CN114451282A - Indoor rapid identification and screening method for salt and alkali-resistant oats - Google Patents
Indoor rapid identification and screening method for salt and alkali-resistant oats Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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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
- 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
- A01C1/02—Germinating apparatus; Determining germination capacity of seeds or the like
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Soil Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physiology (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention discloses a method for indoor rapid identification and screening of saline-alkali tolerant oats, which comprises the following steps: pre-germinating oat seeds; carrying out germination treatment; water culture seedling; nursing hydroponic plants; salt treatment; evaluating saline-alkali resistance; and (5) recovering saline-alkali tolerant strains. The indoor rapid identification and screening method for the saline-alkali tolerant oats has the advantages of high efficiency and accuracy in identifying and screening the saline-alkali tolerant oats, wide application range, capability of recycling germplasm resources with strong saline-alkali tolerance and capability of avoiding resource loss.
Description
Technical Field
The invention relates to a method for indoor rapid identification and screening of saline-alkali tolerant oats.
Background
Oats, which are annual herbaceous plants of the genus Avena of the family Gramineae, are commonly called oats, grow in alpine, arid and barren ecological environments, and have the characteristics of cold resistance, barren resistance, alkali resistance and drought resistance which are unique to the oats after long-term natural selection, so that the oats are called saline-alkali resistant pioneer crops. The soil salinization is one of the most important abiotic stresses, and is a limiting factor of crop production all over the world, according to statistics, about 3.8 hundred million hectares of land all over the world has the problem of salinization in different degrees, the area of the land accounts for about 10 percent of the total cultivated land area of the world, and about 2000 million hectares of salinization land in China accounts for about 25 percent of the total cultivated land area of the whole country. In recent years, the salinization of the land is continuously increased along with the change of the climatic environment and unreasonable irrigation, and becomes an important factor for restricting the current agricultural development, so that the screening of the oat variety of the saline-alkali tolerant crop is beneficial to enriching the diversity of the genetic resources of the saline-alkali tolerant crop, lays a foundation for the research of saline-alkali tolerant molecules of the crop, and further provides a scientific premise for realizing the high yield and income increase of agriculture; meanwhile, the selection and cultivation of a new oat variety with strong saline-alkali stress tolerance have great significance for improving and utilizing the saline-alkali soil.
At present, for research and identification of oat saline-alkali tolerance, most of oat seeds stay in the germination stage of the seeds, only the saline-alkali tolerance of the oat seeds is preliminarily identified, the saline-alkali tolerance of the oat seeds in the seedling stage and the growth and development stage cannot be observed, the efficiency and the accuracy are low, the application range is small, and due to the difference of individual genotypes of the seeds, germplasm resources with strong saline-alkali tolerance in the later stage cannot be recovered, so that the resource loss is caused.
Disclosure of Invention
The invention provides a method for rapidly identifying and screening saline-alkali-tolerant oats indoors, which aims to solve the technical problems that in the prior art, the research and identification efficiency and accuracy of the saline-alkali-tolerant oats are not high, the application range is small, and germplasm resources with strong saline-alkali tolerance cannot be recovered.
A method for indoor rapid identification and screening of saline-alkali tolerant oats comprises the following steps:
pre-germinating oat seeds, selecting the oat seeds with full grains and uniform size, soaking the oat seeds in 0.8% sodium hypochlorite for 20 minutes, washing the oat seeds with sterile water for three times, placing the oat seeds on wet filter paper in a culture dish, and placing the culture dish in an incubator at 4 ℃ for dark culture for 48 hours;
germination treatment, namely putting the dark-treated seeds into sandwich imprinted filter paper, putting the sandwich imprinted filter paper on a support frame, putting the bottom of the filter paper in a tray filled with sterile distilled water, keeping the filter paper moist, transferring the filter paper into a light incubator at the temperature of 25 ℃ for germination, and taking out uniformly-developed seedlings when first leaves grow out from coleoptiles and 3-8 roots after culturing for 5-6 days;
water culture seedling raising, namely taking out seedlings, wrapping the stems of the seedlings in sponge rings, inserting the sponge rings into a water culture system containing Hoagland's solution for culture, then putting the sponge rings into an illumination incubator for culture, setting the temperature to be 20/15 ℃ (day/night), and setting the light cycle to be 16 hours;
nursing the hydroponic plants, measuring the pH value of the hydroponic solution every day, 3 times every 2 days or every week, supplementing Hoagland's solution to the front of the initial test level line, and adjusting the pH value to 6;
salt treatment, namely applying salt and alkali to the seedlings in the full nutrient solution at the 2-3 leaf stage;
evaluating the saline-alkali resistance, evaluating on the 12 th day of saline-alkali treatment, observing and recording the expression symptoms of the oats under saline-alkali stress, wherein the expression symptoms comprise reduced leaf area, whitened lower leaves, dead leaf tips and leaf curl, and obtaining the evaluation conclusion of the saline-alkali resistance of the seedlings according to the expression symptoms;
and (3) recovering saline-alkali tolerant strains, picking out seedlings with strong saline-alkali tolerant evaluation conclusion, keeping the roots of the seedlings intact, continuously growing the seedlings in a hydroponic system containing Hoagland's solution until the seedlings are mature, and harvesting seeds with strong saline-alkali tolerant performance.
Preferably, in the step of pre-germination treatment of oat seeds, less than or equal to 50 seeds are placed in the culture dish.
Preferably, in the pre-germination treatment step of the oat seeds, the seeds are placed on wet filter paper in a culture dish, and water on the wet filter paper cannot completely cover the seeds.
Preferably, the hydroponic system includes test container, supporting platform system, air-breather system and culture solution, the test container sets up to the polyethylene rectangular box that the external diameter of dark colour is 40 x 20 x 10cm, supporting platform system includes supporting platform and sponge strip, and the supporting platform sets up to the polyethylene rectangle of 50 x 25cm, and the drilling is equipped with 30 equidistant holes on the supporting platform, and the sponge strip sets up to the rectangle of 5 x 3cm, and the air-breather system includes air pump and breather pipe, and the air pump is connected to central ring pipe, and the both ends of central ring pipe are equipped with a plurality of foraminiferous steel pipes that 30cm is long through the hose.
Preferably, the saline-alkali resistance evaluation step further includes recording the weight, plant height and tiller number index of oat stem/root/whole plant.
The indoor rapid identification and screening method for the saline-alkali tolerant oats has the advantages of high efficiency and accuracy in identifying and screening the saline-alkali tolerant oats, wide application range, capability of recovering germplasm resources with strong saline-alkali tolerance and capability of avoiding resource loss.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the indoor rapid identification and screening method for the saline-alkali tolerant oats.
Detailed Description
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving equivalent or similar purposes, unless expressly stated otherwise. That is, unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features.
As shown in figure 1, the indoor rapid identification and screening method for the saline-alkali tolerant oats comprises the following steps:
step 1: pre-germination treatment of oat seeds
Selecting full oat seeds with uniform size, soaking in 0.8% sodium hypochlorite (NaClO) for 20 min, washing with sterile water for three times, and placing seeds (at most 50 seeds) in a culture dish
On wet filter paper (note that about 4 ml of sterile water is dropped into each dish, and the water does not completely cover the seeds). The plates were then placed in an incubator at 4 ℃ for 48 hours in the dark, a low temperature which favours uniform germination.
Step 2: germination treatment
After dark culture is finished, putting the pretreated seeds into sandwich imprinting filter paper so as to generate vertical root systems, then putting the sandwich imprinting filter paper on a support frame, and putting the bottom of the filter paper in a sterile distilled water tray filled with the filter paper so as to facilitate germination and water absorption of the seeds; then transferred into a light incubator with the temperature of 25 ℃ for germination. After 5-6 days of culture, when the first leaf grows out from coleoptile and 3-8 roots, taking out the evenly developed seedling, and transferring the seedling into water culture for further culture.
Step 2: hydroponic seedling culture
The seedlings are independently taken out, placed into a sponge ring, inserted into a water culture system containing a full-strength Hoagland's solution for culture, then placed into a light incubator for culture, the temperature is set to be 20/15 ℃ (day/night), the light cycle is 16 hours, so that the seedlings can healthily and robustly grow, and after the seedlings grow to the two-leaf stage, salt treatment is carried out. The water culture system comprises a test container, a supporting platform system, an aeration system and a culture solution, wherein the test container is a polyethylene rectangular box with the dark color and the outer diameter of 40 multiplied by 20 multiplied by 10 cm; the supporting platform system consists of a supporting platform and a sponge strip, the supporting platform is a polyethylene rectangle of 50 multiplied by 25cm, and 30 equidistant holes (the diameter is 1.0cm) are drilled on the supporting platform; the sponge strip is a rectangle of 5 multiplied by 3 cm; the aeration system consists of an air pump and an aeration pipe, wherein the air pump is connected to a central annular pipe, and then a plurality of 30cm long steel pipes with holes (the diameter is about 1 mm) are arranged at two ends of the central ring through hoses so as to allow small bubbles to escape and sink to the bottom of the tank without interfering the growth of roots; the culture solution adopts Hoagland's (Hoagland) nutrient solution formula, and the specific formula of each nutrient solution in the culture solution is shown in tables 1-3.
TABLE 1 Hoagland's (Hoagland) nutrient solution formula
TABLE 2 iron salt solution nutrient solution formula
TABLE 3 formula of nutrient solution for microelement liquid
And 4, step 4: hydroponic plant nursing
Due to evaporation and transpiration, there is a loss of solution volume and a change in pH (algae growth can also lead to fluctuations in pH). The pH of the aqueous culture solution needs to be measured by a pH meter daily, 3 times a day, 2 days or week, before the initial test level needs to be replenished, and the pH is adjusted to 6. The operation is that the supporting platform system is taken down and temporarily placed on an empty tank, all the hydroponic solution can be changed into new solution, the pH value of the volume solution is adjusted, and the supporting system is put back after the whole experiment is completed; alternatively, the pH can be adjusted on separate tanks and more working solution can be added to make up for the capacity of each tank.
And 5: salt treatment
The method is generally used for screening NaCl or NaHCO with salt concentration of 150-200 mmol/l in oat salt tolerance test3. Can be prepared by adding quantitative NaCl solid or NaHCO into the total nutrient solution3Dissolving and mixing the solid by using a stirring rod to prepare the water culture full nutrient solution with higher salt content. When the seedlings in the full nutrient solution are subjected to saline-alkali treatment (the seedling rate is determined) in the 2-3 leaf stage, transferring the seedling support platform system from the control tank to NaCl or NaHCO solution containing full solution3Culturing in a hydroponic tank. The treatment with alkali is carried out once, not stepwise.
Step 6: evaluation of salt and alkali resistance
The visible symptoms of salt and alkali stress are reduced leaf area, lower leaf whitening, dead leaf tip and leaf rolling. The salinity screening technique is based on the ability of seedlings to grow in salinized nutrient solutions. Evaluation is generally carried out on about day 12 of the salt and alkali treatment. At this stage, sensitive seedlings begin to die, while the intermediate genotypes show varying degrees of tolerance. The relative saline alkali tolerance evaluation scores of specific oat seedlings are shown in table 4.
TABLE 4 evaluation score of relative saline alkali tolerance of oat seedlings
The evaluation may be performed every day of treatment, as required by the test. Growth curves can be drawn to investigate the response over time. Seedling biomass can be recorded by recording stem/root/whole plant weight (fresh and dry), plant height and tiller number. However, in general, evaluation should be made under saline-alkali treatment for 12 days or more, because the growth rate of sensitive seedlings is most remarkably decreased, while the growth of saline-alkali tolerant seedlings is increased (but decreased compared with the control seedlings). The saline-alkali tolerant lines showed slight damage on day 12, with brown leaf tips; moderately tolerant lines show more leaf damage, dead old and new leaves are only green at the leaf base; the susceptible strain died, wherein the high resistance, the resistance and the comparative resistance evaluation conclusion of the tolerance evaluation are strong saline-alkali tolerance and weak susceptibility and high sensitivity as shown in table 4.
And 7: recovery of saline-alkali tolerant strains
The selected more tolerant seedlings were picked out of the test chamber and the roots carefully kept intact. The base of the aerial part of the selected seedling was then wrapped lightly with a sponge strip and the seedling was then inserted into a recovery tank. Selected seedlings can continue to grow in the recovery tanks filled with the Hoagland's solution which is replaced every 2 weeks until the seedlings are mature, and seeds with strong saline-alkali resistance are harvested.
The indoor rapid identification and screening method for the saline-alkali tolerant oat has the following advantages:
1. compared with the prior screening under the condition of saline-alkali soil, the screening system is cheaper, quicker and simpler to establish.
2. The screening system can clearly classify the tested plants into sensitive type, moderate type and tolerant type, and can visually see the morphological characteristics of the plants such as leaf color, leaf rolling, dead leaf apex, dead seedling, root damage (growth and browning), biomass and the like.
3. The germplasm resources with strong salt tolerance can be timely recovered, and the loss of resources is avoided.
4. The system is easy to operate, can realize high-flux plant screening, and can be applied to other gramineous crops, such as corn, rice, barley, wheat and other crops.
5. The equipment of the screening system can be repeatedly used, and compared with soil-based salt and alkali resistant screening, the matrix is more uniform and the accuracy is higher.
The indoor rapid identification and screening method for the saline-alkali tolerant oats can be suitable for the whole life cycle of oat screening, including flowering and maturation stages, can visually see plant morphological characteristics such as leaf color, leaf rolling, dead leaf apex, seedling death, root damage (growth and browning) and biomass, can timely harvest germplasm resources with high salt tolerance, avoids resource loss, and provides saline-alkali tolerant selection standards with different concentrations according to different selection targets on the basis of selection.
In conclusion, the indoor rapid identification and screening method for the saline-alkali tolerant oats has the advantages of high efficiency and accuracy in identifying and screening the saline-alkali tolerant oats, wide application range, recovery of germplasm resources with strong saline-alkali tolerance, and avoidance of resource loss.
Finally, it should be noted that: the above examples are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may be implemented or some technical features may be equivalently replaced in the specification, and the modifications or the replacements may not make the essence of the corresponding technical solutions depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. A method for indoor rapid identification and screening of saline-alkali tolerant oats is characterized by comprising the following steps:
pre-germinating oat seeds, selecting the oat seeds with full grains and uniform sizes, soaking the oat seeds in 0.8% sodium hypochlorite for 20 minutes, washing the oat seeds with sterile water for three times, placing the oat seeds on wet filter paper in a culture dish, and placing the culture dish in an incubator at 4 ℃ for dark culture for 48 hours;
germination treatment, namely putting the dark-treated seeds into sandwich imprinted filter paper, putting the sandwich imprinted filter paper on a support frame, putting the bottom of the filter paper in a tray filled with sterile distilled water, keeping the filter paper moist, transferring the filter paper into a light incubator at the temperature of 25 ℃ for germination, and taking out uniformly-developed seedlings when the first leaves grow out of coleoptiles and 3-8 roots after culturing for 5-6 days;
water culture seedling raising, namely taking out seedlings, wrapping the stems of the seedlings in sponge rings, inserting the sponge rings into a water culture system containing Hoagland's solution for culture, then putting the sponge rings into an illumination incubator for culture, setting the temperature to be 20/15 ℃ (day/night), and setting the light cycle to be 16 hours;
nursing the hydroponic plants, measuring the pH value of the hydroponic solution every day, 3 times every 2 days or every week, supplementing Hoagland's solution to the front of the initial test level line, and adjusting the pH value to 6;
salt treatment, namely applying salt and alkali to the seedlings in the full nutrient solution at the 2-3 leaf stage;
evaluating the saline-alkali resistance, evaluating on the 12 th day of saline-alkali treatment, observing and recording the expression symptoms of the oat under saline-alkali stress, wherein the expression symptoms comprise reduced leaf area, whitened lower leaves, dead leaf tips and leaf rolling, and obtaining the evaluation conclusion of the saline-alkali resistance of the seedlings according to the expression symptoms;
and (3) recovering saline-alkali tolerant strains, picking out seedlings with strong saline-alkali tolerant evaluation conclusion, keeping the roots of the seedlings intact, continuously growing the seedlings in a hydroponic system containing Hoagland's solution until the seedlings are mature, and harvesting seeds with strong saline-alkali tolerant performance.
2. The indoor rapid identification and screening method for salt and alkali tolerant oats according to claim 1, wherein in the pre-germination treatment step of oat seeds, less than or equal to 50 seeds are placed in the culture dish.
3. The indoor rapid identification and screening method for salt and alkali tolerant oats according to claim 1, wherein in the pre-germination treatment step of oat seeds, the seeds are placed on wet filter paper in a culture dish, and water on the wet filter paper cannot completely cover the seeds.
4. The indoor rapid identification and screening method for the saline-alkali tolerant oats according to any one of claims 1-3, wherein the water culture system comprises a test container, a support platform system, an aeration system and a culture solution, the test container is configured as a dark polyethylene rectangular box with an outer diameter of 40 x 20 x 10cm, the support platform system comprises a support platform and a sponge strip, the support platform is configured as a 50 x 25cm polyethylene rectangular box, 30 equidistant holes are drilled on the support platform, the sponge strip is configured as a 5 x 3cm rectangular box, the aeration system comprises an air pump and an aeration pipe, the air pump is connected to a central annular pipe, and two ends of the central annular pipe are provided with a plurality of 30cm long steel pipes with holes through hoses.
5. The indoor rapid identification and screening method of saline-alkali tolerant oats according to claim 4, wherein the saline-alkali tolerance evaluation step further comprises recording oat stem/root/whole plant weight, plant height and tiller number indexes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115005034A (en) * | 2022-06-14 | 2022-09-06 | 江苏艾津作物科技集团有限公司 | Device and method for identifying stress resistance of exogenous biostimulant to rice seedling stage |
| CN115119734A (en) * | 2022-07-25 | 2022-09-30 | 河北省农林科学院滨海农业研究所 | Method for rapidly identifying salt tolerance of plants |
| CN116704356A (en) * | 2023-08-08 | 2023-09-05 | 吉林省农业科学院 | Corn salt and alkali tolerance identification method and system based on convolutional neural network |
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