CN106416773A - Screening and identification method of salt tolerance of corns at seedling stage - Google Patents
Screening and identification method of salt tolerance of corns at seedling stage Download PDFInfo
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- CN106416773A CN106416773A CN201610880759.2A CN201610880759A CN106416773A CN 106416773 A CN106416773 A CN 106416773A CN 201610880759 A CN201610880759 A CN 201610880759A CN 106416773 A CN106416773 A CN 106416773A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a screening and identification method of salt tolerance of corns at a seedling stage. According to the screening and identification method disclosed by the invention, the situation that the salt tolerance of the corns is consistent with phosphorus sensibility and zinc sensibility of the corns is discovered and verified; whether a corresponding corn variety is salt-tolerant can be indirectly judged by judging whether the corns at the seedling stage have a phosphorus deficiency symptom under phosphorus deficiency stress treatment; then, zinc stress treatment is carried out, corn seedlings growing from a good state to a bad state before and after the zinc stress treatment are not a salt-tolerant corn variety, and corn seedlings growing from a bad state to a good state before and after the zinc stress treatment are a salt-tolerant corn variety. According to the screening and identification method disclosed by the invention, the successive two steps are combined, thereby improving the accuracy of screening and identification; the method is practicable and effective, is simple and convenient to operate and does not need chemical medicines for detection, therefore, the cost is low, and the method has no toxicity to human bodies and no pollution to the environment and lays the certain theoretical foundation for further screening corn salt-tolerant inbred lines in a large volume and cultivating salt-tolerant crops.
Description
Technical field
The present invention relates to proportion of crop planting technical field, especially relates to a kind of Screening and Identification side of Maize Seedling salt tolerance
Method.
Background technology
The salinization of soil of soil is to cause one of principal element of crop production reduction.According to statistics, the whole world there are about 20% agriculture
Its salinization and alkalization of industry land used is constantly increasing.China has 2.6 × 107hm2Salt-soda soil, wherein have 6.6 × 106hm2It is arable land
Area, is concentrated mainly on the major grain producing areas such as North China, northeast, the regional and North of Yangtze River of Northwest inland.In recent years, due to environment shadow
The impact with unfavorable factors such as mankind's activities is rung, more and more arable lands are faced with the danger that Secondary Saline occurs.Expect
The year two thousand fifty, the whole world will have more than 50% tilth salinization.Therefore, salt-soda soil comprehensive utilizating research becomes scientific research with preventing and treating
Focus, the variety of crops of wherein selection-breeding salt resistance or salt tolerant is most economical effective approach.
Salt stress is an important environment limiting factor for affecting plant growing and crop yield.Salt stress easily cause from
Son stress and osmotic stress, ultimately result in plant leaf area extension is obstructed, photosynthesis and biomass accumulation reduce etc..Infiltration
Stress is the mode that salt toxic action is produced at first.In soil, salinity excessively makes the soil liquid flow of water (Water potential)
Decline, plant water suction can be caused difficult, or even internal water has the danger of extravasation, so as to cause plant physiology arid.
Osmotic stress can cause development of plants slow, suppress growth and the differentiation of plant tissue and organ, make sending out for plant
Educate process and shift to an earlier date aging acceleration, increment reduces, and ultimately results in plant dead because hungry.
Plant is transferred in salt adverse circumstance after a few minutes, and growth rate has declined, and which is declined degree and is permeated with rhizosphere
Pressure (Rhizosphere osmotic pressure) is proportional.Initially salt stress causes plant leaf area spreading rate (The
Expansion rate of leaf area) reduce, with the increase of salinity, leaf area stops increasing, leaf, stem and root
Fresh weight and dry weight reduce.Salinity mainly causes plant carbon assimilation quantity (Carbon by reducing the photosynthetic area of individual plantses
Assimilation minimizing).
In salt-affected soil, certain ion concentration is too high and often absorption of the excluder to other ions, is formed unbalanced
The soil liquid, causes special Ion toxicity to act on.Under salt stress, high concentration Na+The Ca that replaceable cell membrane is combined2+, make
Film combination Na+Increase, the component of membranous system, permeability, transport, ion-flow rate etc. can all be affected and change, and cause matter
The change of membrane permeability, Na+Cell is entered in a large number, intracellular Na+Increase, and K+Extravasation, makes Na+/K+Value increases, suppression tonoplast Jiao
Phosphatase (H+- Ppase) activity and kytoplasm in H+Across vesicular membrane transport, decline across the pH gradient of vesicular membrane transport, vacuole alkali
Change, be unfavorable for Na+Accumulate in vacuole.So as to break original ionic equilibrium, work as Na+/K+When ratio increases to threshold value, plant is i.e.
Aggrieved.
In addition, under the adverse environmental factors such as salt stress, active oxygen (Reactive Oxygen Species, ROS) produce with clear
Dynamic equilibrium between removing is destroyed, so as to cause a large amount of accumulation of active oxygen.The excessive active oxygen of plant interior accumulation, if not
Can remove in time and oxidative stress is will result in, some reactive oxygen species be formed, such as：Singlet oxygen (1O2), hydrogen peroxide (H2O2)、
Hydroxy radical (OH), superoxide anion (O2·-) etc..Active oxygen has very strong oxidability, to unsaturated lipid, egg
The biomolecule such as white matter, nucleic acid have destruction, can cause enzyme inactivation, pigment decolouring, protein degradation and lipid peroxidation
(Membrane lipid peroxidation, LPO) etc. react, seriously can blocking dna replicate and transcribe, cause DNA damage and
Mutation, causes cell death.Active oxygen can also make Lipid peroxidation metabolism (LPO), produce malonaldehyde (Malondialdehyde,
MDA), MDA is one of primary product of peroxidation of membrane lipids, with very strong cytotoxicity, to many in film and cell
Biologically functional molecule such as protein, nucleic acid and enzyme all have very strong destruction.
Plant defines many tolerance regulation mechanisms, such as Na when salt stress environment is adapted to+Outer row, Na+Separating is entered
Vacuole, the accumulation of solable matter and the removing of active oxygen (Reactive Oxygen Species, ROS) etc..Coerce in extraneous salt
Under the conditions of compeling, in soil, salinity can excessively reduce soil water potential, due to the reduction of the flow of water, have impact on plant to moisture and nutrient
Absorb, cause physiological drought and nutrient to wane.This effect is the most important short-term effect of salt damage.For avoiding this injury, carefully
Intracellular can accumulate some solable matters to reduce intracellular penetration gesture (Osmotic potential).On the one hand, plant cell
Inorganic ionss, the such as Na in halophytess is absorbed from extraneous+、K+And Cl-；On the other hand, plant is caused not by osmotic stress
Balance, generally accumulates osmotic protection material (Osmoprotectant) to reduce the osmotic potential of cell, in the cell due to them
Therefore they be not referred to as " compatible solute (Compatibility by normal biochemical reaction in interference cell
solute)”.These compatible solutes mainly include proline (Proline, Pro), glycine betaine (Betaine), polyamines
(Polyamine, PA), glyceryl alcohol (Glycerol), sorbitol (Sorbitol) and inositol (Inositol) and some polysaccharide
Molecule.Inorganic and Organic Osmotic Adjustment, the two carries out jointly Osmoregulation and reduces the cell flow of water, makes the transdermal delivery of moisture
Flow towards the direction for being conducive to cell growth, to ensure the normal physiological metabolism demand of plant.
Additionally, under salt stress, the heteroauxing (Indoleacetic acid, IAA) in plant body, the basic element of cell division
All there is different degrees of change, abscisic acid in the hormones such as (Cytokinin, CTK), gibberellins (Gibberellic acid, GA)
(Abscisic acid, ABA) is a kind of hormone by environmental influence maximum.A lot of research finds, under adverse environmental factor very
ABA level in many plants substantially rises.
Semen Maydiss (Zea mays L.) are both important grain and forage crop, are the important raw material of industry again.Current China
It is the second big country of Maize Production, on market, the contradiction of supply and demand of corn is increasingly sharpened, the Comprehensive Productivity for improving Semen Maydiss has become as
One urgent task.Semen Maydiss are the crops to saline and alkaline medium sensitivity, and salt tolerance is relatively poor, can only exist in NaCl content
Grow in 0.3% soil below.
Maize at Seedling Stage is the critical period of whole growth cycle, stress ratio of the period to various external world's adverse environmental factors
More sensitive, wherein salt stress makes growth of maize be obstructed, and bud gesture is weak, and radicle is few and short, and Seedling is weak, and survival rate is low, has a strong impact on
Its growth promoter and yield.
Therefore, Semen Maydiss show the height of its salt tolerance in seedling stage to the tolerance degree of salt stress, and the salt tolerance of Semen Maydiss is to which
Smoothly can spend growth sensitive period so that whole trophophase all it is critical that, the screening to Maize Seedling salt tolerance will be
Next step haloduric corn selection-breeding lays the foundation.
To sum up research is reported, on the one hand, salt stress causes development of plants slow, and plant leaf area stops increasing, photosynthetic speed
Rate declines, and leaf, the fresh weight of stem and root and dry weight reduce, and accumulates substantial amounts of active oxygen, produces malonaldehyde (MDA), destroys membranous system,
The developmental process of plant is made to shift to an earlier date aging acceleration, so that dead.
Therefore, salt stress early stage can be by determining chlorophyll content, mda content and active oxygen (H2O2) content to be identifying
Plant is by the degree of salt stress damage, and the salt stress later stage can be strong to identify salt tolerance by the withered degree of the blade of observation of plant
Weak.
On the other hand, plant itself can be by accumulating organic osmotic protection material (as soluble sugar, soluble protein and dried meat
Propylhomoserin etc.) adjusting or buffer the osmotic stress of high salt inducement, these solable matters can reduce the cell flow of water, make moisture across
Film transport is flowed towards the direction for being conducive to cell growth.Accordingly, in plant body, the height of these Soluble matter contents can table
The salt tolerance power of its own is levied, so it is resistance to also which can be identified by measure plant by the Soluble matter content after salt stress
Salt.
The each content of material use cost height of said determination, the big and complex operation step of consumption, and have certain danger (institute
If needing misoperation in heating process, liquid easily sprays, skin ambustion).Malonaldehyde, hydrogen peroxide, soluble sugar, proline etc.
Some the required chemical substances that determine of content include thiobarbituricacidα- (TBA), 1,2,3-indantrione monohydrate (Ninhydrin), toluene, anthracene
The high cost of ketone (Anthrone) etc., and have severe toxicity, big to human injury, it is not easily decomposed, pollutes environment.
Therefore, how to provide a kind of low cost, simple and to human body and environmentally friendly, be easy to be grasped on a large scale
The process for screening and identifying of the Maize Seedling salt tolerance of work is the technical problem of those skilled in the art's urgent need to resolve.
Content of the invention
It is an object of the invention to provide a kind of process for screening and identifying of Maize Seedling salt tolerance, the process for screening and identifying becomes
This low, simple and to human body and environmentally friendly, be easy to carry out large-scale operation.
For solving above-mentioned technical problem, the technical scheme that the present invention is provided is：
A kind of process for screening and identifying of Maize Seedling salt tolerance, first by the sprouting of the corn variety of multiple identifications to be screened
Well consistent seedling carries out normal Solution culture method, after waiting corn seedling to grow to one heart stage of SANYE, with nutritional solution to whole
The seedling of the corn variety of identification to be screened all carries out the Low phosphorus stress of 0mM Pi and processes, and after processing certain time, observes seedling
Whether growing way feature, have phosphorus deficiency symptom to judge its whether salt tolerant according to corn seedling, and the corn seedling with phosphorus deficiency symptom is
Salt tolerance corn variety, the corn seedling for not having phosphorus deficiency symptom is non-salt tolerance corn variety；
The height of the salt tolerance of corresponding corn variety, Semen Maydiss are judged according to the order of severity of corn seedling phosphorus deficiency symptom
The more serious salt tolerance for characterizing corresponding corn variety of the phosphorus deficiency symptom of seedling is higher.
Preferably, in Low phosphorus stress processing procedure, nutrient solution used middle each element concentration is：
Preferably, after Low phosphorus stress is processed, the seedling of the corn variety after being processed to whole Low phosphorus stress with nutritional solution is equal
Certain density Zn is carried out again2+Zinc components process, after processing certain time, come according to the change of the growing way of corn seedling further
Judge its whether salt tolerant, after zinc components before processing growing way by the bad corn seedling that takes a turn for the better for non-salt tolerance corn variety, zinc components
After before processing, growing way is salt tolerance corn variety by the corn seedling for badly taking a turn for the better；
In zinc components processing procedure, nutrient solution used in Zn2+Concentration be 20 μM～50 μM, nutrient solution used in Pi dense
Spend for 1mM.
Preferably, in zinc components processing procedure, nutrient solution used middle each element concentration is：
Compared with prior art, the invention provides a kind of process for screening and identifying of Maize Seedling salt tolerance, the present invention sends out
Now with salt tolerance and its phosphorus sensitivity and the relation of zinc sensitivity for demonstrating Maize Seedling；The results showed corn inbred line
Salt patience and its phosphorus sensitivity present consistent, salt tolerance corn inbred line is more sensitive to phosphorus deficiency, i.e. salt tolerance Semen Maydiss
Therefore selfing line, can pass through to judge sensitivity of the corn inbred line to phosphorus, i.e., with phosphorus deficiency symptom under Low phosphorus stress process
Whether there is phosphorus deficiency symptom under Low phosphorus stress is processed, if typical case's " purplish red leaf " performance with phosphorus deficiency symptom, to sentence indirectly
Break corresponding corn variety whether salt tolerant, and the corn seedling with phosphorus deficiency symptom is salt tolerance corn variety, do not have phosphorus deficiency symptom
Corn seedling is non-salt tolerance corn variety；Further, the children of the corn variety after the present invention is processed to whole Low phosphorus stress
Miao Jun carries out certain density Zn again2+Zinc components process, Pi comprising normal proportioning content in nutritional solution and more normally join
The Zn higher than content2+, the corn seedling after Low phosphorus stress is processed being repaired with the Pi of normal proportioning content, is contained with more normal proportioning
The higher Zn of amount2+Zinc components process is carried out to corn seedling, after processing certain time, is entered according to the change of the growing way of corn seedling
One step judges its whether salt tolerant, after zinc components before processing growing way by the bad corn seedling that takes a turn for the better for non-salt tolerance corn variety, zinc
Before and after Stress treatment, growing way is salt tolerance corn variety by the corn seedling for badly taking a turn for the better；Before and after the present invention, two steps are combined, and are improve
The accuracy of Screening and Identification, and the method is effective, easy to operation, detected without using chemical drugss, thus
Low cost, and harmless to the human body, environmentally safe, are to carry out high-volume screening Semen Maydiss salt-tolerant inbred lines, cultivation further
Salt tolerant crop has established certain theoretical basiss.
Description of the drawings
Fig. 1 is growth result of the corn seedling of QXN233 and QXH0121 in the present invention under low-phosphorus stress process；
Fig. 2 is the corn seedling (flowerpot training mode) of QXN233 and QXH0121 in the present invention in salt stress and the without phosphorus side of body
Growth result under compeling to process；
Fig. 3 is the corn seedling (liquid culture pattern) of QXN233 and QXH0121 in the present invention in salt stress and the without phosphorus side of body
Growth result under compeling to process；
Fig. 4 be QXN233 in the present invention corn seedling P deficiency process and under salt+P deficiency is processed under growth
As a result；
Fig. 5 be QXN233 and QXH0121 in the present invention corn seedling under salt+P deficiency is processed under growth result；
Fig. 6 is the overground part (stem) of QXN233 and QXH0121 and underground part (root) weight in the present invention；
In Fig. 7, A is that the corn seedling of QXN233 and QXH0121 in the present invention is processed and salt+P deficiency process in salt stress
Under chlorophyll content；
B is that the corn seedling of QXN233 and QXH0121 in the present invention processes the film under processing with salt+P deficiency in salt stress
Matter peroxide injury degree；
C is that the corn seedling of QXN233 and QXH0121 in the present invention processes the dried meat under processing with salt+P deficiency in salt stress
Histidine content；
D be QXN233 and QXH0121 in the present invention corn seedling salt stress process under processing with salt+P deficiency can
Dissolubility protein content；
E be QXN233 and QXH0121 in the present invention corn seedling salt stress process under processing with salt+P deficiency can
Dissolubility sugared content；
F is that the corn seedling of QXN233 and QXH0121 in the present invention processes surpassing under processing with salt+P deficiency in salt stress
Superoxide dismutase activity SOD；
Fig. 8 is life of the corn seedling of QXN233 and QXH0121 in the present invention under normal Hongland Solution culture method
Long result；
Fig. 9 is growth of the corn seedling of QXN233 and QXH0121 in the present invention under salt stress and high zinc components are processed
As a result；
Figure 10 is growth result of the corn seedling of QXN233 and QXH0121 in the present invention under zinc components process；
Figure 11 is growth result of the corn seedling of QXN233 and QXH0121 in the present invention under salt+zinc components are processed；
Figure 12 is grown junction of the corn seedling of 8 corn varieties in the embodiment of the present invention 1 under Low phosphorus stress process
Really；
Figure 13 is anthocyanidin of the corn seedling of 8 corn varieties in the embodiment of the present invention 1 under Low phosphorus stress process
Content；
Figure 14 is growth result of the corn seedling of 8 corn varieties in the embodiment of the present invention 1 under salt stress process；
Figure 15 is malonaldehyde of the corn seedling of 8 corn varieties in the embodiment of the present invention 1 under salt stress process
(MDA) content.
Specific embodiment
Purpose, technical scheme and advantage for making the embodiment of the present invention is clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
A part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill people
The every other embodiment obtained on the premise of creative work is not made by member, belongs to the scope of protection of the invention.
With reference to Fig. 1～Figure 11, the 1. relation of the salt patience of corn inbred line and its phosphorus sensitivity：
1.1 preliminary identification：
In high 18cm, the flowerpot of diameter 23cm, suitable common garden is filled with Vermiculitum (height 14cm), irrigate first
Hongland nutritional solution (nutrient solution prescription is shown in Table 1), will sprout the non-salt tolerance selfing line QXN233 of well consistent Semen Maydiss and resistance to
Selfing line QXH0121 seedling was carefully transplanted in basin salt, poured 1 fresh normal Hongland nutrition later every 3-5 days
Liquid (nutrient solution prescription is as noted above, and 400ml/ is per basin).
Treat that corn seedling grows to one heart stage of SANYE (about 2 weeks), low-phosphorus stress process is carried out, specially：In low-phosphorous treatment group
Each processes flowerpot and pours containing 0.001mM H2PO4 –The Hongland nutritional solution of (i.e. Pi)；And each flowerpot is poured in matched group
Fill the Hongland nutritional solution containing normal 1mM Pi and slightly lower 0.5mM Pi.After so processing 3 weeks, it is evident that：
(1) matched group of the two will be significantly better than the growth (shown in Fig. 1) of the corn seedling in its treatment group；
(2) and it has been observed that in treatment group, salt tolerance selfing line QXH0121 seedling leaves assume aubergine, and constantly by
Old leaf spreads to young leaves and increases, and is typical Semen Maydiss phosphorus deficiency symptom (Figure 1A)；And not salt tolerance selfing line QXN233 seedling leaves
Normal green is shown, only stem assumes certain aubergine (Figure 1B).
Next, we have carried out salt stress and without phosphorus Stress treatment respectively to corn inbred line QXN233 and QXH0121.
Treat that corn seedling grows to one heart stage of 3 leaf (about 2 weeks), carry out 200mM NaCl and 0mM Pi process respectively.
Concrete scheme is：
In salt treatment group, each processes flowerpot and pours the Hongland nutritional solution containing 200mM NaCl；
In without phosphorus treatment group, each processes flowerpot and pours the Hongland nutritional solution without Pi (i.e. 0mM Pi)；
In matched group, each flowerpot pours normal Hongland nutritional solution.
After so processing 30d, it is evident that：
(1) matched group of the two all grows normal good；
(2), under 200mM NaCl is processed, the two relatively compares growth retardation, plant general performance for short and small, not salt tolerance from
Friendship is that QXN233 seedling stem and leaf is very thin, and blade assumes the withered shape of curling, root system elongated (Fig. 2A)；And salt tolerance selfing line
QXH0121 vane extension, roomy, stem is thicker, root system sturdy, more flourishing (Fig. 2 C), i.e. corn seedling growing way QXN233<
QXH0121 (Fig. 2A and 2C), confirms the salt tolerance of the two；
(3) under without phosphorus process, the corn seedling of QXN233 grows shorter and smaller compared with matched group, but root system slightly increases
(Fig. 2 B)；And QXH0121 is also shorter and smaller than compareing the growth of seedling in strain, and stem presents obvious aubergine, that is, show
Go out typical Semen Maydiss phosphorus deficiency symptom (Fig. 2 D), by comparison, blade and stem still show normal green to QXN233, not purpling
Red (Fig. 2 B), growth situation is substantially better than QXH0121, i.e., by stress order of severity QXH0121>>QXN233 (Fig. 2 B and 2D).
Above-mentioned experimental verification salt tolerance selfing line QXH0121 is more sensitive to phosphorus deficiency, rather than salt tolerance selfing line
QXN233 is insensitive to phosphorus deficiency, a certain degree of Low phosphorus stress of tolerance that can be stronger.This experiment tentatively shows corn inbred line
Salt patience and its phosphorus deficiency sensitivity present consistent, therefore, can be by judging sensitivity of the corn inbred line to phosphorus deficiency, i.e.,
The whether phosphorus deficiency symptom for showing with " purplish red leaf ", to judge indirectly the tolerance degree to salt.
Equally, we are repeated above-mentioned experiment by the way of liquid culture again：
The non-salt tolerance selfing line QXN233 of well consistent Semen Maydiss will be sprouted and salt tolerance selfing line QXH0121 seedling is careful
Be transplanted in conical flask, changed every 1 day later 1 fresh normal Hongland nutritional solution (nutrient solution prescription is as noted above,
Per bottle of 350ml/).
Treat that corn seedling grows to one heart stage of SANYE (about 2 weeks), corn inbred line QXN233 and QXH0121 is carried out respectively
Salt stress and without phosphorus Stress treatment, concrete scheme is：
Hongland nutritional solution containing 25mM NaCl in each process conical flask of salt treatment group；
In without phosphorus treatment group, each processes the Hongland nutritional solution in conical flask without Pi (i.e. 0mM Pi)；
Contain normal Hongland nutritional solution in matched group in each conical flask.
After salt stress is processed 15 days, it is apparent that：
(1) non-salt tolerance selfing line QXN233 blade is presented withered curly (Fig. 3 A and 3B, shown in arrow), and salt tolerance
Selfing line QXH0121 blade remains to keep normal extended configuration (Fig. 3 A and 3C)；
(2) no P deficiency is after 35 days, and salt tolerance selfing line QXH0121 stem assumes obvious aubergine, and to old leaf and
Young leaves spreads (Fig. 3 D and 3F, shown in arrow), and non-salt tolerance selfing line QXN233 still assumes normal green (Fig. 3 D and 3E).
The result of this experiment also indicates that the salt patience of corn inbred line and phosphorus deficiency sensitivity are presented consistent, same table
Bright, can be by judging whether corn inbred line to phosphorus deficiency sensitivity, i.e., has " purplish red leaf " performance to be judged to salt indirectly
Toleration.In addition, accordingly, we are it is also speculated that the selfing line of salt tolerance should be more sensitive to phosphorus deficiency, and high phosphorus should be able to be alleviated
Certain salt stress symptom.Then, we have carried out Late Stage Verification by experiment further.
The normal Hongland nutrient solution prescription of table 1
1.2. Late Stage Verification process：
In high 18cm, the flowerpot of diameter 23cm, suitable common garden is filled with Vermiculitum (height 14cm), irrigate first
Hongland nutritional solution, will sprout the non-salt tolerance selfing line QXN233 seedling of well consistent Semen Maydiss and is carefully transplanted in basin, with
1 fresh nutrient solution was poured afterwards every 3-5 days (400ml/ is per basin).Treat that corn seedling grows to one heart stage of SANYE (about 2 weeks), enter respectively
The process of row P deficiency and salt and P deficiency are jointly processed by, specially：
Phosphorus treatment group sets 3 gradients, respectively 1mM, 3mM, 11mM Pi, and wherein 1mM is normal Hongland battalion
Pi concentration in nutrient solution；
0mM NaCl and 200mM NaCl salt stress process combination of two to each phosphorus gradient respectively, are jointly processed by group, i.e.,
For 0mM NaCl+1mM Pi, 0mM NaCl+3mM Pi, 0mM NaCl+11mM Pi and 200mM NaCl+1mM Pi, 200mM
NaCl+3mM Pi、200mM NaCl+11mM Pi.
After so processing 2 weeks, it will be evident that under 200mM NaCl is processed, corn seedling growing way 1mM<11mM<3mM Pi
(Fig. 4).Therefore, above-mentioned test result indicate that adding to the salt stress that 3mM Pi can effectively alleviate 200mM NaCl in nutritional solution
Injury effect.So, we select 200mM NaCl to process respectively with the combination of two of 1mM and 3mM Pi as the further later stage
The target of checking.
1.3. further Late Stage Verification：
In high 18cm, the flowerpot of diameter 23cm, suitable common garden is filled with Vermiculitum (height 14cm), irrigate first
Hongland nutritional solution, will sprout the non-salt tolerance selfing line QXN233 and salt tolerance selfing line QXH0121 of well consistent Semen Maydiss
In seedling is carefully transplanted into, poured 1 fresh nutrient solution later every 3-5 days (400ml/ is per basin).
Treat that corn seedling grows to one heart stage of 3 leaf (about 2 weeks), carry out salt stress process and salt and P deficiency is jointly processed by,
Specially：
In salt treatment group, each processes flowerpot and pours the Hongland nutritional solution containing 200mM NaCl；
And each flowerpot is poured while the Hongland nutritional solution containing 3mM Pi+200mM NaCl in reply group.
After so processing 2 weeks, it is evident that：
(1) reply group will be significantly better than growth (the corn seedling growing way 3mM Pi+ in Fig. 5 of the corn seedling in treatment group
200mM NaCl>>200mM NaCl)；
(2), after January, in salt treatment group, the corn seedling of non-salt tolerance selfing line QXN233 is almost dead, and tackles
Still major part holds green (Fig. 5 A, shown in red frame) to corn seedling overground part in group；
(3) the corn seedling state and in the reply group of salt tolerance selfing line QXH0121 also has significantly compared with salt treatment group
Improve (Fig. 5 B, shown in red frame).
Therefore, the high salt for adding in nutritional solution that 3mM Pi can effectively alleviate 200mM NaCl has been reconfirmed in this experiment
The salt stress to corn seedling (including salt tolerance Semen Maydiss and non-salt tolerance Semen Maydiss) is divided to injure, meanwhile, this experiment also demonstrate that me
Supposition, the selfing line of salt tolerance is more sensitive to phosphorus deficiency, and serious salt can be significantly alleviated in the supply of certain density Pi
Stress symptom, this may accelerate the growth of corn seedling with higher Pi concentration, promote the whole strain volume of Semen Maydiss to increase, effectively dilute
The salinity that has released in plant body is relevant.
This experiment conclusion also further demonstrate that to the measure of various physical signs：
(1) supply of 3mM Pi is significantly increased non-salt tolerance selfing line QXN233's and salt tolerance selfing line QXH0121
Overground part (stem) and underground part (root) weight (Fig. 6)；For non-salt tolerance selfing line QXN233, under 200mM NaCl stress,
The supply of 3mM Pi significantly improves the overground part (stem) of non-salt tolerance selfing line QXN233 and weight (Fig. 6 A of underground part (root)
And 6B), and the improvement degree of salt tolerance selfing line QXH0121 is not as non-salt tolerance selfing line QXN233 substantially (Fig. 6 C and 6D),
Imply that QXH0121 itself has had stronger salt tolerance, the increase of 3mM Pi is to non-salt tolerance selfing line QXN233 salt tolerance
Improve more notable.
(2) under 200mM NaCl+3mM Pi process, the chlorophyll (Fig. 7 A) of QXN233, proline (Fig. 7 C), solubility
Albumen (Fig. 7 D) and soluble sugar (Fig. 7 E) are significantly higher than only 200mM NaCl treatment group, also, its superoxide dismutase
(Superoxide Dismutase, SOD) (Fig. 7 F) is also significantly greater than 200mM NaCl treatment group, and its membrane lipid peroxidation is damaged
Degree is substantially less than 200mM NaCl treatment group (Fig. 7 B).Equally, except soluble protein (Fig. 7 D), other physiology of QXH0121
The same QXN233 of the change of index.Therefore, Hongland nutritional solution Pi content is brought up to 3mM, can effectively antagonism 200mM NaCl
The salt stress injury caused by non-salt tolerance corn inbred line QXN233 and salt tolerance corn inbred line QXH0121 seedling.
2. the relation of the salt patience of corn inbred line and its zinc sensitivity：
2.1 preliminary identification：
In high 18cm, the flowerpot of diameter 23cm, suitable common garden is filled with Vermiculitum (height 14cm), irrigate first
Hongland nutritional solution (nutrient solution prescription is shown in Table 1), will sprout the non-salt tolerance selfing line QXN233 of well consistent Semen Maydiss and resistance to
Selfing line QXH0121 seedling was carefully transplanted in basin salt, poured 1 fresh normal Hongland nutrition later every 3-5 days
Liquid (nutrient solution prescription is as noted above, and 400ml/ is per basin).
Treat that corn seedling grows to one heart stage of SANYE (about 2 weeks), it has been observed that salt tolerance selfing line QXH0121 seedling leaves
There is yellow spotting, and constantly increase to spread, rather than salt tolerance selfing line QXN233 seedling leaves show normal green, such as
Shown in Fig. 8, after germination and growth January, it is obvious that there is a significantly withered and yellow speckle QXH0121 seedling leaves middle and lower part, and by
Gradual change white spirit is withered, that is, be rendered as " the grey leaf " of Semen Maydiss, is typical Semen Maydiss zinc deficiency symptom.
Next, we have carried out salt stress respectively to corn inbred line QXN233 and QXH0121 and high zinc components are processed.
Treat that corn seedling grows to one heart stage of 3 leaf (about 2 weeks), carry out 200mM NaCl and 100 μM of ZnSO respectively4Process.
Concrete scheme is：
In salt treatment group, each processes flowerpot and pours the Hongland nutritional solution containing 200mM NaCl；
In high zinc treatment group, each processes flowerpot and pours and contains 100 μM of ZnSO4Hongland nutritional solution；
In matched group, each flowerpot pours normal Hongland nutritional solution.
After so processing 30d, it will be evident that the matched group of the two all grows normally well；Under 200mM NaCl is processed,
The two is short and small compared with matched group growth retardation, plant general performance, and non-salt tolerance selfing line QXN233 seedling stem and leaf is very thin, blade
Assume the withered shape of curling, root system elongated (Fig. 9 A)；And salt tolerance selfing line QXH0121 vane extension, roomy, stem is thicker, root
It is sturdy, more flourishing (Fig. 9 C), i.e. corn seedling growing way QXN233<QXH0121 (Fig. 9 A and 9C), confirms the salt tolerant of the two
Property.
Equally in 100 μM of ZnSO4Under process, the corn seedling of QXN233 is relatively compareed and grows short and small, overground part blade appearance
Curling, old leaf withered death, root system serious water shortage, growth retardation, dries up and short (Fig. 9 B)；And although QXH0121 relatively compares children
Seedling is also shorter and smaller, but compared with QXN233, blade is roomy, and stem is sturdy, root system length and bright (Fig. 9 D) in vain, and growth situation is substantially better than
QXN233, i.e., by stress order of severity QXH0121<<QXN233 (Fig. 9 B and 9D).The non-salt tolerance selfing line of this experimental verification
QXN233 is insensitive to zinc element, and Gao Xinke makes which seriously aggrieved；And salt tolerance selfing line QXH0121 is more sensitive to zinc, can
The a certain degree of high zinc components of stronger tolerance.
It is consistent that this experiment tentatively shows that the salt patience of corn inbred line and zinc sensitivity are presented, therefore, can be by sentencing
Whether sensitivity of the disconnected corn inbred line to zinc, i.e., have " grey leaf " performance to judge indirectly the tolerance degree to salt.Separately
Outward, accordingly, we are it is also speculated that the selfing line of salt tolerance should be more sensitive to zinc deficiency, and high zinc should be able to alleviate certain salt
Stress symptom.Then, we have carried out Late Stage Verification by experiment further to this.
2.2 Late Stage Verification：
In high 18cm, the flowerpot of diameter 23cm, suitable common garden is filled with Vermiculitum (height 14cm), irrigate first
Hongland nutritional solution, will sprout the non-salt tolerance selfing line QXN233 and salt tolerance selfing line QXH0121 of well consistent Semen Maydiss
Seedling was carefully transplanted in basin, poured 1 fresh nutrient solution later every 3-5 days (400ml/ is per basin).
Treat that corn seedling grows to one heart stage of SANYE (about 2 weeks), carry out zinc components process and salt respectively and zinc components are common
Process, specially：
Zinc treatment group sets 4 gradients, respectively 0 μM, 1 μM (normal), 20 μM, 50 μM of ZnSO4, wherein 1 μM is in table 1
Zn in normal Hongland nutritional solution2+Concentration；
Each zinc gradient is combined with 200mM NaCl respectively, is jointly processed by group, as 200mM NaCl+0 μM Zn2+、
200mM NaCl+1μM Zn2+、200mM NaCl+20μM Zn2+、200mM NaCl+50μM Zn2+.
After so processing January, it is evident that：
Zn2+Concentration is that under 0 μM (i.e. zinc deficiency), salt tolerance selfing line QXH0121 growing way is substantially not so good as non-salt tolerance selfing line
QXN233, QXH0121 blade is shrivelled, and root system is less, extremely undeveloped, conversely, QXN233 can keep better growth conditions,
Especially root growth is big compared with long and density, therefore, salt tolerance selfing line QXH0121 salt tolerance selfing line QXN233 more non-to zinc deficiency
More sensitive.
With Zn2+The rising of concentration, 20 μM of Zn2+Condition of serious stress of soil is had resulted in QXN233, root system dehydration shortens, overground part
Withered almost dead；Work as Zn2+When rising to 50 μM, QXN233 is completely dead, shows QXN233 intolerant to high zinc components, i.e., 0 μM>20
μM>50 μM (Figure 10 A).
But, salt tolerance selfing line QXH0121 assumes contrary growth conditions, with Zn2+The rising of concentration, QXH0121
Growth performance become better and better (0 μM<20μM<50 μM), upper compared with QXN233 one raises (Figure 10 B), and this is the zinc water sufficient with which
Flat consistent, show in certain limit, sufficient zinc can comparatively fast promote the growth of salt tolerance selfing line QXH0121.
It is shown experimentally that further：
Under 200mM NaCl is processed, 0 μM of non-salt tolerance selfing line QXN233 corn seedling growing way>1μM>20μM>50 μM of (figures
11A and 11B)；
On the contrary, under 200mM NaCl is processed, 0 μM of salt tolerance selfing line QXH0121 corn seedling growing way<1μM<20μM<
50 μM (Figure 11 C and 11D).
It is consistent with the growth situation individually under zinc components are processed that the two growth situation under the combination of salt+zinc is.
Therefore, the result of this experiment shows：
For non-salt tolerance selfing line QXN233, its salt tolerance can not be alleviated or be improved to the supply of zinc, and high zinc even adds
Its salt stress is weighed；
And for salt tolerance selfing line QXH0121, its salt tolerance, such as nutritional solution can be alleviated or be improved to sufficient zinc element
In add to 20 or 50 μM of Zn2+The salt stress injury effect of 200mM NaCl can be relieved.
So, this experiment demonstrates our supposition, the corn inbred line of salt tolerance is more sensitive to zinc deficiency, and Gao Xinneng
Alleviate certain salt stress symptom, this may be with higher Zn2+Concentration accelerates the growth of corn seedling, promotes the whole strain body of Semen Maydiss
Product increases (Figure 11 C), and the salinity for effectively diluting in plant body is relevant.
The present invention can only first carry out Low phosphorus stress process and carry out zinc components process again, it is impossible to first carry out zinc deficiency Stress treatment again
P deficiency process is carried out, is carried out in zinc components process again because first carrying out Low phosphorus stress and processing, after Low phosphorus stress is processed
Corn seedling is first repaired by the Pi of normal proportioning content, eliminates the influence factor that P elements are produced to corn seedling, then by relatively
The normal higher Zn of proportioning content2+Zinc components process is carried out, now, salt tolerance corn seedling can be by the life under Low phosphorus stress process
Length is worse to become that growth is preferable, non-salt tolerance corn seedling can by Low phosphorus stress process lower growth preferably become to grow worse,
All there is such a obvious growing way Umklapp process in the two, change is obvious so that salt tolerance is easy to differentiate with non-salt tolerance,
Very clear；And if in turn, first carrying out zinc deficiency Stress treatment carries out P deficiency process again, now need with comprising normally joining
Zn than content2+And the nutritional solution of the higher Pi of more normal proportioning content carries out P deficiency process, through zinc deficiency Stress treatment
Corn seedling afterwards is first by the Zn of normal proportioning content2+Repair, the influence factor that zinc element is produced to corn seedling is eliminated, then
P deficiency process carried out by the higher Pi of more normal proportioning content, now, salt tolerance corn seedling can be by under zinc deficiency Stress treatment
Growth worse become that growth is preferable, non-salt tolerance corn seedling preferably can still keep life by the growth under zinc deficiency Stress treatment
Long preferable, compared with above-mentioned, at least non-salt tolerance corn seedling formerly carries out zinc deficiency Stress treatment and carries out P deficiency process again
In whole process, growing way is not changed significantly, and is difficult to differentiate, is so allowed for the Screening and Identification of whole Maize Seedling salt tolerance
Accuracy not as first carry out Low phosphorus stress process carry out the so high of zinc components process again.
For a further understanding of the present invention, a kind of Maize Seedling salt tolerance for the present invention being provided with reference to embodiment
Process for screening and identifying is described in detail, and protection scope of the present invention is not limited by the following examples.
Embodiment 1
A kind of process for screening and identifying of Maize Seedling salt tolerance,
First the corn variety of 8 identifications to be screened (is respectively designated as：121、134、137、138、141、142、143、
233) the well consistent seedling that sprouts carries out normal liquid culture, after waiting corn seedling to grow to one heart stage of SANYE, to whole
The seedling of the corn variety of identification to be screened all carries out the Low phosphorus stress of 0mM Pi and processes, and after processing 35 days, observes seedling growth
Feature, has whether phosphorus deficiency symptom, i.e. blade show " purplish red leaf " or " kermesinus " occur judging which according to corn seedling is no
Whether salt tolerant, the corn seedling with phosphorus deficiency symptom is that haloduric corn kind, not have the corn seedling of phosphorus deficiency symptom be non-salt tolerant
Corn variety, as shown in figure 12, it is haloduric corn that can obtain in 8 corn varieties that identifies to be screened 121,141,137 and 138
Kind, therein 134,142,143 and 233 are non-haloduric corn kind.
The height of the salt tolerance of corresponding corn variety, Semen Maydiss are judged according to the order of severity of corn seedling phosphorus deficiency symptom
The more serious salt tolerance for characterizing corresponding corn variety of the phosphorus deficiency symptom of seedling is higher, according to phosphorus deficiency (0mM Pi) stress for determining
The anthocyanidin content (Figure 13) of each kind after processing 35 days, can determine whether that the phosphorus deficiency sensitivity order of above-mentioned 8 kinds of Semen Maydiss is 121>138
>137>141>134>142>143>233, thus speculate that the salt tolerance of these corn varieties should be 121>138>137>141>134>
142>143>233.
Then, zinc components process, institute are all carried out again to the seedling of the above-mentioned 8 kinds of corn varieties after whole zinc deficiency Stress treatments
With the Zn in nutritional solution2+Concentration be 20 μM, nutrient solution used in Pi concentration be 1mM, process 2-3 week after, according to corn seedling
Growing way change judge its whether salt tolerant to verify further, after zinc components before processing growing way by the bad corn seedling that takes a turn for the better be not
Haloduric corn kind, after zinc components before processing growing way by the corn seedling for badly taking a turn for the better be haloduric corn kind, can obtain 8 to be screened
In the corn variety that identifies 121,141,137 and 138 are haloduric corn kind, and therein 134,142,143 and 233 are non-resistance to
Salt corn variety.
Next, these kinds are carried out with 100mM NaCl Stress treatment, by using plant shoots under detection salt stress
The method of malonaldehyde (MDA) content of generation verify to above-mentioned salt tolerance size order result, MDA content is higher to be shown
Cell membrane damage degree is bigger, while sign salt tolerance is lower, under 100mM NaCl stress, after water planting is processed 5 days, experimental result
As shown in FIG. 14 and 15, thus, the salt tolerance of these corn varieties is 121 really>138>137>141>134>142>143>
233.
The method and apparatus of the not detailed description of the present invention is prior art, repeats no more.
Specific embodiment used herein is set forth to the principle of the present invention and embodiment, above example
Illustrate that being only intended to help understands the method for the present invention and its core concept.It should be pointed out that the common skill for the art
For art personnel, under the premise without departing from the principles of the invention, some improvement can also being carried out to the present invention and being modified, these change
Enter and modify to also fall in the protection domain of the claims in the present invention.
Claims (4)
1. a kind of process for screening and identifying of Maize Seedling salt tolerance, it is characterised in that
First seedling well consistent for the sprouting of the corn variety of multiple identifications to be screened is carried out normal Solution culture method, etc.
After corn seedling grows to one heart stage of SANYE, with nutritional solution, 0mM Pi is all carried out to the seedling of all corn varieties of identification to be screened
Low phosphorus stress process, process certain time after, observe seedling growth feature, sentence according to whether corn seedling has phosphorus deficiency symptom
Break its whether salt tolerant, and the corn seedling with phosphorus deficiency symptom is that salt tolerance corn variety, the corn seedling for not having phosphorus deficiency symptom is
Non- salt tolerance corn variety；
The height of the salt tolerance of corresponding corn variety, corn seedling are judged according to the order of severity of corn seedling phosphorus deficiency symptom
The more serious salt tolerance for characterizing corresponding corn variety of phosphorus deficiency symptom higher.
2. process for screening and identifying according to claim 1, it is characterised in that in Low phosphorus stress processing procedure, nutrition used
In liquid, each element concentration is：
3. process for screening and identifying according to claim 1, it is characterised in that after Low phosphorus stress is processed, with nutritional solution to complete
The seedling of the corn variety after portion's Low phosphorus stress process all carries out certain density Zn again2+Zinc components process, process one timing
Between after, determine whether its whether salt tolerant according to the change of the growing way of corn seedling, after zinc components before processing, growing way is bad by taking a turn for the better
Corn seedling be non-salt tolerance corn variety, after zinc components before processing growing way by the corn seedling for badly taking a turn for the better be salt tolerance Semen Maydiss
Kind；
In zinc components processing procedure, nutrient solution used in Zn2+Concentration be 20 μM～50 μM, nutrient solution used in Pi concentration be
1mM.
4. process for screening and identifying according to claim 3, it is characterised in that in zinc components processing procedure, nutrient solution used
Middle each element concentration is：
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| CN116333077A (en) * | 2023-05-22 | 2023-06-27 | 中国农业科学院作物科学研究所 | Application of corn ZmLAC9 gene in plant adaptation to salt stress |
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