CN108243959B - Efficient regeneration method taking stem section of sorghum as explant - Google Patents
Efficient regeneration method taking stem section of sorghum as explant Download PDFInfo
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant, which comprises the following steps: s1: obtaining aseptic seedlings, namely placing the seeds on a constant-temperature shaking table at 37-40 ℃ to shake and soak for 24-36 h; sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 12-15min, washing thoroughly with sterile water for 6 times, and sowing the seeds; s2: elongation and harvesting of the stem segment; s3: induction and elongation of stem sections; s4: rooting culture; s5: hardening and transplanting the seedlings. The efficient tissue culture and plant regeneration method can eliminate the influence of external conditions, and produce strong, tidy and consistent tissue culture seedlings of the sorghum in a large-scale and industrialized manner; compared with the reported method that the hypocotyl, the stem section with the axillary bud and the bud strip are used as explants to construct a regeneration system, the method can meet the requirement of the market on seedlings, can improve and innovate the germplasm resources of the sorghum trees through transgenic research, and lays a foundation for sustainable utilization of the germplasm resources of the sorghum trees.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing callus and regenerating callus by using sorghum stem segments as materials.
Background
Yellowtail (Neomarickia cadamba) is a unique economically valuable plant of the genus Rubiaceae (Rubiaceae) and has been mainly grown in south and southeast Asia and recently introduced in tropical and subtropical countries such as Gossda Li Kao, Bodoku, south Africa, Su-Lian, Venezuela and the like, while it is mainly distributed in the areas of Guangdong, Guangxi, Yunnan, Fujian and the like in China. The plant is a typical multifunctional plant, has good material quality, is light yellow in wood, has straight grains, and can be used as raw materials of furniture, building materials, artificial fibers, plywood and the like; flowers are good honey source, fruits can be eaten, and extracts of barks, stems and roots can be used as essence of spices and for treating snake bite, diabetes, fever, anemia, cholera and other diseases; the branches and leaves can be used as livestock feed; the whole body of the yellow beam wood is precious, and is known as 'precious tree' and 'peculiar tree' by people, and is a local broad-leaved tree species with development prospect.
At present, the sorghum is mainly bred by sowing and seedling raising, but seeds are not storage-resistant, the seed vigor is reduced after one year, the seed germination rate is lower and lower along with the storage time, and the seedlings obtained by the method are slow in growth, uneven in height, large in individual differentiation and long in seedling raising period. In addition, the tender branches and terminal buds of the Huanglianmu plants are sensitive to low temperature, and can generate freeze injury and even die at the low temperature of-2 ℃ for 12h, which seriously affects the growth of the plants and the straightness of the trunks, so that the Huanglianmu is only distributed in south China in China at home. In order to make the distribution of the sorghum wood wider, intensive research for improving the propagation rate of seedlings of the sorghum wood is carried out at home and abroad, the problem of poor cold resistance of the sorghum wood can be solved by a tissue culture mode, along with the completion of whole genome sequencing of the sorghum wood, the breeding of the sorghum wood must enter a molecular breeding era, the construction of a genetic transformation system of the sorghum wood is particularly important, and an efficient regeneration system is a precondition for the construction of the genetic transformation system.
Regarding the tissue culture of the yellow sorghum wood, several successful reports have been made at home and abroad, the stem section with axillary buds and the bud strip are mainly used as explants to respectively induce axillary buds and terminal buds, and then proliferation, rooting and transplantation are carried out, but the stem section and the bud strip of the yellow sorghum wood are both loose fillers, so germs are easily carried, and thorough disinfection is difficult.
Disclosure of Invention
The invention aims to provide a high-efficiency tissue culture breeding method of sorghum vulgare by taking a sorghum vulgare stem section as an explant, which is simpler and more convenient to operate and wider in material obtaining.
The technical scheme adopted by the invention is as follows: a high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant is characterized by comprising the following steps: the method comprises the following steps:
s1: obtaining of sterile seedlings: placing the seeds on a constant temperature shaking table at 37-40 ℃ to shake and soak for 24-36 h; sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 12-15min, washing thoroughly with sterile water for 6 times, and sowing the seeds;
s2: elongation of the stem segment and obtaining: the seeds begin to sprout after 7 days of culture, grow into aseptic seedlings after 15-25 days, cut off the aseptic seedlings, inoculate and culture, after culturing for 15-28 days, cut and take the stem section as the explant;
s3: induction and elongation of stem segments: inducing the explant to heal, and vertically inserting the stem into culture medium during inoculation; after culturing for 15-25 days, when the stem segments begin to change from yellow green callus into green bud points, inducing the explant to elongate adventitious buds;
s4: rooting culture: when the adventitious bud extends to 3-4cm, cutting the adventitious bud for induced rooting;
s5: hardening and transplanting seedlings: after rooting culture for 20 days, growing multiple roots from adventitious buds, digging out the cultured seedlings from a culture bottle, cleaning the culture medium on the roots, soaking the cultured seedlings in tap water for 2 hours, transplanting the cultured seedlings onto sterilized peat soil, covering a plastic bag on a nutrition cup for moisturizing, after 3 days, slowly removing the plastic bag, and watering according to growth requirements. In step S1, the seeds are sown in an MS medium, wherein the MS medium is cultured in an MS medium without any hormone, and the pH of the medium is 5.8.
In step S2, the aseptic seedling is cut off and inoculated into MS culture medium added with BA 1mg/L, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L for culture.
In step S3, specifically, the explant is inoculated into MS culture medium added with TDZ 1-7mg/L + BA 1-2mg/L +2,4-D0.5-0.1mg/L + IBA 0.5-0.1mg/L + sucrose 25g/L + agar 5g/L to induce adventitious buds.
In step S3, transferring the explant to MS culture medium containing BA 1-3mg/L + NAA0.1-0.3mg/L + sucrose 25g/L + agar 5g/L to induce adventitious bud elongation.
In step S1, the culture temperature is 25 + -2 deg.C, the illumination intensity is 2500lx, and the illumination time is 12 h.
In step S3, the culture conditions are 25 + -2 deg.C, illumination intensity is 2500lx, and illumination time is 12 h.
In step S4, the method specifically comprises the step of inoculating the seeds into a rooting culture medium comprising MS, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L to induce rooting, wherein the pH value of the culture medium is 5.8.
In step S4, the pH of the culture medium is 5.8, the culture conditions are 25 + -2 deg.C, the illumination intensity is 2500lx, and the illumination time is 12 h.
Compared with the prior art, the invention has the beneficial effects that: the efficient tissue culture and plant regeneration method can eliminate the influence of external conditions, and produce strong, tidy and consistent tissue culture seedlings of the sorghum in a large-scale and industrialized manner; compared with the reported construction of a regeneration system by taking the hypocotyl, the stem segment with the axillary bud and the bud strip as explants, the regeneration system has the advantages of convenient operation, wide material taking, strong repeatability, more seedlings, high efficiency and the like. The concrete significance lies in that: firstly, 98 seedlings can be generated within 50 days after the callus formed by one stem segment is formed, and the efficient plant regeneration method is the first step of successful genetic transformation; secondly, the seedlings formed by callus are superior to the proliferated seedlings, the growth state of the seedlings is better, if the stems are thick and the leaves are green and large, the stems and the thin leaves cannot grow like the proliferated seedlings; thirdly, placing the plantlets formed by callus into a rooting culture medium, and beginning to sprout roots on the 8 th day, wherein the rooting rate reaches 100%; fourthly, when the stem segments are placed in a callus induction culture medium for 8 th to 15 th days, the stem segments begin to dedifferentiate and present water texture, and the stem segments can generate a yellow-white primary callus stage at the moment, so that the callus in the state is very suitable for being used as a transfection material; fifth, the method has the advantages of convenient operation, wide material selection, strong repeatability, more seedlings and high efficiency.
Therefore, the method can meet the requirement of the market on the seedlings, and can improve and innovate the germplasm resources of the sorghum trees through transgenic research, thereby laying a foundation for sustainable utilization of the germplasm resources of the sorghum trees.
Drawings
FIG. 1 is a diagram showing stem explants obtained after 20 days of culture of the cut seedlings in the four steps S2 of the example;
FIG. 2 shows the four steps of the example S3 for 20 days of stem callus on MS + TDZ 2.5mg/L + BA 1mg/L +2, 4-D0.1 mg/L + IBA 0.1mg/L + sucrose 25g/L + agar 5g/L callus induction culture;
FIG. 3 is a diagram of example four steps S3 of callus pieces on stem explant cultured for 30 days on MS + TDZ 2.5mg/L + BA 1mg/L +2, 4-D0.1 mg/L + IBA 0.1mg/L + sucrose 25g/L + agar 5g/L callus induction culture;
FIG. 4 is a diagram of the four steps S3 of example, culturing the elongated adventitious bud for 7d on the adventitious bud elongation medium BA 3mg/L + NAA 0.3mg/L L + sucrose 25g/L + agar 5 g/L;
FIG. 5 is a diagram of the four steps S3 of example, in the culture of 14d extended adventitious buds on the adventitious bud extension medium of BA 3mg/L + NAA 0.3mg/L + sucrose 25g/L + agar 5 g/L;
FIG. 6 is a diagram showing a four-step procedure S3 of example, in which 50d of extended adventitious buds are cultured on an adventitious bud extension medium containing BA 3mg/L + NAA 0.3mg/L + sucrose 25-30g/L + agar 5-6 g/L;
FIG. 7 is a diagram of the four steps of the example S4 growing 10d of elongated roots on MS + NAA0.1mg/L rooting medium;
FIG. 8 is a four step procedure S5 of example rooting culture of 20d elongated roots on MS + NAA0.1mg/L rooting medium;
Detailed Description
The technical scheme of the invention is further explained by combining specific examples.
Example one
A high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant comprises the following steps:
s1: obtaining of sterile seedlings: placing the seeds on a constant temperature shaking table at 37-40 ℃ to shake and soak for 24-36 h; sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 12-15min, washing thoroughly with sterile water for 6 times, and sowing the seeds;
s2: elongation of the stem segment and obtaining: the seeds begin to sprout after 7 days of culture, grow into aseptic seedlings after 15 days, cut off the aseptic seedlings, inoculate and culture, after culturing for 15 days, cut and take the stem section as the explant;
s3: induction and elongation of stem segments: inducing adventitious buds of the explant, and vertically inserting stem segments into a culture medium during inoculation; after culturing for 15-25 days, callus appears on the stem segment, and the explant induces the adventitious bud to elongate;
s4: rooting culture: when the adventitious bud extends to 3cm, cutting the adventitious bud for induced rooting;
s5: hardening and transplanting seedlings: after rooting culture for 20 days, growing multiple roots from adventitious buds, digging out the cultured seedlings from a culture bottle, cleaning the culture medium on the roots, soaking the cultured seedlings in tap water for 2 hours, transplanting the cultured seedlings onto sterilized peat soil, covering a plastic bag on a nutrition cup for moisturizing, after 3 days, slowly removing the plastic bag, and watering according to growth requirements.
In step S1, the seeds are sown in an MS medium, wherein the MS medium is cultured in an MS medium without any hormone, and the pH of the medium is 5.8.
In step S2, the aseptic seedling is cut off and inoculated into MS culture medium added with BA 1mg/L, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L for culture.
In step S3, specifically, the explant is inoculated into MS culture medium added with TDZ 1-7mg/L + BA 1-2mg/L +2,4-D0.5-0.1mg/L + IBA 0.5-0.1mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious buds.
In step S3, transferring the explant to MS culture medium containing BA 1-3mg/L + NAA0.1-0.3mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious bud elongation.
In step S1, the culture temperature is 25 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h.
In step S3, the culture conditions were 25 deg.C, illumination intensity 2500lx, and illumination time 12 h.
In step S4, the method specifically comprises the step of inoculating the seeds into a rooting culture medium comprising MS, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L to induce rooting, wherein the pH value of the culture medium is 5.8.
In step S4, the pH of the culture medium is 5.8, the culture conditions are 25 + -2 deg.C, the illumination intensity is 2500lx, and the illumination time is 12 h.
Example two
A high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant comprises the following steps:
s1: obtaining of sterile seedlings: placing the seeds on a constant temperature shaking table at 40 ℃ to shake and soak for 36 h; sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 15min, washing thoroughly with sterile water for 6 times, and sowing the seeds;
s2: elongation of the stem segment and obtaining: the seeds begin to sprout after 7 days of culture, aseptic seedlings grow after 25 days, the aseptic seedlings are cut off, inoculated and cultured, and stem segments are cut and taken as explants after 28 days of culture;
s3: induction and elongation of stem segments: inducing adventitious buds of the explant, and vertically inserting stem segments into a culture medium during inoculation; after culturing for 15-25 days, callus appears on the stem segment, and the explant induces the adventitious bud to elongate;
s4: rooting culture: when the adventitious bud extends to 4cm, cutting the adventitious bud for induced rooting;
s5: hardening and transplanting seedlings: after rooting culture for 20 days, growing multiple roots from adventitious buds, digging out the cultured seedlings from a culture bottle, cleaning the culture medium on the roots, soaking the cultured seedlings in tap water for 2 hours, transplanting the cultured seedlings onto sterilized peat soil, covering a plastic bag on a nutrition cup for moisturizing, after 3 days, slowly removing the plastic bag, and watering according to growth requirements.
In step S1, the seeds are sown in an MS medium, wherein the MS medium is cultured in an MS medium without any hormone, and the pH of the medium is 5.8.
In step S2, the aseptic seedling is cut off and inoculated into MS culture medium added with BA 1mg/L, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L for culture.
In step S3, specifically, the explant is inoculated into MS culture medium added with TDZ 1-7mg/L + BA 1-2mg/L +2,4-D0.5-0.1mg/L + IBA 0.5-0.1mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious buds.
In step S3, transferring the explant to MS culture medium containing BA 1-3mg/L + NAA0.1-0.3mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious bud elongation.
In step S1, the culture temperature was 27 ℃, the illumination intensity was 2500lx, and the illumination time was 12 h.
In step S3, the culture conditions were 27 deg.C, illumination intensity 2500lx, and illumination time 12 h.
In step S4, the method specifically comprises the step of inoculating the seeds into a rooting culture medium comprising MS, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L to induce rooting, wherein the pH value of the culture medium is 5.8.
In step S4, the pH of the culture medium was 5.8, the culture condition was 27 deg.C, the illumination intensity was 2500lx, and the illumination time was 12 h.
EXAMPLE III
A high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant comprises the following steps:
s1: obtaining of sterile seedlings: placing the seeds on a constant temperature shaking table at 40 ℃ to shake and soak for 36 h; sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 15min, washing thoroughly with sterile water for 6 times, and sowing the seeds;
s2: elongation of the stem segment and obtaining: the seeds begin to sprout after 7 days of culture, the seedlings grow into aseptic seedlings after 20 days, the aseptic seedlings are cut off, inoculated and cultured, and after 22 days of culture, stem segments are cut and taken as explants;
s3: induction and elongation of stem segments: inducing adventitious buds of the explant, and vertically inserting stem segments into a culture medium during inoculation; after culturing for 20 days, starting callus on the stem segment, and inducing the explant to elongate adventitious buds;
s4: rooting culture: when the adventitious bud extends to 3.5cm, cutting the adventitious bud to induce rooting;
s5: hardening and transplanting seedlings: after rooting culture for 20 days, growing multiple roots from adventitious buds, digging out the cultured seedlings from a culture bottle, cleaning the culture medium on the roots, soaking the cultured seedlings in tap water for 2 hours, transplanting the cultured seedlings onto sterilized peat soil, covering a plastic bag on a nutrition cup for moisturizing, after 3 days, slowly removing the plastic bag, and watering according to growth requirements.
In step S1, the seeds are sown in an MS medium, wherein the MS medium is cultured in an MS medium without any hormone, and the pH of the medium is 5.8.
In step S2, the aseptic seedling is cut off and inoculated into MS culture medium added with BA 1mg/L, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L for culture.
In step S3, specifically, the explant is inoculated into MS culture medium added with TDZ 1-7mg/L + BA 1-2mg/L +2,4-D0.5-0.1mg/L + IBA 0.5-0.1mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious buds.
In step S3, transferring the explant to MS culture medium containing BA 1-3mg/L + NAA0.1-0.3mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious bud elongation.
In step S1, the culture temperature is 26 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h.
In step S3, the culture conditions were 26 deg.C, illumination intensity 2500lx, and illumination time 12 h.
In step S4, the method specifically comprises the step of inoculating the seeds into a rooting culture medium comprising MS, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L to induce rooting, wherein the pH value of the culture medium is 5.8.
In step S4, the pH of the culture medium is 5.8, the culture condition is 26 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h.
Example four
A high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant comprises the following steps:
s1: obtaining of sterile seedlings: placing the seeds on a constant temperature shaking table at 40 ℃ to shake and soak for 36 h; sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 15min, washing thoroughly with sterile water for 6 times, and sowing the seeds;
s2: elongation of the stem segment and obtaining: the seeds begin to sprout after being cultured for 7 days, the seeds grow into aseptic seedlings after 20 days, the aseptic seedlings are cut off and inoculated into MS culture medium of 1mg/L BA, 0.1mg/L NAA, 25-30g/L sucrose and 5-6g/L agar for culturing, and after 20 days of culture, stem segments are cut out to be used as explants (shown in figure 1);
s3: induction and elongation of stem segments: inducing the explant to callus, and vertically inserting stem segments into MS culture medium of TDZ 2.5mg/L + BA 1mg/L +2, 4-D0.1 mg/L + IBA 0.1mg/L + sucrose 30g/L + agar 5g/L during inoculation; after 20 days of culture, callus begins to form on the stem segments (as shown in figure 2), the probability of the stem segments forming the callus reaches 66.7%, after 30 days of culture, the formed callus with the bud points (as shown in figure 3) is placed in MS culture medium with 3mg/L of BA, 0.3mg/L of NAA, 25-30g/L of sucrose and 5-6g/L of agar, the callus starts to sprout (as shown in figure 4) after 7 days, and the sprouts continuously start to sprout and grow (as shown in figure 5) after 14 days; after 50 days, the germination rate reaches 99%, and each callus has 96 buds (as shown in figure 6);
s4: rooting culture: when the adventitious bud extends to 3.5cm, cutting the adventitious bud, placing the adventitious bud in MS culture medium of MS + NAA0.1mg/L + sucrose 25-30g/L + agar 5-6g/L for inducing rooting for 10 days, wherein the adventitious root begins to form, the root rate reaches 98%, the average root length is 8.2, and the average root length is 0.83cm (as shown in figure 7)
S5: hardening and transplanting seedlings: after rooting culture for 20 days, growing multiple roots from adventitious buds (as shown in figure 8), digging out the cultured seedlings from a culture bottle, cleaning the culture medium on the roots, soaking the cultured seedlings in tap water for 2 hours, transplanting the cultured seedlings onto sterilized peat soil, covering a plastic bag on a nutrition cup for moisturizing, slowly removing the plastic bag after 3 days, and watering according to growth requirements.
In step S1, the seeds are sown in an MS medium, wherein the MS medium is cultured in an MS medium without any hormone, and the pH of the medium is 5.8.
In step S2, the aseptic seedling is cut off and inoculated into MS culture medium added with BA 1mg/L, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L for culture.
In step S3, specifically, the explant is inoculated into MS culture medium added with TDZ 1-7mg/L + BA 1-2mg/L +2,4-D0.5-0.1mg/L + IBA 0.5-0.1mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious buds.
In step S3, transferring the explant to MS culture medium containing BA 1-3mg/L + NAA0.1-0.3mg/L + sucrose 25-30g/L + agar 5-6g/L to induce adventitious bud elongation.
In step S1, the culture temperature is 26 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h.
In step S3, the culture conditions were 26 deg.C, illumination intensity 2500lx, and illumination time 12 h.
In step S4, the method specifically comprises the step of inoculating the seeds into a rooting culture medium comprising MS, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L to induce rooting, wherein the pH value of the culture medium is 5.8.
In step S4, the pH of the culture medium is 5.8, the culture condition is 26 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (4)
1. A high-efficiency regeneration method taking a yellow sorghum wood stem section as an explant is characterized by comprising the following steps: comprises the following steps
The method comprises the following steps:
s1: obtaining of sterile seedlings: placing the seeds on a constant temperature shaking table at 37-40 ℃ to shake and soak for 24-36 h; in that
Sterilizing with 75% alcohol for 30s on a sterile super-clean bench, washing with sterile water for 3 times, sterilizing with 10% NaClO solution for 12-15min, washing thoroughly with sterile water for 6 times, sowing the seeds in an MS culture medium, wherein the MS culture medium is an MS culture medium without any hormone, and the pH value of the culture medium is 5.8;
s2: elongation of the stem segment and obtaining: the seeds begin to sprout after being cultured for 7 days, and grow into aseptic seedlings after 15-25 days,
cutting off the aseptic seedling, inoculating the aseptic seedling into an MS culture medium added with 1mg/L of BA, 0.1mg/L of NAA0, 25-30g/L of sucrose and 5-6g/L of agar, culturing for 15-28 days, and cutting stem segments to be used as explants;
s3: induction and elongation of stem segments: inoculating the explant into an MS culture medium added with TDZ 1-7mg/L + BA 1-2mg/L +2,4-D0.5-0.1mg/L + IBA 0.5-0.1mg/L + sucrose 25g/L + agar 5g/L to induce adventitious buds, and vertically inserting stem segments into the culture medium during inoculation; after culturing for 15-25d, when the stem segment begins to change from yellow green callus to green bud point, transferring the explant to MS culture medium added with BA 1-3mg/L, NAA0.1-0.3mg/L, sucrose 25g/L and agar 5g/L to induce adventitious bud elongation;
s4: rooting culture: when the adventitious bud extends to 3-4cm, cutting the adventitious bud, inoculating the adventitious bud into a rooting culture medium of MS, NAA0.1mg/L, sucrose 25-30g/L and agar 5-6g/L for inducing rooting, wherein the pH value of the culture medium is 5.8;
s5: hardening and transplanting seedlings: after rooting culture for 20 days, the adventitious bud grows into multiple roots, and the cultured seedling is taken out from the culture bottle
Digging out, cleaning culture medium on root, soaking the cultured seedling in tap water for 2 hr, and transplanting to sterilized peat
Covering a plastic bag on the nutrient cup on the soil for moisture preservation, slowly removing the plastic bag after 3d, and watering according to growth requirements.
2. The efficient regeneration method of the stem of sorghum kaoliang as explant according to claim 1, wherein said method comprises
Is characterized in that: in step S1, the culture temperature is 25 + -2 deg.C, the illumination intensity is 2500lx, and the illumination time is 12 h.
3. The efficient regeneration method of the stem of sorghum kaoliang as explant according to claim 1, wherein said method comprises
Is characterized in that: in step S3, the culture conditions are 25 + -2 deg.C, illumination intensity 2500lx, and illumination time
12h。
4. The efficient regeneration method of the stem of sorghum vulgare as the explant according to claim 1, wherein the efficient regeneration method comprises the following steps: in step S4, the pH of the culture medium is 5.8, the culture conditions are 25 + -2 deg.C, the illumination intensity is 2500lx, and the illumination time is 12 h.
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| CN110692515A (en) * | 2019-04-25 | 2020-01-17 | 华南农业大学 | Efficient tissue culture propagation method taking phellodendron amurense tender shoots as explants |
| CN113796311B (en) * | 2021-10-12 | 2022-05-17 | 华南农业大学 | Method for inducing tetraploid of phellodendron amurense |
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| CN101032224A (en) * | 2006-08-24 | 2007-09-12 | 厦门涌泉科技有限公司 | Tissue cultivating and seedling method of fast growing anthocephalus |
| CN103782903A (en) * | 2013-11-19 | 2014-05-14 | 华南农业大学 | Efficient regeneration method for anthocephalus chinensis plants |
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| CN101032224A (en) * | 2006-08-24 | 2007-09-12 | 厦门涌泉科技有限公司 | Tissue cultivating and seedling method of fast growing anthocephalus |
| CN103782903A (en) * | 2013-11-19 | 2014-05-14 | 华南农业大学 | Efficient regeneration method for anthocephalus chinensis plants |
| CN104957042A (en) * | 2015-07-23 | 2015-10-07 | 中国林业科学研究院资源昆虫研究所 | Tissue culture and rapid propagation method of luculia yunnanensis |
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