CN114303766A - Application of indoleacetic acid in cultivating spine-free lycium ruthenicum murr - Google Patents
Application of indoleacetic acid in cultivating spine-free lycium ruthenicum murr Download PDFInfo
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Abstract
The invention discloses an application of indoleacetic acid in cultivating spine-free lycium ruthenicum; the application comprises the steps of applying indoleacetic acid to terminal buds of branches of Lycium ruthenicum Murr plants with branches; the concentration of the indoleacetic acid is 0.1 mg/L; the indole acetic acid is applied in a dropping mode; the experimental result shows that after the barbed branches are treated by 0.1mg/L auxin IAA, the thorn yield is reduced from 68.92% to 19.25%, and the thorn yield is obviously reduced. Wherein, when the terminal bud of the branch with thorns is treated by IAA for 15 days, 69.2 percent of newly-sent branches are completely in a thornless state on average; the invention has the advantages that: the method is simple to operate, is beneficial to cultivation of economic forest trees, and the cultivated spine-free lycium ruthenicum murr can improve later-stage picking efficiency.
Description
Technical Field
The invention belongs to the technical field of forest biology, and particularly relates to application of indoleacetic acid in cultivating thornless lycium ruthenicum.
Background
Lycium ruthenicum Murr (Lycium ruthenicum Murr.) (Lycium ruthenicum Murr.) Is a wild excellent plant with economic benefit and ecological benefit, integrates water and soil conservation value, medicinal value and saline-alkali soil greening value, and is mainly distributed in northwest ChinaRegions and desert regions of central asia. China is mainly distributed in Qinghai, Ningxia, Xinjiang and the like, is an ideal plant for preventing and controlling soil desertification and relieving soil salinization in the desert regions of China due to the physiological characteristics of drought resistance and salt and alkali resistance, is also favorable for wind prevention and sand fixation, and has important significance for the ecological system and agriculture of arid regions.
The black fruit medlar is known as 'soft gold'. The fruit is purple black spherical berry, has sweet taste, and contains protein, lycium barbarum polysaccharide, essential fatty acid for human body, 8 essential amino acids for human body and a certain amount of Vc. The lycium ruthenicum contains rich chemical components, mainly comprises organic acid, flavonoid compounds (with high anthocyanin content) and some lycium ruthenicum polysaccharides, and the physiological active components comprise anthocyanin, OPC, polysaccharide, flavone and polyphenol substances. The lycium ruthenicum fruit also contains more reducing sugar and pigment, and is a natural wild plant with the highest procyanidine content found so far. The procyanidin is a bioflavonoid mixture with a special molecular structure, belongs to natural anthocyanin, can be absorbed and utilized by a human body, and can remove free radicals in the human body, thereby having the physiological functions of improving immunity, reducing blood fat and blood pressure, resisting aging, treating cardiovascular and cerebrovascular diseases and the like. In addition, after the toxicological safety of the pigment of the lycium ruthenicum as the edible pigment is researched, the pigment of the lycium ruthenicum is found to be a non-toxic substance and has better edible safety. The pigment has strong tinting strength, good stability and simple processing technology, has great development prospect and development value, and can be widely applied to the industries of medicines, foods and light textiles.
The lycium ruthenicum murr is mainly distributed in arid regions in the northwest of China, and has the characteristics of drought resistance, barren resistance, strong ecological adaptability and high propagation speed. The spine of Lycium ruthenicum Murr is Branch spine. The lycium ruthenicum has high economic value, because wild or artificially planted lycium ruthenicum branches are densely covered with a large number of branches (also called as stem spines) under the drought condition of soil, and the fruit skin of the fruits is extremely thin, the production of the branches greatly reduces the picking efficiency and greatly improves the picking cost in the artificial picking process, and finally the market price of the lycium ruthenicum is extremely high. The method for cultivating the lycium ruthenicum murr with no thorns is feasible and the lycium ruthenicum murr with no thorns is easier to be cultivated in economic forest trees, so that the method for cultivating the excellent lycium ruthenicum murr with no thorns has great significance.
Disclosure of Invention
The invention aims to solve the problem of low picking efficiency caused by thorn of lycium ruthenicum murr, and provides application of indoleacetic acid in cultivating the thorn-free lycium ruthenicum murr.
Application of indoleacetic acid in cultivating the lycium ruthenicum murr without thorn;
the application comprises the steps of applying indoleacetic acid to branches of lycium ruthenicum murr plants;
the application comprises the steps of applying indoleacetic acid to terminal buds of branches of Lycium ruthenicum Murr plants;
the concentration of the indoleacetic acid is 0.02-0.2 mg/L;
the concentration is 0.1 mg/L;
the method for applying the indoleacetic acid is directly dropwise adding to the terminal bud or smearing the mixture of the indoleacetic acid and lanolin on the terminal bud;
the direct dropwise addition is carried out, and the dosage of the indoleacetic acid is 10-6mg；
The indole acetic acid is mixed with lanolin, and 2 ml of the indole acetic acid is mixed with 1 g of the lanolin.
The invention provides an application of indoleacetic acid in cultivating the lycium ruthenicum murr without thorn; the application comprises the steps of applying indoleacetic acid to terminal buds of branches of Lycium ruthenicum Murr plants with branches; the concentration of the indoleacetic acid is 0.1 mg/L; the indole acetic acid is applied in a dropping mode; the experimental result shows that after the barbed branches are treated by 0.1mg/L auxin IAA, the thorn yield is reduced from 68.92% to 19.25%, and the thorn yield is obviously reduced. Wherein, when the terminal bud of the branch with thorns is treated by IAA for 15 days, 69.2 percent of newly-sent branches are completely in a thornless state on average; the invention has the advantages that: the method is simple to operate, is beneficial to cultivation of economic forest trees, and the cultivated spine-free lycium ruthenicum murr can improve later-stage picking efficiency.
Drawings
FIG. 1: the newly born stem sections after 0.1mg/L IAA treatment are marked above the arrows in the pictures.
Detailed Description
Example 1 cultivation of Lycium ruthenicum Murr test Material
The experimental material is a potted seedling of a lycium ruthenicum tissue culture clone planted by Shenyang agriculture university college of forestry (41 degrees 49 '25' 'N; 123 degrees 34' 10 '' E, altitude 25 m), the material is obtained by transplanting G series tissue culture seedlings cultured in the early stage of the subject group, the experimental material is planted in a pot with the diameter of 15 cm, and the soil amount of each pot is about 2/3 of the pot.
Selecting tissue culture clone G series seedlings with height over 7 cm and thick roots, taking out from a tissue culture bottle, washing the culture medium of the roots in water, and then putting the seedlings into carbendazim (Shanxi Qixing pesticide Co., Ltd., effective component content: 80%, wettable powder) with concentration of 1G/L for soaking for 30 min. 'Shenyang agricultural university Van yu gardening seedling raising nutrition substrate' (patent number: ZL 03133591.8; PH: 6.5-6.8; total N, P, K content is more than or equal to 12 g/kg; water content is less than or equal to 40%; organic matter content is more than or equal to 40%; silicon is more than or equal to 0.3 g/kg) sterilized at 121 ℃ under high pressure for 40 min is placed for cooling overnight for use, the substrate in a 1/3 basin is placed in the basin, a proper amount of water is added to moisten the substrate but not to agglomerate, and then the substrate is added into 1/2 of the basin. Digging a small pit with the width of about two fingers and the depth of 6-7 cm in the substrate, fully contacting the root of the tissue culture seedling with the substrate, rotating the rotating disc to wind the root therein, completely burying the root with the substrate, and irrigating with 1 g/L carbendazim. The tissue culture seedlings transplanted into the pot are turned upside down by a perforated and breathable disposable plastic cup and put into an earth culture room (Shenyang agriculture university forest institute 512; temperature 25 +/-1 ℃ and humidity 64 +/-1%) for seedling recovery. Watering is carried out for 2-3 days, the water amount is 130 ml in 100-.
After the seedlings are released in the soil culture period, the experimental materials are placed beside a sunny window of Shenyang agriculture university college of forestry 217 to adapt to the experimental environment, and can be used for experiments after one week of adaptation to the environment. The experiment is carried out in 5-9 months, the time period is that the temperature is 30 +/-5 ℃ in the daytime, the temperature is 21 +/-5 ℃ at night, the indoor sunshine duration is 12 +/-2 hours, the humidity is 55 +/-5 percent, and the growth stage of the lycium ruthenicum is in a vigorous growth stage. According to the climatic conditions, the watering period is defined as watering once in three days, and the watering amount of each pot is 100-130 ml. Pruning branches with poor growth vigor regularly, and selecting plants with thorny/non-thorny characters with similar growth vigor as experimental materials for treatment during experiments.
EXAMPLE 2 determination of endogenous hormones
Taking terminal buds of branches with thorns/no thorns and with consistent growth and development stages as experimental materials, and determining endogenous hormones by using a high performance liquid chromatography-tandem mass spectrometry method.
Instrument, reagent and material
High performance liquid chromatography-tandem mass spectrometry uses Wovert ACQUITY H-class UPLC and XEVO-TQD from Shenyang university of agriculture analysis test center.
50 ml of 80% methanol (containing 1 mmol/L BHT) was prepared: 0.011 g of BHT (product number: R0492-50 g, Nanjing Tolyai Biotechnology Co., Ltd.) in an amount of 50 ml required for the reagent was weighed, 20 ml of 100% methanol was used to dissolve BHT, and the remaining 30 ml of 100% methanol was added to the reagent while stirring after BHT was completely dissolved, to prevent precipitation of BHT. And (3) adding 10 ml of distilled water into the BHT completely dissolved in the methanol to complete preparation, and storing the prepared reagent at normal temperature in a dark place.
Weighing the empty centrifuge tube in advance, recording, and placing the empty centrifuge tube into liquid nitrogen for precooling. Placing the precooled centrifuge tube in liquid nitrogen, cutting terminal buds from the selected plants, and placing the terminal buds into the centrifuge tube for quick freezing. And (3) putting 50-60 terminal buds with the same character into each centrifugal tube, weighing, calculating the weight of each sample, and storing at-80 ℃ for later use, wherein the weight of each sample is more than or equal to 100 mg.
Second, hormone extraction method
And taking out 100mg of the sample, fully grinding the sample in a 1.5ml centrifugal tube pre-cooled by liquid nitrogen by using an adaptive grinding rod, and continuously placing the centrifugal tube in the liquid nitrogen to keep a low-temperature environment in the grinding process.
Grinding, adding 0.6 ml of precooled 80% methanol (containing 1m mol/L BHT), extracting at 4 ℃ in a dark place for 12 h, and centrifuging at 4000 r/min for 15 min at 4 ℃ by a centrifuge; centrifuging and separating supernatant to 2 ml centrifuge tube; 0.2 ml of pre-cooled methanol (containing 1m mol/L BHT) at 80 ℃ was added to the precipitate and leached for 2 h, and the two supernatants were combined and made to volume of 1 ml.
Diluting the supernatant with deionized water at a ratio of 1:1, wherein the diameter of the supernatant is 25 mm; the organic phase filter having a pore size of 0.22 μm was filtered and the detection was carried out according to a standard of 1 ml per sample.
Third, chromatographic-mass spectrum condition
1. Liquid phase
A chromatographic column: agilent Eclipse C182.1 mm 50 mm, 1.8 μm
Mobile phase: column A: 0.01% formic acid water;
column B: methanol
Flow rate: 0.3 ml/min
Gradient: 15% -57.5% B for 0-10 min;
10-15min 57.5% -100% B
15-16min 100%-15% B
16-21min 15% B
the column temperature was 30 ℃ and the injection volume was 3. mu.l.
2. Mass spectrometry
Note: the method comprises the following steps: an electrospray ion source; positive ion mode (ESI +); capillary voltage: 0.8 KV; taper hole voltage: 20V; desolventizing temperature: 650 ℃; desolventizing air flow rate: 1000L/Hr; back blowing of the taper hole: 3L/Hr. Method 2 includes electrospray ion source; negative ion mode (ESI-); capillary voltage: 2.2 KV; taper hole voltage: 25V; desolventizing temperature: 650 ℃; desolventizing air flow rate: 1000L/Hr; back blowing of the taper hole: 3L/Hr.
Fourthly, the result
Endogenous hormone determination shows that the content of the auxin IAA of the terminal bud of the ramose spurge is remarkably higher than that of the terminal bud of the ramose spurge, and the content of SA of the terminal bud of the ramose spurge is remarkably higher than that of the terminal bud of the ramose spurge; top bud IAA and SA contents were negatively correlated with cladosporium development (Table 1) (IAA: indolylacetic acid; SA: salicylic acid; ABA: abscisic acid).
Note: the data in the table are the mean values ± standard errors of three repeated experiments, the difference between the data of the same column marked with different capital letters is very significant (P < 0.01), and the difference between the data of the same column marked with different small letters is significant (P < 0.05);
example 3 Effect of auxin IAA and the auxin inhibitor PCIB on the development of Lycium ruthenicum Murr Branch thorn
First, mother liquor of auxin IAA and auxin inhibitor PCIB and preparation of different concentration gradients
According to the measured content of the endogenous auxin IAA in the terminal bud of the lycium ruthenicum, the concentration gradients of the applied auxin IAA (Beijing Bayer Biotechnology Limited, the product number: DE 0265-10 g, the purity is more than or equal to 98%) and the auxin inhibitor PCIB (Shanghai Meclin Biotechnology Limited, the product number: C830260-100 g, the purity is 98%) are set. According to the determination of the endogenous auxin IAA, the content of the auxin IAA is negatively related to the generation of the spur, so that the auxin IAA is exogenously applied to the lycium ruthenicum plant to verify whether the auxin IAA inhibits the formation of the spur of the lycium ruthenicum. An auxin inhibitor PCIB was applied to Lycium ruthenicum Murr plants to verify whether low auxin promotes the production of spurs. The concentration of auxin IAA applied to the barbed plants was set to 0.2 mg/L, 0.1mg/L, 0.02 mg/L and 0.01 mg/L, respectively. The concentrations of auxin inhibitor PCIB applied to the thornless plants were 0.132. mu.M, 1.32. mu.M and 13.2. mu.M, respectively;
the specific method comprises the following steps: according to the determination result of the concentration gradient of the experimental auxin IAA, setting the concentration of the auxin IAA mother liquor to be 0.1 mg/ml; diluting the mother liquor, preparing 10 ml of IAA solution with each concentration, and preparing an IAA concentration gradient of auxin: 0.2 mg/L was diluted from 20. mu.l of the mother liquor into 10 ml of distilled water, 0.1mg/L was diluted from 10. mu.l of the mother liquor into 10 ml of distilled water, 0.02 mg/L was diluted from 2. mu.l of the mother liquor into 10 ml of distilled water, and 0.01 mg/L was diluted from 1. mu.l of the mother liquor into 10 ml of distilled water. The concentration of the mother liquor of the auxin inhibitor PCIB is 0.01 mg/ml. Concentration gradient of the auxin inhibitor PCIB 0.132. mu.M was diluted from 28.3. mu.l of stock solution to 10 ml of distilled water, 1.32. mu.M from 283. mu.l of stock solution to 10 ml of distilled water, 13.2. mu.M from 2830. mu.l of stock solution to 10 ml of distilled water.
Secondly, processing lycium ruthenicum by auxin IAA and auxin inhibitor PCIB
The method comprises the following steps: and sucking 10 mul of the auxin IAA and the auxin inhibitor PCIB with the concentration gradient from a liquid transfer gun with the maximum range of 10 mul each time, slowly dropwise adding the auxin IAA and the auxin inhibitor PCIB into the terminal bud of a branch of a selected material (an externally applied auxin IAA on a lycium barbarum plant with thorn and an externally applied auxin PCIB on a lycium barbarum plant without thorn), so that the applied reagent is remained at the terminal bud in a water drop state, adding the reagent at 16 pm every day, and adding the same amount of distilled water into the terminal bud by using the same method for a control plant. Morphological index measurements were performed on plants treated with auxin IAA and auxin inhibitor PCIB in the manner described above for 15 days.
The second method comprises the following steps: mixing the above-mentioned auxin IAA or auxin inhibitor PCIB with lanolin (Shanghai Michelin biotechnology Co., Ltd., product number: L812569-500 g) at a ratio of 2:1 (1 g lanolin: 2 ml IAA or PCIB solution), smearing the mixed lanolin on terminal bud, wherein the lanolin can cover the terminal bud thinly, and the amount of lanolin applied to single terminal bud is 0.01 g + -0.005 g. And in order to prevent the terminal bud from being hindered from growing, 0.002-0.005 g of lanolin with the concentration same as that of the first treatment is added again every 7-10 days when the lanolin at the terminal bud of the plant is completely absorbed and no transparent grease exists. The morphological index of the plants was measured 15 days after the first lanolin treatment.
Third, form index detection
Plant shoot length, stem node number, average stem node length, long stab node, average leaf width, average leaf length, apical bud diameter, and length and diameter of the stab were measured and recorded using vernier calipers and tape measures before and after treatment.
Fourth, data analysis
And (3) performing data sorting and preliminary analysis by using Microsoft Office Excel according to the measured data, performing significant difference analysis on branch data with significant branch surface change by using SPSS 20.0, and performing analysis and inspection on different data volumes by using independent sample T inspection and single factor (LSD).
Five results
1. Influence of auxin IAA on pricking rate of lycium ruthenicum
And setting three concentration gradients to carry out auxin IAA treatment on the barbed branches according to the result of the endogenous auxin IAA. The results show that the incidence of branch prick is significantly lower in all treatment groups than in the control group, and the treatment result of 0.1mg/L IAA is significantly better than that of other treatment groups (Table 2). When the top buds of the shoots with the pricks are treated by 0.1mg/L IAA for 15 days, the pricking probability of the new stem nodes (the number of the newly grown stem nodes/the number of the newly grown total stem nodes) is 17.46 percent, and the probability is obviously lower than the pricking rate (79.84 percent) of a control treatment group (Table 2). It is worth mentioning that in the experiment, when 0.1mg/L IAA is used for treating the terminal bud of the branch with thorns for 15 days, the average probability that the new branch completely has no thorns is 50%. In addition, no significant change in the stab type was observed in the 0.01 mg/L IAA treated group.
Note: the data in the table are the mean ± standard error of three replicates, with very significant differences (P < 0.01, LSD) between the same line of data labeled with different upper case letters and significant differences (P < 0.05, LSD) between the same line of data labeled with different lower case letters.
According to the results, the optimal applied concentration of the exogenous auxin IAA for changing the incidence rate of the barbed branches is 0.1 mg/L. Therefore, the subsequent experiments adopt the concentration to repeatedly treat the barbed branches, and the results show that the pricking rate of the new barbed branches is reduced to 19.25% after the barbed branches are treated by 0.1mg/L auxin IAA for 15 days, and the probability is obviously lower than 68.92% before treatment. After the treatment of the IAA with the concentration, the completely non-thorn ratio of the newly born branches reaches 69.2 percent.
Note: the data in the table are the mean ± sem of three replicates, with very significant differences between the data in the same row labeled with different capital letters (P < 0.01) and significant differences between the data in the same row labeled with different lowercase letters (P < 0.05).
2. Influence of inhibitor PCIB on new shoot emergence rate of lycium ruthenicum murr thornless plants
When the non-pricked branches are treated by 13.2 mu M auxin inhibitor PCIB for 15 days, the prick yield of the new-pricked branches is improved to 35.13 percent from 0 percent of the control. No significant promoting effect was observed after treatment with other concentrations of PCIB.
Note: the data in the table are the mean ± sem of three replicates, with very significant differences between the data in the same row labeled with different capital letters (P < 0.01) and significant differences between the data in the same row labeled with different lowercase letters (P < 0.05).
In conclusion, the concentration of IAA (IAA) of the terminal buds of the thorny branches of the same clone of the lycium ruthenicum is obviously lower than that of the terminal buds of the thorny-free branches; applying auxin IAA with proper concentration by an external source can obviously inhibit the generation of the clone spur of the lycium ruthenicum and can cause that a new stem section of the branch with the spur has no spur completely; exogenous application of the auxin inhibitor PCIB with proper concentration can promote branch thorns of the thornless shoots, but the effect on the thorny phenotype is not more obvious than that of IAA treatment. The results show that the externally applied auxin IAA can effectively inhibit the generation of the Lycium ruthenicum Murr spur.
3. Influence of various treatments on growth of Lycium ruthenicum Murr plants and branches
In addition to paying attention to the influence of various treatments on the spur output rate of lycium ruthenicum, the influence of various treatments on the growth of lycium ruthenicum branches and spurs is also determined. After 0.1mg/L auxin IAA is added into the branch with the spur, the growth rate of the spur and the growth rate of the whole branch are obviously reduced compared with the control group with the spur (Table 5); the thornless plants treated with 13.2 μ M of the auxin inhibitor, PCIB, were significantly elevated in all respects compared to the thornless control (Table 5). Therefore, the concentration of the auxin IAA has obvious negative correlation with the growth rate of the branch spines and the growth rate of plants.
Note: the data in the table are the mean ± standard error of three replicates, with very significant differences (P < 0.01, LSD) between the same line of data labeled with different upper case letters and significant differences (P < 0.05, LSD) between the same line of data labeled with different lower case letters.
The measurement of the apical bud node growth amount is to measure and mark the diameter of the apical bud of the plant before the treatment, and after the experimental period is finished, the marked and remained node is the position of the apical bud before the treatment. The diameter of the node is measured, and the difference between the two can be used to obtain the growth amount of the apical bud node.
In addition, similar negative correlations were observed for the elongation of the stem nodes and the stem thickness among other morphological indices of lycium ruthenicum (see table 6).
Note: the data in the table are the mean ± standard error of three replicates, with very significant differences (P < 0.01, LSD) between the same line of data labeled with different upper case letters and significant differences (P < 0.05, LSD) between the same line of data labeled with different lower case letters.
However, no significant correlation with auxin IAA was found in the analysis of the measurement results of other morphological indexes such as leaf.
Claims (7)
1. Application of indoleacetic acid in cultivating Lycium ruthenicum Murr.
2. Use according to claim 1, characterized in that: indoleacetic acid is applied to the branches of lycium ruthenicum plants.
3. Use according to claim 2, characterized in that: the concentration of the indoleacetic acid is 0.02-0.2 mg/L.
4. Use according to claim 3, characterized in that: the concentration is 0.1 mg/L.
5. Use according to claim 2, 3 or 4, characterized in that: the application is directly dripping the indoleacetic acid to the terminal bud or smearing the indolacetic acid and the lanolin after mixing.
6. Use according to claim 5, characterized in that: the direct dropwise addition is carried out, and the dosage of the indoleacetic acid is 10-6mg。
7. Use according to claim 5, characterized in that: the indole acetic acid is mixed with lanolin, and 2 ml of the indole acetic acid is mixed with 1 g of the lanolin.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105875411A (en) * | 2016-04-18 | 2016-08-24 | 黄博 | Method for cultivating thornless Lycium ruthenicum seedlings |
| CN106233880A (en) * | 2016-07-21 | 2016-12-21 | 宾川县杰熙林果专业合作社 | Breeding method without zanthoxylum acanthopodium |
| CN107223563A (en) * | 2017-05-04 | 2017-10-03 | 沈阳农业大学 | A kind of method for cultivating stingless black fruit fructus lycii |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105875411A (en) * | 2016-04-18 | 2016-08-24 | 黄博 | Method for cultivating thornless Lycium ruthenicum seedlings |
| CN106233880A (en) * | 2016-07-21 | 2016-12-21 | 宾川县杰熙林果专业合作社 | Breeding method without zanthoxylum acanthopodium |
| CN107223563A (en) * | 2017-05-04 | 2017-10-03 | 沈阳农业大学 | A kind of method for cultivating stingless black fruit fructus lycii |
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| 太田次郎, 中国农业大学出版社 * |
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