Grafting and seedling raising method for hot-guiding No.1 pepper

Abstract

The invention relates to the technical field of plant culture, in particular to a grafting seedling raising method of hot-guiding No.1 pepper. The method comprises the following steps: taking a bud seedling cultivated by a pepper hemerocallis seed as a stock, taking a somatic embryo cluster bud seedling of the pepper hemerocallis as a scion, and carrying out epicotyl grafting by adopting a top grafting method, so that high seedling reproduction rate and high grafting survival rate are ensured, the interface healing time is also remarkably shortened, and the steps of rooting and seedling hardening are reduced compared with a pure tissue culture method; and the cultivation time of the hot-guiding No.1 pepper resistant seedlings is greatly shortened. In addition, the main roots and fibrous roots of the grafted seedlings cultivated by the method are developed, the growth is rapid, the survival rate is high, and a brand new technical path is provided for efficient breeding of pepper seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, in particular to a grafting seedling raising method for Reyin No. 1 pepper. Background Art

[0002] Pepper (Piper nigrum L.) is a perennial tropical climbing vine of the genus Piper in the family Piperaceae. Its fruit has a spicy aroma. It is the world's most important spice crop and a favorite condiment.

[0003] For a long time, pepper blast, caused by the fungus Phytophthora capsici Leon, has been the most significant pest in production. Currently, there are few resistant varieties and no effective treatments. Prevention is the primary approach, supplemented by integrated control. This has become a bottleneck in the industry due to its complex technical operations, difficult planting and management, high production costs, and low comparative benefits. Grafting is an important means of maintaining the superior characteristics of the parent variety, reducing differentiation and decline, and improving its adaptability.

[0004] Currently, studies have used high-yield, disease-resistant yellow pepper seedlings as rootstocks and branches of the Heat-Induced No. 1 pepper as scions, using grafting to produce high-quality, disease-resistant pepper seedlings. While grafting has yielded survival rates as high as 80%, the limited availability of parent plants has led to low seedling propagation rates. Therefore, effectively improving both grafting survival and seedling propagation rates is crucial for providing technical support for the rapid propagation of resistant Heat-Induced No. 1 pepper seedlings. Summary of the Invention

[0005] In view of this, the present invention provides a method for grafting and raising seedlings of pepper resistant seedlings of Reyin No. 1. The method is efficient in cultivating grafted seedlings, takes a short time, and has simple operation steps. The obtained grafted seedlings can maintain the excellent traits of the mother plant.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The grafting seedling raising method of Reyin No. 1 pepper comprises the following steps:

[0008] (1) Stock cultivation and treatment: sowing and germinating yellow pepper seeds until the stem of the seedlings, which is 1 to 3 cm near the ground, reaches a thickness of 0.2 to 0.5 cm, thereby obtaining seedlings; cross-cutting the epicotyl of the seedlings, removing the top, and making a longitudinal incision in the center of the cross section to obtain stock;

[0009] (2) Scion cultivation and treatment: The outer seed coat of the germinated pepper seeds of Reyin No. 1 was used as the explant, and callus induction and cluster bud induction were carried out in sequence to obtain cluster buds; cluster buds with a stem diameter of 0.2 to 0.5 cm were taken, the terminal bud was cut off, and the base was cut into a wedge surface of 0.5 to 0.9 cm in length to obtain the scion;

[0010] (3) Grafting: inserting the scion into the incision of the rootstock, sealing the joint, and obtaining a grafted seedling.

[0011] In the early stage of the study, the applicant used the heat-induced No. 1 pepper as the scion and the highly resistant pepper blast wild relative, yellow flower pepper, as the rootstock. A breakthrough was made in improving the interspecific grafting survival rate technology, and disease-resistant interspecific grafted seedlings were obtained. Combined with the supporting planting and management technology, the disease resistance of pepper cultivars was improved, and the grafting survival rate was as high as 80%. However, this method was limited by the number of rootstock and scion mother plant materials, resulting in a low seedling propagation rate. The present invention found that the seedlings cultivated with yellow flower pepper seeds were used as the rootstock, and the somatic embryo clustered seedlings induced by the outer seed coat of heat-induced No. 1 pepper were used as the scion, which effectively solved the limitation of the mother plant material and improved the seedling propagation rate. However, the survival rate was low when the conventional grafting method (such as top grafting, oblique belly method, etc.) of traditional pepper grafting was used for grafting, and the seedling raising period was long and the growth condition of the grafted seedlings was poor (such as taproot). The present invention has tried a variety of grafting methods and parameters. After years of research and exploration, it was found that compared with the traditional branch grafting method and the bud grafting method under other parameters, the present invention adopts the epicotyl top grafting bud grafting method, and by reasonably controlling the key factors affecting the grafting survival rate, such as the stem diameter, age, grafting timing and grafting position of the scion and rootstock, the interface healing time is significantly shortened, the grafting survival rate is as high as about 80%, the seedling raising cycle is also significantly shortened, and the cultivated grafted seedlings are in good growth condition, with well-developed main roots, fast growth and high survival rate. It successfully overcomes the technical bottleneck of the difficulty in synchronizing the grafting survival rate and the seedling reproduction rate, and provides a new technical path for the efficient breeding of pepper seedlings.

[0012] In step (1) of the present invention, the germination temperature is 26-30°C, specifically 26°C, 28°C, or 30°C.

[0013] In step (1) of the present invention, the nutrient soil for sowing comprises 6 to 8 parts of topsoil, 2 to 4 parts of commercial organic fertilizer and 2 parts of coconut husk.

[0014] Specifically, the commercial organic fertilizer includes 450-500 g·kg–1 organic matter, 15.3-16.0 g·kg –1 N, 47.0-47.5 g·kg –1 P2O5, 12.6~13.2g·kg –1 K2O, pH 8.4.

[0015] In step (1) of the present invention, the epicotyl is the position of the seed sprout 2 to 3 cm away from the substrate surface, and the specific position can be 2 cm, 3 cm or any value between 2 and 3.

[0016] In step (1) of the present invention, the depth of the incision is 0.5 to 0.8 cm, specifically 0.5 cm, 0.7 cm or 0.8 cm.

[0017] In step (2) of the present invention, the seed coat of the Reyin No. 1 pepper seeds is the sterile seed coat that falls off after the seeds germinate. In some embodiments, the culture medium used for seed germination is composed of: MS + 1.0-2.5 g / L activated carbon + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8-6.0.

[0018] In step (2) of the present invention, the sterile outer seed coat is inoculated into a callus induction medium for culture to induce callus. The callus induction medium comprises MS + 0.25-1.0 mg / L 2,4-D + 0.25-1.0 mg / L IBA + 0.05-1.0 mg / L KT + 30 g / L sucrose + 2.5 g / L phytagel, with a pH of 5.8-6.0.

[0019] In step (2) of the present invention, black callus is induced after culturing for about 30 days. The black, blocky embryonic callus is transferred to a somatic embryo cluster bud induction medium for culturing. White, loosely structured, granular somatic embryos can be differentiated after about 20 days. After culturing for about 26 days, the somatic embryos begin to differentiate into clustered shoots. The composition of the somatic embryo cluster bud induction medium is: MS + 0.05-0.5 mg / L 2,4-D + 0.25-1.0 mg / L IBA + 0.05-1.0 mg / L KT + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8-6.0.

[0020] The MS mentioned in the culture medium mentioned above in the present invention refers to MS basal culture medium, which is a common commercial product.

[0021] In step (2) of the present invention, after obtaining the clustered buds, the individual clustered buds are separated and used as scions for grafting after being processed. First, clustered buds with a thickness equivalent to that of the rootstock are selected. The present invention preferably selects clustered buds with a stem thickness of 0.2 to 0.5 cm, specifically 0.2 cm, 0.3 cm, 0.4 cm, or 0.5 cm. The specific stem thickness depends on the stem thickness of the rootstock.

[0022] In step (2) of the present invention, the process of treating the scion includes the steps of cutting off the terminal bud, retaining the leaves, and cutting the base (morphological lower end) of the scion into a wedge surface.

[0023] Among them, the terminal bud of the cluster bud is cut off. The specific operation is: cut off 2 cm away from the top of the terminal bud. The top here refers to the morphological top part of the cluster bud, that is, remove the part of the terminal bud with a length of 2 cm.

[0024] The retaining of leaves comprises: retaining 2 to 3 leaves of the scion, and retaining 1 / 2 of each leaf.

[0025] The length of the inclined surface is 0.5 cm to 0.9 cm. In some specific embodiments of the present invention, the length is 0.5 cm, 0.8 cm or 0.9 cm.

[0026] The cut scion is inserted into the incision of the rootstock, and the joint is sealed with a film to obtain the grafted seedling of the present invention.

[0027] Furthermore, the sealing film includes: using a transparent film strip with a thickness of 0.01 mm, a width of about 1 cm, and good toughness to wrap it tightly from bottom to top in circles, with the circles overlapping by about 1 / 3, and finally tying a knot above the interface; using a transparent sealing bag to wrap the junction of the scion and the stockwood scion, leaving only a small space near the junction of the stockwood scion for air circulation.

[0028] In the present invention, after obtaining the grafted seedlings, the process also includes water control management after grafting.

[0029] In some embodiments, the water control management includes: maintaining 60% shade for the seedlings, intermittent spraying to maintain air humidity at 80% to prevent dehydration of the scion and promote interface healing; adjusting to 30% shade after 4 weeks and maintaining micro-sprinkler irrigation.

[0030] In the present invention, the grafting seedling raising method further includes fertilization management after grafting.

[0031] In some embodiments, the fertilization management includes: starting to fertilize after the scion grows a complete new leaf, watering in time depending on the dryness and wetness of the soil to keep the soil moist; applying 0.3% urea or potassium dihydrogen phosphate aqueous solution after the scion sprouts, spraying once every 30 days, and normal management.

[0032] The present invention uses yellow pepper seed buds as rootstocks and Reyin No. 1 pepper somatic embryo cluster buds as scions, and performs epicotyl grafting by top grafting. Compared with the prior art, the present invention has the following advantages:

[0033] (1) The defects of the existing pepper cutting method, such as slow growth and small quantity, are eliminated, and the efficiency of cultivating seedlings is greatly improved;

[0034] (2) It reduces the rooting induction and hardening steps of tissue culture seedlings, greatly shortening the cultivation time of Reyin No. 1 pepper seedlings;

[0035] (3) The cultivated Reyin No. 1 pepper seedlings have well-developed taproots, strong disease resistance, and are able to maintain the excellent traits of the mother plant. Compared with traditional grafting and cutting methods, the efficiency of seedling propagation is greatly improved, with an average grafting survival rate of over 80%. The entire process is simple to operate, and the resulting grafted seedlings grow rapidly, providing new technical support for the propagation of Reyin No. 1 pepper seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Rootstock used in the present invention;

[0037] Figure 2 A rootstock having a cross-section epicotyl of the present invention;

[0038] Figure 3 The present invention is a clustered seedling used as a scion;

[0039] Figure 4 The grafted seedlings are obtained by top grafting according to the present invention;

[0040] Figure 5 The invention provides a grafted seedling wrapped in a sealing bag to keep moisture. DETAILED DESCRIPTION

[0041] The present invention provides a method for grafting and raising seedlings of Heat-Induced No. 1 pepper. Those skilled in the art can refer to the contents herein and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately alter and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0042] The present invention uses yellow pepper seedlings as rootstocks and somatic embryo cluster buds of Reyin No. 1 pepper as scions, adopts top grafting to carry out epicotyl grafting, and the grafting process involves the rootstock, scion, and grafted seedlings of cross-cut epicotyls, epicotyl top grafting, and wrapped scions. Figure 1 As shown, the specific steps include:

[0043] (1) Stock cultivation: After cleaning, the yellow pepper seeds for stock cultivation are directly sown into a 12cm×30cm seedling bag filled with nutrient soil and placed in a 28±2℃ environment to germinate. The nutrient soil is prepared by mixing 6-8 parts topsoil, 2-4 parts commercial organic fertilizer, and 2 parts coconut coir. When the yellow pepper seedlings are 1-3 cm above the ground and the stem thickness reaches 0.2-0.5 cm, they can be prepared for grafting.

[0044] The commercial organic fertilizer includes 450-500g kg –1 Organic matter, 15.3-16.0 g·kg –1N, 47.0~47.5g·kg –1 P2O5, 12.6~13.2g·kg –1 of K2O, pH 8.4.

[0045] (2) Cultivation of regenerated scion: Use the pepper seeds of Heat-induced No. 1 pepper as the explant, disinfect the surface with 75% alcohol for 45 seconds, and then rinse with sterile water 3 to 5 times. Then disinfect and sterilize with 95% anhydrous ethanol for 45 seconds, rinse with sterile water 3 to 5 times, and finally soak and disinfect with 0.1% HgCl2 (8 to 12 minutes), and then rinse with sterile water 5 times, each time for about 30 seconds. After absorbing the water with sterile filter paper, inoculate on MS medium containing 2.5g activated carbon. Pepper seeds are cultured on MS medium for about 90 days, and sterile seedlings begin to germinate. Collect the sterile outer seed coat that falls off for use. The sterile seed coats were transferred to MS medium every 30 days. After 30 days of culture, black callus was induced. The embryonic callus was then transferred to MS medium supplemented with 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos were differentiated. After approximately 26 days of culture, the somatic embryos began to differentiate into clustered shoots. The culture room conditions were 28±2°C, with 10-16 hours of light per day, 500-1000 lx for the first seven days, and 1000-1500 lx starting on the eighth day. The callus was inoculated onto somatic embryo induction medium for clustered shoot differentiation and then cultured in the culture room to obtain clustered shoots. The culture room temperature was 28±2°C, and light was applied starting on the second day after inoculation, with a light intensity of 1500-2000 lx for 12 hours per day. When the stems of the clustered buds grow to a thickness of 0.2 to 0.4 cm, the clustered buds are divided to obtain regenerated scions and prepare for grafting operations.

[0046] (3) Grafting and management: Cut the epicotyl 2-3 cm from the surface of the substrate, remove the top, and split it longitudinally in the center of the cross section with a depth of about 0.5-0.8 cm for later use; select a single clustered bud of the same thickness as the rootstock and with a full bud, cut it 2 cm above the top bud, make two oblique cuts at the bottom of the scion to form a wedge shape with a wedge length of 0.3-0.8 cm, leave two leaves on the scion, and trim it to half the size; insert the cut scion into the rootstock cut, and wrap it tightly from bottom to top with a 0.01 mm thick, about 1 cm wide, and tough transparent film tape, overlapping about 1 / 3 between the circles, and finally tie a knot above the interface. Wrap the junction of the scion and the rootstock with a transparent sealing bag, leaving only a small space near the rootstock junction for air circulation. The average daily temperature during the grafting period was between 24 and 30°C, and the grafting was carried out between 7:00 and 9:00 in the morning or 16:00 and 17:30 in the afternoon.

[0047] After grafting, maintain 60% shade for the seedlings and intermittently mist the air to maintain 80% humidity to prevent dehydration of the scion and promote joint healing. After four weeks, adjust the shade to 30% and continue micro-irrigation. Fertilize after the scion sprouts one complete new leaf. Water the soil as needed to keep it moist. After budding, apply 0.3% urea or potassium dihydrogen phosphate solution, spraying every 30 days. Maintain normal management.

[0048] In a specific embodiment of the present invention, the coconut bran used in rootstock cultivation is produced by Wenchang Yongxing Industrial Co., Ltd., with the product number YK0124.

[0049] In a specific embodiment of the present invention, the callus induction medium and cluster bud induction medium used in step 2) are as follows:

[0050] The composition of the seed germination culture medium is: MS + 1.0-2.5 g / L activated carbon + 30 g / L sucrose + 2.5 g / L plant gel, pH 5.8±0.2, and then high-temperature sterilized for use.

[0051] The composition of the callus induction medium is: MS + 0.25-1.0 mg / L 2,4-D + 0.25-1.0 mg / L LIBA + 0.05-1.0 mg / L KT + 30 g / L sucrose + 2.5 g / L phytagel, adjusted to pH 5.8 ± 0.2, and then sterilized at high temperature for later use. The composition of the somatic embryo cluster bud induction medium is: MS + 0.05-0.5 mg / L 2,4-D + 0.25-1.0 mg / L LIBA + 0.05-1.0 mg / L KT + 30 g / L sucrose + 2.5 g / L phytagel, adjusted to pH 5.8 ± 0.2, and then sterilized at high temperature for later use.

[0052] Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

[0053] The present invention will be further described below in conjunction with the embodiments:

[0054] Example 1

[0055] (1) Rootstock cultivation

[0056] Seed cleaning and sowing: After cleaning the yellow pepper seeds, sow them in a seedling bag (12 cm × 30 cm) filled with nutrient soil, and then place them in an environment controlled at a temperature of 28±2℃ for germination.

[0057] Germination conditions: The seeds begin to germinate at a suitable temperature. When the seedlings grow to a height of 1 cm from the ground and the stem reaches 0.2 cm in diameter, they can be prepared for grafting.

[0058] Stock Preparation: Cut the epicotyl of the stock transversely 2 cm from the substrate surface and remove the tip. Make a longitudinal incision about 0.5 cm deep in the center of the cross section and set aside.

[0059] (2) Scion cultivation

[0060] (1) Scion seed disinfection and inoculation surface disinfection: Use the pepper seeds of Heat Induction No. 1 as the explant and disinfect the surface with 75% alcohol for 45 seconds.

[0061] Sterile water rinse: Rinse 3 to 5 times with sterile water to remove residual alcohol.

[0062] Further disinfection: Then disinfect and sterilize with 95% anhydrous ethanol for 45 seconds, and then wash again with sterile water 3 to 5 times.

[0063] Final disinfection: Finally, soak and disinfect in 0.1% mercuric chloride (HgCl2) solution for 8 to 12 minutes, then rinse with sterile water 5 times, each washing time is about 30 seconds.

[0064] Inoculation: After absorbing the moisture on the surface of the seeds with sterile filter paper, the seeds were inoculated on MS medium containing 2.5 g of activated carbon.

[0065] (2) Seed culture and seed coat treatment

[0066] Culture conditions: Culture on MS medium for about 90 days, during which time the seeds begin to germinate and form sterile seedlings.

[0067] Seed coat collection: Collect the sterile seed coats that fall off the sterile seedlings for subsequent use.

[0068] Seed coat culture: Transfer the sterile seed coat to MS medium every 30 days. After 30 days of culture, black callus will be induced.

[0069] Callus induction: Embryonic callus was transferred to MS medium containing 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos were differentiated.

[0070] Somatic embryo culture: The somatic embryos are cultured for about 26 days and begin to differentiate into clustered sprouts.

[0071] (3) Treatment of clustered seedlings and preparation for grafting

[0072] Screening of clustered buds: When the stem thickness of the clustered buds grows to 0.2 cm, the clustered buds are divided to obtain regenerated scions for grafting.

[0073] Scion Preparation: Select a single, clustered bud of comparable thickness to the rootstock, with plump buds, as the scion. Cut the scion 2 cm above the terminal bud and make two diagonal cuts below it, forming a wedge shape with a length of 0.5 cm. Leave the two leaves on the scion and trim them to half their size.

[0074] (3) Grafting operation

[0075] Insert the prepared scion into the cut of the rootstock, ensuring the cambium of the scion and the rootstock are aligned to obtain the grafted seedling. Using a 0.01 mm thick, approximately 1 cm wide transparent film tape, wrap it from bottom to top, overlapping by about one-third. Finally, tie a knot above the joint to secure.

[0076] Moisturize and protect: Wrap the junction of the scion and stockwood with a transparent sealable bag, leaving a small amount of space near the junction to ensure air circulation.

[0077] (4) Management after grafting

[0078] Shading and humidity control: After grafting, maintain 60% shade for the seedlings and keep the air humidity at around 80% through intermittent spraying to prevent dehydration of the scion and promote interface healing.

[0079] Environmental adjustment: After 4 weeks, adjust the shade to 30% and use micro-sprinkler irrigation to maintain the air humidity at 70%.

[0080] Fertilization and Irrigation: Start fertilizing after the scion sprouts a complete new leaf. Water the soil as needed to keep it moist. After the scion sprouts, apply 0.3% urea or a 0.3% potassium dihydrogen phosphate solution. Spray every 30 days and continue normal management.

[0081] Example 2

[0082] (1) Rootstock cultivation

[0083] Seed cleaning and sowing: After cleaning the yellow pepper seeds, sow them in a seedling bag (12 cm × 30 cm) filled with nutrient soil, and then place them in an environment controlled at a temperature of 28±2℃ for germination.

[0084] Germination conditions: Seeds begin to germinate at a suitable temperature. When the seedlings grow to 2 cm above the ground and the stem reaches 0.4 cm in diameter, they can be prepared for grafting.

[0085] Stock Preparation: Cut the epicotyl of the stock transversely 2 cm from the substrate surface and remove the tip. Make a longitudinal incision about 0.7 cm deep in the center of the cross section and set aside.

[0086] (2) Scion cultivation

[0087] (1) Disinfection and inoculation of scion seeds

[0088] Surface disinfection: Use the Heat-Induced No. 1 pepper seeds as explants and perform surface disinfection with 75% alcohol for 45 seconds.

[0089] Sterile water rinse: Rinse 3 to 5 times with sterile water to remove residual alcohol.

[0090] Further disinfection: Then disinfect and sterilize with 95% anhydrous ethanol for 45 seconds, and then wash again with sterile water 3 to 5 times.

[0091] Final disinfection: Finally, soak and disinfect in 0.1% mercuric chloride (HgCl2) solution for 8 to 12 minutes, then rinse with sterile water 5 times, each washing time is about 30 seconds.

[0092] Inoculation: After absorbing the moisture on the surface of the seeds with sterile filter paper, the seeds were inoculated on MS medium containing 2.5 g of activated carbon.

[0093] (2) Seed culture and seed coat treatment

[0094] Culture conditions: Culture on MS medium for about 90 days, during which time the seeds begin to germinate and form sterile seedlings.

[0095] Seed coat collection: Collect the sterile seed coats that fall off the sterile seedlings for subsequent use.

[0096] Seed coat culture: Transfer the sterile seed coat to MS medium every 30 days. After 30 days of culture, black callus will be induced.

[0097] Callus induction: Embryonic callus was transferred to MS medium containing 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos were differentiated.

[0098] Somatic embryo culture: The somatic embryos are cultured for about 26 days and begin to differentiate into clustered sprouts.

[0099] (3) Treatment of clustered seedlings and preparation for grafting

[0100] Screening of clustered buds: When the stem thickness of the clustered buds grows to 0.3 cm, the clustered buds are divided to obtain regenerated scions for grafting.

[0101] Scion Preparation: Select a single, clustered bud of comparable thickness to the rootstock, with plump buds, as the scion. Cut the scion 2 cm above the terminal bud and make two diagonal cuts below it, forming a wedge shape with a wedge length of 0.9 cm. Leave the two leaves on the scion and trim them to half their size.

[0102] (3) Grafting operation

[0103] Insert the prepared scion into the cut of the rootstock, ensuring the cambium of the scion and the rootstock are aligned to obtain the grafted seedling. Using a 0.01 mm thick, approximately 1 cm wide transparent film tape, wrap it from bottom to top, overlapping by about one-third. Finally, tie a knot above the joint to secure.

[0104] Moisturize and protect: Wrap the junction of the scion and stockwood with a transparent sealable bag, leaving a small amount of space near the junction to ensure air circulation.

[0105] (4) Management after grafting

[0106] Shading and humidity control: After grafting, maintain 30% shade for the seedlings and keep the air humidity at around 80% through intermittent spraying to prevent dehydration of the scion and promote interface healing.

[0107] Environmental adjustment: After 4 weeks, adjust the shade to 60% and use micro-sprinkler irrigation to maintain the air humidity at 70%.

[0108] Fertilization and Irrigation: Start fertilizing after the scion sprouts a complete new leaf. Water the soil as needed to keep it moist. After the scion sprouts, apply 0.3% urea or a 0.3% potassium dihydrogen phosphate solution. Spray every 30 days and continue normal management.

[0109] Example 3

[0110] (1) Rootstock cultivation

[0111] Seed cleaning and sowing: After cleaning the yellow pepper seeds, sow them in a seedling bag (12 cm × 30 cm) filled with nutrient soil, and then place them in an environment controlled at a temperature of 28±2℃ for germination.

[0112] Germination conditions: Seeds begin to germinate at a suitable temperature. When the seedlings grow to 3 cm from the ground and the stem thickness reaches 0.5 cm (see Figure 1 ) can be used for subsequent grafting preparations.

[0113] Stock treatment: Cut the epicotyl of the stock transversely at a distance of 2 cm from the substrate surface and remove the top. Make a longitudinal incision of about 0.8 cm in depth in the center of the transverse section and set aside (see Figure 2 ).

[0114] (2) Scion cultivation

[0115] (1) Disinfection and inoculation of scion seeds

[0116] Surface disinfection: Use the Heat-Induced No. 1 pepper seeds as explants and perform surface disinfection with 75% alcohol for 45 seconds.

[0117] Sterile water rinse: Rinse 3 to 5 times with sterile water to remove residual alcohol.

[0118] Further disinfection: Then disinfect and sterilize with 95% anhydrous ethanol for 45 seconds, and then wash again with sterile water 3 to 5 times.

[0119] Final disinfection: Finally, soak and disinfect in 0.1% mercuric chloride (HgCl2) solution for 8 to 12 minutes, then rinse with sterile water 5 times, each washing time is about 30 seconds.

[0120] Inoculation: After absorbing the moisture on the surface of the seeds with sterile filter paper, the seeds were inoculated on MS medium containing 2.5 g of activated carbon.

[0121] (2) Seed culture and seed coat treatment

[0122] Culture conditions: Culture on MS medium for about 90 days, during which time the seeds begin to germinate and form sterile seedlings.

[0123] Seed coat collection: Collect the sterile seed coats that fall off the sterile seedlings for subsequent use.

[0124] Seed coat culture: Transfer the sterile seed coat to MS medium every 30 days. After 30 days of culture, black callus will be induced.

[0125] Callus induction: Embryonic callus was transferred to MS medium containing 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos were differentiated.

[0126] Somatic embryo culture: The somatic embryos are cultured for about 26 days and begin to differentiate into clustered sprouts.

[0127] (3) Treatment of clustered seedlings and preparation for grafting

[0128] Selection of clustered buds: When the stems of clustered buds grow to 0.4 cm in diameter, the clustered buds are divided to obtain regenerated scions for grafting (see Figure 3 ).

[0129] Scion Preparation: Select a single, clustered bud of comparable thickness to the rootstock, with plump buds, as the scion. Cut the scion 2 cm above the terminal bud and make two diagonal cuts below it, forming a wedge shape with a wedge length of 0.8 cm. Leave the two leaves on the scion and trim them to half their size.

[0130] (3) Grafting operation

[0131] Insert the treated scion into the cut of the rootstock, making sure that the cambium of the scion and the rootstock are aligned to obtain the grafted seedling (see Figure 4 Use a 0.01mm thick, 1cm wide transparent film tape to wrap the joint from bottom to top, overlapping by about one-third each time. Finally, tie a knot above the joint to secure it.

[0132] Moisturizing and protecting: Wrap the junction of scion and stock with a transparent sealable bag, leaving a small amount of space near the junction to ensure air circulation. Figure 5 .

[0133] (4) Management after grafting

[0134] Shading and humidity control: After grafting, maintain 60% shade for the seedlings and keep the air humidity at around 80% through intermittent spraying to prevent dehydration of the scion and promote interface healing.

[0135] Environmental adjustment: After 4 weeks, adjust the shade to 30% and use micro-sprinkler irrigation to maintain the air humidity at 70%.

[0136] Fertilization and Irrigation: Start fertilizing after the scion sprouts a complete new leaf. Water the soil as needed to keep it moist. After the scion sprouts, apply 0.3% urea or a 0.3% potassium dihydrogen phosphate solution. Spray every 30 days and continue normal management.

[0137] Comparative Example 1 (the thickness of the clustered bud stems is different from that of the present application)

[0138] After cleaning, yellow pepper seeds are directly sown into 12cm x 30cm seedling bags and placed at 28±2°C for germination. When the stem thickness of the seedlings, 1cm above the ground, reaches 0.2cm, they are ready for grafting. Using the heat-induced pepper No. 1 as an explant, surface-sterilize the seeds with 75% alcohol for 45 seconds, then rinse 3-5 times with sterile water. Sterilize and sterilize them with 95% anhydrous ethanol for 45 seconds, rinse 3-5 times with sterile water, and finally, soak and disinfect them with 0.1% HgCl2 for 8-12 minutes. Rinse five times with sterile water, each rinse lasting approximately 30 seconds. After absorbing the moisture with sterile filter paper, the seeds are inoculated onto MS medium containing 2.5g of activated carbon. After incubating on MS medium for approximately 90 days, sterile seedlings begin to germinate. Collect the shed sterile outer seed coats for later use. The sterile seed coats were transferred to MS medium every 30 days. After 30 days of culture, black callus was induced. The embryonic callus was transferred to MS medium containing 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos were differentiated. After about 26 days of culture, the somatic embryos began to differentiate into clustered shoots. When the clustered shoots grew to 0.1 cm in diameter, the clustered shoots were divided to obtain regenerated scions for grafting. Cut the epicotyl transversely 2 cm from the substrate surface, remove the tip, and make a longitudinal incision approximately 0.7 cm deep in the center of the cross section for later use. Select a single, clustered bud of comparable thickness to the rootstock and with a full bud. Cut the scion 2 cm above the terminal bud. Make two diagonal cuts at the base of the scion, forming a wedge-shaped cut with a length of 0.9 cm. Leave two leaves on the scion and trim it to half its original size. Insert the trimmed scion into the cut in the rootstock. Wrap it tightly with a 0.01 mm thick, approximately 1 cm wide, flexible transparent film tape from bottom to top, overlapping by about one-third. Finally, tie a knot above the joint. Wrap the scion and stock-scion joint in a transparent, airtight bag, leaving only a small space near the joint for air circulation. After grafting, maintain 60% shade for the seedlings and intermittent misting to maintain an air humidity of 80% to prevent dehydration and promote healing of the joint. After four weeks, adjust the shade to 30% and use micro-sprinkler irrigation to maintain an air humidity of 70%. Start fertilizing after the scion grows a complete new leaf, and water the soil in time depending on the dryness and wetness of the soil to keep the soil moist; after the scion sprouts, apply 0.3% urea or potassium dihydrogen phosphate aqueous solution, spray once every 30 days, and manage normally.

[0139] Comparative Example 2 (the stock bud seedling stem thickness is different from that of the present application)

[0140] After cleaning, yellow pepper seeds are directly sown into 12cm x 30cm seedling bags and placed at 28±2°C for rapid germination. When the stem diameter of the seedlings, 2cm above the ground, reaches 0.6cm, they are ready for grafting. Using Heat-Induced No. 1 pepper seeds as explants, surface-sterilize them with 75% alcohol for 45 seconds and rinse them 3-5 times with sterile water. Sterilize and sterilize them with 95% anhydrous ethanol for 45 seconds, rinse them 3-5 times with sterile water, and finally, soak and disinfect them with 0.1% HgCl2 for 8-12 minutes. Rinse them five times with sterile water, each rinse lasting approximately 30 seconds. After absorbing the moisture with sterile filter paper, the seeds are inoculated onto MS medium containing 2.5g of activated carbon. After incubating on MS medium for approximately 90 days, sterile seedlings begin to germinate. The shed sterile outer seed coats are collected and used for future use. The sterile seed coats were transferred to MS medium every 30 days. After 30 days of culture, black callus was induced. The embryonic callus was transferred to MS medium containing 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos were differentiated. After about 26 days of culture, the somatic embryos began to differentiate into clustered shoots. When the clustered shoots grew to 0.6 cm in diameter, the clustered shoots were divided to obtain regenerated scions for grafting. Cut the epicotyl transversely 2-3 cm from the surface of the substrate, remove the tip, and make a longitudinal incision approximately 0.7 cm deep in the center of the cross section for later use. Select a single, clustered bud of comparable thickness to the rootstock and with a full bud. Cut the scion 2 cm above the terminal bud. Make two diagonal cuts at the base of the scion, forming a wedge-shaped cut 0.9 cm long. Leave two leaves on the scion and trim it to half its original size. Insert the trimmed scion into the cut of the rootstock. Wrap it tightly with a 0.01 mm thick, approximately 1 cm wide, flexible transparent film tape from bottom to top, overlapping by about one-third. Finally, tie a knot above the joint. Wrap the scion and stock-scion joint in a transparent, airtight bag, leaving only a small space near the joint for air circulation. After grafting, maintain 60% shade for the seedlings and intermittent misting to maintain an air humidity of 80% to prevent dehydration and promote healing of the joint. After four weeks, adjust the shade to 30% and use micro-sprinkler irrigation to maintain an air humidity of 70%. Start fertilizing after the scion grows a complete new leaf, and water the soil in time depending on the dryness and wetness of the soil to keep the soil moist; after the scion sprouts, apply 0.3% urea or potassium dihydrogen phosphate aqueous solution, spray once every 30 days, and manage normally.

[0141] Comparative Example 3 Oblique Belly Connection Method

[0142] After cleaning, sow yellow pepper seeds directly into a 12cm x 30cm seedling bag filled with nutrient soil. When the stems, 3cm below the ground, reach a thickness of 1.5cm, they are ready for grafting. Cut 1- to 2-year-old lignified or semi-lignified branches from a high-yielding, high-quality, and excellent Reyin No. 1 pepper mother tree that has been bearing fruit for more than two years. Use branches with a thickness of 0.7-1cm, a yellowish-brown skin, and plump buds as scions for the grafting procedure. Adopt oblique belly grafting method. On the day of grafting, select a 1-2 year old, strong and mechanically undamaged main vine from the scion mother tree; retain the 0.5-1.0 cm diameter part of the main vine and cut it into 6-10 cm scions. Each scion has 1-2 nodes and 1-2 axillary buds, and keep it moist; before grafting, cut off the leaves, retain the axillary buds and petioles; first cut a 1-1.6 cm long bevel at the lower end of the scion, with the end deep to 1 / 2-2 / 3 of the scion thickness, and then cut a 0.7-1.2 cm long short bevel at the back. Keep the surface of the rootstock's main vine dry before bud grafting. Make a straight, oblique incision about 20 cm from the soil surface, as the grafting site, reaching 1 / 3 to 1 / 2 of the xylem and equal in length to the long cut surface. Grafting should be performed between 7:00 AM and 9:00 AM or 4:00 PM and 5:30 PM, with an average daily temperature of no less than 25°C. Insert the cut scion into the incision, aligning and closely contacting the cambium of the scion and the rootstock. Wrap the scion-stock junction tightly with plastic film from bottom to top. After grafting, maintain 60% shade and maintain a humidity of 70% using micro-sprinkler irrigation. Fertilize after the bud develops two complete new leaves. Water the soil as needed to keep it moist. Apply 0.3% urea or potassium dihydrogen phosphate solution after budding, spraying every 30 days. Maintain normal management.

[0143] Comparative Example 4 Tissue Culture Method

[0144] This method involves tissue culture. Pepper seeds (Heat-Induced No. 1 pepper) are used as explants. After surface disinfection with 75% alcohol for 45 seconds, they are rinsed 3 to 5 times with sterile water. Sterilize and sterilize them with 95% anhydrous ethanol for 45 seconds, rinse with sterile water 3 to 5 times, and finally, soak and disinfect them with 0.1% HgCl2 (8 to 12 minutes). Afterwards, they are rinsed 5 times with sterile water, each rinse lasting about 30 seconds. After absorbing the moisture with sterile filter paper, the seeds are inoculated onto MS medium containing 2.5g of activated carbon. After culturing on MS medium for about 90 days, sterile seedlings begin to germinate. The shed sterile outer seed coats are collected and set aside. Transfer the sterile seed coats to MS medium every 30 days. After 30 days of culture, black callus will be induced. Embryogenic callus will be transferred to MS medium supplemented with 0.4 mg / L 2,4-D and 0.6 mg / L KT. After 20 days of culture, white, loosely structured, granular somatic embryos will differentiate. After approximately 26 days of culture, the somatic embryos will begin to differentiate into clustered shoots. Clustered shoots are divided and inoculated onto rooting medium. After 30 days of culture, the clustered shoots begin to root and grow into full plants. Fully grown seedlings of the Reyin No. 1 pepper are placed outdoors in 50% shade for 20 days, then adjusted to 30% shade for another 20 days. The hardened seedlings are then transplanted into seedling bags for cultivation. Water the soil regularly to maintain moisture, depending on soil dryness. Apply 0.3% urea or potassium dihydrogen phosphate solution after scion germination, spraying every 30 days and maintaining normal care. The culture chamber conditions were 28±2°C and 2000 lx illumination for 12 h.

[0145] Comparative Example 5 Cutting Propagation Method

[0146] Cut the root system of the parent plant of the Reyin No. 1 pepper plant near the surface into 15cm cuttings, leaving them intact with the top cut flat and the bottom cut at an angle. Insert the cuttings at a 45-degree angle into the seedling bed. Cover the surface with a 1cm thick layer of fine coconut coir and spray the bed once with a 500x dilution of carbendazim. Leave the upper 2-3cm of the roots exposed to air. Build a 50-80cm high plastic film arch over the seedling bed, topped with a shade net to maintain 50% shade. Use micro-sprinkler irrigation to maintain an air humidity of 80% and a temperature between 24-30°C. After 30 days, the tillers will begin to swell. After 60 days, when the tillers reach 25cm and have their own independent root systems, separate the tillers and transplant them into seedling bags for cultivation. Water the soil as needed to keep it moist. After scion budding, apply 0.3% urea or potassium dihydrogen phosphate solution, spraying every 30 days. Maintain normal care.

[0147] Comparing the growth of pepper seedlings using different methods (Table 1), the methods of Examples 1-3 of the present invention and the modified methods of Comparative Examples 1 and 2 showed shorter seedling establishment times, shorter times for breeding 1,000 seedlings, and shorter interface healing times. The average number of root tillers was higher, and the seedling survival rate was higher, averaging over 80%. The seedlings cultivated had strong taproots and developed root systems. Examples 1-3 were the most optimal methods. Compared with the patch budding method of Comparative Example 3, Examples 1-3 significantly shortened the time required to breed 1,000 seedlings and the interface healing time. Compared with the tissue culture method of Comparative Example 4, Examples 1-3 significantly shortened the seedling formation time and the time required to breed 1,000 seedlings. The cultivated seedlings had taproots, were stronger, and had more developed root systems. Compared with the root tiller propagation method of Comparative Example 5, Examples 1-3 significantly shortened the time required to breed 1,000 seedlings, had higher average root number and survival rate, cultivated seedlings had taproots, had more developed root systems, and were less susceptible to restrictions on the amount of mother plant material. Although the root tiller propagation method has an advantage in seedling formation time, the number of seedlings formed by cutting tillers during batch propagation is far less than that formed by tissue culture and grafting, and is limited by the amount of mother plant material, resulting in the longest time required to breed 1,000 seedlings.

[0148] Table 1 Growth of pepper seedlings of Reyin No. 1 under different seedling raising methods

[0149]

[0150]

[0151] Note: Seedling formation time refers to the time from sowing or preparing cuttings to the formation of seedlings for transplanting. The time required to breed 1,000 plants includes the time required to prepare the mother plant cutting materials, seed seedling breeding, grafting, tissue culture, seedling hardening, etc., and finally form seedlings for transplanting.

[0152] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The grafting seedling raising method of heat-induced No. 1 pepper is characterized in that, The steps include: (1) Stock cultivation and treatment: sowing and germinating yellow pepper seeds until the stem of the seedlings, which is 1 to 3 cm near the ground, reaches a thickness of 0.2 to 0.5 cm, thereby obtaining seedlings; cross-cutting the epicotyl of the seedlings, removing the top, and making a longitudinal incision in the center of the cross section to obtain stock; (2) Scion cultivation and treatment: The outer seed coat of the germinated pepper seeds of Reyin No. 1 was used as the explant, and callus induction and cluster bud induction were carried out in sequence to obtain cluster buds; cluster buds with a stem diameter of 0.2 to 0.5 cm were taken, the terminal bud was cut off, and the base was cut into a wedge surface of 0.5 to 0.9 cm in length to obtain the scion; (3) Grafting: inserting the scion into the incision of the rootstock, sealing the joint, and obtaining a grafted seedling.

2. The grafting seedling raising method according to claim 1, wherein In step (1), the epicotyl is the position of the seed sprout 2 to 3 cm away from the substrate surface.

3. The grafting seedling raising method according to claim 1, wherein In step (1), the depth of the incision is 0.5 to 0.8 cm.

4. The grafting seedling raising method according to claim 1, wherein In step (1), the germination temperature is 26-30°C; The nutrient soil for sowing includes 6 to 8 parts of topsoil, 2 to 4 parts of commercial organic fertilizer and 2 parts of coconut husk; The commercial organic fertilizer includes 450-500g kg –1 Organic matter, 15.3-16.0 g·kg –1 N, 47.0-47.5 g·kg –1 P2O5, 12.6~13.2g·kg –1 K2O, pH 8.

4.

5. The seedling raising method according to claim 1, wherein In step (2), the scion retains 2 to 3 leaves, and 1 / 2 of each leaf is retained.

6. The grafting seedling raising method according to claim 1, wherein In step (2), the length of the cut terminal bud is 2 cm.

7. The grafting seedling raising method according to claim 1, wherein In step (2), The seed germination culture medium is: MS+1.0-2.5g / L activated carbon+30g / L sucrose+2.5g / L plant gel, pH 5.8-6.0; The callus induction culture medium is MS+0.25-1.0 mg / L 2,4-D+0.25-1.0 mg / LIBA+0.05-1.0 mg / L KT+30 g / L sucrose+2.5 g / L phytagel, pH 5.8-6.0; The culture medium for inducing clustered buds is: MS+0.05-0.5 mg / L 2,4-D+0.25-1.0 mg / L IBA+0.05-1.0 mg / L KT+30 g / L sucrose+2.5 g / L plant gel, with a pH of 5.8-6.

0.

8. The grafting seedling raising method according to claim 6, wherein: The sealing film comprises: using a transparent film strip with a thickness of 0.01 mm, a width of about 1 cm and good toughness to wrap it tightly from bottom to top in circles, with the circles overlapping by about 1 / 3, and finally tying a knot above the interface; using a transparent sealing bag to wrap the junction of the scion and the stockwood scion, leaving only a small space near the junction of the stockwood scion for air circulation.

9. The grafting seedling raising method according to any one of claims 1 to 8, characterized in that: It also includes water control management after grafting; The water management includes: maintaining 60% shade for the seedlings, intermittent spraying to maintain 80% air humidity to prevent dehydration of the scion and promote interface healing; adjusting to 30% shade after 4 weeks and maintaining micro-sprinkler irrigation.

10. The grafting seedling raising method according to any one of claims 1 to 9, characterized in that: It also includes post-grafting fertilization management; The fertilization management includes: starting to fertilize after the scion grows a complete new leaf, irrigating in time according to the dryness and wetness of the soil to keep the soil moist; applying 0.3% urea or potassium dihydrogen phosphate aqueous solution after the scion sprouts, spraying once every 30 days, and normal management.

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