CN111616054B - Method for obtaining regenerated plants by using Ledebouriella seseloides leaves - Google Patents
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Abstract
The invention discloses a method for obtaining a regeneration plant by using a windproof leaf, which comprises the following steps: 1) culturing the divaricate saposhnikovia root explant to obtain callus, 2) carrying out induction culture on the callus to obtain somatic embryos, and 3) further culturing the somatic embryos to obtain the divaricate saposhnikovia root regeneration plants. The key point of the test is to obtain a scheme of quickly utilizing the leaves and obtaining the regenerated seedlings through a somatic cell embryo way, and the number of the obtained regenerated seedlings can be controlled by controlling the number of the subcultured and propagated cells.
Description
Technical Field
The invention relates to the field of plant cultivation methods, in particular to a method for obtaining a regenerated plant by using windproof leaves.
Background
The divaricate saposhnikovia root is an important traditional Chinese medicinal plant, the divaricate saposhnikovia root is an umbelliferae plant, seeds naturally contain 20-30% of germ-free seeds, and the seeds are light with thorns and hairs, so that the use of the seeds is greatly limited, and the current planting mode in production is mainly that the seeds are mixed with fine soil for broadcast sowing; the life of the divaricate saposhnikovia root seeds is short, the germination rate of the fresh seeds in the current year is generally 60-80%, and the germination rate of the seeds stored for more than 1 year is obviously reduced and even lost.
At present, the planting modes of the divaricate saposhnikovia root in production mainly comprise 2 types: firstly, sowing seeds by using new seeds in the current year; and secondly, root division propagation, namely selecting root strips which are more than two years old, strong in growth and 0.7 cm thick and have no plant diseases and insect pests when the divaricate saposhnikovia roots are harvested, and cutting the root strips into small sections with the length of 3-5 cm for planting. The invention can directly obtain the seedlings for production in an industrial manner.
Research on rapid propagation of tissue culture, research on culture of Leishaohua and other plant physiological communications, research on culture of Ledebouriella seseloides callus, northern horticulture, no. 2, 183-185 in 2011; ledebouriella seseloides callus induction and plant regeneration system research, zhang Jia Jing, etc., no. 8 of 2012, shandong agricultural science 13-16; the establishment of a divaricate saposhnikovia root callus induction and plant regeneration system, and the development of a plant regeneration system, chinese herbal medicines, 2018,49 (13), all disclose methods for obtaining a plant regeneration system by inducing the divaricate saposhnikovia root callus to generate cluster buds. However, the above scheme is cluster bud induction, and requires separation of single bud and induction of root and seedling, and the steps are complicated. The patent "a tissue culture and rapid propagation method of Ledebouriella root" describes a method of using Ledebouriella root seeds to cultivate callus, further inducing germination, obtaining differentiated bud and raising seedling.
Meanwhile, in the prior art, the method for hardening the obtained test-tube plantlets adopts a direct bottle opening method, but the bottle cap is directly opened by the method, so that the culture medium in the bottle can pollute growing bacteria in about 3 days, and the root of the tissue culture plantlet can be seriously polluted and blackened.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a method for rapidly utilizing the windproof blades to obtain regenerated seedlings through a somatic embryo way.
The invention provides a method for obtaining a regeneration plant by using a windproof leaf, which comprises the following steps:
1) Culturing the divaricate saposhnikovia root explant to obtain a callus,
2) Inducing and culturing the callus to obtain somatic embryo,
3) And further culturing the somatic embryo to obtain a divaricate saposhnikovia root regeneration plant.
Wherein, in the step 1), the process of taking the windbreaker leaf as the explant and culturing the windbreaker explant to the callus specifically comprises the following steps:
11 Carrying out dark culture on the explant, and carrying out dark culture on the leaf blade in a dark culture medium for 15-21 days at the temperature of 25-28 ℃, wherein the dark culture medium is a mixture of MS, 2, 4-dichlorophenoxyacetic acid, 6-benzylamino adenine, glucose and plant gel, and each liter of MS culture medium in the dark culture medium contains 1.0-1.5 mg2, 4-dichlorophenoxyacetic acid and 0.5-1.0 mg6-benzylamino adenine; the mass fraction of the glucose is 1.5-3%, the mass fraction of the plant gel is 0.35-0.4%, and the pH value of the dark culture medium is 6.5-7.5;
12 Culturing the cell tissue after dark culture in a dark culture medium for 30 to 35 days at the temperature of between 25 and 28 ℃ under 16h of illumination/8 h of darkness;
13 Culturing the cell tissue obtained in the step 12) in a callus culture medium under the conditions of 28-30 ℃ and 16h illumination/8 h dark, transferring the cell tissue once every 25-30 days, and transferring the cell tissue for 3-4 times to obtain the callus, wherein the callus culture medium is a mixture of MS, 2, 4-dichlorophenoxyacetic acid, 6-benzylaminopurine, glucose and plant gel, and each liter of the MS culture medium in the dark culture medium contains 0.2-0.5mg2, 4-dichlorophenoxyacetic acid and 0.4-0.6 mg6-benzylaminopurine; the mass fraction of the glucose is 1.5-3%, the mass fraction of the plant gel is 0.35-0.4%, and the pH value of the callus culture medium is 6.5-7.5.
Wherein, the illumination intensity in the step 12) in the step 1) is 1000 to 3000Lx, and the illumination intensity in the step 13) is 3000 to 5000Lx.
Wherein, the inducing and culturing the callus to obtain the somatic embryo comprises the following steps: culturing the callus in a differentiation culture medium under the conditions of 25-28 ℃ and 16h of illumination/8 h of dark, and transferring for 0-1 time, wherein the differentiation culture medium is a mixture of N6, 2.4-D, NAA, ABA, glucose and plant gel; each liter of N6 culture medium contains 2.4-D0.05-0.2 mg, NAA0.1-0.5mg and ABA0.1-0.5mg, the mass fraction of the glucose is 1.5-3%, the mass fraction of the plant gel is 0.35-0.4%, and the pH value of the callus culture medium is 6.5-7.5.
Wherein, the illumination intensity in the step 2) in the step 1) is 1000 to 3000Lx.
Wherein, the step 3) in the step 1) comprises culturing the somatic embryos in a seedling culture medium for 40-50 days at the temperature of 25-28 ℃ under the condition of 16h light/8 h dark, wherein the seedling culture medium is as follows: MS + KT0.5-1.0mg/L + IAA0.05-0.1mg/L + glucose 1.5-3% + plant gel 0.35-0.4%.
Wherein the illumination intensity in the step 3 in the step 1) is 1000 to 3000Lx.
Wherein, still include, the hardening off and transplanting of ledebouriella root regenerated plant specifically includes: and when the height of the windproof seedlings is more than 5cm, culturing the windproof seedlings by using a closed culture medium, and transplanting after culturing for at least 7 days.
Wherein the closed culture medium is a sodium benzoate aqueous solution, and the concentration of the sodium benzoate aqueous solution is 50mg/L.
Wherein, before dark culture is carried out on the divaricate saposhnikovia root explant to obtain callus, disinfection and pretreatment of the explant are carried out.
The invention has the beneficial effects that: the culture process of the ledebouriella root somatic embryo needs more complicated culture medium sequence change, and proper culture medium needs to be discussed in different stages of explant selection and disinfection, initial culture callus starting, embryogenic callus induction, somatic embryo development, embryo mature germination and the like. In the process of proliferation and subculture, the callus multiple can be increased, generally by 100-500 times, one bottle of the callus can be induced to obtain one bottle of embryonic callus, and one bottle of the embryonic callus, and 5-8 regenerated seedlings can be obtained because the tissue culture bottle is a 100 ml triangular flask. Therefore, the final regenerated seedling number can be obtained by controlling the number of subculture bottles according to the target regenerated seedling number. The key point of the test is to obtain a scheme of quickly utilizing the leaves and obtaining the regenerated seedlings through a somatic embryo way, and the number of the obtained regenerated seedlings can be controlled by controlling the number of the subcultured and proliferated cells.
Drawings
FIG. 1 is a photograph of leaf-induced callus.
FIG. 2 is a photograph of embryogenic callus and somatic embryos.
FIG. 3 is a photograph of somatic embryo germination and growth.
FIG. 4 is a photograph of somatic embryo germination into a seedling.
FIG. 5 is a photograph showing the survival of the transplanted regenerated seedlings.
FIG. 6 mold contamination occurred on day 3 of decapping and acclimatizing.
FIG. 7 shows that the regenerated plantlets died after drying up on the 5 th day of direct bottle opening and seedling hardening.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Taking the divaricate saposhnikovia root explant: the windproof blades are collected from Guanshan national geological parks in New county City 7.21.2018.
2, 4-Dichlorophenoxyacetic acid (2, 4-D for short), kinetin (KT), auxin (IAA), naphthylacetic acid (NAA), 6-benzylaminopurine (6-BA) and abscisic acid (ABA) (Zhengzhou Dingguo Biotech Co., ltd.)
The MS culture medium is prepared according to the following steps, and the solvent is pure water, so that the MS culture medium is obtained:
1. preparing MS stock solution macroelement R1, preparing the macroelement into 20 times of mother liquor, and diluting 50 ml to 1L when in use. Separately weighing NH 4 NO 3 33 g, KNO 3 38 g, mgSO 4 ·7H 2 O7.4 g, KH 2 PO 4 3.4 g of the solutions are combined into 1 liter of mother liquor after being dissolved, and the mother liquor is stored in a reagent bottle and stored in a refrigerator at 4 ℃ for standby.
2. Stock solution macroelement R2, caCI 2 ·2H 2 O8.8 g or anhydrous CaCI 2 6.6 g of the extract is dissolved in 1L of pure water, the volume is fixed to 1L, and 50 ml of the extract is diluted to 1L when in use.
3. Stock solution (inorganic trace element) R3, preparing the trace element into 1000 times of mother solution, and diluting 1 ml to 1L when in use. Sequentially weighing 0.83 g of KI and MnSO 4 ·H 2 O6.2 g, H 3 BO 3 16.9 g, znSO 4 ·7H 2 O8.6 g, naMoO 4 ·2H 2 0.25 g of O, cuSO 4 ·5H 2 O0.025 g, coCI 2 ·6H 2 Dissolving 0.025 g of O, metering the volume to 1 liter, and storing in a refrigerator at 4 ℃ for later use.
4. Stock solution (FeSO) 4 ) R4, preparing 200 times of mother liquor, and diluting 5 ml to 1L when in use. Sequentially weighing Na 2 EDTA·2H 2 O3.73 g, feSO 4 ·7H 2 O2.78 g, dissolved separatelyMixing, metering to 500 ml, and storing in a refrigerator at 4 ℃ for later use.
5. The organic component R5 of the stock solution is prepared into 200 times of mother solution, and 5 milliliters of the mother solution is diluted to 1 liter when in use. Sequentially weighing nicotinic acid (VB) 3 ) 0.1 g of pyridoxine hydrochloride (VB) 6 ) 0.1 g of thiamine hydrochloride (VB) 1 ) 0.08 g and 0.4 g of glycine, preparing 1 liter of mother liquor, and storing the mother liquor in a refrigerator with constant volume and 4 ℃ for later use.
6. Stock solution (inositol) R6, 20 g of inositol is weighed to prepare 1L of mother liquor, and 5 ml of the mother liquor is diluted to 1L when in use.
An N6 culture medium is prepared according to the following steps, and the solvent is pure water, so that the N6 culture medium is obtained:
n6 macroelement mother liquor 1, preparing macroelement into 10 times of mother liquor, and diluting 100 ml of mother liquor to 1L when in use. Respectively weighing KNO 3 28.3 g (NH) 4 ) 2 SO 4 4.63 g, caCI 2 ·2H 2 O1.66 g, KH 2 PO 4 4 g, respectively dissolving, combining into 1L of mother liquor, storing in a reagent bottle, and storing in a refrigerator at 4 ℃ for later use.
Preparing 2 N6 microelement mother liquor, preparing 100 times of microelement mother liquor, and diluting 10 ml to 1L when in use. Respectively weighing MnSO 4 ·H 2 O0.22 g, znSO 4 ·7H 2 O0.08 g, H 3 BO 3 0.04 g and 0.08 g of KI, dissolving and combining in turn, metering to 500 ml, and storing in a refrigerator at 4 ℃ for later use.
Preparing 3 N6 ferric salt mother liquor into 100 times of mother liquor, and diluting 10 ml of mother liquor to 1L when in use. Sequentially weighing Na 2 EDTA·2H 2 0.745 g of O, feSO 4 ·7H 2 And 0.557 g of O, mixing the solution after separately dissolving the solution until the volume is 200 ml, and storing the solution in a refrigerator at 4 ℃ for later use.
4, preparing 4 times of mother liquor of N6 organic components, diluting 10 ml of mother liquor to 1L when in use, and sequentially weighing nicotinic acid (VB) 3 ) 0.01 g of pyridoxine hydrochloride (VB) 6 ) 0.01 g of thiamine hydrochloride (VB) 1 ) 0.02 g and 0.04 g of glycine are prepared into 200 ml of mother liquor, and the mother liquor is stored in a refrigerator with constant volume and 4 ℃ for later use.
B5 culture medium: the preparation method comprises the following steps that the solvent is pure water, and the B5 culture medium is obtained:
preparing 1 by using the B5 macroelement mother liquor, preparing 10 times of macroelement mother liquor, and diluting 100 milliliters of the mother liquor to 1 liter when in use. Respectively weighing KNO 3 25 g, caCI 2 ·2H 2 O1.5 g, mgSO 4 ·7H 2 O2.5 g, (NH) 4 ) 2 SO 4 1.34 g, after dissolving, combining into 1L mother liquor, storing in a reagent bottle, and storing in a refrigerator at 4 ℃ for standby.
Preparing a mother solution of the trace elements 2 by using the B5, preparing the trace elements into a mother solution of which the volume is 100 times that of the mother solution, and diluting 10 milliliters of the mother solution into 1 liter when in use. Sequentially weighing 0.0375 g of KI and H 3 BO 3 0.15 g, mnSO 4 ·H 2 0.5 g of O, znSO 4 ·7H 2 O,1.0 g, na 2 MoO 4 ·2H 2 O0.0125 g, coCI 2 ·6H 2 0.00125 g of O and CuSO 4 ·5H 2 0.00125 g of O, dissolving and mixing in turn, metering the volume to 500 ml, and storing in a refrigerator at 4 ℃ for later use.
Preparing 3 times of the mother liquor of B5 iron salt, and diluting 5 ml of the mother liquor to 1 liter when in use. Sequentially weighing Na 2 EDTA·2H 2 O3.73 g, feSO 4 ·7H 2 And (3) dissolving O2.78 g separately, combining the solutions until the volume is 500 ml, and storing the solution in a refrigerator at 4 ℃ for later use.
4, preparing 4 times of mother liquor by using the B5 organic component mother liquor, and diluting 10 milliliters of the mother liquor to 1 liter when in use. Nicotinic acid (VB) 3 ) 0.02 g, pyridoxine hydrochloride (VB) 6 ) 0.02 g of thiamine hydrochloride (VB) 1 ) 0.2 g, preparing 200 ml of mother liquor, and storing in a refrigerator with constant volume at 4 ℃ for later use.
5, preparing 5 by using B5 inositol mother liquor, weighing 2 g of inositol, preparing 200 ml of inositol, and diluting 10 ml of inositol to 1L when in use.
Example 1
Constructing a divaricate saposhnikovia root regeneration plant by taking the divaricate saposhnikovia root leaves as explants by the following method:
step 1, explant disinfection and pretreatment:
11. soaking the wind-proof blades in 75% ethanol for 30 s for disinfection, shaking up continuously during the soaking process, and then washing with sterile water for 1 time;
12. the washed Ledebouriella seseloides leaves were treated at 0.1% HgCI 2 Soaking in the solution for 5 min while shaking, and washing with sterile water for more than 4 times.
13. The leaf washed in step 12 was divided into leaf blocks of about 0.5cm × 0.5 cm.
The invention adopts leaves as explants, the materials are easy to obtain, the disinfection procedure and the disinfection time are generally recorded as 70 percent or 75 percent ethanol for 30 seconds, the percent of the leaf buds is 0.1 percent 2 Soaking for 5-7 min, the invention proposes 70-75% ethanol disinfection for 20-30 s, 0.1% HgCI 2 Soaking for 3-5 minutes, and the disinfection time prolongs the callus of the explant to appear slowly, even die, and no callus appears. According to data record, 0.1 percent of HgCI2 is soaked for 3-5 minutes, the callus forming time is 20-25 days and 6-7 minutes, the callus forming time is 30-40 days, and the callus forming rate is 60-90 percent; when the time is more than 8 minutes, the leaves basically do not generate callus, and the callus generation rate is about 5 to 10 percent.
Step 2, culture of callus:
21. dark culture: inoculating the leaf blocks in the step 13 into a dark culture medium, and culturing in the dark for 15 days at the culture temperature of 28 ℃. The components of the dark culture medium are MS +2, 4-D1.0 mg/L +6-BA 1.0mg/L + glucose 3% + plant gel 0.4%, and the pH is 7.5; namely, a dark medium prepared by adding 1.0mg of 2,4-D, 1.0mg of 6-BA, 3% by mass of glucose and 0.4% by mass of plant gel per liter of MS medium and adjusting the pH to 7.5. Wherein, the culture conditions can be selected according to requirements within the range of full dark culture for 15-21 days and the culture temperature of 25-28 ℃, and the formula of the culture medium can be selected according to requirements within the range of MS +2, 4-D1.0-1.5 mg/L +6-BA 0.5-1.0mg/L + glucose 1.5-3% + plant gel 0.35-0.4% and pH of 6.5-7.5.
22. Callus induction: when the callus appears in the dark culture step, the culture medium is switched to alternate culture (16 hours of illumination/8 hours of darkness), the culture medium is still the dark culture medium, the temperature is 28 ℃, and all the callus appears after 30 days of culture. (the light intensity at this time is 3000 Lx). Wherein the above culture conditions may be at a temperature of 25-28 ℃ for 30-35 days, the light intensity is: selected in the range of 1000-3000Lx according to the requirement
Compared with the prior scheme, the method has the advantages that the temperature, the components and the dosage are different from those of the method, so that the callus induction influence factors are more, a best combination scheme is provided in the experiment, and the induction rate is 100%. The content of glucose and the pH value of the culture medium influence the generation of callus.
23. And (3) callus rapid proliferation: in order to obtain more callus in a short time, the callus in the second step is transferred to a callus culture medium and cultured alternately at the temperature of 28-30 ℃ (16 hours light/8 hours dark) for 30 days each time, and the transfer culture is carried out for 4 times. Can obtain over 100 times of cell growth. (100-fold increase of cells means that 1 g of tissue is transferred and cultured for 30 days to reach 100 g or so.) the callus medium comprises the following components: MS +2,4-D0.5mg/L +6-BA0.6mg/L + glucose 3% + plant gel 0.4%, pH 7.5, namely adding 0.5mg of 2,4-D0.6 mg of 6-BA, 3% of glucose by mass fraction and 0.4% of plant gel by mass fraction to each liter of MS culture medium, and adjusting the pH value to 7.5 to prepare the callus culture medium, wherein the illumination intensity is 3000Lx. The resulting callus is shown in FIG. 1. Wherein, the culture conditions can be switched for 3-4 times in 25-30 days per switching culture, the illumination intensity is selected according to requirements within the range of 3000-5000Lx, the formula of the culture medium can be selected according to requirements within the range of MS +2, 4-D0.2-0.5 mg/L +6-BA0.4-0.6mg/L + glucose 1.5-3% + plant gel 0.35-0.4%, and the pH is selected according to requirements within the range of 6.5-7.5.
Step 3, induction of somatic embryos:
transferring the callus into a differentiation medium, and performing alternate culture (16 hours of light/8 hours of darkness) at the temperature of 28 ℃ for 25 days each time, wherein somatic embryos are obtained after 1 transfer. The components of the differentiation medium are as follows: n6+2.4-D0.2mg/L + NAA0.5mg/L + ABA0.1-0.5mg/L + glucose 3% + plant gel 0.4%, pH 7.5, i.e., each liter of N6 medium was supplemented with 0.2mg of 2,4-D, 0.5mg of NAA0.5mg of ABA, 3% by mass of glucose and 0.4% by mass of plant gel, and the pH was adjusted to 7.5 to prepare a differentiation medium. Wherein, the illumination intensity is 3000Lx. The somatic embryos prepared in step 3 are shown in FIG. 2. Wherein the culture conditions can be that the alternate culture (16 hours light/8 hours dark) is carried out at the temperature of 28-30 ℃ for 20-25 days in each transfer culture, and the 0-1 transfer to obtain somatic embryo is carried out for 0 time, namely, the callus is transferred to a differentiation culture medium to be cultured for 20-25 days to obtain the somatic embryo; transferring 1 time refers to transferring callus to differentiation culture medium for culturing for 20-25 days, then transferring callus to differentiation culture medium for culturing for 20-25 days to obtain somatic embryo), and selecting according to requirement, wherein the formula of the culture medium can be selected according to requirement in the range of N6+ 2.4-D0.05-0.2 mg/L + NAA0.1-0.5mg/L + ABA0.1-0.5mg/L + glucose 1.5-3% + plant gel 0.35-0.4%, and pH is 6.5-7.5.
Step 4, somatic embryo seedling formation:
adjusting the hormone proportion and concentration of a culture medium to prepare a seedling culture medium, wherein the seedling culture medium comprises the following components: MS + KT1.0 mg/L + IAA0.1mg/L + glucose 3% + plant gel 0.4%, pH is 7.5, namely add 1.0mg KT, 0.1mg IAA, mass fraction 3% glucose and mass fraction 0.4% plant gel in MS culture medium each liter, and adjust pH to the seedling culture medium that 7.5 made. (wherein, the formulation of the culture medium can be selected according to requirements in the range of 0.5-1.0mg/L of MS + KT + 0.05-0.1mg/L of IAA + 0.5-3% of glucose + 0.35-0.4% of plant gel and pH 6.5-7.5.)
Transferring the somatic embryo to seedling culture medium, alternately culturing at 28 deg.C (16 hr light/8 hr dark) under illumination intensity of 3000Lx for 40 days to obtain regenerated plant of radix Saposhnikoviae. Wherein when the culture is carried out for 15 days, the somatic embryo germinates and grows, as shown in figure 3, and when the culture is carried out for 30 days, the divaricate saposhnikovia root regeneration plant is obtained, as shown in figure 4. Wherein the culture conditions can be selected according to requirements at 25-28 deg.C, illumination intensity of 1000-3000Lx, and culture time of 40-50 days.
Step five, hardening and transplanting seedlings:
when the height of the seedlings of the windproof regenerated plants is more than 5cm, opening a bottle sealing film, adding 50mg/L sodium benzoate aqueous solution into a culture bottle to seal a culture medium, checking the integrity of the water film every day, incompletely supplementing the water film completely, replacing the 50mg/L sodium benzoate aqueous solution once every 2 days, taking out test-tube seedlings of the windproof regenerated plants from the culture bottle after 7 days or more, washing off a root culture medium, avoiding damaging the roots as much as possible, planting the test-tube seedlings into a matrix formed by mixing loam and nutrient soil, and planting the test-tube seedlings in a field after 7 days of survival, wherein the survival rate is 100%. (as shown in FIG. 5)
Comparative example: directly opening bottles for hardening seedlings of the saposhnikovia divaricata regenerated plants in the step 4, culturing for 3 days under the same conditions as shown in figure 6, and continuously culturing for 5 days as shown in figure 7.
The comparison of the data shows that the culture medium in the bottle can pollute and grow bacteria after the bottle cap is directly opened for about 3 days, and the root of the tissue culture seedling can be seriously polluted and blackened, and the experiment adopts a method of sealing the culture medium by a 50mg/L sodium benzoate solution, so that the pollution problem of bottle opening and seedling hardening is effectively solved.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Claims (5)
1. A method for obtaining a regenerated plant by using Ledebouriella seseloides leaves, comprising:
1) Culturing the divaricate saposhnikovia root explant to obtain a callus,
2) Inducing and culturing the callus to obtain somatic embryo,
3) Further culturing the somatic embryo to obtain a divaricate saposhnikovia root regeneration plant;
in the step 1), the process of culturing the divaricate saposhnikovia root explant to the callus by taking the divaricate saposhnikovia root leaves as the explant specifically comprises the following steps:
11 Carrying out dark culture on explants, and carrying out dark culture on leaves in a dark culture medium at 25 to 28 ℃ for 15 to 21 days, wherein the dark culture medium is a mixture of MS, 2, 4-dichlorophenoxyacetic acid, 6-benzylamino adenine, glucose and plant gel, and each liter of MS culture medium in the dark culture medium contains 1.0 to 1.5mg2, 4-dichlorophenoxyacetic acid and 0.5 to 1.0 mg6-benzylamino adenine; the mass fraction of the glucose is 1.5 to 3 percent, the mass fraction of the plant gel is 0.35 to 0.4 percent, and the pH value of the dark culture medium is 6.5 to 7.5;
12 Culturing the cell tissue subjected to dark culture in a dark culture medium for 30 to 35 days at the temperature of 25 to 28 ℃ under the condition of 16h of illumination/8 h of darkness;
13 Culturing the cell tissue obtained in the step 12) in a callus culture medium under the conditions of 28 to 30 ℃ and 16h of illumination/8 h of dark, transferring the cell tissue once every 25 to 30 days, and transferring the cell tissue for 3 to 4 times to obtain a callus, wherein the callus culture medium is a mixture of MS, 2, 4-dichlorophenoxyacetic acid, 6-benzylaminopurine, glucose and plant gel, and each liter of MS culture medium in the dark culture medium contains 0.2 to 0.5mg2, 4-dichlorophenoxyacetic acid and 0.4 to 0.6 mg6-benzylaminopurine; the mass fraction of the glucose is 1.5 to 3 percent, the mass fraction of the plant gel is 0.35 to 0.4 percent, and the pH value of the callus culture medium is 6.5 to 7.5;
the induction and culture of the callus to obtain somatic embryos comprises the following steps: culturing the callus in a differentiation culture medium under the conditions of 25 to 28 ℃ and 16h of illumination/8 h of dark, and transferring for 0-1 time, wherein the differentiation culture medium is a mixture of N6, 2.4-D, NAA, ABA, glucose and plant gel; each liter of N6 culture medium contains 2.4-D0.05-0.2mg, NAA0.1-0.5mg and ABA0.1-0.5mg, the mass fraction of the glucose is 1.5-3%, the mass fraction of the plant gel is 0.35-0.4%, and the pH value of the callus culture medium is 6.5-7.5;
the step 3) comprises culturing the somatic embryos in a seedling culture medium for 40 to 50 days at the temperature of 25 to 28 ℃ under the condition of 16h of light/8 h of dark, wherein the seedling culture medium is as follows: 0.5-1.0mg/L of MS + KT + 0.05-0.1mg/L of IAA + 1.5-3% of glucose + 0.35-0.4% of plant gel;
hardening seedlings and transplanting the divaricate saposhnikovia root regeneration plants, which specifically comprises the following steps: when the height of the windproof seedlings is more than 5cm, carrying out closed culture medium culture on the windproof seedlings, and transplanting after at least 7 days of culture; the closed culture medium is a sodium benzoate aqueous solution, and the concentration of the sodium benzoate aqueous solution is 50mg/L.
2. The method according to claim 1, wherein the illumination intensity in the step 1) in the step 12) is 1000 to 3000Lx, and the illumination intensity in the step 13) is 3000 to 5000Lx.
3. The method according to claim 1, wherein the illumination intensity in the step 2) is 1000 to 3000Lx.
4. The method according to claim 1, wherein the illumination intensity in step 3 is 1000 to 3000Lx.
5. The method of any one of claims 1 to 4, wherein the explants are sterilized and pretreated before dark culture to obtain callus.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6150587A (en) * | 1997-06-27 | 2000-11-21 | The Penn State Research Foundation | Method and tissue culture media for inducing somatic embryogenesis, Agrobacterium-mediated transformation and efficient regeneration of cacao plants |
| CN104686346A (en) * | 2015-02-24 | 2015-06-10 | 陈桂容 | Tissue culture and rapid propagation method for divaricate saposhnikovia root |
| CN104686347A (en) * | 2015-02-24 | 2015-06-10 | 陈桂容 | Ultralow-temperature preservation technique of callus of divaricate saposhnikovia root |
-
2020
- 2020-06-16 CN CN202010547395.2A patent/CN111616054B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6150587A (en) * | 1997-06-27 | 2000-11-21 | The Penn State Research Foundation | Method and tissue culture media for inducing somatic embryogenesis, Agrobacterium-mediated transformation and efficient regeneration of cacao plants |
| CN104686346A (en) * | 2015-02-24 | 2015-06-10 | 陈桂容 | Tissue culture and rapid propagation method for divaricate saposhnikovia root |
| CN104686347A (en) * | 2015-02-24 | 2015-06-10 | 陈桂容 | Ultralow-temperature preservation technique of callus of divaricate saposhnikovia root |
Non-Patent Citations (4)
| Title |
|---|
| Somatic Embryogenesis and in vitro Flowering in Saposhnikovia divaricata;Qi Qiao et al.;《J Plant Growth Regul》;20080925;第28卷;第81-86页 * |
| 外源激素对防风体细胞胚发生和发育的影响;乔琦等;《西北大学学报(自然科学版)》;20050630;第35卷(第03期);第316-319页 * |
| 防风体细胞胚发生发育的研究;乔琦;《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》;20051015(第6期);第10-16页 * |
| 防风愈伤组织诱导研究;毕博等;《中国现代中药》;20110831;第13卷(第08期);第22-24页 * |
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