Preparation technology of special inoculation block for cistanche deserticola with function of self-inducing seed dormancy breaking

Abstract

The invention discloses a preparation technology of a special cistanche inoculation block with a seed dormancy breaking self-induction function by combining a cistanche seed dormancy breaking inducer and hydrogel, and relates to the technical field of cistanche cultivation. The specific technical process is as follows: preparation of a dormancy breaking inducer solution, preparation of a hydrogel block containing the dormancy breaking inducer and cistanche seeds, and preparation of an inoculation block. According to the method, gibberellin and cytokinin are used as dormancy breaking inducers, and a yeast aqueous extract is used as an auxiliary agent. The problems of seed dormancy breaking, germination promoting and water retention inoculation are effectively solved, seed dormancy breaking does not need to be conducted in advance before inoculation, meanwhile, hydrogel has high water absorption and water retention capacity, the problem that dormancy breaking seeds are inactivated due to the fact that the dormancy breaking seeds cannot germinate and parasitize in time after being inoculated due to water shortage is effectively solved, and the survival rate of the seeds is increased. The inoculation efficiency is improved to 75% or above from about 20% of existing inoculation paper inoculation, and the method has a good popularization prospect and has very important significance in promoting the industrial development of cistanche deserticola.

Description

Technical Field

[0001] The invention adopts gibberellins and cytokinins as dormancy-breaking inducers, yeast water extract as an adjuvant, sodium alginate-carboxymethyl cellulose hydrogel as a water retaining agent and dormancy-breaking inducer and a Cistanche deserticola seed carrier, and invents a preparation technology of a special Cistanche deserticola inoculation block capable of inducing dormancy-breaking of Cistanche deserticola seeds and directional growth of Haloxylon ammodendron roots, relating to the technical field of Cistanche deserticola cultivation. Background Art

[0002] Cistanche deserticola is a plant of the genus Cistanche in the family Orobanchaceae in the order Lamiaceae. The 2020 edition of the Chinese Pharmacopoeia lists both Cistanche deserticola YCMa and Cistanche tubulosa (Schenk) Wight as authentic medicinal materials, believing that they have the effects of tonifying kidney yang, replenishing essence and blood, and moistening the intestines and relieving constipation. In the medicinal material market, Cistanche deserticola is usually referred to as soft dayun and Cistanche tubulosa as hard dayun. In January 2020, the National Health Commission and the State Administration for Market Regulation issued the "Notice on Piloting the Management of Nine Substances, Including Codonopsis, as Traditional Food and Medicinal Materials." The notice clearly proposed desert Cistanche as a pilot variety for the production and operation of materials that are both traditional Chinese medicinal materials and food. In November 2023, the National Health Commission and the State Administration for Market Regulation officially issued a document confirming that Cistanche deserticola is a medicinal and edible substance. Since then, Cistanche deserticola can be legally used as a raw material in food production.

[0003] Cistanche deserticola is primarily grown in Inner Mongolia, Gansu, and Xinjiang. It parasitizes desert plants such as Haloxylon ammodendron and Tamarix chinensis, making it a parasitic psammophyte. The host of Cistanche deserticola is Haloxylon ammodendron, while the host of Cistanche tubulosa is Tamarix chinensis. With Cistanche deserticola's inclusion in the list of medicinal and edible plants, market demand has rapidly increased. Since both wild Haloxylon ammodendron and Cistanche deserticola are classified as Class II protected plants in China, their harvesting is prohibited. In recent years, with the implementation of the "Three Norths Shelterbelt Project" and other large-scale desertification control projects in Xinjiang and Inner Mongolia, the area of ​​artificial Haloxylon ammodendron forests has rapidly increased. The widespread adoption of artificial inoculation techniques to parasitize Cistanche deserticola in Haloxylon ammodendron forests has not only met consumer demand but also generated substantial economic benefits for the desertification control industry, significantly increasing the enthusiasm of residents in sandy areas to combat desertification.

[0004] However, a common problem with artificial inoculation of Cistanche deserticola is its low inoculation efficiency. This not only wastes seed resources, but also increases labor costs due to repeated inoculation, leading to rising planting costs. Improving inoculation efficiency has long been a technical bottleneck in artificial inoculation of parasitic Cistanche deserticola. Traditionally, inoculation methods use inoculation paper: Cistanche deserticola seeds are glued to a sheet of paper, such as newspaper or straw paper, with mud. The paper is then cut into 8-10 cm square pieces and buried at the base of a Haloxylon ammodendron tree. However, this inoculation technique has several inherent technical issues: First, the seeds must first be broken from dormancy. Cistanche deserticola seeds have a strong dormancy characteristic, requiring induction treatment. However, if the seeds do not germinate for a long time after being broken from dormancy, they lose vitality. Second, the seed quantity required is large, with approximately 200-300 seeds per sheet. Third, the inoculation paper does not retain water. While the use of water-inoculated seeding has improved inoculation rates in recent years, it remains at only around 20%. This is due to poor water retention in sand and limited water availability. Water-inoculated seeding requires only 500-1000 mL of water per hole. If the seeds fail to germinate within a short period of time, the water loss will leave them dehydrated. Upon germination, Cistanche deserticola seeds release allelopathic substances that induce Haloxylon ammodendron roots to grow toward the seeds. However, if the seeds fail to germinate or germinate only slightly, a strong induction effect is not achieved, and successful parasitism is unlikely, hindering successful seed-root contact. Consequently, increasing inoculation rates remains difficult.

[0005] To address the above problems, the present invention uses plant hormones as dormancy-breaking inducers, adds yeast water extract as an inducer, and uses sodium alginate and carboxymethyl cellulose as hydrogel raw materials to develop a technology for preparing hydrogel inoculation blocks for Cistanche deserticola inoculation. The prepared inoculation blocks have strong water absorption and water retention capabilities and can also induce dormancy-breaking of seeds, thus solving the two major technical bottlenecks of seed dormancy-breaking treatment and water retention to promote germination in artificial inoculation of Cistanche deserticola. The specific invention content is as follows: Summary of the Invention

[0006] The purpose of the present invention is to solve the problems of low inoculation efficiency, large seed dosage and high cost when inoculating Cistanche deserticola in artificial Haloxylon ammodendron forests. To achieve the above object, the specific technical solutions provided by the present invention are as follows:

[0007] Using gibberellins and cytokinins as dormancy-breaking inducers, yeast extract as an adjuvant, and sodium alginate-carboxymethyl cellulose hydrogel as a water-retaining agent and carrier for the dormancy-breaking inducer and seeds, we optimized the preparation conditions to prepare inoculation blocks that can induce seed dormancy-breaking, induce directional growth of Haloxylon ammodendron roots, and retain water. The specific preparation process is as follows:

[0008] Step 1: Preparation of sleep-breaking inducing agent solution

[0009] According to the results of previous experimental studies, the sleep-breaking inducer uses two plant hormones, gibberellin and cytokinin, and the adjuvant uses yeast water extract. The ratio of gibberellin to cytokinin is 2:1-4:1, and the concentration of gibberellin is 80-120 mg L -1 The concentration of cytokinin is 20-60 mg L -1 , the dosage of the additive is 8-12gL -1 According to the above conditions, the preparation method of 1L of sleep-destroying agent solution is to weigh 80-120mg of gibberellin and 20-60mg of cytokinin respectively, add them to 1L of water and fully dissolve them; then weigh 8-12g of yeast aqueous extract and add them to the above solution, stir at 200-300rpm and 40-60℃ on a heated magnetic stirrer for 10-20min, and then centrifuge at 800-1000×g for 10-15min.

[0010] The second step is the preparation of Cistanche deserticola seed hydrogel

[0011] The hydrogel is prepared using sodium alginate and carboxymethyl cellulose as raw materials. After experimental optimization, the optimal ratio of sodium alginate and carboxymethyl cellulose is 3:1-5:1, and the concentrations of sodium alginate and carboxymethyl cellulose are 1.2-2.5% and 0.4-0.5%, respectively. According to the above conditions, a hydrosol is first prepared by weighing 12-25g of sodium alginate and 4-5g of carboxymethyl cellulose respectively and adding them to 1L of the prepared sleep-breaking inducer solution, and stirring at 500-700rpm and 60-80°C on a heated magnetic stirrer for 10-20 minutes. After stirring, the aqueous solution is cooled to room temperature, 10-12mg of Cistanche deserticola seeds are weighed and added to the above solution, and stirred with a glass rod until the seeds are evenly distributed in the solution. Then, 8-12g of calcium chloride is added, and stirred at 1000-1200rpm on a magnetic stirrer for 5-10 minutes. The stirred solution is then quickly poured into a mold with a length, width, and height of 2cm, 2cm, and 4cm, respectively, and the solution is allowed to naturally solidify into a hydrogel.

[0012] The third step is the preparation of the inoculation block

[0013] The naturally solidified hydrogel block containing the sleep-breaking inducer and Cistanche deserticola seeds is taken out of the mold, and then dehydrated and solidified at 35-45° C. for 10-14 hours to allow the hydrogel block to dehydrate, become hard and elastic, and have a water content below 20%, and then can be used as an inoculation block.

[0014] The invention, based on previous research, uses gibberellins and cytokinins as dormancy-breaking inducers and yeast water extract as an auxiliary agent, optimizes their ratio, and achieves the best dormancy-breaking induction effect; in order to solve the water retention problem, uses sodium alginate and carboxymethyl cellulose as raw materials, optimizes the preparation technology of hydrogel with high water retention capacity, uses hydrogel as a water retention agent and dormancy-breaking inducer and a carrier for seeds, develops a preparation technology of a special inoculation block for Cistanche deserticola with the function of self-inducing seed dormancy-breaking, prepares a special inoculation block for Cistanche deserticola with the function of self-inducing seed dormancy-breaking, and provides a new inoculation material for artificial inoculation of Cistanche deserticola. The invention effectively solves the problems of seed dormancy-breaking, germination-promoting, and water-retention inoculation, does not require seed dormancy-breaking in advance before inoculation, and the hydrogel has strong water absorption and water-retention capacity, effectively solves the problem of dormancy-breaking seeds being unable to germinate and parasitize in time due to lack of water after inoculation, and improves the inoculation efficiency from about 20% of the current inoculation paper inoculation to more than 75%. The invention has good promotion prospects and is of great significance for promoting the development of the Cistanche deserticola industry.

[0015] The advantages and positive effects of the present invention are:

[0016] (1) The step of breaking dormancy of seeds separately is omitted. The dormancy breaking inducer and seeds are buried together in the inoculation block. The dormancy breaking inducer can be used to induce dormancy breaking under water conditions. At the same time, the seeds can germinate after breaking dormancy, which effectively avoids the loss of vitality of seeds after breaking dormancy due to long-term non-germination and ensures the survival rate of seeds.

[0017] (2) The inoculation block has a strong water absorption and water retention capacity. The inoculation block can absorb water equivalent to three times its own volume and can maintain water retention for 15-20 days, fully ensuring the water demand of the seeds for germination after dormancy, and promoting rapid seed germination. After the seeds germinate quickly, the release of allelopathic substances from the seeds induces the roots of Haloxylon ammodendron to grow rapidly towards the inoculation block, greatly improving the inoculation efficiency.

[0018] (3) The inoculated block has a dual induction effect. One is that it can induce the seeds to break dormancy, and the other is that it can induce the directional growth of the Haloxylon ammodendron root system.

[0019] (4) It significantly saves seed and water usage and improves inoculation efficiency. Each inoculation block contains about 50 seeds. Compared with the traditional inoculation paper with about 200-300 seeds per sheet, it only uses one-fourth to one-sixth of the seed amount of inoculation paper. Each inoculation hole only needs 150-200mL of water, but the inoculation efficiency is increased from about 20% of the inoculation paper to more than 75%, achieving the goal of saving seeds, saving water, improving inoculation efficiency, and reducing the cost of Cistanche deserticola cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Attachment Figure 1The inoculation block is prepared using a 7×7 mold and can be cut directly along the cutting line when in use. DETAILED DESCRIPTION

[0021] Example 1

[0022] 1. Preparation of sleep-breaking inducing agent solution

[0023] According to the ratio of gibberellin and cytokinin in the sleep-breaking inducer of 4:1, the concentrations were 80 and 20 mg L -1 Prepare the sleep-breaking inducing agent solution.

[0024] 80 mg of gibberellin and 20 mg of cytokinin were weighed separately and added to 1 L of water to fully dissolve, then 8 g of yeast water extract was added, stirred on a heated magnetic stirrer at 200 rpm and 40°C for 10 minutes, and then centrifuged at 800×g for 10 minutes.

[0025] 2. Preparation of Hydrogel Containing Sleep-Reducing Inducers and Cistanche Deserticola Seeds

[0026] Sodium alginate and carboxymethyl cellulose were prepared in a 3:1 ratio, with concentrations of 1.2% and 0.4%, respectively. According to the above conditions, 12g of sodium alginate and 4g of carboxymethyl cellulose were weighed and added to the prepared sleep-breaking inducer solution. The mixture was stirred on a heated magnetic stirrer at 500 rpm and 60°C for 10 minutes. After stirring, the aqueous solution was cooled to room temperature. 10mg of Cistanche deserticola seeds were weighed and added to the solution. A glass rod was used to stir until the seeds were evenly distributed in the solution. 8g of calcium chloride was then added and stirred on a magnetic stirrer at 1000 rpm for 5 minutes. The stirred solution was then quickly poured into a mold with a length, width, and height of 2 cm, 2 cm, and 4 cm, respectively, and allowed to solidify naturally into a hydrogel.

[0027] 3. Preparation of Inoculation Blocks

[0028] The naturally solidified hydrogel block containing the sleep-breaking inducer and Cistanche deserticola seeds is taken out of the mold, placed in a vacuum drying oven, and dehydrated and solidified at 35°C for 10 hours to allow the hydrogel block to dehydrate, become hard and elastic, and the water content is reduced to below 20%, and then it can be used as an inoculation block.

[0029] Example 2

[0030] 1. Preparation of sleep-breaking inducing agent solution

[0031] According to the ratio of gibberellin and cytokinin in the sleep-breaking inducer of 3:1, the concentrations were 90 and 30 mg L -1 Prepare the sleep-breaking inducing agent solution.

[0032] 90 mg of gibberellin and 30 mg of cytokinin were weighed separately and added to 1 L of water to fully dissolve, then 10 g of yeast water extract was added, stirred on a heated magnetic stirrer at 250 rpm and 50°C for 15 minutes, and then centrifuged at 900×g for 12 minutes.

[0033] 2. Preparation of Hydrogel Containing Sleep-Reducing Inducers and Cistanche Deserticola Seeds

[0034] Sodium alginate and carboxymethyl cellulose were prepared in a 4:1 ratio, with concentrations of 1.8% and 0.45%, respectively. According to the above conditions, 18g of sodium alginate and 4.5g of carboxymethyl cellulose were weighed and added to the prepared sleep-breaking inducer solution. The mixture was stirred on a heated magnetic stirrer at 600 rpm and 70°C for 15 minutes. After stirring, the aqueous solution was cooled to room temperature. 11mg of Cistanche deserticola seeds were weighed and added to the solution. A glass rod was used to stir the seeds until the seeds were evenly distributed in the solution. 10g of calcium chloride was then added. The mixture was stirred on a magnetic stirrer at 1100 rpm for 8 minutes. The stirred solution was then quickly poured into a mold with a length, width, and height of 2 cm, 2 cm, and 4 cm, respectively, and allowed to solidify naturally into a hydrogel.

[0035] 3. Preparation of Inoculation Blocks

[0036] The naturally solidified hydrogel block containing the sleep-breaking inducer and Cistanche deserticola seeds is taken out of the mold, placed in a vacuum drying oven, and dehydrated and solidified at 40°C for 12 hours to allow the hydrogel block to dehydrate, become hard and elastic, and the water content is reduced to below 20%, and then it can be used as an inoculation block.

[0037] Example 3

[0038] 1. Preparation of sleep-breaking inducing agent solution

[0039] The sleep-breaking inducer solution is prepared according to the ratio of gibberellin to cytokinin in the sleep-breaking inducer being 2:1.

[0040] 120 mg of gibberellin and 60 mg of cytokinin were weighed separately and added to 1 L of water to fully dissolve, then 12 g of yeast water extract was added, stirred on a heated magnetic stirrer at 300 rpm and 60°C for 20 min, and then centrifuged at 1000×g for 15 min.

[0041] 2. Preparation of Hydrogel Containing Sleep-Reducing Inducers and Cistanche Deserticola Seeds

[0042] Sodium alginate and carboxymethyl cellulose were used in a ratio of 5:1, with concentrations of 2.5% and 0.5%, respectively. According to the above conditions, 25g of sodium alginate and 5g of carboxymethyl cellulose were weighed and added to the prepared sleep-breaking inducer solution. The mixture was stirred on a heated magnetic stirrer at 700 rpm and 80°C for 20 minutes. After stirring, the aqueous solution was cooled to room temperature, 12mg of Cistanche deserticola seeds were weighed and added to the solution. A glass rod was used to stir the seeds until the seeds were evenly distributed in the solution. 12g of calcium chloride was then added, and the mixture was stirred on a magnetic stirrer at 1200 rpm for 10 minutes. The stirred solution was then quickly poured into a mold with a length, width, and height of 2 cm, 2 cm, and 4 cm, respectively, and allowed to solidify naturally into a hydrogel.

[0043] 3. Preparation of Inoculation Blocks

[0044] The naturally solidified hydrogel block containing the sleep-breaking inducer and Cistanche deserticola seeds is taken out of the mold, placed in a vacuum drying oven, and dehydrated and solidified at 45°C for 14 hours to allow the hydrogel block to dehydrate, become hard and elastic, and the water content is reduced to below 20%, and then it can be used as an inoculation block.

Claims

1. A technology for preparing a special inoculation block for Cistanche deserticola with the function of self-inducing seed dormancy breaking, characterized in that: The invention is prepared by using gibberellins and cytokinins as sleep-breaking inducers, sodium alginate and carboxymethyl cellulose hydrogel as water retaining agents, and yeast water extract as an auxiliary agent.

2. The preparation technology of a special inoculation block for Cistanche deserticola with the function of self-inducing seed dormancy breaking according to claim 1 is characterized in that The ratio of gibberellin and cytokinin in the sleep-breaking inducer is 2:1-4:1, and the concentration is 80-120 mg L -1 and 20-60 mg L -1 The dosage of yeast extract is 8-12g L -1 The ratio of sodium alginate and carboxymethyl cellulose in the hydrogel is 3:1-5:1, and the concentration is 12-25g L -1 and 4-5gL -1 .

3. The preparation technology of a special inoculation block for Cistanche deserticola with the function of self-inducing seed dormancy breaking according to claim 1 is characterized in that The hydrogel containing the sleep-breaking inducer and Cistanche deserticola seeds is prepared by the following steps: Step 1: Weigh 80-120 mg of gibberellin and 20-60 mg of cytokinin respectively, add them into 1 L of water and fully dissolve them; Step 2: Weigh 8-12 g of yeast water extract and add it to the solution prepared in step 1. Stir the mixture on a heated magnetic stirrer at 200-300 rpm and 40-60°C for 10-20 minutes, then centrifuge at 800-1000 × g for 10-15 minutes. Step 3: Weigh 12-25 g of sodium alginate and 4-5 g of carboxymethyl cellulose respectively and add them to the solution prepared in step 2. Stir on a heated magnetic stirrer at a speed of 500-700 rpm and 60-80°C for 10-20 minutes. Step 4: After the solution prepared in step 3 is cooled to room temperature, 10-12 mg of Cistanche deserticola seeds are weighed and added to the above solution. Stir with a glass rod until the seeds are evenly distributed in the solution. Then, 8-12 g of calcium chloride is added and stirred on a magnetic stirrer at 1000-1200 rpm for 5-10 minutes. Step 5: Pour the solution prepared in step 4 into a mold with a length, width, and height of 2 cm, 2 cm, and 4 cm, respectively, and allow the solution to solidify naturally.

4. The preparation technology of a special inoculation block for Cistanche deserticola with the function of self-inducing seed dormancy breaking according to claim 1 is characterized in that After the solidified hydrogel block is taken out from the mold, it is placed in a vacuum drying oven at 35-45°C for dehydration and curing for 10-14 hours.

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