CN114303766B - Application of indoleacetic acid in cultivation of thornless lycium ruthenicum - Google Patents
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Abstract
The invention discloses an application of indoleacetic acid in cultivation of thornless lycium ruthenicum; the application is that indoleacetic acid is applied to the top buds of branches of the black fruit matrimony vine plants with branches; the concentration of the indoleacetic acid is 0.1 mg/L; the mode of applying the indoleacetic acid is dropwise adding; experimental results show that after the thorn 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 IAA is treated with the terminal bud of the thorn branch for 15 days, 69.2 percent of new branches are in a completely thorn-free state on average; the invention has the advantages that: the operation is simple, the economic forest cultivation is facilitated, and the later picking efficiency of cultivated thornless lycium ruthenicum can be improved.
Description
Technical Field
The invention belongs to the technical field of forest biology, and particularly relates to application of indoleacetic acid in cultivation of thornless lycium ruthenicum.
Background
Lycium ruthenicum MurrLycium ruthenicumMurr.) is a wild excellent plant with economic and ecological benefits, integrates water and soil conservation value, medicinal value and saline-alkali soil greening value, and is mainly distributed in northwest regions and middle-asia desert regions of China. The plant is mainly distributed in Qinghai, ningxia, xinjiang and other places in China, is an ideal plant for preventing and controlling soil desertification and relieving soil salinization in the desert areas of China due to the physiological characteristics of drought resistance and salt and alkali resistance, is beneficial to wind prevention and sand fixation, and has important significance for ecosystems and agriculture in arid areas.
Lycium ruthenicum is known as "soft gold". The fruit is purple black spherical berries, has sweet fruit taste, and contains protein, lycium barbarum polysaccharide, essential fatty acids of human body, 8 essential amino acids of human body and a certain amount of Vc. The lycium ruthenicum contains rich chemical components, mainly comprises organic acid, flavonoid compounds (high in anthocyanin content) and some lycium ruthenicum polysaccharide, and the physiological active components comprise anthocyanin, OPC, polysaccharide, flavone and polyphenol substances. The fruit of lycium ruthenicum also contains more reducing sugar and pigment, and is a natural wild plant with highest procyanidine content discovered so far. Procyanidins are bioflavonoids with special molecular structures, belong to natural anthocyanins, can be absorbed and utilized by human bodies, and can remove free radicals in the human bodies, thereby playing 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 researching the toxicological safety of the pigment of the lycium ruthenicum as the edible pigment, the pigment is found to belong to non-toxic substances, and has better edible safety. The pigment has strong tinting strength, good stability, simple processing technology, great development prospect and development value, and can be widely applied to the medicine, food and light spinning industries.
The lycium ruthenicum is mainly distributed in arid areas in northwest of China, and has the characteristics of drought resistance, barren resistance, strong ecological adaptability and high propagation speed. The thorns of Lycium ruthenicum are branch thorns. 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 stems), the fruit peel is extremely thin, in the artificial picking process, the picking efficiency is greatly reduced, the picking cost is greatly improved due to the generation of the branches, and finally, the market price of the lycium ruthenicum is extremely high. The cultivation of the thornless variety of the lycium ruthenicum is feasible and the thornless variety is easier to be cultivated as economic forest, so that the cultivation of the excellent thornless lycium ruthenicum has great significance.
Disclosure of Invention
The invention aims to solve the problem of low picking efficiency caused by thorn of lycium ruthenicum, and provides an application of indoleacetic acid in cultivation of thorn-free lycium ruthenicum.
The application of indoleacetic acid in culturing thornless lycium ruthenicum;
the application comprises the steps of applying indoleacetic acid to branches of lycium ruthenicum plant;
the application is that indoleacetic acid is applied to the top buds of branches of the lycium ruthenicum plant;
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 to directly drop the indoleacetic acid to terminal buds or mix the indoleacetic acid with lanolin and then smear the mixture on the terminal buds;
the dosage of the direct dropwise adding indoleacetic acid is 10 -6 mg；
The indoleacetic acid is mixed with lanolin, and 2 ml indoleacetic acid is mixed with 1 g lanolin.
The invention provides an application of indoleacetic acid in cultivation of thornless lycium ruthenicum; the application is that indoleacetic acid is applied to the top buds of branches of the black fruit matrimony vine plants with branches; the concentration of the indoleacetic acid is 0.1 mg/L; the mode of applying the indoleacetic acid is dropwise adding; experimental results show that after the thorn 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 IAA is treated with the terminal bud of the thorn branch for 15 days, 69.2 percent of new branches are in a completely thorn-free state on average; the invention has the advantages that: the operation is simple, the economic forest cultivation is facilitated, and the later picking efficiency of cultivated thornless lycium ruthenicum can be improved.
Drawings
Fig. 1: the arrow mark in the picture is the nascent stem segment after 0.1mg/L IAA treatment.
Description of the embodiments
Example 1 cultivation of Lycium ruthenicum Experimental Material
The experimental material is a basin seedling of a lycium ruthenicum tissue culture clone planted by the Shenyang agricultural university forest college (41 degrees 49 '25' 'N; 123 degrees 34' 10'' E, altitude 25 m), the experimental material is obtained by transplanting G series tissue culture seedlings which are cultivated in advance of the subject tissue culture, the experimental material is planted in a basin with the diameter of 15 cm, and the soil amount of each basin is about 2/3 of that of the basin.
Selecting a group G series seedling of a clone with a height of more than 7 cm and a thick root, taking out from a group culture bottle, washing a culture medium at the root in water, and then soaking the culture medium in carbendazim (Shanxi star pesticide Co., ltd., active ingredient content: 80% and wettable powder) with a concentration of 1G/L for 30 min. The nutrient substrate for the Van horticultural seedling culture of Shenyang agricultural university (patent number: ZL03133591.8; PH:6.5-6.8; total content of N and P, K is not less than 12 g/kg; water content is not more than 40%, organic matter content is not less than 40%, silicon is not less than 0.3 g/kg) is placed for one night and used after being cooled, 1/3 of the substrate of the basin is placed in the basin, a proper amount of water is added to moisten the substrate but not agglomerate, and then the substrate is added to 1/2 of the basin. A small pit with the width of about two fingers and the depth of 6-7 cm is dug in the matrix, the root of the tissue culture seedling is fully contacted with the matrix, is spirally wound in the small pit, the root is fully buried by the matrix, and is irrigated with 1 g/L carbendazim. The tissue culture Miao Yong transplanted into the basin is perforated and ventilated, and is reversely buckled in a disposable plastic cup, and then placed into a soil culture room (512 of the university of Shenyang agriculture, forest college; the temperature is 25+/-1 ℃ and the humidity is 64+/-1%) for seedling reviving. Watering is carried out for 2-3 days, the water quantity is 100-130 ml each time, and the plastic cup is uncovered according to the transplanting seedling state in 7-12 days.
After seedling is recovered in the soil culture room, the experimental materials are placed on the sunlight sufficient window side of the forest college 217 of Shenyang agricultural university to adapt to the experimental environment, and the experimental materials can be used for experiments after being adapted to the environment for one week. The experiment is carried out in 5-9 months, the time period is that the daytime air temperature is 30+/-5 ℃, the night air temperature is 21+/-5 ℃, the indoor sunlight duration is 12+/-2 h, and the humidity is 55+/-5%, and the experiment is a growth vigorous stage of lycium ruthenicum. According to the climate conditions, the watering period is set to be that water is watered once every three days, and the watering amount of each basin is 100-130 ml. Pruning branches with poor growth vigor at regular intervals, and selecting plants with similar growth vigor and with or without thorns as experimental materials for treatment.
Example 2 measurement of endogenous hormone
And taking the terminal buds of branches with thorn/no thorn characteristics and consistent growth stages as experimental materials, and measuring endogenous hormones by using a high performance liquid chromatography-tandem mass spectrometry method.
1. Instrument, reagent and material
High performance liquid chromatography-tandem mass spectrometry used Volterray ACQUITY H-class UPLC and XEVO-TQD at the analysis test center of Shenyang agricultural university.
50 ml 80% methanol (BHT containing 1 mmol/L) preparation method: the BHT (Nanjing Dulai Biotechnology Co., ltd., product number: R0492-50 g) required for 50.011 g was weighed out, 20 ml of 100% methanol was used for dissolving BHT, and after the BHT was completely dissolved, the remaining 30 ml of 100% methanol was added with stirring to prevent precipitation of BHT. And (3) completely dissolving the BHT in methanol, adding 10 ml distilled water to complete the preparation, and preserving the prepared reagent at normal temperature in a dark place.
The empty centrifuge tube is weighed and recorded in advance, and placed in liquid nitrogen for precooling. And placing the precooled centrifuge tube in liquid nitrogen, shearing terminal buds from the selected plants, and placing the terminal buds into the centrifuge tube for quick freezing. Placing about 50-60 terminal buds with the same characteristics into each centrifuge tube, weighing, calculating the weight of each group of samples, wherein the weight of each group of samples is equal to or greater than 100mg, and storing at-80 ℃ for later use.
2. Hormone extraction method
100mg of sample is taken out and fully ground by an adaptive grinding rod in a 1.5ml centrifuge tube pre-cooled by liquid nitrogen, and the centrifuge tube is continuously placed in the liquid nitrogen to keep a low-temperature environment during the grinding process.
Adding 80% methanol (containing 1m mol/L BHT) precooled by 0.6. 0.6 ml after grinding, leaching at 4deg.C in the dark for 12 h, centrifuging at 4deg.C in a centrifuge for 15 min at 4000 r/min; separating the supernatant to a 2 ml centrifuge tube after centrifugation; 0.2. 0.2 ml precooled methanol (containing 1m mol/L BHT) at 80 ℃ is added to the precipitate for leaching 2 h, and the two supernatants are combined and fixed to a volume of 1 ml.
Diluting the supernatant with deionized water in a ratio of 1:1, wherein the diameter of the supernatant is 25 to mm; the organic phase filter with a pore size of 0.22 μm was filtered and tested according to the standard of 1 ml for each sample.
3. Chromatographic-mass spectrometry conditions
1. Liquid phase
Chromatographic column: agilent Eclipse C18 2.1 mm by 50 mm,1.8 μm
Mobile phase: column a: 0.01% formic acid water;
b column: methanol
Flow rate: 0.3 ml/min
Gradient: 15% -57.5% of B in 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 sample volume was 3. Mu.l.
2. Mass spectrometry
Note that: method 1: an electrospray ion source; positive ion mode (esi+); capillary voltage: 0.8 KV; taper hole voltage: 20V; desolventizing temperature: 650. the temperature is lower than the temperature; desolventizing gas flow rate: 1000 L/Hr; reversely blowing by the taper hole: 3L/Hr. The method 2 comprises electrospray ion source; negative ion mode (ESI-); capillary voltage: 2.2 KV; taper hole voltage: 25: 25V; desolventizing temperature: 650. the temperature is lower than the temperature; desolventizing gas flow rate: 1000 L/Hr; reversely blowing by the taper hole: 3L/Hr.
4. Results
Endogenous hormone measurement shows that the content of the growth hormone IAA of the non-branch thorn terminal bud is extremely higher than that of the branch thorn terminal bud, and the SA content of the non-branch thorn terminal bud is remarkably higher than that of the branch thorn terminal bud; the content of terminal bud IAA and SA was inversely related to the occurrence of thorns (Table 1) (IAA: indoleacetic acid; SA: salicylic acid; ABA: abscisic acid);
note that: the data in the table are the average value of three repeated experiments plus or minus standard error, the difference between the same row of data marked with different capital letters is extremely remarkable (P < 0.01), and the difference between the same row of data marked with different lowercase letters is remarkable (P < 0.05);
example 3 effects of auxin IAA and auxin inhibitor PCIB on the occurrence of Lycium ruthenicum branch thorns.
1. Mother liquor of auxin IAA and auxin inhibitor PCIB and preparation of different concentration gradients
The concentration gradient of the applied auxin IAA (product number: DE 0265-10 g, purity. Gtoreq.98%) and the auxin inhibitor PCIB (product number: C830260-100 g, purity 98%) was set according to the measured content of the endogenous auxin IAA in the terminal buds of Lycium ruthenicum. From the measurement of endogenous auxin IAA, the auxin IAA content was inversely related to the occurrence of thorns, and therefore, exogenous application of auxin IAA to the thorn-bearing Lycium ruthenicum plant was performed to verify whether auxin IAA inhibited the formation of thorns of Lycium ruthenicum. The auxin inhibitor PCIB was applied to the branch-free Lycium ruthenicum plants to verify whether low auxin promoted branch-thorn production. The concentrations of auxin IAA applied to the barbed plants were set at 0.2mg/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: setting the concentration of the auxin IAA mother solution to be 0.1 mg/ml according to the concentration gradient measurement result of the experimental auxin IAA; diluting the mother solution, preparing IAA solution 10 ml with each concentration, and preparing auxin IAA concentration gradient: 0.2mg/L was diluted from 20. Mu.l of mother liquor to 10 ml distilled water, 0.1mg/L was diluted from 10. Mu.l of mother liquor to 10 ml distilled water, 0.02 mg/L was diluted from 2. Mu.l of mother liquor to 10 ml distilled water, and 0.01 mg/L was diluted from 1. Mu.l of mother liquor to 10 ml distilled water. The mother liquor concentration of the auxin inhibitor PCIB was 0.01 mg/ml. The concentration gradient of the auxin inhibitor PCIB was formulated to be 0.132. Mu.M diluted from 28.3. Mu.l of mother liquor to 10 ml distilled water, 1.32. Mu.M diluted from 283. Mu.l of mother liquor to 10 ml distilled water, and 13.2. Mu.M diluted from 2830. Mu.l of mother liquor to 10 ml distilled water.
2. Treatment of Lycium ruthenicum Murr with auxin IAA and auxin inhibitor PCIB
The method comprises the following steps: and (3) sucking 10 mu l of the auxin IAA and the auxin inhibitor PCIB with the concentration gradient each time by a maximum range of 10 mu l of a pipetting gun, slowly dripping the auxin IAA and the auxin inhibitor PCIB at the top buds of branches of selected materials (the auxin IAA is exogenously applied to the lycium ruthenicum plant without the auxin inhibitor PCIB applied to the lycium ruthenicum plant), enabling the applied reagent to remain at the top buds in a water drop state, adding the reagent at 16 pm every day, and adding equal amount of distilled water at the top buds by a control plant in the same method. When the terminal buds are treated by the auxin IAA and the auxin inhibitor PCIB for 15 days in the method, morphological indexes of plants are measured.
The second method is as follows: the auxin IAA or auxin inhibitor PCIB with the concentration is mixed with lanolin (Shanghai microphone Biotechnology Co., ltd., product number: L812569-500 g) in a ratio of 2:1 (1 g lanolin: 2 ml IAA or PCIB solution), the mixed lanolin is smeared on the terminal buds, the lanolin can cover the terminal buds thinly, and the lanolin is applied to the terminal buds of single branches in an amount of 0.01 g +/-0.005 g. In order to prevent the growth of the terminal buds from being blocked, 0.002-0.005 g of lanolin with the same concentration as that of the first treatment is added again every 7-10 days when lanolin at the terminal buds of the plants is completely absorbed and no transparent grease exists. At 15 days of the first lanolin treatment, the plants were subjected to morphological index determination.
3. Morphological index detection
The length of the plant branches, the number of stem nodes, the average length of stem nodes, the length of thorns, the average leaf width, the average leaf length, the diameter of the terminal buds and the length and diameter of thorns were measured and recorded before and after the treatment using a vernier caliper and a tape measure.
4. Data analysis
Data sorting and preliminary analysis were performed using Microsoft Office Excel on the measured data, significant difference analysis was performed using SPSS 20.0 on shoot data with significant change in shoot phenotype, and analysis was performed on different data amounts using independent sample T-test and single factor (LSD).
5. Results
1. Effect of auxin IAA on the thorn-out Rate of Lycium ruthenicum Murr
According to the result of endogenous auxin IAA, three concentration gradients are set to treat the thorn branches with auxin IAA. The results showed that the incidence of thorns was significantly lower in all the treatment groups than in the control group and that the treatment results with 0.1mg/L IAA were significantly better than in the other treatment groups (Table 2). 0.1 When 15 days of mg/L IAA treated with the terminal buds of the barbed branches, the probability of thorn emergence of the new nodes (the number of thorns of the new nodes/the number of total new nodes) was 17.46%, which is significantly lower than that of the control treated group (79.84%) (Table 2). It is worth mentioning that in this experiment, when 0.1mg/L IAA is treated with the terminal bud of the thorn branch for 15 days, the average probability of the new branch being completely thorn-free is 50%. In addition, no significant change in the thorn phenotype was found in the 0.01 mg/L IAA treated group;
note that: the data in the table are the average value of three repeated experiments plus or minus standard error, the difference between the same column of data marked with different capital letters is extremely remarkable (P < 0.01 and LSD), and the difference between the same column of data marked with different lowercase letters is remarkable (P < 0.05 and LSD).
According to the results, the optimal application concentration of the exogenous auxin IAA for changing the thorn incidence rate of the thorn branches is 0.1 mg/L. Thus, subsequent experiments with this concentration repeated treatment of the barbed shoots showed that when the barbed shoots were treated with 0.1mg/L auxin IAA for 15 days, the barbed rate of the new shoots was reduced to 19.25% and this probability was significantly lower than 68.92% prior to treatment. After the IAA treatment, the proportion of the new branches to be completely free from thorns reaches 69.2 percent;
note that: the data in the table are the average value of three repeated experiments plus or minus standard error, the difference between the same column of data marked with different capital letters is extremely remarkable (P < 0.01), and the difference between the same column of data marked with different lowercase letters is remarkable (P < 0.05).
2. Effect of inhibitor PCIB on the shoot emergence rate of thorn-free plants of Lycium ruthenicum
When the non-thorn branches are treated by 13.2 mu M auxin inhibitor PCIB for 15 days, the thorn emergence rate of the new branches is increased from 0% of the control to 35.13%. No significant promoting effect was observed after treatment with other concentrations of PCIB;
note that: the data in the table are the average value of three repeated experiments plus or minus standard error, the difference between the same column of data marked with different capital letters is extremely remarkable (P < 0.01), and the difference between the same column of data marked with different lowercase letters is remarkable (P < 0.05).
In conclusion, the concentration of IAA in the terminal buds of the thorn-free branches of the same clone of lycium ruthenicum is obviously lower than that of the terminal buds of the thorn-free branches; exogenous application of auxin IAA at a proper concentration can significantly inhibit thorns of the asexual lines of lycium ruthenicum and can lead to complete thornless of the newly developed stem segments of thorn branches; exogenous application of the auxin inhibitor PCIB at appropriate concentrations can promote shoot penetration in non-shoots, but the effect on the penetration phenotype is less pronounced than IAA treatment. The above results demonstrate that exogenous application of auxin IAA is effective in inhibiting the development of Lycium ruthenicum branch thorn.
3. Effect of various treatments on Lycium ruthenicum plant and shoot growth
In addition to focusing on the effect of various treatments on the thorn yield of Lycium ruthenicum, we also determined the effect of various treatments on the growth of Lycium ruthenicum branches and thorns. After 0.1mg/L auxin IAA is added to the branches with the branches, the growth rate of the branches with the branches is obviously reduced compared with that of a control group with the branches (table 5); following treatment of the thornless plants with 13.2 μm auxin inhibitor PCIB, the above aspects were significantly elevated compared to the thornless control group (table 5). From this, it can be obtained that the auxin IAA concentration has a significant negative correlation with the shoot growth rate and the plant growth rate;
note that: the data in the table are the average value of three repeated experiments plus or minus standard error, the difference between the same column of data marked with different capital letters is extremely remarkable (P < 0.01 and LSD), and the difference between the same column of data marked with different lowercase letters is remarkable (P < 0.05 and LSD).
The measurement of the increment of the terminal bud node is to measure and mark the diameter of the terminal bud of the plant before the treatment, and after the experimental period is ended, the remained node is marked as the terminal bud before the treatment. And measuring the diameter of the node, and obtaining the increment of the terminal bud node by the difference of the diameter and the diameter.
In addition, similar negative correlations were also seen with respect to elongation of the nodes and stem thickness in other morphological indexes of lycium ruthenicum (see table 6);
note that: the data in the table are the average value of three repeated experiments plus or minus standard error, the difference between the same column of data marked with different capital letters is extremely remarkable (P < 0.01 and LSD), and the difference between the same column of data marked with different lowercase letters is remarkable (P < 0.05 and LSD).
However, in analysis of measurement results of other morphological indexes such as leaf, no significant correlation with auxin IAA was found.
Claims (1)
1. The method for cultivating thornless lycium ruthenicum by using indoleacetic acid is characterized by comprising the following steps of:
ss1, preparing indoleacetic acid with the concentration of 0.1 mg/L;
ss2, dropwise adding indoleacetic acid to the terminal buds of the lycium ruthenicum plant, or mixing the indoleacetic acid with lanolin and then coating the mixture on the terminal buds of the lycium ruthenicum plant;
the dosage of the directly added indoleacetic acid is 10 mu L;
the indoleacetic acid is specifically mixed with lanolin: 2 ml indoleacetic acid was mixed with 1 g lanolin;
after the barbed shoots were treated with 0.1. 0.1mg/L indoleacetic acid, the barbed rate was reduced from 68.92% to 19.25%, wherein the average 69.2% of new shoots had been in a completely non-barbed state when the barbed shoots were treated with indoleacetic acid for 15 days.
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| 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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