Open type tissue culture rooting method for sugarcane virus-free seedling without transplanting and false planting

Abstract

The present application belongs to the field of sugarcane seedling cultivation, and particularly relates to an open type tissue culture rooting method for sugarcane virus-free seedling without transplanting and false planting. The present application makes the seedling strong before culture, so that the leaf of the seedling reaches a certain growth amount, and the self-sustaining ability of the plant is ensured; rooting and false planting are combined, and the seedling is in contact with the outside air, sunlight and moisture through the open type rooting promoting culture medium without adding organic matter, so that the seedling reaches full self-sustaining growth while rooting, reaches a certain growth amount in a short period, and meets the demand of field planting. The seedling can be directly planted in the field without transplanting and false planting, the artificial and material costs of the traditional false planting link are omitted, and the growth cycle is shortened.

Description

Technical Field

[0001] This invention belongs to the field of sugarcane seedling cultivation, specifically relating to an open tissue culture rooting method for virus-free sugarcane seedlings that does not require transplanting. Background Technology

[0002] Sugarcane, as an important sugar crop and energy crop, has a high biomass yield and sucrose content, and can be used for the production of sugar and alcohol. Sugar production depends on the production of sugarcane seedlings. Therefore, the quality and efficiency of sugarcane seedling propagation directly affect the large-scale development of the sugarcane industry. Currently, the main process for improving sugarcane varieties is "superior varieties - virus elimination - tissue culture - hardening off - transplanting and temporary planting - field planting".

[0003] 1. Sugarcane is an asexually propagated crop. Conventional propagation easily leads to varietal degeneration. Years of asexual reproduction make it highly susceptible to carrying various pathogens, including viral viruses such as sugarcane mosaic virus and yellow leaf virus, and bacterial pathogens such as root dwarfing pathogens. This reduces sugarcane yield and sugar content. Currently, using heat treatment and thermotherapy combined with shoot tip meristem culture of sugarcane axillary buds can efficiently obtain virus-free, healthy seedlings.

[0004] In tissue culture, explant preparation and subculture all require a sterile environment, necessitating autoclaves, laminar flow hoods, and skilled aseptic operators. This process is extremely resource-intensive. While tissue culture can meet the requirements for crops with significant economic value, the average price of sugarcane is around 400 yuan per ton. For large-scale propagation of seedlings of this low-value crop, only by significantly reducing production costs can virus-free seedlings be widely adopted and sugar production further guaranteed. Therefore, if a certain stage of the culture could be conducted in a natural, open environment where explants can grow normally, it would not only reduce the requirements for equipment, operators, and propagation sites, but also fundamentally simplify the tissue culture process, shorten the seedling stage, and effectively reduce the production cost of virus-free sugarcane seedlings.

[0005] 2. Traditional tissue culture and transplanting have long production cycles. Traditional tissue culture requires aseptic techniques for rooting, with a rooting cycle of approximately 30-45 days, followed by a 5-day hardening-off period. The transplanting environment is a semi-closed, moist environment, making it susceptible to pests and diseases, extending the growth cycle to 40-65 days. In summary, depending on the variety and season, the total time for traditional tissue culture rooting, hardening-off, and transplanting is approximately 75-115 days, resulting in a long seedling production cycle that is not conducive to rapid seedling propagation.

[0006] 3. Complex process and high production costs. The cost of tissue culture includes independent inoculation rooms, culture medium preparation rooms, sterilization rooms, sterilizers, ultra-clean workbenches, pure water systems, and other equipment. It also requires high labor costs, such as skilled aseptic operators. Furthermore, the operation of the workplace and equipment requires high electricity costs. The transplanting stage, as a crucial step connecting seedling cultivation and field planting, not only has a long production cycle but also faces the following production cost issues: The first step after emergence, hardening off the seedlings, requires a sufficiently large, well-ventilated, and well-lit area. The transplanting process requires renting land, building separate seedling sheds, and manual labor for transplanting and transplanting operations, resulting in high land rental and labor management costs per acre of transplanted seedlings. High risk of pests and diseases: The semi-enclosed transplanting environment has high humidity, making it prone to diseases such as damping-off and root rot, as well as pests such as snails. Frequent spraying of fungicides and insecticides is required, increasing pesticide and management costs and posing a risk of pesticide residues.

[0007] To address these issues, the industry has attempted various improvements, such as shortening the transplanting period and optimizing substrate ratios, and has also implemented an open-type tissue culture rooting process. However, none of these solutions have broken through the traditional process of "from transplanting and transplanting to field planting," failing to fundamentally solve the problems of high costs and long cycles. Therefore, developing an open culture technology that eliminates the need for transplanting and allows for simultaneous open-type rooting and transplanting of seedlings has become a key requirement in the field of sugarcane industrial propagation. Summary of the Invention

[0008] The purpose of this invention is to overcome the high cost and long cycle of indoor tissue culture rooting and greenhouse transplanting in existing sugarcane breeding technologies. It provides an open tissue culture rooting method for virus-free sugarcane seedlings that eliminates the need for transplanting. By optimizing seedling conditions, the rooting environment, and seedling treatment processes, it achieves open rooting of seedlings, eliminating the need for a sterile environment. Simultaneously, it promotes seedling growth, completing the growth requirements for hardening and transplanting while simultaneously promoting rooting. This integrated process of rooting, hardening, and transplanting ensures consistent seedling quality and transplant survival rates comparable to traditional methods, while shortening the breeding cycle and reducing production costs.

[0009] The technical solution of this invention is implemented as follows:

[0010] An open-culture rooting method for virus-free sugarcane seedlings without transplanting includes the following steps:

[0011] (1) Strong seedlings

[0012] The proliferating seedlings obtained from sugarcane tissue culture are divided into small pieces, with 8-12 seedlings in each piece. Culture medium is added to the tissue culture bags, and the openings of the tissue culture bags are left open. The seedlings are cultured in an open environment with bacteria until they grow to 10-15cm in height. The culture medium is prepared by adding 1mL of isothiazolinone to every 1L of culture medium.

[0013] (2) Seedling pretreatment

[0014] Take tissue culture seedlings that have undergone robust seedling treatment, disinfect them, and drain them for later use;

[0015] (3) Preparation before rooting

[0016] The culture medium consists of a nutrient solution and a fixation substrate; wherein the nutrient solution is: 1 / 2MS + naphthaleneacetic acid (NAA) 2.0-3.0 mg / L, pH 5.8-6.5, after removing organic matter and sugar components; the prepared culture medium is filled into the planting trough to a height of about 6-8 cm;

[0017] (4) Open rooting culture

[0018] Seedling transplanting: Transplant the pretreated seedlings onto the culture medium in the planting trough;

[0019] Management: For the first 1-3 days after transplanting, spray with fungicide solution once a day to keep the culture medium moist; thereafter, water the leaves and the surface of the culture medium 2-3 times a day, each time spraying enough to moisten the surface of the culture medium but not to make it waterlogged; cultivate until the plants meet the standards for field transplanting.

[0020] Further, in step (1), the culture medium composition is as follows: KNO3 1.9 g / L, NH4NO3 1.65 g / L, MgSO4·7H2O 0.37 g / L, KH2PO4 0.17 g / L, CaCl2 0.332 g / L, MnSO4·4H2O 0.0223 g / L, ZnSO4·7H2O 0.0086 g / L, H3BO3 0.0062 g / L, KI 0.00083 g / L, Na2MoO4·2H2O 0.00025 g / L, CuSO4·5H2O 0.000025 g / L, CoCl2 2.6H2O 0.000025g / L, FeSO4·7H2O 0.0278g / L, Na2-EDTA 0.0373g / L, glycine 0.002g / L, vitamin B1 0.0005g / L, vitamin B6 0.001g / L, niacin 0.0005g / L, inositol 0.1g / L, sucrose 30g / L, carrageenan 8g / L, isothiazolinone 1ml / L, distilled water balance, pH 6.0; open culture conditions: 28-30℃, light intensity 12000-16000lx, light duration 12h / d; culture for 15-20 days; culture until stem diameter 0.2-0.3cm.

[0021] Furthermore, in step (2), the disinfection procedure is as follows: open the tissue culture bag, take out the seedlings and rinse the base of the seedlings with tap water to remove any remaining culture medium, and soak them in a 50% wettable powder of carbendazim at a dilution of 800-1000 times for 15-20 minutes; or in a 600-800% wettable powder of mancozeb at a dilution of 600-800 times for 15-20 minutes.

[0022] Further, in step (3), the nutrient solution composition is as follows: KNO3 0.95 g / L, NH4NO3 0.825 g / L, MgSO4·7H2O 0.185 g / L, KH2PO4 0.085 g / L, CaCl2 0.166 g / L, MnSO4·4H2O 0.01115 g / L, ZnSO4·7H2O 0.0043 g / L, H3BO3 0.0031 g / L, KI 0.000415 g / L, Na2MoO4·2H2O 0.000125 g / L, CuSO4·5H2O 0.0000125 g / L, CoCl2 2. 6H2O 0.0000125g / L, FeSO4·7H2O 0.0139g / L, Na2-EDTA 0.01865g / L, naphthaleneacetic acid (NAA) 2.0-3.0mg / L, water balance, pH 5.8-6.5; the fixation substrate is: commercial soil, fully decomposed coconut coir or sawdust and / or carrageenan.

[0023] Furthermore, in step (3), the carrageenan needs to be prepared together with the nutrient solution. Tap water can be used for preparation. 8g of carrageenan is needed for 1L of nutrient solution. The nutrient solution and carrageenan should be mixed evenly first, and then heated until the carrageenan is completely melted. After solidification and cooling, it can be used.

[0024] Furthermore, in step (3), the planting trough is a rooting trough or a seedling tray; the dimensions of the rooting trough are: length × width × height = 35cm × 20cm × 10cm; the rooting trough is divided into two types: sealed bottom and bottom with holes. Seedling trays can also be used as planting troughs, with dimensions of 25cm × 47cm, and 50 or 32 holes are acceptable.

[0025] Furthermore, in step (4), the seedlings are planted at a spacing of 3-5cm between plants and 5-7cm between rows, and the base of the tissue culture seedlings is inserted 1-2cm below the culture medium. The fungicide solution is 50% wettable powder of carbendazim at 800 times dilution; or 80% wettable powder of mancozeb at 600 times dilution. The culture conditions are to maintain a relative humidity of 65%-80%, a temperature of 28-30℃, a light duration of 12-14h / d, and a light intensity of 20000-30000lx. When the root length of the seedling reaches 8-10cm, the number of fibrous roots is ≥5, the stem diameter reaches 5-8mm, the plant height reaches 20cm or more, and the pseudostem is 5cm or more, then open rooting is completed.

[0026] The above-described open tissue culture rooting method for sugarcane without transplanting has been applied to improve the root length, number of fibrous roots, stem diameter, overall plant height, and / or pseudostem length of sugarcane tissue culture seedlings.

[0027] The above-described open tissue culture rooting method for sugarcane without transplanting is applied to sugarcane seedling cultivation.

[0028] The above-described open tissue culture rooting method for sugarcane without transplanting and temporary planting is applied to shorten the sugarcane seedling cultivation cycle.

[0029] The beneficial effects of this invention are:

[0030] The open tissue culture rooting method without transplanting described in this invention ensures the plant's self-sufficiency by strengthening the seedlings before cultivation and achieving a certain level of leaf growth. It then fully leverages the advantages of sugarcane as a high-light-efficiency C4 crop, combining rooting and transplanting. Using an open rooting-promoting culture medium without added organic matter, the seedlings are exposed to air, sunlight, and water while rooting, achieving sufficient self-sufficiency and reaching a certain growth level in a short period, meeting the requirements for field planting. This eliminates the need for transplanting and transplanting, saving labor and material costs associated with traditional transplanting, shortening the growth cycle, and maintaining rooting and survival rates comparable to conventional tissue culture.

[0031] 1. Increased Efficiency: Traditional tissue culture methods have a rooting cycle of approximately 30-45 days (the time varies depending on the variety), followed by 5 days of hardening-off, and a transplanting / temporary planting cycle of approximately 40-65 days (shorter in spring and summer, longer in autumn and winter), totaling about 75-115 days. The open-type tissue culture rooting method of this invention, which eliminates the need for transplanting / temporary planting, shortens the rooting cycle to 35-40 days, eliminating the need for subsequent hardening-off and temporary planting. This reduces the rooting and transplanting / temporary planting time by approximately 50% compared to traditional tissue culture methods.

[0032] 2. Cost Reduction: This invention allows for an open environment during the rooting process, requiring only a small amount of substrate and a relatively inexpensive plastic film greenhouse. Compared to traditional greenhouses or semi-enclosed temporary planting environments with small arched sheds in the open air, air circulation is improved, significantly reducing the breeding of pests and diseases. It eliminates the large costs associated with substrate procurement and manual management in the temporary planting stage, reducing the propagation cost per acre by 35%-45%; pesticide use is reduced by more than 80%. The nutrient solution used during rooting is 1 / 2 MS, and tap water is sufficient instead of filtered pure water. Sucrose is also eliminated. The solid substrate can be carrageenan, inexpensive commercial soil, or even cheaper coconut coir or sawdust, further reducing production costs. The invention eliminates the need for sterilization equipment, reducing equipment and electricity investment. There is no need to purchase traditional disposable seedling trays; the planting troughs are small transport baskets made of plastic, which are inexpensive, have a long service life, and can be reused multiple times. Using planting troughs allows for full utilization of space, enabling three-dimensional cultivation without the need for traditional transplanting greenhouses or field land, saving the cost of renting land for the transplanting process.

[0033] 3. Simple Operation: No aseptic operation techniques are required. Traditional tissue culture rooting methods require trained aseptic operators, and improper operation can lead to contamination and significant losses. The method described in this invention does not require specialized tissue culture rooting or transplanting techniques, simplifying the process. Large-scale application allows for mechanized spray management, adapting to large-scale factory propagation, saving at least 80% of labor costs and facilitating widespread adoption.

[0034] The method described in this invention improves the culture medium during the rooting stage, enabling its antibacterial function to be used in an open environment. It also fully utilizes space in existing sites to achieve three-dimensional cultivation, ensuring normal rooting while simultaneously promoting vegetative growth. During rooting, the plants reach the growth level required for direct field transplanting after transplanting. This method simplifies the tissue culture process and eliminates the need for a series of sterile workshops (inoculation room, sterilization room, culture medium preparation room) required by traditional tissue culture, reducing the aseptic operation requirements for tissue culture workers. It also makes full use of space, eliminating the need for large areas of land and manual planting during transplanting, and reducing manual field management after transplanting. This significantly increases the speed of seedling cultivation, thereby significantly reducing production costs. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The height of seedlings after conventional tissue culture treatment before rooting culture: the left picture shows seedlings with a height of 1-2.5cm, the middle picture shows seedlings with a height of 5-8cm, and the right picture shows seedlings with a height of 10-15cm.

[0037] Figure 2 The images show the growth of seedlings after cultivation using different seedling strengthening methods, where a represents the seedling strengthening method described in this invention, and b represents the conventional tissue culture method.

[0038] Figure 3 The effects of different methods and cultivation substrates on seedling growth were investigated. Among them, a) was the conventional tissue culture method (tissue culture bag size 10cm×10cm); b) 32-cell tray (with holes at the bottom) + sawdust + nutrient solution; c) rooting trough (with holes at the bottom) + commercial soil + nutrient solution; d) rooting trough (without holes at the bottom) + carrageenan + nutrient solution. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] An open-culture rooting method for virus-free sugarcane seedlings without transplanting includes the following steps:

[0042] (1) Seedling strengthening: The proliferating seedlings (Zhongtang No. 1 variety) obtained from sugarcane tissue culture were cut into pieces about the size of a fingernail, with about 10 seedlings per piece. Four pieces were placed in a culture bag, which was pre-filled with culture medium. The culture medium retained all the components of MS (including 30 g / L sucrose) but removed all exogenous hormones, with a pH of 5.8-6.5. At the same time, 1 ml of isothiazolinone stock solution was added to 1 L of MS medium as an antibacterial component. The specific composition of the culture medium is as follows: KNO3 1.9 g / L, NH4NO3 1.65 g / L, MgSO4·7H2O 0.37 g / L, KH2PO4 0.17 g / L, CaCl2 0.332 g / L, MnSO4·4H2O 0.0223 g / L, ZnSO4·7H2O 0.0086 g / L, H3BO3 0.0062 g / L, KI 0.00083 g / L, Na2MoO4·2H2O 0.00025 g / L, CuSO4·5H2O 0.000025 g / L, CoCl2 2. The following ingredients were added: 6H₂O 0.000025 g / L, FeSO₄·7H₂O 0.0278 g / L, Na₂-EDTA 0.0373 g / L, glycine 0.002 g / L, vitamin B1 0.0005 g / L, vitamin B6 0.001 g / L, niacin 0.0005 g / L, inositol 0.1 g / L, sucrose 30 g / L, carrageenan 8 g / L, isothiazolinone 1 ml / L, distilled water balance, pH 6.0. The tissue culture bags were left open, and seedlings were encouraged to grow vigorously in a sterile environment. Culture conditions were: 28-30℃, light intensity 12000-16000 lx, light duration 12 h / d. After 15-20 days of open culture, when the seedlings reached 10-15 cm in height, they could proceed to the next step of open rooting culture without transplanting.

[0043] (2) Seedling pretreatment: Select strong sugarcane virus-free seedlings, soak the roots in 800 times diluted carbendazim (50% wettable powder) solution for 18 minutes, drain and set aside.

[0044] (3) Preparation before rooting: The culture medium consists of nutrient solution and carrageenan. The nutrient solution is: the basic culture medium is 1 / 2 MS + naphthaleneacetic acid with organic matter and sugar components removed. The specific composition of the culture medium is: KNO3 0.95 g / L, NH4NO3 0.825 g / L, MgSO4·7H2O 0.185 g / L, KH2PO4 0.085 g / L, CaCl2 0.166 g / L, MnSO4·4H2O 0.01115 g / L, ZnSO4·7H2O 0.0043 g / L, H3BO3 0.0031 g / L, KI 0.000415 g / L, Na2MoO4·2H2O 0.000125 g / L, CuSO4·5H2O 0.0000125 g / L, CoCl2 2. The nutrient solution contains 0.0000125 g / L 6H₂O, 0.0139 g / L FeSO₄·7H₂O, 0.01865 g / L Na₂-EDTA, 2.5 mg / L naphthaleneacetic acid (NAA), 8 g / L carrageenan, water balance, and pH 6.0. The nutrient solution and carrageenan are first mixed thoroughly, then heated until the carrageenan is completely dissolved. Water is then added to bring the volume to a final level.

[0045] Rooting troughs are selected for planting. The dimensions of the rooting troughs are: length × width × height = 35cm × 20cm × 10cm. The bottom of the rooting trough is sealed and has no holes. Fill the rooting troughs with the prepared culture medium to a height of approximately 6-8cm. Existing tissue culture rooms can be used as rooting sites. Place the rooting troughs on culture racks to create three-dimensional rooting racks, making full use of the space. Pour culture medium into each rooting trough to a height of approximately 6-8cm (total volume approximately 2L), and allow it to cool and solidify before use.

[0046] (4) Open-type rooting cultivation: Pre-treated virus-free sugarcane seedlings are planted in rooting troughs at a spacing of 4cm between plants and 7cm between rows. For the first 1-3 days, spray once daily with a 50% wettable powder of carbendazim (800x dilution), simply moistening the substrate and seedling surface. Afterward, spray water 2-3 times daily to keep the substrate and seedling surface moist. Maintain a relative humidity of 65%-80%, a temperature of 28-30℃, and a light intensity of 25000 lx for 13 hours / day. After 10 days of cultivation, the root length reaches over 4cm, with 6 fibrous roots. After 35-40 days of cultivation, the seedlings can be directly transplanted into the field.

[0047] Example 2

[0048] An open-culture rooting method for virus-free sugarcane seedlings without transplanting includes the following steps:

[0049] (1) Strong seedlings: Same as in Example 1.

[0050] (2) Seedling pretreatment: Select tissue culture seedlings of virus-free sugarcane with a plant height of 10cm and a stem diameter of 0.2cm, soak the roots in a 600-fold dilution of mancozeb (80% wettable powder) for 15 minutes, drain and set aside.

[0051] (3) Preparation before rooting: Select rooting troughs. Existing tissue culture rooms can be used as rooting sites. Place the rooting troughs (with holes at the bottom) on the culture rack to form a three-dimensional rooting rack, making full use of the space. The dimensions of the rooting troughs are: length × width × height = 35cm × 20cm × 10cm. The culture medium consists of nutrient solution and commercial soil. The nutrient solution is: 1 / 2MS (removed organic matter and sugar components) + 2.5mg / L naphthaleneacetic acid (NAA), pH 6.0 (tap water is used to prepare the nutrient solution). The solid substrate is commercial soil, which is mixed with the nutrient solution and then filled into the rooting troughs.

[0052] (4) Open-type rooting cultivation: Pretreated virus-free sugarcane seedlings were planted in the rooting trough at a spacing of 4 cm between plants and 7 cm between rows. For the first 1-3 days, spray once daily with 80% wettable powder of mancozeb (800x dilution), simply moistening the substrate and seedling surface. Afterward, ensure adequate ventilation in the rooting chamber by keeping the windows open and supplementing with natural light. Spray water 5-6 times daily, closing the windows at night to maintain humidity. Supplement with 4 hours of light (20000 lx) between 7:00-9:00 AM and 5:00-7:00 PM.

[0053] Example 3

[0054] An open-culture rooting method for virus-free sugarcane seedlings without transplanting is described. The steps are the same as in Example 2, except that the commercial soil is replaced with lower-cost coconut coir or sawdust, and the planting troughs are replaced with lower-cost seedling trays instead of rooting troughs. The trays are 25cm × 47cm in size, with either 50 or 32 cells. The solid substrate is sawdust or coconut coir, which, after fully absorbing the nutrient solution, is filled into the rooting troughs to a depth of 7cm for later use.

[0055] In the above embodiments, the virus-free sugarcane seedlings can be common varieties used in sugarcane cultivation, such as Zhongtang No. 1, Xintaitang No. 22, Zhongtang No. 2, Zhongtang No. 3, etc., and this invention does not make any specific limitations.

[0056] I. Effects of different cultivation methods on seedling physiological parameters

[0057] The same batch of virus-free sugarcane seedlings (Zhongtang No. 1) that had undergone propagation culture were randomly divided into two groups. The two groups were cultivated using the open tissue culture rooting method without transplanting as described in this invention and the traditional method of transplanting after tissue culture rooting. The effects of different cultivation methods on the growth of seedlings were statistically analyzed.

[0058] The rooting method described in this invention is operated in accordance with Example 2, and the open rooting culture is carried out for 40 days.

[0059] The traditional method of transplanting and temporarily planting after tissue culture rooting (hereinafter referred to as the "conventional method") (Yang Benpeng, Zhang Shuzhen, Zhao Tingting, et al. Sugarcane Improved Variety Propagation and Virus-Free Seedling Technology [M]. Beijing: China Agriculture Press, 2019: 121-167) is as follows:

[0060] (1) Strengthening seedlings: The conventional seedling culture medium is MS + 1.5 mg / L 6-BA + 30 g / L sucrose (pH 5.8-6.0). The culture conditions are: 25-30℃, 12 h light per day, and closed aseptic culture for 15-20 days.

[0061] (2) Rooting: Remove organic matter and sugar components from 1 / 2MS + 2.0-3.0mg of naphthaleneacetic acid (NAA), pH 5.8-6.5; fill the prepared culture medium into the seedling bag to a height of about 6-8cm; maintain a relative humidity of 65%-80%, a temperature of 28-30℃, a light duration of 12-14h / d, and a light intensity of 12000-15000lx.

[0062] (3) Hardening off seedlings: Harden off seedlings for 5 days using the conventional method.

[0063] (4) Temporary transplanting: The conventional method requires transplanting and temporary transplanting for 57-62 days after hardening off the seedlings.

[0064] The chlorophyll content, soluble protein content, soluble sugar content, and superoxide dismutase (SOD) activity of seedlings cultured using both Example 2 and conventional methods were statistically analyzed. The determination methods are as follows:

[0065] Chlorophyll content: Acetone colorimetric method

[0066] Soluble protein content: Coomassie brilliant blue colorimetric method

[0067] Soluble sugar content: anthrone colorimetric method

[0068] SOD: Activity: Superoxide Dismutase (SOD) Kit, Brand: Solarbio

[0069] The measurement results are shown in Table 1-4.

[0070] Table 1 Chlorophyll content

[0071]

[0072] Note:

[0073] Table 2 Soluble Protein Content

[0074]

[0075] Note: C: Protein content per tube obtained from the standard curve (mg); V: Total volume of extract (mL); A: Sample volume taken during the assay (mL); W: Sample volume (g)

[0076] Table 3 Soluble sugar content

[0077]

[0078] Note:

[0079] Table 4 SOD Activity

[0080]

[0081] Note: A0 - Optical density of the control tube; As - Extinction value of the sample tube; V1 - Total volume of sample solution (mL); V2 - Sample volume used during measurement (mL); Fw - Fresh weight of sample (g)

[0082] As shown in Tables 1 to 4, there are no significant differences in chlorophyll content, soluble protein content, soluble sugar content, and SOD activity between traditional tissue culture rooting followed by transplanting and the open rooting method without transplanting described in this invention. The measured parameters are comparable to those of sugarcane seedlings cultivated by conventional tissue culture, and their physiological states are basically consistent.

[0083] Traditional tissue culture rooting takes approximately 30-45 days (the time varies depending on the variety), plus 5 days of hardening-off, and a transplanting period of approximately 40-65 days (shorter in spring and summer, longer in autumn and winter). The total time required for rooting and transplanting using tissue culture is approximately 75-115 days to reach the standard for field transplanting. This invention combines the rooting and transplanting processes, eliminating the intermediate hardening-off stage, shortening the cycle to 35-40 days (approximately 50% shorter than the traditional transplanting period). The rooting rate remains at 95%-98%, comparable to traditional methods. Seedling physiological indicators show no significant difference from traditional tissue culture methods, and the seedlings also meet the standards for field transplanting. Therefore, the method described in this invention significantly shortens the rooting cycle while ensuring seedling survival rate and quality, achieving rooting in only 35-40 days and meeting the standards for field transplanting.

[0084] II. The Influence of Sugarcane Virus-Free Seedling Size on Rooting Efficiency

[0085] Seedlings of different heights and stem diameters were subjected to conventional closed-system seedling strengthening treatment. Figure 1After 40 days of rooting using the open rooting method of this invention (using nutrient solution + commercial soil as the substrate), the growth was statistically analyzed. The results are shown in Table 5. Significant differences in growth were observed among the virus-free sugarcane seedlings of different heights, with seedlings of 10-15cm in height showing significantly higher rooting and survival rates than other groups. Therefore, subsequent experiments were conducted using virus-free sugarcane seedlings with a height of 10-15cm and a stem diameter of 0.2-0.3cm.

[0086] Rooting rate = (Number of rooted seedlings / Total number of seedlings) × 100%

[0087] Survival rate = (Number of surviving seedlings / Total number of seedlings) × 100%

[0088] Table 5 Comparison of seedling growth

[0089]

[0090] III. The Impact of Different Seedling Strengthening Methods on Seedling Growth

[0091] Seedling strengthening was performed using both conventional tissue culture methods (closed seedling strengthening) and the seedling strengthening method described in this invention (refer to Example 1, open culture with the bag opening cut open). Figure 2 After 15 days of vigorous seedling growth, the seedlings underwent open rooting culture (using nutrient solution and commercial soil as the culture medium) for 40 days. Growth was then assessed. The results are shown in Table 6.

[0092] Table 6. Effects of different seedling strengthening methods on seedling growth

[0093]

[0094] As shown in Table 6, although there was no significant difference in plant height and stem diameter before cultivation using the seedling strengthening method described in this invention, after 40 days of cultivation using the open seedling strengthening method described in this invention, not only can the rooting rate and survival rate of the seedlings be significantly improved, but also the indicators such as the length of the main root and the number of fibrous roots are significantly higher than those of the conventional root strengthening method. This indicates that the seedling strengthening method described in this invention has a significant promoting effect on the growth of sugarcane seedlings and is beneficial to subsequent open rooting cultivation.

[0095] IV. The Influence of Different Culture Media on Seedling Growth

[0096] According to the method described in this invention, seedlings were cultivated using different culture media, with the conventional method serving as a control group. Seedling growth was compared after cultivation. In the table, the conventional method measured various indicators once after rooting in tissue culture, and then measured a set of data after hardening (5 days) and transplanting (40 days) following emergence. The method described in this invention integrates open rooting and transplanting, simultaneously achieving the growth expected after transplanting, thus requiring only one set of data. Results are shown in Table 7 and... Figure 3 .

[0097] Table 7. Effects of different culture media on seedling growth

[0098]

[0099] As shown in Table 7, the culture media used in this invention can all ensure the normal growth of sugarcane seedlings, and the seedling survival rate (93.2%, 95.4%, 94.6%) is basically the same as the survival rate after conventional transplanting (94.5%); the rooting rate (95.3%, 97.8%, 96.4%) is also basically the same as the rooting rate after conventional transplanting (95.5%). The measured parameters are not significantly different from those of sugarcane seedlings cultivated using conventional methods plus transplanting, and some indicators are even better, meeting the requirements for field transplanting.

[0100] Compared to conventional rooting culture methods, the method described in this invention can significantly improve the root length, number of fibrous roots, stem diameter, overall plant height, and pseudostem length of seedlings. After rooting culture, the effect of conventional transplanting culture can be achieved, and the seedling propagation cycle is shortened by 35-70 days compared to the traditional process. It saves the land rent for greenhouses or open-air sites required for transplanting and transplanting, as well as the transportation costs of seedlings, and reduces the amount of pesticides used.

[0101] Using nutrient solution and carrageenan saves about 80% on material costs and reduces labor input by about 70%.

[0102] Using nutrient solution and commercial soil, the nutrient solution can be prepared with tap water, eliminating the need for distilled water. Compared to Example 1, this method further eliminates the costs associated with expensive carrageenan and electricity usage from 10:00 AM to 4:00 PM, fully utilizing the strongest natural sunlight of the day (except on cloudy or rainy days). No cultivation room is required; a relatively inexpensive plastic film greenhouse is used, saving approximately 82% on materials and reducing labor input by about 70%. Compared to carrageenan, the cost is significantly lower.

[0103] Using nutrient solution and sawdust (coconut coir), the nutrient solution can be prepared with tap water, eliminating the need for distilled water. Compared with Example 1, this method further eliminates the use of expensive carrageenan, and compared with Example 2, it eliminates the cost of commercial soil, saving about 85% of materials and reducing labor input by about 70%, thus further reducing costs.

[0104] V. Comparison of different rooting methods

[0105] Referring to the method described in this invention, virus-free sugarcane seedlings with a plant height of 10-15cm and a stem diameter of 0.2-0.3cm were used. The culture medium consisted of nutrient solution and commercial soil. Open rooting culture was carried out according to the rooting methods in Table 8, and the seedling growth was statistically analyzed. The results are shown in Table 8.

[0106] Table 8. Effects of different rooting methods on seedling growth

[0107]

[0108] Different rooting methods significantly impact seedling growth. Failure to disinfect before rooting and the absence of fungicide spraying three days prior to rooting culture not only affects seedling growth but also significantly increases contamination rates, resulting in significantly lower rooting and survival rates. The absence of NAA in the substrate not only hinders seedling growth and slows rooting but also causes slight wilting, with rooting and survival rates significantly lower than those of the method described in this invention, and significantly lower than treatments 1 and 2. Therefore, the open rooting method described in this invention not only improves seedling rooting and survival rates but also results in healthy overall seedling growth and a significantly reduced contamination rate.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of an open-type tissue culture rooting method of sugarcane virus-free seedlings free of transplanting and false planting in improving root length, rootlet number, stem diameter, overall plant height and / or pseudostem length of sugarcane seedlings, the open-type tissue culture rooting method of sugarcane virus-free seedlings free of transplanting and false planting comprising the following steps: (1) strengthening seedlings divide the proliferation seedlings obtained by tissue culture into small blocks, 8-12 proliferation seedlings per block, add culture medium into the tissue culture bag, open the tissue culture bag, and open-culture in a bacterial environment until the seedlings grow to a height of 10-15 cm; the culture medium comprises KNO3 1.9 g / L, NH4NO3 1.65 g / L, MgSO4·7H2O 0.37 g / L, KH2PO4 0.17 g / L, CaCl2 0.332 g / L, MnSO4·4H2O 0.0223 g / L, ZnSO4·7H2O 0.0086 g / L, H3BO3 0.0062 g / L, KI 0.00083 g / L, Na2MoO4·2H2O 0.00025 g / L, CuSO4·5H2O 0.000025 g / L, CoCl2·6H2O 0.000025 g / L, FeSO4·7H2O 0.0278 g / L, Na2-EDTA 0.0373 g / L, glycine 0.002 g / L, vitamin B1 0.0005 g / L, vitamin B6 0.001 g / L, nicotinic acid 0.0005 g / L, myo-inositol 0.1 g / L, sucrose 30 g / L, 8 g / L carrageenan, isothiazolinone 1 ml / L, and distilled water in the rest, and the pH is 6.0; (2) seedling pretreatment take the seedlings treated by strengthening seedlings, perform disinfection treatment, and drain for standby use; (3) preparation before rooting the culture medium is composed of nutrient solution and fixed substrate; the nutrient solution comprises KNO3 0.95 g / L, NH4NO3 0.825 g / L, MgSO4·7H2O 0.185 g / L, KH2PO4 0.085 g / L, CaCl2 0.166 g / L, MnSO4·4H2O 0.01115 g / L, ZnSO4·7H2O 0.0043 g / L, H3BO3 0.0031 g / L, KI 0.000415 g / L, Na2MoO4·2H2O 0.000125 g / L, CuSO4·5H2O 0.0000125 g / L, CoCl2·6H2O 0.0000125 g / L, FeSO4·7H2O 0.0139 g / L, Na2-EDTA 0.01865 g / L, naphthalene acetic acid 2.0-3.0 mg / L, and water in the rest, and the pH is 5.8-6.5; fill the prepared culture medium into the planting groove until the height is 6-8 cm; (4) open-type rooting cultivation seedling planting: plant the pretreated seedlings on the culture medium in the planting groove at a plant distance of 3-5 cm and a row distance of 5-7 cm. Management: 1-3 days after planting, need to spray fungicide liquid once a day, to ensure the moisture state of the culture medium; thereafter, need to spray water 2-3 times a day on the leaf surface and the culture medium surface, the spraying amount each time is to make the culture medium surface wet but not waterlogged; culture to the standard of field planting; the culture conditions are to keep the relative humidity 65%-80%, the temperature 28-30℃, the light time 12-14h / d, and the light intensity 20000-30000lx; when the root length reaches 8-10cm, the number of fibrous roots is ≥5, the stem diameter reaches 5-8mm, the plant height is above 20cm, and the pseudostem is above 5cm, the open-rooting is completed.

2. Application of the open-rooting method of sugarcane virus-free seedling free transplanting and false planting in the cultivation of sugarcane seedlings, wherein the open-rooting method of sugarcane virus-free seedling free transplanting and false planting comprises the following steps: (1) Strengthening seedlings Divide the proliferation seedlings obtained by tissue culture into small blocks, 8-12 proliferation seedlings per block, add culture medium into the tissue culture bag, open the tissue culture bag, and culture in an environment with bacteria until the seedlings grow to 10-15cm high; the culture medium comprises KNO3 1.9g / L, NH4NO3 1.65g / L, MgSO4·7H2O 0.37g / L, KH2PO4 0.17g / L, CaCl2 0.332g / L, MnSO4·4H2O 0.0223g / L, ZnSO4·7H2O 0.0086g / L, H3BO3 0.0062g / L, KI 0.00083g / L, Na2MoO4·2H2O 0.00025g / L, CuSO4·5H2O 0.000025g / L, CoCl2·6H2O 0.000025g / L, FeSO4·7H2O 0.0278g / L, Na2-EDTA 0.0373g / L, glycine 0.002g / L, vitamin B10.0005g / L, vitamin B60.001g / L, nicotinic acid 0.0005g / L, myo-inositol 0.1g / L, sucrose 30g / L, 8g / L carrageenan, isothiazolinone 1ml / L, and distilled water in the rest, and the pH is 6.0; (2) Seedling pretreatment Take the seedlings treated by strengthening seedling, perform disinfection treatment, and drain for standby use; (3) Preparation before rooting The culture medium is composed of nutrient solution and fixed medium; the nutrient solution comprises: KNO3 0.95 g / L, NH4NO3 0.825 g / L, MgSO4·7H2O 0.185 g / L, KH2PO4 0.085 g / L, CaCl2 0.166 g / L, MnSO4·4H2O 0.01115 g / L, ZnSO4·7H2O 0.0043 g / L, H3BO3 0.0031 g / L, KI 0.000415 g / L, Na2MoO4·2H2O 0.000125 g / L, CuSO4·5H2O 0.0000125 g / L, CoCl2·6H2O 0.0000125 g / L, FeSO4·7H2O 0.0139 g / L, Na2-EDTA 0.01865 g / L, naphthalene acetic acid 2.0-3.0 mg / L, water in remainder, pH 5.8-6.5; the prepared culture medium is filled into the planting groove to a height of 6-8 cm; (4) open rooting cultivation Seedling planting: the pretreated seedlings are planted on the culture medium in the planting groove at a plant distance of 3-5 cm and a row distance of 5-7 cm; Management: after planting, the culture medium is sprayed with fungicide once a day for 1-3 days to keep the culture medium moist; thereafter, the culture medium and the seedling leaves are sprayed with water 2-3 times a day, and the amount of water sprayed each time is controlled such that the culture medium is moist but not waterlogged; the seedlings are cultivated until the seedlings reach the standard for field planting; the cultivation conditions are as follows: the relative humidity of the environment is kept at 65%-80%, the temperature is kept at 28-30℃, the light time is kept at 12-14 h / d, and the light intensity is kept at 20000-30000 lx; when the root length of the seedlings reaches 8-10 cm, the number of fibrous roots is greater than or equal to 5, the stem diameter reaches 5-8 mm, the plant height is greater than or equal to 20 cm, and the height of the tiller is greater than or equal to 5 cm, the open rooting is completed.

3. Application of an open rooting method for sugarcane virus-free seedlings in shortening the seedling raising period of sugarcane, wherein the open rooting method for sugarcane virus-free seedlings comprises the following steps: (1) strong seedling raising The cane tissue culture proliferation seedlings are divided into small pieces, each piece has 8-12 proliferation seedlings, culture medium is added into the tissue culture bag, the tissue culture bag is opened, and open culture is carried out in the environment with bacteria until the seedlings grow to 10-15 cm; the culture medium comprises KNO3 1.9 g / L, NH4NO3 1.65 g / L, MgSO4·7H2O 0.37 g / L, KH2PO4 0.17 g / L, CaCl2 0.332 g / L, MnSO4·4H2O 0.0223 g / L, ZnSO4·7H2O 0.0086 g / L, H3BO3 0.0062 g / L, KI 0.00083 g / L, Na2MoO4·2H2O 0.00025 g / L, CuSO4·5H2O 0.000025 g / L, CoCl2·6H2O 0.000025 g / L, FeSO4·7H2O 0.0278 g / L, Na2-EDTA 0.0373 g / L, glycine 0.002 g / L, vitamin B1 0.0005 g / L, vitamin B6 0.001 g / L, nicotinic acid 0.0005 g / L, myo-inositol 0.1 g / L, sucrose 30 g / L, 8 g / L carrageenan, isothiazolinone 1 ml / L, and the rest is distilled water, and pH is 6.0; (2) Seedling pretreatment The seedlings treated by the seedling treatment are subjected to disinfection treatment, and then are drained and reserved; (3) Preparation before rooting The culture medium is composed of nutrient solution and fixing matrix; the nutrient solution comprises KNO3 0.95 g / L, NH4NO3 0.825 g / L, MgSO4·7H2O 0.185 g / L, KH2PO4 0.085 g / L, CaCl2 0.166 g / L, MnSO4·4H2O 0.01115 g / L, ZnSO4·7H2O 0.0043 g / L, H3BO3 0.0031 g / L, KI 0.000415 g / L, Na2MoO4·2H2O 0.000125 g / L, CuSO4·5H2O 0.0000125 g / L, CoCl2·6H2O 0.0000125 g / L, FeSO4·7H2O 0.0139 g / L, Na2-EDTA 0.01865 g / L, naphthalene acetic acid 2.0-3.0 mg / L, and the rest is water, and pH is 5.8-6.5; the prepared culture medium is filled into the planting groove until the height is 6-8 cm; (4) Open rooting cultivation Seedling planting: the seedlings after pretreatment are planted on the culture medium in the planting groove at a plant distance of 3-5 cm and a row distance of 5-7 cm. Management: 1-3 days after planting, need to spray fungicide liquid once a day, to ensure the moisture state of the culture medium; thereafter, need to spray water 2-3 times a day on the leaf surface and the culture medium surface, the spraying amount of each time is to keep the culture medium surface wet but not waterlogged; culture to the standard of field planting; the culture conditions are to keep the relative humidity 65%-80%, the temperature 28-30℃, the light time 12-14h / d, and the light intensity 20000-30000lx; when the root length reaches 8-10cm, the number of fibrous roots is ≥5, the stem diameter reaches 5-8mm, the plant height is more than 20cm, and the pseudostem is more than 5cm, the open rooting is completed.

4. Use according to any one of claims 1 to 3, wherein In step (1), the open culture conditions are: the temperature is 28-30℃, the light intensity is 12000-16000lx, and the light time is 12h / d; the culture time is 15-20 days; the stem diameter is 0.2-0.3cm.

5. Use according to any one of claims 1 to 3, wherein the compound is ###0002### In step (2), the disinfection treatment method is: open the tissue culture bag, take out the seedling, wash the culture medium remaining at the base of the seedling with tap water, and soak in 800-1000 times fungicide 50% wettable powder liquid for 15-20min; or soak in 600-800 times mancozeb 80% wettable powder liquid for 15-20min.

6. Use according to any one of claims 1 to 3, wherein In step (3), the fixed substrate is: commercial soil and / or carrageenan.

7. Use according to claim 6, wherein In step (3), the carrageenan needs to be configured with nutrient solution, which can be configured with tap water, 8g of carrageenan is needed for 1L of nutrient solution, the nutrient solution and carrageenan are mixed uniformly first, then heated until the carrageenan is completely melted, and used after solidification and cooling.

8. Use according to any one of claims 1 to 3, wherein In step (3), the planting groove is a rooting groove or a plug tray; the size of the rooting groove is: length×width×height=35cm×20cm×10cm; the rooting groove is divided into two types of bottom sealing and bottom with holes.

9. Use according to any one of claims 1 to 3, wherein In step (4), the seedling base is inserted 1-2cm below the culture medium; the fungicide liquid is 800 times fungicide 50% wettable powder liquid, or 600 times mancozeb 80% wettable powder liquid.

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