CN104604555A - Method for screening saline-alkaline resistant/tolerant cotton - Google Patents

Abstract

The invention relates to the field of plant physiology, in particular to a method for screening saline-alkaline resistant/tolerant cotton. The method comprises the steps that (1), a cotton culture solution is prepared, and the pH value of the cotton culture solution is adjusted between 6.5 and 7.0; (2), the culture solution serves as a solution to prepare NaCl solutions with the concentration being 0.0 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt% and 0.8 wt%; (3), a sand sprout method is adopted, when cotton seeds sprout and grow until all cotyledons are spread and turn into green and no root is generated, the cotton seeds are moved in the NaCl solutions; (4), cultivation is carried out in a shading condition; (5), in the ninth to tenth day, seedlings are taken out for surveying the root hair number of each cotton seedling, and the saline-alkaline resistant/tolerant degree of cotton is judged according to the number of the root hairs. The method excludes the interference of the factors that salinity gradient is uneven and rainfall wets salt in the field identification process, the duration is short, the identification result is stable and reliable, and method is high in practicability and maneuverability.

Description

A kind of Resistance Strain of Cotton/Salt And Alkali Tolerance screening method

Technical field

The present invention relates to plant physiology field, be specifically related to a kind of Resistance Strain of Cotton/Salt And Alkali Tolerance screening method.

Background technology

China has large area saline land, and along with the continuous deterioration of ecotope, comprise the modernization of industry, irrigated land and Protectorate cultivation enlarged areas, soil secondary salinization area increases year by year.The soil salinization and shortage of fresh water have become a worldwide problem, cotton is the important economic crops of China, also be the pioneer crop of saline land plantation, the development principle that exploitation inland and beach saline land plant cotton meet " not striving ground with grain and cotton ", has broad application prospects.

Along with developing rapidly of China's agricultural transgenic technology, under China's Eleventh Five-Year Plan, " 12 " and the support by " 13 " the genetically modified plants rearing new variety key special subjects prepared, the novel transgene cotton such as drought resisting, Salt And Alkali Tolerance continues to bring out.Domestic and international concerned plant Advances in salt tolerance is very fast, but because plant growth is greatly affected by environment, and plant salt endurance is by multiple gene cooperation control, so up to the present still do not cultivate comparatively ideal salt-tolerant plant, also not yet inherently illustrate plant salt endurance mechanism.The salt resistance of different plant is different with Mechanisms of Salt Resistance, and the physiological change trend that therefore salt stress is foretold differs greatly.

Relatively stable relevant not only with osmotic adjustment ability and Protective substances of the salt resistance of cotton, more mainly depends on that root system is to the Selective absorber of salinity and the compartmentalization at organ, tissue and cell three levels thereof.If the salt ion absorbed can morely stay in root, reduce the transport to overground part and distribution, then can show stronger Salt-endurance.Simultaneously because the Saline alkali tolerance of plant is by controlled by multiple genes, resistance mechanism more complicated, and root system is that cotton is injured the earliest in saline and alkaline adverse circumstance, the most direct position, so the fresh weight of root system, dry weight, root are long, radical is all morphological index important in Salt And Alkali Tolerance qualification, but find in these indexs of root system in research, the change of quantity to salt concentration of coring is the most responsive, and therefore it can be used as the ideal indicator of the Forepart identification of Salt And Alkali Tolerance cotton Forepart identification system and screening this system applicable.

For a long time, the screening of antagonism/Salt And Alkali Tolerance cotton material or qualification are all adopt saline land, field, artificial salt pond to test, or the parameter of some Physiology and biochemistries of indoor measurement, as comparing reference.Existing method carries out salt damage investigation according to cotton material upgrowth situation in saline land or salt pond to carry out judging whether to have anti-/ patience to saline and alkaline.Although the method is directly at field test, qualification result is relatively direct, and annual cotton only has a season of growth, namely can only carry out a collection of test every year; To higher required by verification field, as salinity will have certain gradient, salt pond or place, saline land cannot be changed, because be not available to through the saline land of this kind of approval test or salt pond plant other plant, in case bring salt damage to other crops, floor space is large, and determination rates is low; Salt damage symptom is individual scope qualitatively, does not have concrete digital quantization, and the result difference that different testing persons observes is comparatively large, and the conclusion drawn has difference and instability; Saline land, field is nature, and the not easily unification of the degree of mixedness of soil and saline and alkaline, although the salinity in salt pond is the NaCl be manually sprinkled into, but after entering soil, the factors such as the content of soil, pH value and weather rainfall all can cause the change at any time of salinity level, between year and parallel between the comparativity of result of the test poor.

Because Salt-resistant of Cotton alkalescence has a lot of complicated gene jointly to regulate and control, at present to transgenosis resist/Saline alkali tolerance cotton is many to think the Salt Strees Condition of coercing its root system from its indoor, study by measuring more biochemical substances, secondary metabolite, enzyme system etc., but generally can only initial reaction under the coercing of NaCl solution, the change of Cucumber, the impact that can produce after NaCl coerces can only be received as this plant, but can not carry out as the technical parameter of qualification.Because the change of these parameter quantities is very faint, still can not as parameter and the value evaluating whether Saline alkali tolerance, can only be as a reference.

Cotton, because growth cycle is long, spatial content is large, brings very large difficulty to its salt tolerant breeding work.The salt resistance of qualification institute seed selection plant is success or failure and the efficiency that the quality of an important step, particularly Forepart identification in Salt Tolerance directly can have influence on final seed selection work.The objective trait of the cotton new material newly emerged as the performance of anti-/ Salt And Alkali Tolerance and degree how, also there is no good appraisal procedure at present.

Water planting is the one of soilless culture, it is a new and high technology in modern agriculture science and technology, because it instead of soil environment with the root system environment of artificial creation, because it has many advantages in Tolerant salt, it provide a homogeneous root system ecotope especially, thus the reaction of plant to salt damage can be gone out by Accurate Determining, so be widely applied in plant salt tolerance qualification.

The present invention cultivates cotton plants by utilizing ciltivating process, in nutrient solution, field arranges different salinity gradients, transplanted by specific seedling raising manners and specific time, and investigate specific parameter in the specific time, according to specific, can directly observe, simple parameter carries out judging the resistance level of this cotton material.Can utilize on the one hand the method to existing anti-/ Salt And Alkali Tolerance material identifies, Evaluating and screening can be carried out on the other hand to the Saline alkali tolerance of cotton inbred lines or crossbreed, the germplasm resource for cotton finding high Salt And Alkali Tolerance is the prior development direction of cotton Saline alkali tolerance from now on, lays the foundation for creating excellent germ plasm resource.

Summary of the invention

The object of this invention is to provide a kind of Resistance Strain of Cotton/Salt And Alkali Tolerance screening method.

Novel transgenosis is anti-/ after Salt And Alkali Tolerance cotton material occurs, in order to improve the efficiency of screening further faster, improve the better Breeding and application of the antagonism/floral material of resistance to new cotton, assess the application prospect of such cotton material in actual production, in indoor by short-term and multiple batches of anti-/ Saline alkali tolerance screening, for breeding man can make the going or staying suggestion of this material in advance, the prospect simultaneously can assessing the practical application afterwards of this material makes technological guidance and theories integration.Therefore, according to feature and the demand of the objective trait of current new cotton floral material, the present invention takes the lead in studying and propose indoor transgenosis and resist/and Salt And Alkali Tolerance cotton is to the resistance level screening technique of NaCl.This method eliminates the interference of the factors such as field weather, salinity level natural trend, the bad control of salt pond salinity gradient, qualification last greatly shorten, result is stable, reliable, practicality and workable.

According to Resistance Strain of Cotton of the present invention/Salt And Alkali Tolerance screening method, comprise the following steps:

(1) according to 1/2Hoagland complete nutrition formula of liquid preparation cotton culture fluid, and by the adjustment of the pH value of nutrient solution between 6.5-7.0;

(2) with the culture fluid of step (1) for solution, be mixed with respectively 0.0% (CK), 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, the NaCl solution of 0.6% and 0.8%, be distributed into in the Clear glass bottles and jars of high 60mm.The saline land in land for growing field crops under normal conditions, be divided into low salt pan (<0.2%), middle salt pan (>0.2% but <0.4%) and high salt pan (>0.4%), within the scope of less salt, cotton planting can directly germinate and grow, middle salt state lower part is germinateed and growth, does not substantially grow in high salt situation.The situation in land for growing field crops is all included by the NaCl concentration of the present invention's design, if too concentrate or do not comprise, approval test all can cause gross error or failure; Salinity needs indoor are in addition adjusted to higher, because the cotton seedling that indoor approval test uses germinates, do not have suitable concentration, not remarkable to its inhibitory action.

(3) " sandy soil sproutung method " is adopted, when Cotton Seed grow to cotyledon all launch and become green and need not root occur time, the root of cotton seedling is rinsed well with clear water in leakage basin, divide individual plant to move into step (2) configure and load in the vial of different N aCl solution, cotton cotyledon is stayed outside bottleneck.Seedling raising manners selects sandy soil seedling method, because communications centre root when transplanting cotton seedling, sandy soil in clear water and the impulse force of water is very little can remove the sandy soil on root system surface, other nursery carrier such as vermiculite, soil etc. are all not easy to rinse well, cause washing time long, the intensity of flushing water is large, easily makes root receive damage, affects result of the test.The time of transplanting cotton seedling is very crucial, if cotton seedling is too little, its main root does not also grow, transplant in the adverse circumstance entered with NaCl solution, cotton seedling is easy to death, if the time too late, the coring of cotton seedling grows, move in the adverse circumstance of NaCl solution, its resistivity is strengthened, and experiment also can be caused not have effect again.

(4) vial appearance wrap up in that one deck is tight can light screening material, and guarantee not make light penetrate in bottle, every vial is for once to repeat, and each NaCl solution gradient of each screening material repeats 10 times; Place (25 scholar 2) DEG C, cultivate under light intensity 2000lx, photoperiod 12/12h condition, in vial, timing every day supplements corresponding NaCl solution in cotton seedling whole root immersion culture fluid;

Within (5) 9-10 days, take out the radical amount of the every strain cotton of investigation, comprise the fibrous root quantity on taproot and lateral root and side root, be referred to as coring number; With the coring quantity of the cotton material to NaCl sensitivity for contrast, according to how many judgement cotton materials of coring quantity to saline and alkaline anti-/ patience degree.The selection of control time is very important, time period, the coring ateliosis of cotton seedling, because 9-10 days just cotton true leaf growth launch period, the nutrition of seed is to provide the nutrition library of cotton seedling cotyledon growth, select not occur at coring, cotton seedling cotyledon all launches to be because now seed nutritional is used up completely, show seedling in good health, be implanted in nutrient solution, the nutrition that cotton seedling now utilizes is all nutrient solution, thus be effective to salinity absorption, the coring number occurred, the normal grade of true leaf all comes from nutrient solution, thus absorb the salinity affecting its growth.

Root system is the critical organ of plant growth source of nutrition, and the nutrition of 90%, all from root system, does not have the growth of its each side of root system all can be obstructed.Therefore when cotton root system is after absorbing the nutrient solution containing NaCl solution, do not have the cotton material of resistance, its root system is obstructed naturally, and along with the rising of NaCl solution concentration, is obstructed more serious, even occurs death.And the suppression of salt-resistance cotton material to NaCl is not obvious, can with not the same containing growth in NaCl solution in routine, coring quantity is more, illustrates that its root system reaches all the more, thus more favourable to the growth of overground part, thus the low anti-salinity ability of whole plant is strengthened.

It is uneven that method of the present invention eliminates field test salinity gradient, and rainfall, to the interference of the factors such as salinity drenches, lasts short, and qualification result is stable, reliable, practicality and workable.

Embodiment

Example 1:

1, experimental technique:

(1) according to 1/2Hoagland complete nutrition formula of liquid preparation cotton culture fluid, and by the adjustment of the pH value of nutrient solution between 6.5-7.0;

(2) with the culture fluid of step (1) for solution, be mixed with respectively 0.0% (CK), 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, the NaCl solution of 0.6% and 0.8%, be distributed into in the Clear glass bottles and jars of high 60mm;

(3) " sandy soil sproutung method " is adopted, when Cotton Seed grow to cotyledon all launch and become green and need not root occur time, the root of cotton seedling is rinsed well with clear water in leakage basin, divide individual plant to move into step (2) configure and load in the vial of different N aCl solution, cotton cotyledon is stayed outside bottleneck;

(4) vial appearance wraps up in the tight masking foil of one deck or can light screening material, and guarantees not make light penetrate in bottle; Every vial is for once to repeat, and each NaCl solution gradient of each screening material repeats 10 times; Place (25 scholar 2) DEG C, cultivate under light intensity 2000lx, photoperiod 12/12h condition; In vial, timing every day supplements corresponding NaCl solution in cotton seedling whole root immersion culture fluid;

Within (5) 9-10 days, take out the radical amount of the every strain cotton of investigation, comprise the fibrous root quantity on taproot and lateral root and side root, be referred to as coring number; With the coring quantity of the cotton material to NaCl sensitivity for contrast, according to how many judgement cotton materials of coring quantity to saline and alkaline anti-/ patience degree.

2, experimental result:

From following table test data, in Low Concentration NaCl nutrient solution (<0.4%) two type cotton all can normal growth, its coring quantity is basically identical, be respectively 79.4 and 81.0, when in solution, NaCl concentration raises gradually, there is obvious difference in the coring of two type cottons, if 13018 cotton materials are 116.8, and the coring number of cotton No. 3 of nasal mucus is 66.8, difference reaches extremely significant level.

In table, data are visible, in the NaCl nutrient solution of 0.3%, the two coring numbers grown cotton are basically identical, show that the NaCl nutrient solution of this scope is not enough to Levant Cotton Root must be obstructed, cause harm, but after reaching the even high salt of salt, the coring of the cotton material of different resistance counts and has showed rightabout development, the coring number normal growth of resistant material, do not receive restriction, and the coring number of susceptibility cotton material in being implanted in salt and high salt concentration nutrition after, growing of its root system is seriously obstructed, just part creates coring, quantity does not reach normal level.

Claims (1)

1. Resistance Strain of Cotton/Salt And Alkali Tolerance screening method, is characterized in that, said method comprising the steps of:

(1) according to 1/2Hoagland complete nutrition formula of liquid preparation cotton culture fluid, and by the adjustment of the pH value of nutrient solution between 6.5-7.0;

(2) with the culture fluid of step (1) for solution, be mixed with the NaCl solution of 0.0wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt% and 0.8wt% respectively;

(3) " sandy soil sproutung method " is adopted, when Cotton Seed grow to cotyledon all launch and become green and need not root occur time, the root clear water of cotton seedling is rinsed well, divide individual plant to move in the vial of the NaCl solution that step (2) configures, cotton cotyledon is stayed outside bottleneck;

(4) vial appearance wraps up in light screening material, and guarantees not make light penetrate in bottle, and every vial is for once to repeat, and each NaCl solution gradient of each screening material repeats 10 times; Place 25 scholar 2 DEG C, light intensity 2000lx, cultivate under photoperiod 12/12h condition, in vial, timing every day supplements corresponding NaCl solution in cotton seedling whole root immersion culture fluid;

Within (5) 9-10 days, take out the radical amount of the every strain cotton of investigation, comprise the fibrous root quantity on taproot and lateral root and side root, be referred to as coring number; With the coring quantity of the cotton material to NaCl sensitivity for contrast, according to how many judgement cotton materials of coring quantity to saline and alkaline anti-/ patience degree.