Background
At present, in the aspect of plant seed initiation technology, seed is mainly subjected to initiation treatment through a plurality of chemical reagent aqueous solutions. If plant hormone and analogues, potassium permanganate, hydrogen peroxide, calcium chloride, potassium nitrate, polyethylene glycol (PEG) and other substances are used for seed soaking, the germination and seedling emergence of the seeds are promoted in order by improving the vigor, the water absorption capacity and other aspects of the seeds, and the effect of increasing the yield is further achieved.
Although, these existing methods of initiation are capable of enhancing the germination capacity of seeds to some extent, as well as increasing the resistance of plants to abiotic and biotic stresses during the germination phase. However, these methods have disadvantages, such as a salt reagent such as calcium chloride and potassium nitrate, which is a substance having stress capability for seed germination, and which can inhibit the subsequent growth of plants when the dosage is not controlled, a chemical reagent such as hydrogen peroxide and potassium permanganate, which has strong oxidizing property and can decompose in a short time, but the oxidizing capability may cause a damage effect on seeds which is difficult to reverse when applied, and a common plant hormone such as gibberellic acid, which has a slightly efficient characteristic but has a high synthetic cost due to a complex chemical structure, and is not commonly used in agricultural production.
The chemical control technique has space-time limitation, and the same chemical control means has different or even completely opposite effects on different plant growth periods, such as poor plant growth vigor, growth promotion of auxin chemical substances, inhibition of germination during germination, inhibition of growth inhibition of auxin chemical substances, promotion of seed germination by gibberellin chemical substances, inhibition of flower bud differentiation, and delay of flowering time. The above example is the same and the results are exactly opposite, so that the person skilled in the art will not trade off the existing chemical control technique for other growth phases. The prior published technical patent document CN105076136B, namely the novel application of melatonin in improving the resistance of plants to waterlogging stress and the periodical document, namely the influence of melatonin soaking seeds on the growth of rice seedlings under flooding stress, illustrate the application of melatonin in resisting waterlogging in the growth process of the seedlings, and aiming at the condition that the germination rate is obviously reduced after the seeds are flooded, how to initiate the treatment of the seeds to quickly and efficiently recover the phenotype, and the prior study is very few.
In addition, the related published patent document technology has certain defects and shortcomings that the published patent needs to be subjected to one technical treatment for two days, and the total treatment time is 5-10 times, namely the total treatment time is at least 10 days, and the total treatment time can reach 20 days. Objectively, flooding can cause irreversible damage to plants in a short period of time (hours to days), and the disclosed patent cannot save damage caused by flooding events in a short period of time. In the sowing period, when unforeseen flooding disasters (such as storm, damage of water conservancy facilities and artificial accidents) occur, the person skilled in the art needs to complete sowing preparation within a few hours or a few days without the time required by the technology to be disclosed for advance preparation, and furthermore, the technical means such as drainage and the like can not remove flooding caused by ponding to apply objective climate, especially when sowing is performed in a short time caused by temperature change, the person skilled in the art can not delay the sowing time until the sowing is completed according to the technology of the disclosed patent under objective conditions. Accordingly, the prior art has certain drawbacks and limitations.
Disclosure of Invention
The invention aims to provide a method for promoting the germination of crop seeds under flooding stress so as to solve the problems existing in the prior art, the invention adopts melatonin aqueous solution to trigger crop seeds, has the advantages of quick aging, low cost and good effect, and provides a new idea for resisting waterlogging stress and promoting germination and growth of the crop seeds.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for promoting germination of crop seeds under flooding stress, which comprises the step of initiating the crop seeds by adopting melatonin aqueous solution before sowing.
Further, the concentration of the melatonin aqueous solution is 10-50 mu M, and the initiation time is 1-6h.
Further, the concentration of the melatonin aqueous solution is 30 mu M, and the initiation time is 6 hours.
Further, the initiation is carried out by completely submerging the crop seeds with the melatonin aqueous solution, and the liquid level is 1-3cm above the crop seeds.
Further, the crop seeds are normally harvested, naturally air-dried, uniform in size, full in grain and disease-free.
Further, the crop seeds include soybean seeds.
The invention discloses the following technical effects:
In the existing initiation technology for promoting germination and emergence, other additional operations such as back drying and the like are needed after initiation, the number of the additional operations is more than several days and less than several hours, and long waiting time is still needed under the condition that actual agricultural production needs 'rushed planting' and other rush time. The research of the invention discovers that the soybean seeds initiated by melatonin do not need to be subjected to back drying and other treatments, and can directly resist flooding stress and promote the germination capacity of the soybean seeds under flooding.
Compared with the initiation modes of salt, potassium permanganate and the like, the melatonin has the advantages of wide application dosage and no obvious damage to the plants at the seed and seedling stages by initiation treatment. In addition, the optimum concentration to be searched for in the present invention is low in cost (about 4-membered (water-free cost) in preparing 100L of melatonin solution), and has the advantage of low cost and good effect.
The invention also explores the mechanism of melatonin in the germination process of seeds, researches show that the melatonin improves the activity of the flooded soybean seeds, the melatonin initiates and improves the synthesis of endogenous melatonin in the soybean seeds at the molecular level, the synthesis of abscisic acid is inhibited and the synthesis of gibberellin is promoted in the germination process of the seeds initiated by the melatonin, meanwhile, the damage of cell membranes is reduced in the melatonin initiating process, the enzyme activities of peroxidase and catalase in the germination process of the seeds are lower than those of the non-initiated flooded soybean seeds, and the malondialdehyde content in the soybean seeds initiated by the melatonin is lower than that of the non-initiated flooded soybean seeds. Multiple factors together promote germination under flooding stress of soybean seeds.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
The invention adopts a mode of triggering seeds by aqueous solution to improve the germination rate of the seeds under the condition of flooding, and the main operation process is as follows:
and selecting soybean seeds which are normally harvested, naturally air-dried and stored, uniform in size, full in grain and free of diseases. As shown in fig. 1, the following processes are performed:
1. the dry seeds were germinated directly in a petri dish containing 20mL of water (where the water level was approximately 1/2 of the seed), denoted CK20;
2. The dry seeds were germinated directly in petri dishes containing 45mL of water (at which time the water completely floods the seeds), denoted CK45;
The method comprises the steps of initiating dry seeds in water, taking out the dry seeds, directly placing the seeds in 20mL of water for germination, marking the seeds as H20, initiating the dry seeds in water, taking out the dry seeds, directly placing the seeds in 45mL of water for germination, marking the seeds as H45, initiating the dry seeds in an aqueous solution containing melatonin, taking out the dry seeds, directly placing the seeds in 45mL of water for germination, marking the seeds as MT45. Wherein the initiation operation is that the selected seeds are placed in a beaker with the volume of 250mL, distilled water or aqueous solution containing melatonin is added until the liquid level completely drops out of the upper section of the seeds and is at least 1cm higher than the uppermost end of the seeds. Through preliminary studies, melatonin priming for 6 hours was optimal, with an optimal concentration of 30 μm. The following are all presented with melatonin priming for 6 hours.
The soybean seeds initiated by aqueous solution (pure water, melatonin aqueous solution) and normal, non-initiated soybean seeds were germinated in different waters. The specific operation is that the normal soybean seeds which are not subjected to initiation treatment and 20 soybean seeds which are subjected to water initiation are respectively placed in a 9cm culture dish paved with double-layer filter paper, and different water amounts are added into the culture dish (the water amount for normal germination is 20mL per dish, and the flooding stress is 45 mL). And counting the germination rate of the seeds in the process of seed swelling.
As shown in the result of FIG. 2, the germination rate of normal soybean seeds is remarkably reduced under the condition of flooding and almost no germination occurs (CK 45), the phenotype that the seeds cannot germinate under the condition of flooding cannot be recovered by water initiation (H45), and the germination rate of the soybean seeds under the condition of flooding can be greatly improved by melatonin initiation (MT 45). In fig. 2a is a phenotype graph of soybean seed germination at different water levels and after different treatments, and B is statistical data. The test is carried out in 9cm culture dishes, 20 soybean seeds which are uniform in size and free of obvious diseases and insect pests are selected from each culture dish, different pretreatment is carried out, different water is added, statistical data are obtained after germination is finished (120 hours after swelling) and photographing is carried out.
Example 2
The related principles and mechanisms are studied as follows:
1. The experimental process comprises the following steps:
Soybeans subjected to various treatments were germinated under normal water or flooded water for 6 hours, soybeans with complete appearance were selected, cut along the umbilicus direction, and an aqueous solution (ph=7.4) containing 1% (w/v) of TTC (2, 3, 5-triphenyltetrazolium chloride, CAS No. 298-96-4, purchased from Shanghai source foliar organisms, cat No. S19026) was added and the seeds were completely submerged. After treatment for 12 hours in the dark at 25 ℃, the seeds were rinsed with sterile water until there was no change in red color after the liquid was blotted with paper. Shooting was performed using a single-lens reflex camera (nikon D3500) (a in fig. 3).
Seeds after germination (120 hours of imbibition) were counted for radicle length (B in FIG. 3) by peeling all radicle of the germinated seeds with a scalpel, shooting the radicle length with a single-phase camera, and counting each radicle length using the "Analyze" option in the software "ImageJ" software. After completion, excel was used for data processing, including the average of radicle length, standard error of radicle length, etc. And the significance analysis was performed by SPSS software. Similarly, the radicles of all germinated seeds were weighed for the radicles of the samples (fig. 3C) using a precision balance (certolis, BSA 223S) and analyzed by data processing using Excel and SPSS software. The above "radicle length" and "radicle weight" refer to germinated seeds, and the non-germinated seeds are not counted, and the length or weight of all non-germinated seeds is recorded as "0".
Subsequently, the seeds were subjected to gene expression analysis. Firstly, 8 seeds (selection standard: no injury, full seeds, seeds with appearance representative of the moment) of each of the dry seeds, the melatonin-initiated seeds at 3 hours and the melatonin-initiated seeds at 6 hours are selected, ground into fine powder in liquid nitrogen, and the Beijing full type gold biology company is used "PLANT RNAKIT "kit (cat# ER 311), for extracting total RNA from soybean seeds, the extraction method is completely according to the kit instruction. Subsequently, beijing full gold Co "The One-Step gDNARemoval AND CDNA SYNTHESIS Supermix "kit (product number: AT 311) was subjected to the operations of genome removal and reverse transcription, and the method of operation was completely in accordance with the kit instructions. After completion, a fluorescent quantitative PCR experiment was performed. The reference gene is GmACT11, and the primer sequence is F ATCTTGACTGAGCGTGGTTATTCC and R GCTGGTCCTGGCTGTCTCC. The quantitative gene GmCOMT, the primer F CTTCTGTCCTTCCAATGATCCTCA and the primer R ACGGTCCAACATAACGGGTG. The quantitative gene GmASMT, the primer F AATGTGCGATTGAGCTTGGC and the primer R TTGAGAATAGCCCCTGCTGC. The enzyme used for performing fluorescent quantitative PCR was "Beijing full gold Co., ltd"GREEN QPCR Supermix "(cat# AQ 601), the method of use is fully according to instructions. The instrument was StepOnePlusTM Real-TIME PCR SYSTEM from Applied Biosystems. The experimental results are shown in FIG. 4A-B.
Then, soybean seeds of 0, 3, 6 and 9 hours during germination of the seeds were selected, and gene expression change detection during germination was performed (fig. 5). RNA extraction, genome DNA removal, reverse transcription and qPCR are performed as described above, the internal reference gene is GmACT11, the internal reference primer sequences are as described above, and the other primer sequences are shown in Table 1.
TABLE 1 primers used in experiments
Finally, the malondialdehyde content of the peroxidase, catalase active agent during germination was determined (fig. 6). The experiments are all determined by using a Beijing Soy Bao technology Co., ltd. The experimental procedure was performed fully in accordance with the instructions of the peroxidase kit (cat No. BC 0090), the catalase kit (cat No. BC 0205), the Malondialdehyde (MDA) kit (cat No. BC 0025). The quantitative microplate reader was BioTek company Synergy Neo2. The results are shown in FIG. 6A-C.
2. Experimental results
As shown in fig. 2-3, the germination rate and seed vigor of normal soybean seeds are significantly improved under the flooding condition after melatonin initiation. From fig. 2, it can be seen that the germination rate of the seed induced by water is significantly improved in the case of flooding, while the root length and fresh weight are significantly improved (B-C in fig. 3). The experiment adopts 9cm culture dishes, each dish selects 20 healthy seeds without obvious diseases and insect pests, different water is added, and germination rate is counted in the germination process. FIG. 2A is a photograph showing phenotypes of aged seeds after germination in different water with and without an initiation treatment, wherein "CK20" and "H20" are phenotypes of normal seeds after germination in 20mL water with and without an initiation treatment, respectively, "CK45" is a phenotypes of non-initiated soybean seeds after germination in 45mL water, "H45" is a phenotypes of initiated soybean seeds after germination in 45mL water, and "MT45" is a phenotypes of melatonin-initiated soybean seeds after germination in 45mL water. Data statistics of radicle length and radicle weight after germination of different treated seeds in different waters are shown in fig. 3B and fig. 3C, respectively.
As shown in fig. 4, endogenous melatonin synthesis genes GmCOMT and GmASMT gene expression were up-regulated in melatonin-induced soybean seeds. This result shows that melatonin priming can induce synthesis of endogenous melatonin in plants, so that the available melatonin content of plants is further increased, and soybean seeds can further undergo seed germination processes.
As shown in FIG. 5, in the germination process of soybean seeds under different treatments, the negative regulation hormone abscisic acid synthesis and signal of seed germination and the positive regulation hormone gibberellin negative regulation transcription factor, synthesis and other related genes are changed. Specifically, in untreated materials subjected to flooding stress, the expression of abscisic acid synthesis and signal related genes is up-regulated compared with that of a control group, and the abscisic acid synthesis (GmNCED, gmNCED 9) and the signal related genes (GmABI 5) of soybean seeds are down-regulated compared with that of the control group through melatonin. Among gibberellin-related genes, melatonin-induced treatment up-regulates expression of gibberellin-synthesizing genes (GmKAO, gmGA3ox1 genes) and down-regulates expression of gibberellin negative-control transcription factor genes (GmRGL genes). The result shows that after the normal soybean is subjected to melatonin initiating treatment, the synthesis of gibberellin is promoted, the synthesis of abscisic acid is inhibited, and the biological process of seed germination is promoted at the hormone level.
As shown in fig. 6, the melatonin-induced biofilm damage of soybean seeds was lower. Compared with normal soybeans under flooding stress, the enzyme activities of the soybean seed peroxidase and the catalase initiated by melatonin are lower than those of the soybean seeds which are not initiated and germinate under flooding stress by water, which indicates that the seeds have fewer peroxides under flooding stress. Peroxide is capable of oxidizing and destroying cell membranes, while malondialdehyde is an indicator of the extent to which cell membranes are destroyed, the higher the malondialdehyde content, the greater the cell membrane damage. According to the invention, the malondialdehyde content is further detected, and the melatonin-induced seeds are found to have less cell damage under flooding stress. The melatonin reduces the damage of flooding stress to cell membranes, maintains the integrity of the cell membranes, and further promotes the germination process of seeds.
In summary, melatonin-initiated soybean seeds were able to resist flooding stress, promoting germination of soybean seeds under flooding (fig. 2), as melatonin increased vigor of the flooded soybean seeds (fig. 3). At the molecular level, melatonin triggers soybean seeds to improve germination under the flooding stress of the seeds, the melatonin triggers to improve the synthesis of endogenous melatonin in the soybean seeds (figure 4), the synthesis of abscisic acid is inhibited in the germination process of the seeds triggered by the melatonin, the synthesis of gibberellin is promoted (figure 5), and meanwhile, the melatonin triggers to reduce the damage of cell membranes, the enzyme activities of peroxidase and catalase in the germination process of the seeds are lower than those of the non-triggered flooded soybean seeds, and the malondialdehyde content in the soybean seeds triggered by the melatonin is lower than those of the non-triggered flooded soybean seeds (figure 6). Multiple factors together promote germination under flooding stress of soybean seeds.
In this embodiment, soybean seeds are selected as the seed material, and other crop seeds, such as cucumber, wheat, and mucuna pruriens, may also be selected as the seed material.
The use method of the melatonin for inducing the germination of plant seeds is inconsistent with the prior art, the growth period of the used plant is inconsistent, and the person skilled in the art cannot associate or routinely adjust the germination method according to the prior experimental experience and the prior art, so that the technical operation of the germination method is obtained, and the germination method is adjusted and used according to the prior achievements, the experimental experience and the prior art, and unexpected risks (such as yield reduction, harvest failure and the like) exist. Therefore, the technical scheme of the invention should be protected by the technology in the patent.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.