4th purpose of the invention is to provide a kind of breeding method of haloduric corn cenospecies.
To achieve the above object, the present invention adopts the following technical scheme that:
Application of the corn allopolyploid on haloduric corn kind is cultivated.
Described corn allopolyploid (Tripsazea creammaize T.) refers to corn allohexaploid MTF-1
(Tripsazea creammaize T., 2n=76) or its Derivative line.
Described MTF-1 (Tripsazea creammaize T.；It is 2n=76) that Sichuan Agricultural University passes through distant hybridization
What is be bred as contains corn (Zea mays L.), tetraperennial teosinte (Zea perennis) and friction standing grain
(the expensive sichuan agricultures of Su Yue are big for the corn allohexaploid material of a full set of chromosome of (Tripsacum dactyloides L.)
Master's thesis, 2009).
MTF-1 to be perennial, can vegetative propagation, plant is healthy and strong, well developed root system.MTF-1 can be obtained on a small quantity with corn hybridization
Seed, seed glume is hard, brown or taupe.Plant high-output stress-resistance, remained to just under 0-5 DEG C and soil obigotrophic condition
It is frequently grown and grows new tiller.
MTF-1 is mainly numerous by dividing the asexual reproduction methods such as pocket breeding, stalk cuttage or other vegetative manners to carry out
Grow.
The public can be obtained from Sichuan Agricultural University MTF-1 (Tripsazea creammaize are T.2n=76) (Su Yuegui,
Sichuan Agricultural University's Master's thesis, 2009) biomaterial.
Described Derivative line refers to using MTF-1 as female parent, with corn (Zea mays L.), tetraperennial teosinte
(Zea perennis) or friction standing grain (Tripsacum dactyloides L.) be paternal hybrid, selection include corn, four times
The offspring (2n=76) of the perennial class corn of body and friction three species full set chromosomes of standing grain, the corn allohexaploid of gained
MTF-1 Derivative line.
Present invention also offers the method that haloduric corn kind is cultivated using corn allopolyploid, comprise the following steps：
(1), in the early spring, when local temperature rise is to more than 10 DEG C, by corn allohexaploid (Tripsazea creammaize
T, 2n=76) a point root and stem of certain plants, cuttage or other vegetative manners acquisition seedling are carried out, it is that 2m × 2m carries out individual plant kind by seeding row spacing
Plant；Meanwhile point 10 batches of maize planting self-mating systems (Zea mays L., 2n=20), 28 plants every batch, every batch is spaced 3 days；First
The corn inbred line of plantation should be sowed for two weeks earlier than corn allohexaploid；
(2) before spinning, by corn allohexaploid female fringe bagging, and its filigree is cut short to 2~3cm before pollination,
Then pollinated using corn inbred line as male parent to corn allohexaploid female fringe, each female fringe at least repeats pollination 3 times, awards every time
All marked after powder on bagging, seed is harvested when ripe, obtain F1Generation；
(3) by F1Planted for seed according to spacing in the rows 40cm, line-spacing 80cm kinds；Meanwhile same step (1) equally divides 10 batches of plantations beautiful
Rice self-mating system；With F1On behalf of female parent, it is returned by male parent of corn inbred line, seed is harvested when ripe, obtains BC1F1Generation；
(4) Salt-Tolerance Identification screens：(a) seed disinfection：Choose full seed, intact sunken BC1F1For seed, use
75% alcohol disinfecting 8min, then with distilled water flushing 2 times；(b) by the BC after sterilization1F1For seed by be pressed into using vermiculite as hair
The germination box of bud bed, makes vermiculite not have seed, then pours the NaCl solution that concentration is 250mmol/L, makes to reach vermiculite maximum
Water-holding capacity is 75%, covers germination lid, avoids the too fast evaporation of moisture from causing salinity to change；(c) seed germination box is put
In illumination box, temperature be 25 DEG C, relative humidity 85%, 12h illumination on daytime, light intensity 2000-3000Lx, at night
Cultivated 7 days under the conditions of dark 12h；This body length of seed is exceeded as the standard normally germinateed using bud length, with parental maize self-mating system
As control, the BC normally to germinate and bud length and root system are not significantly affected is selected1F1Plant, first restore it normal condition and treat
It restore normal growth, and then transplants to field, and with selected BC1F1For female parent, it is returned by male parent of corn inbred line, into
Seed is harvested when ripe, obtains BC2F1Generation；
(5) repeat step (4) 2-4 times, obtains BC4F1~BC6F1Generation；In addition to salt resistance ability, remaining character substantially returns to institute
The character for the corn inbred line stated；
(6) material selected by step (5) is selfed, selfed seed is harvested when ripe；According to step (4) methods described pair
Self progeny carries out Salt-Tolerance Identification, the offspring for selecting salt-tolerance character no longer to separate, as haloduric corn self-mating system.
Corn allohexaploid described in above method step (1) refer to MTF-1 (Tripsazeacreammaize T,
2n=76) or its Derivative line.
Described MTF-1 (Tripsazea creammaize T, 2n=76) (Su Yuegui, Sichuan Agricultural University master opinion
Text, 2009) it is one comprising a full set of maize chromosome group (20), finger-like friction standing grain genome (36) and tetraploid is for many years
The intergeneric hybrid of raw class maize chromosome group (20).MTF-1 is Sichuan Agricultural University with the tetraploid corn quoted from the U.S.
(Zea mays L, 4n=40) and finger-like friction standing grain (or tetraploid gama grass, Tripsacum dactyloides
L, 2n=72) between intergeneric hybrid F1For female parent, with tetraperennial teosinte (Zea perennis, 2n=40) for father
The corn allohexaploid of this cross breeding.
Described Derivative line refers to using MTF-1 as female parent, with corn (Zea mays L.), tetraperennial teosinte
(Zea perennis) or friction standing grain (Tripsacum dactyloides L.) be paternal hybrid, selection include corn, four times
The offspring (2n=76) of the perennial class corn of body and friction three species full set chromosomes of standing grain, the corn allohexaploid of gained
MTF-1 Derivative line.
Corn inbred line described in above method step (1) refers to the self-mating system commonly used in production, such as Mo17, B73, Zheng
58th, prosperous 7-2, PH4CV, PH6WC etc..
Seed germination box described in above method step (4), including plastic box body, there are lid, lid on plastic box body
Corner stamp have aperture, one layer of vermiculite is covered with plastic box body bottom, the thickness of vermiculite layers is 4~5cm；Wherein described plastics
Box body is rectangle, and plastic box body is transparent.
Described seed germination box can be bought in the market, i.e., conventionally used expendable transparent plastic lunch box.As described in
The length of plastic box body of seed germination box be 17.2cm, width 11.5cm, be highly 5.6cm, can according to germination needs
Adjust the size of seed germination box.
A diameter of 1~3mm of vermiculite described in above method step (4).
The preparation method of above-mentioned seed germination box：(1) 1~3mm of diameter clean vermiculite, is selected, is sterilized in 80 DEG C of baking ovens
30min, it is standby；(2), by the clean transparent plastic box of in the market purchase in 80 DEG C of sterilizing 30min, the corner stamp aperture of lid；
The box body specification of wherein described germination box can be：Length is 17.2cm, width 11.5cm, is highly 5.6cm；(3), will
(1) sterilized vermiculite is put into (2) in the transparent plastic box after sterilizing in, and the thickness of vermiculite layers is 4~5cm, floating stand-by.
A kind of back cross breeding method of haloduric corn self-mating system, including the salt-tolerant inbred lines cultivated in method described above are non-
Recurrent parent, using the excellent self-mating system of economical character as recurrent parent, it is returned by 4~5 generations, selects to have in backcross progeny
The material of salt tolerance, finally it was selfed for 1 generation, selects the unseparated offspring of salt tolerance, as new salt-tolerant inbred lines.
A kind of cross breeding method of salt-tolerant inbred lines, including the salt-tolerant inbred lines cultivated in method described above be parent it
One, with another hybridization between selfed lines, F must be hybridized1Generation；Then selfing is carried out continuously, selects economical character excellent and there is salt tolerance
Offspring, be selfed for 4~6 generations, that is, be bred as the strong new self-mating system of salt tolerance.
A kind of breeding method of haloduric corn cenospecies, including the haloduric corn self-mating system being bred as in method described above are parent
This, assembles cenospecies.
The present invention has the advantage that and beneficial effect；(1) the inventive method by using corn allohexaploid MTF-1 as
Bridge material, the difficulty of distant hybridization between corn and its Related species is overcome, will be excellent present in corn Related species
Resistant gene of salt is imported into corn, has widened the Germplasm Bases of corn salt tolerant breeding.(2) corn cultivated using the inventive method
The good salt tolerance of self-mating system or the corn variety assembled, suitable for being planted in varieties in saline-alkali areas, corn planting scope is expanded, and in fact
The utilization and improvement to salt-soda soil are showed.(3) caused in the seed germination box in the inventive method with the vermiculite for pouring salting liquid
Salt environment be more nearly true salt environment, it is and loose ventilative, be advantageous to seed and root, it is good for Tolerant salt effect.
(4) vermiculite in the seed germination box of the inventive method has fixation, can fix seed putting position, is easy to move and tests
Investigation statisticses.(5) the vermiculite Water-saving effect in the inventive method is good, it is ensured that salinity is consistent in certain period of time, is salt tolerant
Identification provides stable salt environment.(6) the vermiculite bud bed in the inventive method is slim and graceful compared with sand bed quality, good permeability, cost
Cheap, it is convenient to rinse, and convenient storage is carried, also reusable.(7) breeding method of the present invention is simple, efficient, cost is low.
Brief description of the drawings
Fig. 1 is the proline of Zheng 58 and Improved lines 47 (Pro) content column diagram before and after salt stress；Wherein 1 is that Zheng 58,2 is
Improved lines 47.
Fig. 2 is the peroxidase of Zheng 58 and Improved lines 47 (POD) active column diagram before and after salt stress；Wherein 1 is Zheng 58,
2 be Improved lines 47.
Fig. 3 is the seedling stage photo of Zheng 58 and Improved lines 47 under condition of salt stress.
Fig. 4 is the germination photo of Zheng 58 and Improved lines 47 under condition of salt stress.
Embodiment
The present invention is made further explanation and description with specific embodiment below, but the invention is not restricted to this.
Embodiment 1：The method that haloduric corn self-mating system is cultivated using corn allopolyploid
Carry out in accordance with the following steps：
(1), 2013 spring, in Sichuan Agricultural University proving ground, when ground temperature rises to more than 10 DEG C, by MTF-1
(Tripsazea creammaize are T.2n=76；Su Yuegui, Sichuan Agricultural University's master thesis, 2009.The public can be from
Sichuan Agricultural University obtains MTF-1 biomaterials) a point root and stem of certain plants is carried out, it is that 2m × 2m carries out single-strain planting by seeding row spacing；Meanwhile divide 10
Plantation Zheng 58 is criticized, every batch is spaced 3 days, and the sowing time of first Zheng 58 was than MTF-1 early sowing two weeks.
(2), before spinning, MTF-1 female fringe baggings cut short filigree to 2-3cm after spinning, the loose powder phase is with the flower of Zheng 58
Powder is pollinated to MTF-1 female fringes, is pollinated again once every 2 days after pollinating for the first time, is pollinated 3 times altogether, is done every time after pollination on paper bag
Mark, harvests seed, obtains F when ripe1Generation, threshing are stored in -20 DEG C of refrigerator.
(3), winter in 2013, by all F1Planted for seed according to spacing in the rows 40cm, line-spacing 80cm kinds in Yunnan Province Jinghong City
Xishuangbanna Sichuan Agricultural University breeding experiment base；Meanwhile same step (1) is equally, interval sowing Zheng 58；With F1For it is maternal,
It is that male parent is returned with Zheng 58, seed is harvested when ripe, obtains BC1F1Generation.
(4) making of seed germination box：(I) the clean vermiculite of diameter 1-3mm specifications, is selected, is sterilized in 80 DEG C of baking ovens
30min, it is standby；(II) be, 17.2cm by length, width 11.5cm, be highly 5.6cm clean transparent plastic box (lunch box,
In the market is bought) sterilize 30min at 80 DEG C, the corner stamp aperture of lid；(III) vermiculite sterilized in (I), is put into (II)
In sterilized transparent plastic box, vermiculite layers thickness is 4cm, floating stand-by.
(5) 2014 year spring, to BC1F1Salt-Tolerance Identification screening is carried out for seed, only retains salt-tolerant plants, specific practice
It is as follows：(a) full seed, intact sunken BC are chosen1F1For seed seed, with 75% alcohol disinfecting 8min, distillation is then used
Water rinses 2 times；(b) seed after being sterilized in (a) is pressed into the vermiculite layers of the seed germination box of step (4), to germination box
Middle to add the Nacl solution that concentration is 250mmol/L, it is 75% to make to reach vermiculite maximum water-holding capacity, is closed the lid；(c) by seed
Germination box be placed in illumination box, temperature be 25 DEG C, relative humidity 85%, 12h illumination on daytime, light intensity 2000-
3000Lx, cultivated 7 days under the conditions of dark 12h at night；This body length of seed is exceeded as the standard normally germinateed using bud length, with parent
Inbred Zheng 58 plant for selecting normal germination, bud length and root traits not to be significantly affected, recovers normal cultivation as control
Condition treats that it restore normal growth, and is transplanted to Sichuan Agricultural University Experimental Base Tanaka, and with selected BC1F1For plant
For female parent, it is that male parent is returned with Zheng 58, seed is harvested when ripe, obtains BC2F1Generation.
(6) repeat step (5) 2 times, obtain BC4F1Generation；Now BC4F1In generation in addition to salt resistance ability retains, remaining character base
Originally return to consistent with the trait expression of Zheng 58.
(7) by step (6) obtained by BC4F1For material in Sichuan Agricultural University's Experimental Base plantation, and it is selfed, when ripe
Harvest selfed seed.
(8) self progeny is planted, and Salt-Tolerance Identification, choosing are carried out to gained self progeny according to step (5) methods described
Salt-tolerance character is selected no longer to separate, and other economical characters show consistent self progeny with Zheng 58, and Zheng 58 of as salt tolerant changes
Good system, numbered and named with its field：47.
Embodiment 2：The Improved lines of Zheng 58 that the embodiment of the present invention 1 is bred as carry out Seedling Salt-tolerance qualification test (one) and test material
Material
(1) handle：(a) Zheng 58；(b) Improved lines 47 cultivated in embodiment 1.
(2) compare：(a) Zheng 58；(b) Improved lines 47 cultivated in embodiment 1.
(2) test method
(1) 80 disease-free, 58 seed of Zheng of full seed, 80,47 seed of Improved lines, with step in embodiment 1 are selected
(5) method described in is normally germinateed, that is, pours running water.Treat that seedling was grown to the heart stage of two leaf one, it is each to choose the consistent children of growing way
Transplantation of seedlings is to vermiculite in the flowerpot of matrix, if 1 group is control, another 1 group is processing, and experiment is all provided with 3 repetitions, often repeatedly 10 plants
Seedling.1/2 Gram nutrient solution (conventionally preparation) suddenly is poured after transplanting, 2 days, seedling is spent the laundering period, adapts to
Phase terminates rear control group pouring 1/2, and Gram nutrient solution, treatment group pour the Gram nutrition suddenly of the Nacl containing 250mmol/L suddenly
Liquid, poured once, poured every time to 2 times of vermiculite water-holding capacity every 2 days, water content is handled 15 days 75% or so.Take within 16th day
Material, photograph to record, determine index of correlation.Testing index is as follows：Plant height, blade dry weight and fresh weight of plant seedlings, root dry weight and fresh weight of plant seedlings, leaf water content, phase
To electrical conductivity, chlorophyll content, Pro (proline), POD (peroxidase).All data use IBM SPSS below
The softwares of Statistics 20.0 carry out statistical analysis and use one-way analysis of variance (One Way Anova), insolation level α
=0.05, significance p ＜ 0.05, pole level of signifiance p ＜ 0.01, * represents the level of signifiance, and * * represent the pole level of signifiance.
(2) growth indexes determine：Plant height, aerial part fresh weight, dry weight, under ground portion fresh weight, dry weight
Take out seedling and rinse well, moisture is dried with filter paper, randomly select 5 plant, measurement seedling length (basal part of stem to leaf
The distance of point), aerial part fresh weight, under ground portion fresh weight are weighed immediately after.
Dry weight is the weight after aerial part or under ground portion drying, and specific method is：The root or seedling of fresh weight will be weighed
It is encapsulated in paper bag, in the fixing 30 minutes of 105 DEG C of baking oven, then turns to 80 DEG C of drying until weight no longer changes, taking-up, which is used, to divide
Analysis balance weighs dry weight and obtains aerial part dry weight, under ground portion dry weight.
Leaf fresh weight comparative test result before and after the salt stress of table 1
Root fresh weight comparative test result before and after the salt stress of table 2
Aerial part dry weight comparative test result before and after the salt stress of table 3
Under ground portion dry weight comparative test result before and after the salt stress of table 4
Salt stress can influence photosynthesis of plant, and carbon assimilation amount is reduced, so as to cause growth rate to decline, when serious, and leaf
Area stops increase, and biomass accumulation is reduced, and showing as the dry weight and fresh weight of plant seedlings reduction of above and below ground part, (the molecule plants such as Yang Shaohui educate
Kind, 2006,4 (s1):139-142.).From figure 3, it can be seen that the seedling of Zheng 58 shows blade wilting under salt stress, and improve
Be 47 under salt stress growth of seedling it is normal, do not influenceed by salt stress.In addition, as can be seen from Table 1 and Table 2, the salt side of body
Aerial part fresh weight, the under ground portion fresh weight of Zheng 58 and 47 has different degrees of reduction after compeling, and wherein Zheng 58 substantially reduces, and
The relative reduction of Improved lines 47 is not notable, and the ground of Improved lines 47, under ground portion fresh weight, all apparently higher than Zheng 58, explanation changes
Good is that 47 salt tolerance is obtained apparently higher than Zheng 58.
From table 3 and table 4 as can be seen that the ground of Zheng 58 and 47, under ground portion dry weight have and subtracted in various degree after salt stress
Few, Zheng 58 substantially reduces, and the especially root dry weight of Zheng 58 pole after salt stress substantially reduces, and the reduction of the dry weight of Zheng's Improved lines 47 is not
Significantly, illustrate that Zheng 58 is more sensitive for salt stress, compared with Zheng 58, the salt tolerance of Improved lines 47 significantly improves.
(2) leaf r elative water content determines
Reference《The experiment instruction of science of plant life》Described in method mutually carry out moisture determination.Specific method is：Will
Seedling leaves put distilled water immersion a few hours into, tissue water suction is reached saturation state, until weight no longer changes to obtain saturation
Fresh weight, then relative water content (%)=(fresh weight-dry weight)/(saturation fresh weight-dry weight), the measurement of fresh weight dry weight is the same as in (1) in formula
Described method.
Leaf r elative water content comparative test result before and after the salt stress of table 5
Under salt stress, osmotic stress can be caused to plant, absorption of the root system to moisture is hindered when serious, plant can accelerate
Dehydration, leaf water content reduce, and show wilting symptom (Wang Hong oceans Yangzhou Universitys Master's thesis, 2016.).It can be seen by table 5
Going out, the leaf r elative water content of Zheng 58 and Improved lines 47 reduces under salt stress, and the water content of Zheng 58, which reduces, reaches the level of signifiance,
And the relative water content decrement of Improved lines 47 is not notable, illustrate the salt tolerances of Improved lines 47 apparently higher than Zheng 58.
(4) relative conductivity determines
Unified to choose second leaf measure electrical conductivity, conductance measurement is with reference to Hou Fulin chief editors'《Plant physiology experiment》
Described in method.Specific method is：Sample 0.3g is weighed, with deionized water rinsing 3-4 times, is shredded, loads clean scale examination
Pipe, accurate to add 30ml deionized waters, immersion starts to survey electrical conductivity E1 after 12 hours, and standby one glass of deionized water is before each measurement
Rinse electrode, after boiling water bath 30min, room temperature is cooled to, then survey electrical conductivity E2.Relative conductivity=E1/E2.
Relative conductivity comparative test result before and after the salt stress of table 6
| Test material | Relative conductivity | The significance of difference |
| Zheng 58 | 0.69 | ** |
| 47 | 0.52 |
When plant is forced, different degrees of increase can occur for plasmalemma permeability, and paraplasm has different journeys
The extravasation of degree, so that dielectric conductance rate has different degrees of increasing, electrical conductivity is bigger, shows in Plant Under The Stress membranous system
Undermined degree is more serious.Because different plants have electrolyte abundance difference, initial conductivity value often covers electrical conductivity change
Change value, it is (old to build white tropical agricultures science and technology, 1999 (1) so as to cover the degree that is hurt of species:26-28.), thus frequently with
Relative conductivity (processing electrical conductivity/boil processing electrical conductivity) is used as indices.As can be seen from Table 6, under stress conditions,
The relative conductivity of Improved lines 47 illustrates Improved lines 47 under stress conditions, plasmalemma damage situations are obvious significantly less than Zheng 58
Less than Zheng 58, the salt tolerance for further relating to Improved lines 47 is significantly higher than Zheng 58.
(4) measuring chlorophyll content
Chlorophyll content is determined using SPAD-502 types Chlorophyll meter, is surveyed in the case of fine at noon
Amount, measures second leaf of number from top to bottom, and every leaf surveys three blade tip, Ye Zhong, leaf base positions.
The salt stress chlorophyll content comparative test result of table 7
Under salt stress, often plant moisture is caused to wane, and moisture serves not only as the reactant of metabolic process, Er Qieshi
The medium of metabolic response, waning for moisture can cause the decline of chlorophyll content, while salt stress can cause Ion toxicity, influence
Chlorophyll synthesis (the Agriculture of Anhui science such as horse east, 2012,40 (34)：16518-16520).Therefore, chlorophyll content can be made
For plant economic stress and the index of anti-stress ability.As can be seen from Table 7, Zheng 58 and Improved lines 47 under condition of salt stress
Change in various degree occurs for the two material chlorophyll contents, and the wherein chlorophyll content of Zheng 58 is remarkably decreased, and the leaf of Improved lines 47
Chlorophyll contents do not decline not only, however rise.Illustrate that injury of the salt stress to Zheng 58 becomes apparent from, the salt tolerant of Improved lines 47
Property is significantly higher than Zheng 58.
(5) physiological index determining：Proline (Pro), peroxidase (POD)
Proline, peroxidase determination are using Suzhou section inscription kit, and concrete operation step is with reference to kit explanation
Book is carried out.
Pro is widely present in animal, plant, microorganism, and under adverse environmental factor, Pro contents dramatically increase in plant.
Pro incrementss reflect resistance to a certain extent, and the plant of strong stress resistance often accumulates more dried meat under environment stress
Propylhomoserin.Under environment stress, the accumulation of proline can increase the stability of enzyme, the activity of protective enzyme, prevent that film is damaged, protection
Polysome normal function (entirely first celebrating etc. biotechnology communications, 2007,18 (1):159-162.).Therefore proline increase
Measure one of physical signs that can be as breeding for stress tolerance.As seen from Figure 1, the proline content after salt stress of Improved lines 47 is bright
Aobvious relatively control increase, and Zheng 58 then increases unobvious, illustrates that the salt tolerance of Improved lines 47 has more apparent raising.
POD can be catalyzed peroxide, hydroxide, phenols and aminated compounds, have and eliminate hydrogen peroxide and phenols, amine
The double action of class toxicity.Many vital movement processes of plant are participated in, are plant Enzyme-recovery-systems under adverse environmental factor
One of key enzyme, the free radical of surplus can be removed, interior free yl is maintained 1 normal dynamic level, planted with improving
Resistance (Liang Yanrongdeng Agricultural University of the Inner Mongol journal (natural science edition), 2003,24 (2) of thing:110-113.), it is lived
Property can represent plant stress-resistance ability to a certain extent.As seen from Figure 2, the POD activity after salt stress of Improved lines 47 is aobvious
Write and improve, and Zheng 58 does not have significant change then, illustrates Improved lines 47 compared with Zheng 58, salt tolerance significantly improves.
Embodiment 3：Sprouting stage salt tolerance contrast test (one) the experiment material for the corn inbred line 47 that the embodiment of the present invention 1 is bred as
Material
(1) handle：The Improved lines of Zheng 58 cultivated in embodiment 1：47
(2) compare：Zheng 58.
(2) test method
(1) seed disinfection：Full seed, 90, the seed of intact sunken Zheng 58 and 47 are chosen respectively, with 75% wine
Essence sterilization 8min, then with distilled water flushing 2 times；
(2) seed after sterilization is made vermiculite not have seed, is poured concentration by the germination box being pressed into using vermiculite as germinating bed
For 250mmol/L NaCl solution, it is about 75% to make to reach vermiculite maximum water-holding capacity, covers germination lid, avoids moisture mistake
Fast evaporation causes salinity to change, and Zheng 58 and 47 is all provided with 3 repetitions, repeats to include 30 seeds every time；
(3) seed germination box is placed in illumination box, temperature be 25 DEG C, relative humidity 85%, 12h light on daytime
According to, light intensity 2000-3000Lx, cultivate 7 days under the conditions of dark 12h at night, this body length of seed is exceeded for the mark that germinates using bud length
Standard, start within the 3rd day record germination percentage daily, calculate germination index.Data statistics uses IBM SPSS Statistics
20.0 softwares carry out statistical analysis and use one-way analysis of variance (One Way Anova), insolation level α=0.05, significantly
Property horizontal p ＜ 0.05, pole level of signifiance p ＜ 0.01, * represent the level of signifiance, and * * represent the pole level of signifiance.
The germination test result of Zheng 58 and 47 under the salt stress of table 8
| Germinating energy | Germination percentage | Germination index | The significance of difference | |
| Zheng 58 | 0% | 82% | 3.52 | |
| 47 | 71% | 92% | 11.06 | ** |
From Fig. 4 and table 8, salt stress has had a strong impact on the normal germination of the seed of Zheng 58, and 47 have no and significantly affect, its
Germinating energy, germination index are and Zheng 58 reaches the pole level of signifiance；Illustrate that 47 salt tolerance is significantly higher than Zheng 58.
Claims (10)
1. application of the corn allopolyploid on haloduric corn kind is cultivated.
2. application according to claim 1, it is characterised in that described corn allopolyploid (Tripsazea
Creammaize T.) refer to corn allohexaploid MTF-1 (Tripsazea creammaize T., 2n=76) or its derivative
System.
3. the method for haloduric corn kind is cultivated using corn allopolyploid, it is characterised in that comprise the following steps：
(1), in the early spring, when local temperature rise is to more than 10 DEG C, by corn allohexaploid (Tripsazea creammaize T,
2n=76) carry out a point root and stem of certain plants, cuttage or other vegetative manners and obtain seedling, be that 2m × 2m carries out single-strain planting by seeding row spacing；
Meanwhile point 10 batches of maize planting self-mating systems (Zea mays L., 2n=20), 28 plants every batch, every batch is spaced 3 days；First plantation
Corn inbred line should be sowed earlier than corn allohexaploid two weeks；
(2) before spinning, by corn allohexaploid female fringe bagging, and its filigree is cut short to 2~3cm before pollination, then
Pollinated using corn inbred line as male parent to corn allohexaploid female fringe, each female fringe at least repeats pollination 3 times, every time after pollination
All marked on bagging, seed is harvested when ripe, obtain F1Generation；
(3) by F1Planted for seed according to spacing in the rows 40cm, line-spacing 80cm kinds；Meanwhile same step (1) equally divides 10 batches of maize plantings certainly
Hand over system；With F1On behalf of female parent, it is returned by male parent of corn inbred line, seed is harvested when ripe, obtains BC1F1Generation；
(4) Salt-Tolerance Identification screens：(a) seed disinfection：Choose full seed, intact sunken BC1F1For seed, with 75%
Alcohol disinfecting 8min, then with distilled water flushing 2 times；(b) by the BC after sterilization1F1For seed by being pressed into using vermiculite as germinating bed
Germination box, vermiculite was not had seed, then pour concentration be 250mmol/L NaCl solution, make to reach vermiculite maximum water holding
Measure as 75%, cover germination lid, avoid the too fast evaporation of moisture from causing salinity to change；(c) seed germination box is placed in light
According in incubator, temperature be 25 DEG C, relative humidity 85%, 12h illumination on daytime, light intensity 2000-3000Lx is dark at night
Cultivated 7 days under the conditions of 12h；Using bud length exceed this body length of seed as the standard normally germinateed, using parental maize self-mating system as
Control, select the BC normally to germinate and bud length and root system are not significantly affected1F1Plant, first restore it normal condition and treat that its is extensive
Multiple normal growth, is then transplanted to field, and with selected BC1F1For female parent, it is returned by male parent of corn inbred line, when ripe
Seed is harvested, obtains BC2F1Generation；
(5) repeat step (4) 2-4 times, obtains BC4F1~BC6F1Generation；In addition to salt resistance ability, remaining character substantially returns to described
The character of corn inbred line；
(6) material selected by step (5) is selfed, selfed seed is harvested when ripe；According to step (4) methods described to selfing
Offspring carries out Salt-Tolerance Identification, the offspring for selecting salt-tolerance character no longer to separate, as haloduric corn self-mating system.
4. according to the method for claim 3, it is characterised in that the corn allohexaploid described in its step (1) refers to
MTF-1 (Tripsazea creammaize T, 2n=76) or its Derivative line.
5. according to the method for claim 3, it is characterised in that it is upper normal that the corn inbred line described in its step (1) refers to production
Self-mating system.
6. according to the method for claim 3, it is characterised in that the seed germination box described in its step (4), including plastics
Box body, there is lid on plastic box body, the corner stamp of lid has aperture, one layer of vermiculite is covered with plastic box body bottom, vermiculite layers
Thickness is 4~5cm；Wherein described plastic box body is rectangle, and plastic box body is transparent.
7. according to the method for claim 3, it is characterised in that a diameter of 1~3mm of the vermiculite described in its step (4).
A kind of 8. back cross breeding method of haloduric corn self-mating system, it is characterised in that to be cultivated in the method described in claim 3
Salt-tolerant inbred lines be nonrecurrent parent, using the excellent self-mating system of economical character as recurrent parent, by 4-5 generation be returned, return
Material of the selection with salt tolerance in offspring is handed over, was finally selfed for 1 generation, selects the unseparated offspring of salt tolerance, as new salt tolerant
Self-mating system.
9. a kind of cross breeding method of salt-tolerant inbred lines, it is characterised in that with including being cultivated in the method described in claim 3
Salt-tolerant inbred lines one of for parent, the excellent hybridization between selfed lines with another economical character, F must be hybridized1Generation；Then to hybridization after
In generation, is carried out continuously selfing, and selects economical character excellent and have the offspring of salt tolerance, was selfed for 4~6 generations, that is, is bred as salt tolerance
Strong corn inbred line.
10. a kind of breeding method of haloduric corn cenospecies, including the haloduric corn to be bred as in the method described in claim 3
Self-mating system is parent, assembles cenospecies.
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