Application of KAR1 in regulation and control of apple root development

Abstract

The invention discloses application of KAR1 in regulation and control of apple root development. The chemical name of the KAR1 is 3-methyl 2H-furo [2, 3-c] pyran-2-ketone, and the CAS number of the KAR1 is 857054-02-5. The Gala apple tissue culture seedling strain is cultured in the rooting culture medium containing KAR1, and it is found that 1 nM KAR1, 5 nM KAR1, 50 nM KAR1 and 500 nM KAR1 can remarkably enhance root development of the apple tissue culture seedling, growth of main roots and lateral roots of apples is promoted, and a theoretical basis is laid for field application of KAR1.

Description

Technical Field

[0001] The present invention relates to the fields of plant biotechnology and biochemistry, and in particular to the application of KAR1 in regulating the development of apple root system. Background Art

[0002] KARs (Karrikins) are a new type of butenolide plant growth regulators present in the smoke of burning plant materials. Their mechanism of being specifically recognized by plants and activating resistance responses is widely present in the plant kingdom and is biologically active at extremely low concentrations.

[0003] From 2004 to 2009, six different molecular structures of Karrikins were discovered and their chemical structures were analyzed. They were named KAR1-KAR6 according to the order of their discovery. The biological activities of various molecules are different. KAR1 was first discovered in 2004. In terms of chemical structure, the six different KARs all contain a five-membered lactone ring and a six-membered heterocycle. The main difference is the different positions of the methyl groups on the six-membered heterocycle.

[0004] At present, the research on the biological function of KARs mainly focuses on regulating the germination of plant seeds and photomorphogenesis. In previous studies, it was found that KARs can promote the germination of seeds of species such as Arabidopsis, wild oats, early brome, gray brome, southern chrysanthemum, wild mallow, poppy, white mustard, stone grass and chickweed. But it is worth noting that in addition to promoting seed germination, KARs also have an inhibitory physiological effect on the seed germination process of some species, such as the germination process of seeds of species such as galium, shepherd's purse, poor brome and large-eared oats is inhibited by KARs. The functions of the same KARs molecule in different species are not necessarily the same. For example, KAR2 can promote the germination of Arabidopsis seeds, but KAR2 has a significant inhibitory effect on the germination of soybean seeds. However, there is no report on what function KARs have in apples. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to regulate the development of apple root system.

[0006] In order to solve the above technical problems, the present invention first provides a new use of KAR1.

[0007] The present invention provides the use of KAR1 in any of the following a1) or a2): a1) Promote the development of apple root system; a2) Preparing products for promoting root development of apples.

[0008] Furthermore, the promoting the development of apple root system is promoting the development of apple taproot and / or lateral root.

[0009] Furthermore, the promotion of the development of the main root and lateral roots of apple is manifested in increasing the relative number of apple roots and / or the total root length.

[0010] In order to solve the above technical problems, the present invention provides an apple rooting medium.

[0011] The apple rooting medium provided by the invention is a rooting medium containing KAR1.

[0012] Further, the concentration of KAR1 in the rooting medium may be 1-500 nM or 1-50 nM or 1-5 nM or 5-500 nM or 5-50 nM or 50-500 nM.

[0013] The concentration of KAR1 in the rooting medium is specifically 1 nM, 5 nM, 50 nM or 500 nM.

[0014] The concentration of KAR1 in the rooting medium is preferably 1 nM.

[0015] Furthermore, the apple rooting medium is a rooting medium containing KAR1 and IAA.

[0016] The concentration of the IAA in the rooting medium is specifically 0.1 mg / L.

[0017] Furthermore, the rooting medium is 1 / 2MS medium. The solvent of the 1 / 2MS culture medium is water, and the solutes and their concentrations are: 950 mg / L potassium nitrate; 825 mg / L ammonium nitrate; 85 mg / L potassium dihydrogen phosphate; 185 mg / L magnesium sulfate heptahydrate; 115 mg / L calcium chloride; 0.83 mg / L potassium iodide; 6.2 mg / L boric acid; 17 mg / L manganese sulfate monohydrate; 8.6 mg / L zinc sulfate heptahydrate; 0.25 mg / L sodium molybdate dihydrate; 0.025 mg / L copper sulfate pentahydrate; 0.025 mg / L cobalt chloride hexahydrate; 37.3 mg / L disodium ethylenediaminetetraacetic acid; 27.8 mg / L ferrous sulfate heptahydrate; 100 mg / L inositol; 2 mg / L glycine; 0.1 mg / L thiamine hydrochloride; 0.5 mg / L pyridoxine hydrochloride; 0.5 mg / L nicotinic acid; 30 g / L sucrose; and 7 g / L agar. The pH of the 1 / 2MS medium is 5.80.

[0018] In a specific embodiment of the present invention, the apple rooting medium consists of IAA, KAR1 and 1 / 2MS medium.

[0019] In order to solve the above technical problems, the present invention also provides a new use of the above apple rooting medium.

[0020] The present invention provides the use of the apple rooting medium in any of the following a1) or a2): a1) Promote the development of apple root system; a2) Preparing products for promoting root development of apples.

[0021] Furthermore, the promoting the development of apple root system is promoting the development of apple taproot and / or lateral root.

[0022] Furthermore, the promotion of the development of the main root and lateral roots of apple is manifested in increasing the relative number of apple roots and / or the total root length.

[0023] In order to solve the above technical problems, the present invention finally provides a method for promoting the development of apple root system.

[0024] The method for promoting apple root development provided by the present invention comprises the following steps: culturing apple tissue culture seedlings in the apple rooting medium.

[0025] In the above method, the culture conditions are a temperature of 25° C., a photoperiod of 16 hours of light / 8 hours of darkness, and a light intensity of 2000-3000 lx.

[0026] In the above method, the culturing time may be at least 58 days, specifically 58 days.

[0027] The method also includes the step of subculturing the apple tissue culture seedlings.

[0028] Furthermore, the subculture comprises the following steps: cutting off the tissue containing the growth point and 2-3 cotyledons from the apple tissue culture seedling, inserting the lower stem segment of the tissue into the MS subculture medium (3-4 mm) for culture, and subculturing once every 30 days.

[0029] Furthermore, the solvent of the subculture medium is water, and the solutes and their concentrations are: potassium nitrate 1900 mg / L; ammonium nitrate 1650 mg / L; potassium dihydrogen phosphate 170 mg / L; magnesium sulfate heptahydrate 370 mg / L; calcium chloride 330 mg / L; potassium iodide 0.83 mg / L; boric acid 6.2 mg / L; manganese sulfate monohydrate 17 mg / L; zinc sulfate heptahydrate 8.6 mg / L; sodium molybdate dihydrate 0.25 mg / L; copper sulfate pentahydrate 0.025 mg / L; cobalt chloride hexahydrate 0.025 mg / L; disodium ethylenediaminetetraacetic acid 37.3 mg / L; ferrous sulfate heptahydrate 27.8 mg / L; inositol 100 mg / L; glycine 2 mg / L; thiamine hydrochloride 0.1 mg / L; pyridoxine hydrochloride 0.5 mg / L; nicotinic acid 0.5 mg / L; 6-BA 0.5 mg / L; IAA 0.2 mg / L; sucrose 30 g / L; agar 7 g / L. The pH of the subculture medium is 5.80.

[0030] In the above method, the apple tissue culture seedlings are apple tissue culture seedlings 'GL-3'.

[0031] Any of the above apples is a 'Gala' apple.

[0032] The structural formula of any of the above-mentioned KAR1 is shown in Formula I, the chemical name is 3-methyl 2H-furo[2,3-c]pyran-2-one, the molecular formula is C8H6O3, and the CAS number is 857054-02-5.

[0033]

[0034] Formula Ⅰ.

[0035] The present invention uses the 'Gala' apple tissue culture seedling strain, which is currently widely used and has relatively stable genetic traits, as the test material, adds KAR1 into an ordinary rooting medium for treatment, and finds that 1 nM, 5 nM, 50 nM, and 500 nM KAR1 can significantly enhance the root development of apple tissue culture seedlings and promote the occurrence of main roots and lateral roots, which lays a theoretical foundation for the field application of KAR1. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effect of KAR1 on the development of taproot and lateral roots of apple tissue culture seedlings.

[0037] Figure 2 The effect of KAR1 on the relative number and total root length of apple tissue culture seedlings. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] The 'Gala' apple tissue culture seedling 'GL-3' in the following example is recorded in the document "Hu DG, Sun CH, ZhangQ Y, Gu KD, Hao Y J. The basic helix-loop-helix transcription factor MdbHLH3modulates leaf senescence in apple via the regulation of dehydratase-enolase-phosphatase complex 1[J]. Horticultural Research, 2020, 7: 50."

[0041] The structural formula of KAR1 in the following embodiments is shown in Formula I, the chemical name is 3-methyl 2H-furo[2,3-c]pyran-2-one, the molecular formula is C8H6O3, the CAS number is 857054-02-5, and it is a product of Canadian TRC Chemical Company with the product number M305480.

[0042]

[0043] Formula Ⅰ.

[0044] The solvent of the MS subculture medium in the following examples is water, and the solutes and their concentrations are: potassium nitrate 1900 mg / L; ammonium nitrate 1650 mg / L; potassium dihydrogen phosphate 170 mg / L; magnesium sulfate heptahydrate 370 mg / L; calcium chloride 330 mg / L; potassium iodide 0.83 mg / L; boric acid 6.2 mg / L; manganese sulfate monohydrate 17 mg / L; zinc sulfate heptahydrate 8.6 mg / L; sodium molybdate dihydrate 0.25 mg / L; copper sulfate pentahydrate 0.025 mg / L; cobalt chloride hexahydrate 0.025 mg / L; disodium ethylenediaminetetraacetic acid 37.3 mg / L; ferrous sulfate heptahydrate 27.8 mg / L; inositol 100 mg / L; glycine 2 mg / L; thiamine hydrochloride 0.1 mg / L; pyridoxine hydrochloride 0.5 mg / L; nicotinic acid 0.5 mg / L; 6-BA 0.5 mg / L; IAA 0.2 mg / L; sucrose 30 g / L; agar 7 g / L. Adjust pH to 5.80, sterilize by high pressure steam (121℃, 20min), and use after cooling.

[0045] The solvent of the 1 / 2MS culture medium in the following examples is water, and the solutes and their concentrations are: potassium nitrate 950 mg / L; ammonium nitrate 825 mg / L; potassium dihydrogen phosphate 85 mg / L; magnesium sulfate heptahydrate 185 mg / L; calcium chloride 115 mg / L; potassium iodide 0.83 mg / L; boric acid 6.2 mg / L; manganese sulfate monohydrate 17 mg / L; zinc sulfate heptahydrate 8.6 mg / L; sodium molybdate dihydrate 0.25 mg / L; copper sulfate pentahydrate 0.025 mg / L; cobalt chloride hexahydrate 0.025 mg / L; disodium ethylenediaminetetraacetic acid 37.3 mg / L; ferrous sulfate heptahydrate 27.8 mg / L; inositol 100 mg / L; glycine 2 mg / L; thiamine hydrochloride 0.1 mg / L; pyridoxine hydrochloride 0.5 mg / L; nicotinic acid 0.5 mg / L; sucrose 30 g / L; agar 7 g / L. Adjust pH to 5.80, sterilize by high pressure steam (121°C, 20 min), and use after cooling.

[0046] Example 1: Effect of KAR1 on rooting of apple tissue culture seedlings 1. Cultivation of apple tissue culture seedlings The 'Gala' apple tissue culture seedling 'GL-3' was subcultured using MS subculture medium. The specific steps were as follows: the tissue containing the growth point and 2-3 cotyledons was cut from the 'Gala' apple tissue culture seedling 'GL-3' in a sterile clean bench, and the lower stem segment of the tissue was inserted into the MS subculture medium 3-4 mm, and cultured at a temperature of 25°C, a photoperiod of 16 h light / 8 h dark, and a light intensity of 2000-3000 lx. The subculture was performed every 30 days.

[0047] 2. Effect of KAR1 on rooting of tissue culture seedlings of 'Gala' apple Gala apple tissue culture seedlings 'GL-3' were cultured in rooting medium under the following conditions: temperature of 25°C, photoperiod of 16 h light / 8 h dark, and light intensity of 2000-3000 lx.

[0048] The rooting medium was a 1 / 2MS medium containing KAR1 and IAA, wherein the concentration of IAA in the 1 / 2MS medium was 0.1 mg / L. The cells were divided into the following groups according to the different concentrations of KAR1 in the 1 / 2MS medium: 0 nM group: The concentration of KAR1 in 1 / 2MS medium was 0 nM.

[0049] 1 nM group: The concentration of KAR1 in 1 / 2MS medium was 1 nM.

[0050] 5 nM group: The concentration of KAR1 in 1 / 2MS medium was 5 nM.

[0051] 50 nM group: The concentration of KAR1 in 1 / 2MS medium was 50 nM.

[0052] 500 nM group: The concentration of KAR1 in 1 / 2MS medium was 500 nM.

[0053] After 58 days of cultivation, photos were taken to record the phenotype. Image J (version 1.52V) was used to calculate the root area in the image by the scale bar, and the calculated root area reflected the relative number of roots. WinRHIZO (version 2013) was used to calculate the total root length. Microsoft Excel software was used to calculate significant differences (Student's t-test, * indicates a significant difference, P < 0.05; ** indicates an extremely significant difference, P < 0.01). Three replicates were set for each treatment, and the results were averaged.

[0054] The results are as follows Figure 1 and Figure 2 The results showed that KAR1 treatment significantly promoted the development of taproot and lateral root system of Gala seedlings ( Figure 1). Compared with the 0 nM group, the relative number of roots and total root length in the 1 nM group, 5 nM group, 50 nM group, and 500 nM group increased significantly ( Figure 2 ). The average relative number of roots in the 0 nM group, 1 nM group, 5 nM group, 50 nM group, and 500 nM group were 2.11±0.26, 6.44±2.28, 4.66±0.81, 6.25±0.84, and 6.87±2.86, respectively, and the differences were significant or extremely significant; the average total root length in the 0 nM group, 1 nM group, 5 nM group, 50 nM group, and 500 nM group were 24.06±5.02, 56.53±13.41, 42.78±5.62, 48.21±6.09, and 49.85±12.69, respectively, and the differences were significant.

[0055] The above results show that different concentrations of KAR1 significantly promoted the root development of the main root and lateral root of apple tissue culture seedlings.

[0056] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of KAR1 in any of the following a1) or a2): a1) Promote the development of apple root system; a2) Preparing products for promoting root development of apples.

2. The use according to claim 1, characterized in that: The promoting the development of apple root system is promoting the development of apple taproot and / or lateral root.

3. The use according to claim 2, characterized in that: The promotion of the development of the main root and lateral roots of apple is manifested in increasing the relative number of apple roots and / or the total root length.

4. An apple rooting medium, wherein the apple rooting medium is a rooting medium containing KAR1.

5. The apple rooting medium according to claim 4, characterized in that: The concentration of KAR1 in the rooting medium is 1-500 nM.

6. The apple rooting medium according to claim 4 or 5, characterized in that: The apple rooting medium consists of IAA, KAR1 and 1 / 2MS medium.

7. Use of the apple rooting medium according to any one of claims 4 to 6 in any one of the following a1) or a2): a1) Promote the development of apple root system; a2) Preparing products for promoting root development of apples.

8. The use according to claim 7, characterized in that: The promoting the development of apple root system is promoting the development of apple taproot and / or lateral root.

9. The use according to claim 8, characterized in that: The promotion of the development of the main root and lateral roots of apple is manifested in increasing the relative number of apple roots and / or the total root length.

10. A method for promoting the development of apple root system, comprising the following steps: culturing apple tissue culture seedlings in the apple rooting medium according to any one of claims 4 to 6.

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