Simple and efficient sugarcane tissue culture seedling transplanting method

Abstract

The invention discloses a simple and efficient sugarcane tissue culture seedling transplanting method, and belongs to the technical field of plant rapid propagation. The sugarcane tissue culture seedling transplanting method provided by the invention comprises the following steps: taking tissue culture seedlings subjected to rooting treatment, cleaning a culture medium, directly planting without dividing into single plants, culturing for 20 days, then planting into single plants, culturing for 10 days, and collecting the seedlings; wherein the culture is carried out in an artificial climate chamber with set illumination, temperature and humidity. Experimental results show that the transplanting survival rate of the tissue culture seedlings is 100%, and a large number of healthy single-plant seedlings with excellent growth traits can be efficiently obtained in a short time. Compared with a conventional tissue culture seedling transplanting method, the transplanting method provided by the invention does not need the steps of seedling hardening, root cutting, leaf cutting, sterilization, disinfection and the like, is simple to operate and is green and environment-friendly, and the transplanted tissue culture seedlings have high transplanting survival rate and propagation rate. The invention provides a new transplanting method for rapid propagation of sugarcane, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid plant propagation, and particularly to a simple and efficient method for transplanting tissue-cultured sugarcane seedlings. Background Art

[0002] Sugarcane (Saccharum.ssp) is an important sugar crop and also an important energy plant in the world. Sugarcane is a vegetatively propagated crop, and in production, it is mostly propagated by seed stems. Although the tissue culture technology of sugarcane has long been mature, its application in production has been insufficient for a long time, and tissue-cultured sugarcane seedlings have not been widely planted in many sugarcane production areas due to backward production technology.

[0003] At present, planting virus-free and healthy sugarcane seedlings is an important technical measure to maintain excellent varietal characteristics, increase sugarcane yield and sugar content. One of the main techniques for producing healthy virus-free sugarcane seedlings is to adopt an integrated virus elimination technology. After constant-temperature heat treatment, shoot tips are taken for tissue culture to achieve the rapid propagation of healthy sugarcane seedlings. Transplanting tissue-cultured seedlings is a key link in the rapid propagation of healthy seedlings. However, due to the fragility of sugarcane tissue-cultured seedlings, their poor root absorption ability and weak stress resistance, they cannot be directly planted in the field and need to be temporarily planted for a period of time. This process includes hardening off the seedlings, cleaning, separating and temporarily planting single plants in a sand bed, transplanting to the field, etc. In this series of operations, due to the excessive tenderness of tissue-cultured seedlings, they are easily injured during cleaning, and the sugarcane seedlings are also easily broken during separating single plants, resulting in a direct loss of most healthy seedlings. Due to the damage caused by cleaning, pesticides such as bactericides and disinfectants need to be used for maintenance during the transplanting process, which is a cumbersome process, and the survival rate of the seedlings cannot be guaranteed 100%, which is not conducive to the rapid propagation and popularization speed of healthy seedlings. The use of bactericides and disinfectants is also not conducive to the agricultural ecological environment. In addition, the transplanting survival rate of sugarcane tissue-cultured seedlings is also a key link for phenotypic analysis in molecular breeding. Therefore, there is an urgent need to provide a transplanting method that is simple to operate, environmentally friendly and can effectively improve the survival rate of sugarcane tissue-cultured seedlings. Summary of the Invention

[0004] The purpose of the present invention is to provide a simple and efficient method for transplanting sugarcane tissue-cultured seedlings to solve the problems existing in the above-mentioned prior art. The method for transplanting sugarcane tissue-cultured seedlings provided by the present invention does not need to go through steps such as hardening off the seedlings, root pruning, leaf pruning and disinfection, and can obtain a large number of tissue-cultured seedlings with high transplanting survival rate and high propagation rate.

[0005] To achieve the above purpose, the present invention provides the following scheme:

[0006] The present invention provides a method for transplanting sugarcane tissue-cultured seedlings, including the following steps:

[0007] Take the pre-treated sugarcane tissue-cultured seedlings;

[0008] Wash the tissue-cultured sugarcane seedlings, and directly plant the washed tissue-cultured sugarcane seedlings in the seedling-raising substrate without separating them into individual plants to obtain clustered tissue-cultured seedlings;

[0009] Cultivate the clustered tissue-cultured seedlings in an artificial climate chamber for 20 days to obtain the clustered tissue-cultured seedlings after temporary planting;

[0010] Plant the clustered tissue-cultured seedlings after temporary planting into individual plants in the seedling-raising substrate, cultivate them in an artificial climate chamber for 10 days, and collect the seedlings, thus completing the transplantation of the tissue-cultured sugarcane seedlings.

[0011] Preferably, the conditions of the artificial climate chamber include:

[0012] The photoperiod is 12 h light / 12 h dark, and the light intensity is 2000 lx; the temperature during the light time is 28 °C, and the temperature during the dark time is 25 °C; the air humidity is 60% RH.

[0013] Preferably, the cultivation in the artificial climate chamber includes the following steps:

[0014] Cover the tissue-cultured seedlings to be cultivated with a film for moisture preservation for 3 days, and remove the film after 3 days; place the tissue-cultured seedlings to be cultivated under weak light conditions for the first 6 days, and place them under the above-mentioned light intensity after 6 days.

[0015] Preferably, the light intensity under the weak light conditions is 500 lx.

[0016] Preferably, the washing is to wash the culture medium on the tissue-cultured sugarcane seedlings with flowing clean water.

[0017] Preferably, the pretreatment includes the following steps: Take sugarcane seed stems, retain 3 - 4 buds, incubate in a constant temperature water bath at 52 °C, disinfect, raise seedlings, germinate, take the shoot apical meristem from the single bud after germination, induce and culture it in the dark until callus grows, transfer, and culture.

[0018] The present invention also provides an application of sugarcane seedlings obtained according to the above transplantation method in sugarcane breeding.

[0019] The present invention also provides an application of sugarcane seedlings obtained according to the above transplantation method in the study of sugarcane traits.

[0020] The present invention discloses the following technical effects:

[0021] 1. The present invention simplifies the process of temporary planting and transplanting of sugarcane tissue culture seedlings. Traditional transplanting of tissue culture seedlings requires acclimatization, washing, root trimming, leaf trimming, and the simultaneous use of bactericidal disinfectants, and the survival rate cannot be guaranteed to be 100%. However, the tissue culture seedlings cultivated in an artificial climate chamber with controllable temperature and humidity are selected in the present invention. Whether it is single-plant transplanting or cluster-seedling transplanting, after simplifying the transplanting method, the survival rate has reached 100%. The acclimatization time is saved, the time for root and leaf trimming and the labor input cost are reduced. In addition, the use of pesticides is eliminated, protecting the agricultural ecological environment.

[0022] 2. The present invention improves the rapid propagation speed of healthy seedlings. Through experiments, it is found that after flushing the cluster tissue culture seedlings with running water and directly planting them in an artificial climate chamber, they are separated into single plants after 20 days and then transplanted. After another 10 days, a large number of single-plant healthy seedlings can be obtained. Separating single plants 20 days after planting is easier than separating single plants from tissue culture seedlings. At this time, the stems of the seedlings are thick, strong and firm, the roots are developed, and the loss of seedlings is not easy to occur. Each tissue culture seedling is efficiently utilized, and a large number of healthy single-plant seedlings can be obtained in a short period, saving the costs and seedling raising cycle generated by mass-producing tissue culture seedlings in the laboratory.

[0023] 3. The present invention can be used for transplanting tissue culture seedlings in the natural environment. Through grouped experiments, the simplified transplanting method of tissue culture seedlings is also applicable to the natural environment. Relatively speaking, the survival rate of single-plant seedling transplanting in the natural environment will decrease, but the difference is not significant. Cluster seedlings can be selected for transplanting in the natural environment, and the survival rate can reach 100%. However, compared with the cluster seedlings cultivated in the artificial climate chamber, the number of single-plant seedlings obtained later is less and the growth rate is slow. Therefore, the best way to transplant tissue culture seedlings is to transplant cluster seedlings in the artificial climate chamber. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 They are growth state diagrams of single-plant and cluster tissue culture seedlings before and after temporary planting; among them, A1 is a single-plant seedling cultivated in an artificial climate chamber on the day of transplanting; B1 is a cluster seedling cultivated in an artificial climate chamber on the day of transplanting; C1 is a single-plant seedling cultivated in the natural environment on the day of transplanting; D1 is a cluster seedling cultivated in the natural environment on the day of transplanting; A2 is a single-plant seedling cultivated in an artificial climate chamber 20 days after transplanting; B2 is a cluster seedling cultivated in an artificial climate chamber 20 days after transplanting; C2 is a single-plant seedling cultivated in the natural environment 20 days after transplanting; D2 is a cluster seedling cultivated in the natural environment 20 days after transplanting;

[0026] Figure 2It is a growth state diagram of single - plant transplanted from a transplanted clump of seedlings; among them, B2 is the clump of seedlings divided into single plants cultured in an artificial climate chamber 20 days after transplantation; D2 is the clump of seedlings divided into single plants cultured in the natural environment 20 days after transplantation; A3 is a single - plant seedling cultured in an artificial climate chamber 30 days after transplantation; B3 is a single - plant seedling 10 days after single - plant transplantation of the clump of seedlings cultured in an artificial climate chamber 20 days after transplantation; C3 is a single - plant seedling cultured in the natural environment 30 days after transplantation; D3 is a single - plant seedling 10 days after single - plant transplantation of the clump of seedlings cultured in the natural environment 20 days after transplantation. Detailed implementation mode

[0027] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only used to describe specific implementation modes and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation modes of the present invention's specification, which are obvious to those skilled in the art. Other implementation modes obtained from the present invention's specification are obvious to those skilled in the art. The present invention's specification and examples are only exemplary.

[0031] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open - ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Material preparation

[0034] The yellow - skinned sugarcane after warm - water treatment is germinated, and the meristem is peeled off for tissue culture, then differentiated and rooted to obtain healthy rooted tissue - cultured seedlings of yellow - skinned sugarcane as experimental materials. The specific experimental method refers to the virus - free seedling production method in the literature "Wu Zhuandi, Zhao Peifang, Yang Hongchang, et al. Detection of the pathogen of the new high - sugar variety Yunzhe 1640 mosaic and production of virus - free seedlings [J]. Sugar Crops of China, 2024, 46(02): 1 - 8.".

[0035] 2. Potting of rooted tissue - cultured seedlings

[0036] The potting time of the rooted tissue - cultured seedlings is in February. Without hardening - off, directly wash the culture medium on the tissue - cultured seedlings with running water. Without cutting the leaves and roots, directly divide the tissue - cultured seedlings into single plants and non - single plants (clustered tissue - cultured seedlings), and plant them in plug trays filled with a general seedling - raising substrate (purchased from Yanli Laoqian Flower Shop in Kaiyuan City). Place a tray of single - plant seedlings and a tray of clustered seedlings in an artificial climate chamber for cultivation. Set the photoperiod to 12h:12h (light: dark), the light intensity to 2000lx, the temperature during the light period to 28°C, the temperature during the dark period to 25°C, and the air humidity to 60.0%RH. Mark the two treatments as group A1 and group B1 respectively; then place a tray of single - plant seedlings and a tray of clustered seedlings in the natural environment for cultivation, and mark the two treatments as group C1 and group D1 respectively.

[0037] Cover the tissue - cultured seedlings in all the above - mentioned treatments with a film to keep moisture. Remove the film after three days. Keep them in a low - light environment (500lx) for the first six days, and then cultivate them in the corresponding environments set in the experiment. After 20 days, mark the treatment groups A1, B1, C1, and D1 as A2, B2, C2, and D2 respectively, and statistically analyze their survival rate, plant height, number of leaves, leaf length of the +1 leaf, number of roots (the number of roots of clustered seedlings is recorded as the average number of roots per single plant), and root length. Among them, the statistical methods for plant height and leaf length of the +1 leaf are shown in Figure 1 A2 in

[0038] 3. Transplanting single plants from clustered potting seedlings

[0039] Separate the clustered seedlings in treatment groups B2 and D2 into single plants and plant them on plug trays. Similarly, cover them with a film for the first three days, and keep them in a low - light environment (500lx) for three days after removing the film, and then cultivate them in the original corresponding artificial climate chamber and natural environment. After 10 days of transplanting, mark the originally single - transplanted seedlings as A3 and C3, and the newly transplanted single - plant seedlings as B3 and D3, and analyze their survival rate, plant height, number of leaves, leaf length of the +1 leaf, number of roots, and root length for comparative analysis.

[0040] 4. Analysis of experimental results

[0041] 4.1 Observation and analysis of the growth status of single - plant and clustered tissue - cultured seedlings after transplantation

[0042] As Figure 1 shown in Table 1, the tissue culture seedlings were transplanted from the tissue culture bottles to the nursery substrate without hardening, root pruning, leaf pruning, or application of bactericidal disinfectants. After 20 days, both single seedlings and clump seedlings had survived. Among them, the growth of Group A2 and Group B2 cultured in the artificial climate chamber was better than that of Group C2 and Group D2 in the natural environment. For the tissue culture seedlings cultured in the artificial climate chamber, whether single seedlings or clump seedlings were transplanted, the survival rate was 100%. The single-transplanted seedlings were stronger than the clump seedlings, and the heights of the seedlings were basically the same. The survival rate of single seedlings grown in the natural environment was 93.33%, and the survival rate of clump seedlings was 100%. There was no significant difference in growth between the two, but overall, compared with the seedlings of Group A2 and Group B2 cultured in the artificial climate chamber, there was a significant difference in growth. The seedlings of Group C2 and Group D2 in the natural environment were short, with shorter leaves, and the number and length of roots were significantly lower than those of the tissue culture seedlings cultured in the artificial climate chamber.

[0043] Therefore, by using the simplified nursery procedure, the present invention can still ensure the survival rate of transplanted tissue culture seedlings. When planted in an artificial climate chamber with temperature and humidity control, not only can a 100% survival rate be achieved, but also the growth of healthy seedlings can be accelerated.

[0044] Table 1 Comparison of the growth status of single seedlings and clump seedlings in different environments during nursery

[0045]

[0046] Note: Different lowercase letters in the same column in the table indicate significant differences in data, p < 0.05.

[0047] 4.2 Observation and analysis of the growth status of single seedlings after transplanting clump seedlings during nursery

[0048] After separating the clump seedlings B2 and D2 after nursery into single seedlings, from Figure 2 it can be seen from B2 and D2 that the number of single seedlings of the clump seedlings D2 grown in the natural environment was significantly lower than that of the clump seedlings B2 in the artificial climate chamber.

[0049] As shown in Table 2 and Figure 2 A3, B3, C3, and D3 therein, after 10 days of single-planting, the survival rates of B3 and D3 were both 100%. Compared with the tissue culture seedlings A3 and C3 in the corresponding environment that were single-planted and transplanted from the beginning, there was little difference in plant height and the number of leaves, there was a certain gap in leaf length, and there were also significant differences in the number and length of roots. The reason for these gaps is that root damage will occur during the process of separating into single seedlings, and 10 days is not enough time for recovery; but generally speaking, tissue culture seedlings are first planted in clumps and then separated into single seedlings, which does not affect the survival rate of the seedlings, and their growth can catch up with that of the tissue culture seedlings that were single-planted from the beginning in the corresponding environment in a short time.

[0050] Therefore, for the transplanting of tissue-cultured seedlings, the clustered seedlings can be temporarily planted in an artificial climate chamber with temperature and humidity control, and then planted individually after 20 days. In this way, more individual healthy seedlings can be obtained in a short period.

[0051] Table 2 Comparison of the growth status between the individually transplanted clustered seedlings after temporary planting and the individually transplanted tissue-cultured seedlings

[0052]

[0053] Note: Different lowercase letters in the same column of the table indicate significant differences in data, p < 0.05.

[0054] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for transplanting tissue culture seedlings of sugarcane, characterized in that, It includes the following steps: Take the pre-treated sugarcane tissue culture seedlings; Wash the sugarcane tissue culture seedlings, and directly plant the washed sugarcane tissue culture seedlings without separating them into individual plants in a seedling-raising substrate to obtain clustered tissue culture seedlings; Cultivate the clustered tissue culture seedlings in an artificial climate chamber for 20 days to obtain the clustered tissue culture seedlings after temporary planting; Plant the clustered tissue culture seedlings after temporary planting into individual plants in a seedling-raising substrate, cultivate them in an artificial climate chamber for 10 days, and collect the seedlings, thus completing the transplantation of the sugarcane tissue culture seedlings.

2. The transplanting method according to claim 1, characterized in that, The conditions of the artificial climate chamber include: The photoperiod is 12 h light / 12 h darkness, and the light intensity is 2000 lx; the temperature during the light time is 28 °C, and the temperature during the dark time is 25 °C; the air humidity is 60% RH.

3. The transplanting method according to claim 1, wherein The cultivation in the artificial climate chamber includes the following steps: Cover the tissue culture seedlings to be cultivated with a film for moisture preservation for 3 days, and remove the film after 3 days; place the tissue culture seedlings to be cultivated under weak light conditions for the first 6 days, and place them under the light intensity described in claim 2 after 6 days.

4. The transplanting method according to claim 3, characterized in that, The light intensity under the weak light conditions is 500 lx.

5. The transplanting method according to claim 1, characterized in that, The washing is to wash the culture medium on the sugarcane tissue culture seedlings with flowing clean water.

6. The transplanting method according to claim 1, characterized in that, The pre-treatment includes the following steps: take sugarcane seed stems, retain 3 - 4 buds, incubate in a constant temperature water bath at 52 °C, disinfect, germinate the seedlings, take the shoot apical meristem from the single bud after germination, induce and culture it in the dark until callus grows, transfer and culture.

7. Application of sugarcane seedlings obtained by the transplantation method according to any one of claims 1 - 6 in sugarcane breeding.

8. Application of sugarcane seedlings obtained by the transplantation method according to any one of claims 1 - 6 in sugarcane trait research.

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