A method for inducing high-quality sugarcane callus that overcomes seasonal limitations

Abstract

The present invention discloses a method for inducing high-quality sugarcane callus that overcomes seasonal limitations. This method relates to the field of plant tissue culture technology and includes the following steps: 1) material selection; 2) germination in an incubator; 3) callus induction; and 4) subculture. By selecting the season when sugarcane callus induction is most difficult and explants are most scarce for experimentation, and then germinating the seeds in an incubator, using the heart leaves of seedlings as explants for callus induction, the present invention can produce large quantities of high-quality callus in a short period of time. This overcomes the seasonal limitations of sugarcane callus induction and shortens the experimental period for obtaining embryonic callus.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for inducing high-quality sugarcane callus tissue that overcomes seasonal limitations. Background Art

[0002] Sugarcane callus culture is an important biotechnology tool with extensive applications in sugarcane molecular breeding, genetic transformation, virus removal and rapid propagation, germplasm conservation and utilization, and physiological and biochemical research. Callus tissue is an ideal recipient material for genetic engineering operations. Transgenic sugarcane plants can be obtained through Agrobacterium-mediated introduction or gene gun bombardment, followed by screening and regeneration, effectively achieving genetic improvement of sugarcane varieties. By applying specific selective pressures during callus induction, such as environmental stresses like drought and high salt levels, calli with superior traits can be selected and subsequently regenerated into plants with corresponding stress tolerance. Sugarcane callus culture can preserve high-quality sugarcane germplasm resources over the long term, preventing damage to sugarcane germplasm resources caused by natural disasters and human factors. Sugarcane callus culture can achieve virus removal and rapid propagation of seedlings to meet production needs. Furthermore, sugarcane callus culture allows for precise control of environmental conditions indoors, facilitating research on physiological and biochemical processes in sugarcane.

[0003] High-quality sugarcane callus is the material foundation for conducting research related to sugarcane tissue culture. Low-quality callus reduces the efficiency of exogenous gene transformation and severely impacts the differentiation, growth, and development of tissue culture seedlings, leading to extended experimental periods or even failure. Besides genotype and tissue culture technique, the explant is a key factor in determining callus quality. Explants commonly used for callus induction are derived from the heart leaves of sugarcane shoots. However, seasonal variations can significantly affect the ability of sugarcane donor materials with different physiological states to dedifferentiate and form callus. Within the sugarcane growth cycle, the jointing stage is the most suitable stage for callus collection. Explants collected at 7 to 8 months of age have a low browning rate and a high embryonic callus induction rate. Explants collected at other times have a significantly increased browning rate and a decreased callus induction rate. Consequently, the scarcity of explant donor material and the poor quality of callus induction during winter and spring are key limitations to sugarcane tissue culture and related research. Summary of the Invention

[0004] The present invention aims to provide a method for inducing high-quality sugarcane callus that overcomes seasonal limitations. By germinating seedlings in an incubator and selecting the heart leaves of seedlings as explants for callus induction, large quantities of high-quality callus can be obtained. Compared to traditional methods, this method overcomes the seasonal limitations of sugarcane callus induction, while also increasing embryonic callus yield and shortening experimental cycles. This method lays a solid foundation for rapid propagation of sugarcane virus-free plants, molecular breeding, and physiological and molecular biological research.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for inducing high-quality sugarcane callus that overcomes seasonal limitations comprises the following steps:

[0007] Step 1) Material selection: Select sugarcane stalks from January;

[0008] Step 2) Cultivation: After retrieving the sugarcane stems, separate the upper buds from the lower buds, rinse with running water, place them on a tray, add clean water, and then place them in a light incubator for cultivation;

[0009] Step 3) Sugarcane callus induction: When sugarcane buds reach the four-leaf stage, remove the sugarcane seedlings and remove the leaves and outer old leaf sheaths. After surface disinfection, continue removing the leaf sheaths in a sterile laminar flow hood, leaving the heart leaves. These are cut into slices and inoculated onto solid culture medium for dark culture. Observe and record the induction rate and browning rate, and select tissue pieces showing callus proliferation for subculture.

[0010] Step 4) Subculture: Inoculate 24-30 callus pieces into each culture dish, record the growth of induced embryonic callus, and calculate the embryonic callus acquisition rate.

[0011] Furthermore: in the step 1), the sugarcane variety selected is Yunzhe 08-1609.

[0012] Furthermore: in step 2), the depth of the clean water in the tray is 2-3 mm.

[0013] Furthermore: in the step 2), in the light incubator, the light cycle is set to 12 hours of light culture time, 12 hours of dark culture time, the light intensity is 2000 lx, the light culture temperature is 28°C, and the dark cycle temperature is 25°C.

[0014] Furthermore: in the step 2), the light incubator is rinsed with running water once every morning and evening; or cephalosporin is added to clean water and rinsed once every 1-2 days.

[0015] Furthermore: in step 3), the heart leaves with a length of 3-5 cm above the growth point and a diameter of 2-4 mm are left and cut into 1-2 mm slices.

[0016] Furthermore: in step 3), the solid culture medium is MS+2,4-D 1.5 mg / L+KT 0.5 mg / L+sucrose 30 g / L+activated carbon 0.2 g / L+carrageenan 8 g / L.

[0017] Furthermore: in step 3), the dark culture is cultured at 28° C. for 15 days.

[0018] Further: in step 3), the induction rate = the number of explants induced to produce callus tissue / the number of inoculations × 100%;

[0019] Browning rate = number of explants that browned / number of inoculated plants × 100%.

[0020] Furthermore: in the step 4), the yield of embryonic callus = number of embryonic callus / number of explants from which callus is induced × 100%.

[0021] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0022] (1) The present invention overcomes the seasonal limitations of sugarcane callus induction. The sugarcane growing period is generally 12 months, but the most suitable stage for collection is the jointing stage (generally in July and August each year). The callus induced at other times is of poor quality due to physiological differences in sugarcane. The present invention selects sugarcane stems for germination to become excellent explant donors. Therefore, sugarcane callus induction can be carried out all year round, and the callus quality is not affected by the season, ensuring a sufficient supply of high-quality callus.

[0023] (2) The present invention improves the efficiency of sugarcane callus induction. Compared with traditional methods, the efficiency of sugarcane callus induction increased by 23.4%. The present invention can effectively reduce tissue browning and increase the proliferation rate of callus. Traditional callus induction requires two to three subcultures to obtain the required embryonic callus, while the present method only requires one subculture to obtain sufficient high-quality embryonic callus tissue, greatly shortening the sugarcane callus induction process.

[0024] (3) The present invention saves production costs. The traditional method only uses the growing point and heart leaf of the sugarcane tip for stem tip detoxification culture and callus induction. One sugarcane can only obtain one growing point and young heart leaf. Therefore, a large amount of land is required to plant sugarcane materials to ensure the supply of explants. However, the present method uses sugarcane buds as donors. One sugarcane can have 10-20 sugarcane buds. The amount of sugarcane required to be planted does not need to be too large, thus saving a lot of planting and management costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the upper bud and lower bud of the sugarcane stem in the present invention.

[0026] Figure 2 The effects of sugarcane explants in different states on callus induction in the present invention; wherein, Figure 2 A is the callus induction of sugarcane buds, Figure 2 B is the callus induced by tillering seedlings in the field. Figure 2 C is the induced callus of mature sugarcane shoots in the field. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Example 1:

[0029] In this example, the induction process of sugarcane callus was affected by the natural environment and the artificial environment, and some experimental conditions adopted range values.

[0030] The sampling time is January, which is the season when explants are most scarce and callus induction is the worst. The sugarcane variety selected is Yunzhe 08-1609. After the sugarcane stems are retrieved from the field, the leaf sheaths are removed and the middle part of the sampled sugarcane stems is separated. The upper half of the 1 / 2 of the sugarcane stem is the upper bud, and the lower half of the 1 / 2 of the sugarcane stem is the lower bud. Figure 1 As shown. Upper buds have short internodes and sprout quickly, so they can be cut into multi-bud segments. Lower buds can be cut into single buds. Wash and rinse under running water for 12 hours. Place the single-bud segments with the buds facing upward, and the multi-bud segments on their sides, in a tray. Add an appropriate amount of clean water to a depth of 2-3 mm. Then, incubate in a lighted incubator. Set the photoperiod to 12L:12D (light:dark), meaning the light cycle is 12 hours light and 12 hours dark, with a light intensity of 2000 lx, a light cycle temperature of 28°C, and a dark cycle temperature of 25°C. Rinse with running water every morning and evening (8:00 AM and 8:00 PM) to moisturize and prevent bacterial growth. Alternatively, add cephalosporin to the water at a concentration of 300 mg / L and rinse every 1-2 days. This method reduces the number of washes, effectively inhibits bacteria, and ensures normal growth of the buds. When the sugarcane buds grow to the 4-leaf stage, they can be used for sugarcane callus induction.

[0031] Sugarcane seedlings were removed, their leaves and outer sheaths removed, and their surfaces disinfected with 75% (v / v) ethanol. The sheaths were then removed in a sterile laminar flow hood, leaving heart leaves 3-5 cm above the growing point and 2-4 mm in diameter. These leaves were then cut into 1-2 mm slices and inoculated onto a solid culture medium containing MS, 1.5 mg / L 2,4-D, 0.5 mg / L KT, 30 g / L sucrose, 0.2 g / L activated carbon, and 8 g / L carrageenan (pH 5.8). The culture was dark-cultured at 28°C for 15 days. Callus blocks were then selected from the explants with callus proliferation and subcultured. The calli were observed and recorded. Calli with good growth, pale yellow color, dense texture, smooth surface, and good differentiation ability were considered embryonic calli. In addition, field tillering seedlings and mature sugarcane tips were used as experimental materials. Heart leaves at the growing point of the sugarcane tip were peeled and used as explants. Callus culture was performed under the same induction conditions for comparison with the present invention. Each culture dish was inoculated with 24-30 explants, and 15 dishes were inoculated for recording and analysis.

[0032] Observation of callus tissue induced from explants of three different materials: germination in a light incubator (A), sugarcane tillering seedlings (B), and mature sugarcane shoots (C) ( Figure 2 ), the results showed that after 15 days of induction, the explants of Group A showed no callus except for the outer circle of leaf sheaths, while a large number of calli were formed on the inner explants, which were white and loose. The calli in good condition were selected for subculture. After one subculture and 30 days of callus induction, a large amount of light yellow, compact embryonic callus could be obtained. The explants of Group B underwent severe browning during the induction process. At 15 days, the innermost explants had calli with dotted proliferation. The tissue blocks with calli were selected for subculture. At 30 days, the callus morphology was mostly loose and transparent non-embryonic callus, and there was also a small amount of browned tissue. After 15 days of induction, the explants of Group C showed no callus formed in the two outer circles of the explants, and a small amount of callus proliferation was found inside. After one subculture, most of its callus tissue was also loose and transparent non-embryonic callus. In general, callus induction in groups B and C needs to be subcultured again before it can be used in subsequent experiments, while explants germinated in an incubator take the shortest time and have the best callus quality, and can be used for other scientific research after one subculture.

[0033] Record callus growth during callus induction, and calculate the induction rate and browning rate. Induction rate = number of explants induced to produce callus / number of inoculated cells × 100%, and browning rate = number of explants exhibiting browning / number of inoculated cells × 100%. Select high-quality calli, subcultured once, and then classify them based on their quality into embryonic and non-embryonic calli. Embryonic calli exhibit good growth, pale yellow color, dense texture, and a smooth surface, indicating excellent differentiation capacity. Non-embryonic calli exhibit translucent, soft, and mushy tissue, lacking embryonic capacity and lacking tissue culture value. The embryonic callus yield rate is calculated as: number of embryonic calli / number of explants induced to produce callus × 100%.

[0034] Table 1 Analysis of callus induction from explants from different sources

[0035]

[0036] The statistical results are shown in Table 1. The order of callus induction rate and embryonic callus acquisition rate from high to low is incubator-germinated seedlings > shoots > tillering seedlings, while the order of browning rate is the opposite, indicating that the callus induction effect of explants germinated in the incubator is the best and the embryonic callus acquisition rate is the highest.

[0037] Although the present invention has been described herein with reference to a number of illustrative embodiments thereof, it will be understood that numerous other modifications and implementations may be devised by those skilled in the art that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, the drawings, and the claims, numerous variations and modifications may be made to the components and / or layout of the subject combination arrangement. In addition to variations and modifications to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A method for inducing high-quality sugarcane callus that overcomes seasonal restrictions, characterized by: The following steps are involved: Step 1) Material selection: Select sugarcane stalks of the sugarcane variety Yunzhe 08-1609 in January; Step 2) Incubator Germination: After retrieving the sugarcane stems, separate the upper buds from the lower buds. Cut the upper buds into multi-bud segments and the lower buds into single buds. Wash and rinse with running water for 12 hours, then place them in a tray with the single bud segments facing upward and the multi-bud segments placed sideways. Add 2-3 mm of clean water to the tray and place in a light incubator for incubation. Step 3) Sugarcane callus induction: When sugarcane buds reach the four-leaf stage, remove the sugarcane seedlings and remove the leaves and outer old leaf sheaths. After surface disinfection with 75% ethanol, continue removing the leaf sheaths in a sterile laminar flow hood, leaving the heart leaves 3-5 cm above the growing point and 2-4 mm in diameter. Cut them crosswise into 1-2 mm slices and inoculate them onto solid culture medium. Incubate in the dark at 28°C for 15 days. Observe and record the induction rate and browning rate, and select tissue pieces showing callus proliferation for subculture. Step 4) Subculture: Inoculate 24-30 callus pieces per culture dish, record the growth of induced embryonic callus, and calculate the embryonic callus acquisition rate; In the step 2), the light cycle in the light incubator is set to a light culture time of 12 hours, a dark culture time of 12 hours, a light intensity of 2000 lx, a light culture temperature of 28°C, and a dark cycle temperature of 25°C; In the step 2), the light incubator is rinsed with running water once every morning and evening; or cephalosporin is added to clean water at a concentration of 300 mg / L and rinsed once every 1-2 days; In step 3), the solid culture medium is MS + 2,4-D 1.5 mg / L + KT 0.5 mg / L + sucrose 30 g / L + activated carbon 0.2 g / L + carrageenan 8 g / L, with a pH of 5.8; The induction rate = the number of explants induced to produce callus tissue / the number of inoculations × 100%; The browning rate = the number of explants that browned / the number of inoculations × 100%; The embryonic callus acquisition rate=the number of embryonic callus / the number of explants from which callus was induced×100%.

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