High-quality sugarcane callus induction method capable of overcoming season limitation

Abstract

The invention discloses a high-quality sugarcane callus induction method capable of overcoming seasonal restrictions, and relates to the technical field of plant tissue culture, the high-quality sugarcane callus induction method comprises the following steps: 1) material selection; 2) accelerating germination in an incubator; (3) callus induction; and step 4) subculture. According to the method, experiments are carried out in seasons where sugarcane callus induction is most difficult and explants are most scarce, germination accelerating is carried out through an incubator, and seedling core leaves are selected as explants for callus induction, so that a large number of high-quality calluses can be obtained in a short time. The seasonal limitation of sugarcane callus induction is overcome, and the experimental period for obtaining the embryogenic callus is shortened.

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 restrictions. Background Art

[0002] Sugarcane callus culture is an important biotechnology method, which has a wide range of applications in sugarcane molecular breeding, genetic transformation, detoxification and rapid propagation, germplasm resource conservation and utilization, physiological and biochemical research, etc. Callus is an ideal receptor material for genetic engineering operations. Foreign genes are introduced into callus by Agrobacterium-mediated method or gene gun bombardment method, and then transgenic sugarcane plants are obtained after screening and regeneration, which can effectively achieve genetic improvement of sugarcane varieties. By applying specific selection pressures during callus induction, such as environmental stresses such as drought and high salt, callus with excellent traits are screened out, and then plants with corresponding stress resistance are regenerated. Through sugarcane callus culture, the excellent germplasm resources of sugarcane can be preserved for a long time, avoiding the destruction of sugarcane germplasm resources by natural disasters and human factors. Sugarcane callus culture can achieve seedling detoxification and rapid propagation to meet production needs. In addition, sugarcane callus culture can accurately control environmental conditions indoors, which provides convenience for studying the physiological and biochemical processes of sugarcane.

[0003] High-quality sugarcane callus is the material basis for conducting research related to sugarcane tissue culture. Low-quality callus will reduce the transformation efficiency of exogenous genes, and will also seriously affect the differentiation, growth and development of tissue culture seedlings, resulting in extended experimental cycles or failure. In addition to genotype and tissue culture technology, explants are the key factors that determine the quality of callus. Explants usually used for callus induction are derived from the heart leaves of sugarcane tips. Under the influence of seasonal changes, sugarcane donor materials in different physiological states have significant differences in their ability to dedifferentiate and form callus. From the perspective of the growth cycle of sugarcane, the jointing stage is a more suitable stage for material collection, that is, the browning rate of explants collected when the sugarcane grows for 7 to 8 months is low, and the embryonic callus induction rate is high, while the browning rate of explants collected at other times is significantly increased, and the callus induction rate decreases. Therefore, there is a severe shortage of explant donor materials during winter and spring, and the callus induction quality is poor, which is also a key factor restricting sugarcane tissue culture and related research. Summary of the invention

[0004] The purpose of the present invention is to provide a method for inducing high-quality sugarcane callus that overcomes seasonal restrictions. By germinating in a culture box and selecting seedling heart leaves as explants for callus induction, a large amount of high-quality callus can be obtained. Compared with traditional methods, the present invention overcomes the seasonal restrictions of sugarcane callus induction, while also increasing the yield of embryonic callus and shortening the experimental cycle. This lays a good foundation for sugarcane detoxification and rapid propagation, molecular breeding, physiological and molecular biological research, etc. In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for inducing high-quality sugarcane callus that overcomes seasonal restrictions comprises the following steps: Step 1) Material selection: Select sugarcane stalks from January; Step 2) Cultivation: After the sugarcane stems are retrieved, the upper buds are separated from the lower buds, rinsed with running water, placed in a tray, clean water is added, and then placed in a light incubator for cultivation; Step 3) Sugarcane callus induction: When the sugarcane buds grow to the 4-leaf stage, take the sugarcane seedlings and remove the sugarcane leaves and the outer old leaf sheaths. After surface disinfection, continue to remove the leaf sheaths in a sterile clean bench, leaving the heart leaves, cut them into slices, inoculate them on solid culture medium for dark culture, observe and record, and calculate the induction rate and browning rate. Select tissue blocks with callus proliferation for subculture; Step 4) Subculture: 24-30 callus blocks were inoculated into each culture dish, and the growth of induced embryonic callus was recorded, and the embryonic callus acquisition rate was calculated.

[0005] Further: in the step 1), the sugarcane variety selected is Yunzhe 08-1609.

[0006] Further: in step 2), the depth of the clean water in the tray is 2-3 mm.

[0007] Further: in the step 2), in the light incubator, the light cycle 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.

[0008] Further: in the step 2), the light incubator is rinsed with running water once in the morning and evening every day; or cephalosporin is added to clean water and rinsed once every 1-2 days.

[0009] Further: in the 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.

[0010] Further: in the 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.

[0011] Furthermore: in step 3), the dark culture is carried out at 25° C. for 15 days.

[0012] Further: in step 3), the induction rate = the number of explants induced with callus tissue / the number of inoculations × 100%; Browning rate = number of explants that browned / number of inoculations × 100%.

[0013] Furthermore: in the step 4), the rate of obtaining embryonic callus = the number of embryonic callus / the number of explants from which callus is induced × 100%.

[0014] Compared with the prior art, the present invention has at least one of the following beneficial effects: (1) The present invention overcomes the seasonal limitation of sugarcane callus induction. The growth period of sugarcane is generally 12 months, but the suitable material collection stage is the jointing period of sugarcane (generally in July and August each year). The callus induced at other times is of poor quality, which is caused by the physiological differences of sugarcane. The present invention selects sugarcane stems to become excellent explant donors after germination, so sugarcane callus induction can be carried out all year round, and the callus quality is not affected by the season, ensuring sufficient supply of high-quality callus.

[0015] (2) The present invention improves the callus induction efficiency of sugarcane. Compared with the traditional method, the callus induction efficiency of sugarcane is 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.

[0016] (3) The present invention saves production costs. The traditional method only uses the growth point and heart leaf of the sugarcane tip for stem tip detoxification culture and callus induction. One sugarcane can only obtain one growth point and young heart leaf. Therefore, a large amount of land is needed to plant sugarcane materials to ensure the supply of explants. The present method uses sugarcane buds as donors. One sugarcane can have 10-20 sugarcane buds. The number of sugarcanes 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

[0017] Figure 1 It is a schematic diagram of the upper bud and the lower bud of the sugarcane stem in the present invention.

[0018] 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 induction of tillering seedlings in the field. Figure 2 C is the induced callus of mature sugarcane shoots in the field. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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.

[0020] Embodiment 1: 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.

[0021] The material collection 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. The upper buds have short internodes and sprout quickly, so they can be cut into multi-bud segments, and the lower buds can be cut into single buds, washed and rinsed with running water for 12 hours. The single bud segment of the sugarcane buds is placed upward and the multi-bud segment of the sugarcane buds is placed on the tray sideways, and an appropriate amount of clean water is added. The depth of the clean water in the tray is 2-3mm, and then placed in a light incubator for cultivation. The photoperiod is set to 12L:12D (light: dark), that is, the photoperiod is set to 12 hours of light cultivation time, 12 hours of dark cultivation time, the light intensity is 2000lx, the light cycle temperature is 28℃, and the dark cycle temperature is 25℃. Rinse with running water once every morning and evening (8 am and 8 pm) to keep moisture and prevent bacteria growth; or add cephalosporin to clean water, the concentration of cephalosporin is 300 mg / L, and rinse once every 1-2 days. This method can reduce the number of cleaning times, effectively inhibit bacteria, and to a certain extent ensure the normal growth of sugarcane buds. When the sugarcane buds grow to the 4-leaf stage, they can be used for sugarcane callus induction.

[0022] The sugarcane seedlings were removed, the sugarcane leaves and the outer old leaf sheaths were removed, and the surface was disinfected with 75% (V / V) ethanol, and then the leaf sheaths were removed in a sterile clean bench, leaving the heart leaves with a length of 3-5 cm above the growth point and a diameter of 2-4 mm, which were cut into 1-2 mm slices, inoculated on a solid culture medium of 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 (pH 5.8), and cultured in the dark at 28°C for 15 days, and then callus blocks were selected from the sheet explants with callus proliferation for subculture, and observation and recording were made. The callus with good growth, light yellow color, dense texture, smooth surface, and good differentiation ability was embryonic callus. At the same time, field tillering seedlings and mature sugarcane tips were used as test materials, the heart leaves at the top growth point of sugarcane were peeled off as explants, and callus was cultured under the same induction conditions for comparison with the present invention. Each dish was inoculated with 24-30 explants, and 15 dishes were inoculated for recording and analysis.

[0023] Observation of callus 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 of the explants in group A, except for the outer circle of leaf sheaths, there was no callus proliferation, and a large number of calli were formed in the internal explants, which were white and loose. Select the callus tissue in good condition for subculture. After one subculture and 30 days of callus induction, a large amount of light yellow and compact embryonic callus can be obtained. The explants in group B were severely browned during the induction process. At 15 days, the innermost explants had point-like proliferative calli. The tissue blocks with long callus 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 of the explants in group C, no callus was 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 tissues were 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.

[0024] Record the callus induction callus growth status, and count the induction rate and browning rate. The induction rate = the number of explants induced callus / the number of inoculations × 100%, and the browning rate = the number of explants that browned / the number of inoculations × 100%. After selecting excellent callus for subculture once, they are classified into embryonic callus and non-embryonic callus according to the quality of the callus. The former has good growth, light yellow color, dense texture, smooth surface, and good differentiation ability; the latter is translucent, soft and sticky, has no embryonic ability, and has no tissue culture value. The acquisition rate of embryonic callus = the number of embryonic callus / the number of explants induced callus × 100%.

[0025] Table 1 Analysis of callus induction from explants from different sources The statistical results are shown in Table 1. The callus induction rate and embryonic callus acquisition rate are in the order of incubator germination > shoot > tillering seedling from high to low, while the browning rate is in the opposite order, indicating that the callus induction effect of explants germinated in the incubator is the best and the embryonic callus acquisition rate is the highest.

[0026] Although the present invention is described herein with reference to a number of illustrative embodiments of the present invention, it will be appreciated that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, drawings, and claims, a variety of variations and modifications may be made to the components and / or layout of the subject combination layout. In addition to the variations and modifications made 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 in that: The following steps are involved: Step 1) Material selection: Select sugarcane stalks from January; Step 2) Germination in an incubator: After the sugarcane stems are retrieved, the upper buds are separated from the lower buds, rinsed with running water, placed in a tray, and clean water is added, and then placed in a light incubator for cultivation; Step 3) Sugarcane callus induction: When the sugarcane buds grow to the 4-leaf stage, take the sugarcane seedlings and remove the sugarcane leaves and the outer old leaf sheaths. After surface disinfection, continue to remove the leaf sheaths in a sterile clean bench, leaving the heart leaves, cut them into slices, inoculate them on solid culture medium for dark culture, observe and record, and calculate the induction rate and browning rate. Select tissue blocks with callus proliferation for subculture; Step 4) Subculture: 24-30 callus blocks were inoculated into each culture dish, and the growth of induced embryonic callus was recorded, and the embryonic callus acquisition rate was calculated.

2. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In the step 1), the sugarcane variety selected is Yunzhe 08-1609.

3. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In the step 2), the depth of the clean water in the tray is 2-3 mm.

4. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: 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.

5. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In the step 2), the light incubator is rinsed with running water once in the morning and evening every day; or cephalosporin is added to clean water and rinsed once every 1-2 days.

6. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: 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.

7. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In the 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.

8. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In step 3), dark culture is carried out at 25° C. for 15 days.

9. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In step 3), the induction rate = the number of explants induced to produce callus tissue / the number of inoculations × 100%; Browning rate = number of explants that browned / number of inoculations × 100%.

10. The method for inducing high-quality sugarcane callus that overcomes seasonal restrictions according to claim 1, characterized in that: In the step 4), the acquisition rate of embryonic callus = the number of embryonic callus / the number of explants from which callus is induced × 100%.

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