CN111713405A - Method for culturing regenerated plant by pepper protoplast and special protoplast culture medium - Google Patents
Method for culturing regenerated plant by pepper protoplast and special protoplast culture medium Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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 method for culturing regenerated plants by using pepper protoplasts, which comprises the steps of suspending protoplasts derived from pepper sterile test-tube seedling leaves in a protoplast culture medium, adjusting the density, and performing static culture on a liquid shallow layer to form cell clusters and small callus tissues; after the callus is cultured in a proliferation mode, the callus is inoculated to a differentiation culture medium to be cultured and differentiated to obtain adventitious buds, and then the adventitious buds are transferred to a rooting culture medium to be cultured to form a complete plant. The invention carries out deep research on important links in the pepper protoplast culture process, and particularly gropes out a special protoplast culture medium, thereby greatly improving the cell division frequency and the cell cluster formation frequency of the protoplast. The invention successfully obtains protoplast regeneration plants, initially establishes a complete pepper protoplast regeneration system, and has important significance for developing related biotechnology researches such as pepper somatic cell crossbreeding, protoplast genetic transformation and the like.
Description
Technical Field
The invention relates to a method for culturing regenerated plants by pepper protoplasts and a special protoplast culture medium, belonging to the technical field of plant tissue culture.
Background
Chinese prickly ash (zanthoxylum bungeanum Maxim.)Zanthoxylunm bungeanumMaxim, also called Zanthoxylum piperitum, Zanthoxylum bungeanum Maxim, is of genus Zanthoxylum (Rutaceae) of RutaceaeZanthoxylum) Plants, distributed in tropical and subtropical regions of asia, america, africa, and the continental continents, have about 250 varieties worldwide, and 39 14 varieties in our country, and belong to perennial deciduous shrubs or small trees. The pepper is a native plant in China, has a long cultivation history and is widely distributed, and besides a few areas such as northeast, inner Mongolia, Tibet, Xinjiang and the like, other provinces are planted, so that the pepper is an important economic tree species in China. The most main purpose of the pepper is to be used as spice, is mainly used for skillful cooking and seasoning of food, and has the functions of perfuming, increasing appetite,Covering up peculiar smell and preventing corrosion. The pricklyash peel has unique medicinal value. Zanthoxylum bungeanum has pungent, hot and small toxic properties, and has effects of warming middle energizer, relieving pain, killing parasites and relieving itching as recorded in Chinese pharmacopoeia, and can be used for treating abdominal psychroalgia, emesis, diarrhea, abdominal distention, ascariasis, eczema, pruritus, etc. The pepper is rich in active ingredients, mainly comprises volatile oil, numb-taste component substances, alkaloid and flavonoid substances, and has the effects of inhibiting bacteria, resisting inflammation, improving immunity and the like. The pepper also has the characteristics of early fruiting, fast growing, drought resistance, easy cultivation, strong adaptability, good management and the like, and the pepper has a very developed root system and strong soil fixation capability, so that the pepper can be used as an ecological tree species with greening and water and soil conservation effects.
China has abundant pepper germplasm resources, the cultivation area and the yield are the first in the world, but the pepper breeding research is lagged, and a part of resources are still in a wild or scattered state. With the expansion of the planting scale of the zanthoxylum bungeanum and the development of the zanthoxylum bungeanum industry, the traditional zanthoxylum bungeanum variety can not meet the requirements of the current development. Common methods for plant breeding include selective breeding, cross breeding, mutation breeding, ploidy breeding, and cell engineering breeding and molecular breeding based on biotechnology. The pepper is parthenocarpic, belongs to apomixis and is not easy to perform cross breeding, so the breeding work of the pepper is concentrated on the aspect of selective breeding, and the defects that the genetic improvement mainly depends on natural variation, the variation rate is low, the time for breeding new materials is too long, and the breeding effect is not good, so the germplasm innovation and the fine variety breeding work of the pepper have not been developed in a breakthrough way.
Plant protoplasts are naked, viable cells of a plant that have been obtained by removing the cell wall from the plant cell. It can regenerate into complete plant through continuous cell division, namely it has cell totipotency; can take in foreign substances such as DNA, plasmids, viruses, bacteria, organelles and the like, and is a good material for genetic transformation of plants; and the protoplast fusion of the same species and different species plants can generate heterokaryons to realize somatic cell hybridization, thus providing possibility for realizing distant hybridization, overcoming incompatibility obstacle of sexual hybridization and cultivating new plant varieties. Therefore, the protoplast culture has wide application prospect in the aspects of plant rapid propagation, plant distant genetic recombination, transgenosis, variety improvement, new type creation and the like, and opens up a new way for pepper breeding.
The isolation of large numbers of viable protoplasts is one of the prerequisites for protoplast culture. Currently, the conventional method for isolating protoplasts is an enzymatic method. The selection of suitable materials for isolating protoplasts is fundamental and critical to the success of protoplast culture. Leaves, cotyledons, callus, embryogenic cell suspensions, etc. can be used as starting materials for protoplast separation, and different materials have advantages and disadvantages and are different according to plant species. After the plant protoplast is successfully separated, cell clusters, cell fragments, organelles and the like are often mixed in the obtained protoplast, the protoplast needs to be separated from other impurities and enzyme liquid and purified, the purification method mainly comprises a centrifugal precipitation method, a floating method, an interface method and the like, and the principle of the purification method is that the protoplast and the impurities have different specific gravities and are layered. The plant protoplast culture is carried out by transferring the separated and purified protoplast to an appropriate condition and culturing the protoplast in a predetermined direction. The main factors influencing the culture of the protoplast comprise plant genotype, culture density of the protoplast, osmotic pressure stabilizer, culture medium type, hormone, culture mode and the like, and the protoplast to plant regeneration is subjected to the stages of protoplast division, callus growth, plant regeneration and the like. The protoplast culture requires different culture media at different stages, and usually requires replacing the culture media several times, namely, the culture medium for promoting the protoplast to restore cell walls, start division and maintain cell division, the culture medium for promoting callus to grow and the culture medium for inducing each organ to differentiate, wherein the initial culture medium is directly related to whether the protoplast can regenerate cell walls, and the mitosis is restarted, thereby influencing whether plants can be successfully regenerated at last.
At present, few research reports about pepper protoplast are reported. In the existing research on the separation and culture of pepper protoplasts, better test results have been obtained by the separation and purification of protoplasts, but the culture of protoplasts has the problems of low cell division frequency, low cell mass forming frequency, low culture working efficiency and the like, and no people obtain regenerated plants by the culture of pepper protoplasts, so that further research is urgently needed, and the blank in the aspect is filled.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for culturing regenerated plants by pepper protoplasts and a special protoplast culture medium.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the protoplast culture medium special for the pepper is characterized by comprising the following components: k2SO4650~750mg/L，NH4NO3180~220mg/L，Ca(NO3)2·4H2O 415~455mg/L，CaCl2·2H2O 150~190mg/L， MgSO4·7H2O 320~360mg/L， KH2PO4160~200mg/L，Fe-EDDHA 28.3~28.7mg/L，MnSO4·4H2O 17.6~18.0mg/L，ZnSO4·7H2O 8.1~8.5mg/L，H3BO33.3~3.7mg/L，KI 0.79~0.83mg/L，Na2MoO4·2H2O 0.23~0.27mg/L，CoCl2·6H2O 0.02~0.03mg/L，CuSO4·5H20.02-0.03 mg/L of O, 0.8-1.2 mg/L of thiamine hydrochloride, 0.8-1.2 mg/L of pyridoxine hydrochloride, 0.8-1.2 mg/L of nicotinic acid, 0.3-0.5 mg/L of choline chloride, 0.01-0.03 mg/L of biotin, 1.6-2.0 mg/L of ascorbic acid, 20-25 mg/L of sodium pyruvate, 100-150 mg/L of inositol, 120-160 mg/L of proline, 200-250 mg/L of bovine serum albumin, 50-100 mg/L of rhamnose, 50-100 mg/L of fructose, 50-100 mg/L of xylose, 50-100 mg/L of ribose, 225-275 mg/L of cellobiose, 4.5-5.5 mmol/L of gamma-aminobutyric acid, 1.9-2.1 mg/L of picloram, 0.4mg/L of NAA, 0.9-1.1 mg/L of NANA, 0.8-0.8 mg/L of spermidine, 90-110 mu mol/L of sodium nitroprusside, 1.5-1.7 mg/L of furbenuron and 15-25 mL/L of pea leaching liquor; osmotic pressure regulator: 0.6-0.7 mol/L of sucrose.
The pH value of the culture medium is adjusted to 5.6-6.0.
The method comprises the following steps:
1) protoplast culture: suspending pepper protoplasts in a protoplast culture medium, adjusting the density to a proper value, sucking the protoplast suspension by using a rubber-tipped dropper, dripping the protoplast suspension into a culture dish (the diameter is 3 cm), dripping about 15-20 drops into each dish to form a liquid shallow layer, sealing by using a sealing film, and standing and culturing at the temperature of 25 +/-2 ℃ in the dark; supplementing a protoplast culture medium without an osmotic pressure regulator once after culturing for 5-8 days, and supplementing the protoplast culture medium once every 10 days;
2) and (3) callus proliferation culture: when protoplasts are cultured to form small callus with the diameter of about 1-2 mm, picking out and inoculating the callus to a callus proliferation culture medium (WPM basic culture medium +2, 4-D1.0 mg/L + ZT 0.5mg/L + coconut milk 20ml/L + sucrose 50g/L + agar 7 g/L), and culturing at the temperature of 25 +/-2 ℃ under the condition of scattered light; cutting the callus with a scalpel at intervals to form smaller tissue blocks, and transferring the tissue blocks to a fresh multiplication culture medium for subculture;
3) differentiation culture of callus: selecting callus with diameter of about 5mm, inoculating to differentiation medium (WPM minimal medium + ZT1.0mg/L +6-BA 1.0mg/L + NAA0.1mg/L + H)2O280 mu mol/L + asparagine 200mg/L + polyvinyl alcohol 1g/L + coconut milk 20ml/L + sucrose 30g/L + agar 7 g/L), culturing for 3-4 days under the condition of weak light, and culturing under the condition of normal light, wherein the culture temperature is 25 +/-2 ℃;
4) rooting culture: when the adventitious bud differentiated from the callus grows to about 1-2 cm, cutting and transferring the adventitious bud to a rooting culture medium (1/4 MS minimal medium + IBA2.0mg/L + NAA0.2mg/L + activated carbon 1.0g/L + sucrose 20g/L + agar 6 g/L) for culturing to form a complete plant.
The pepper protoplast in the step 1) is obtained by separating and purifying pepper sterile test-tube seedling leaves.
The density of the biomass in the step 1) is adjusted to 1 × 105~2×105one/mL.
The protoplast culture medium without the osmotic pressure regulator supplemented in the step 1) is about 30% of the protoplast culture medium.
In the step 3), the illumination intensity is 300-500 lx and the illumination time is 12-14 h/d under the low-light condition, and the illumination intensity is 1500-2000 lx and the illumination time is 12-14 h/d under the normal illumination condition.
The culture conditions in the step 4) are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 14-16 h/d.
The invention has the beneficial effects that:
1) the invention discloses a special culture medium suitable for pepper protoplast culture, which adjusts the proportion of macroelements and microelements, adds choline chloride, biotin, ascorbic acid, sodium pyruvate, proline, bovine serum albumin, rhamnose, cellobiose and other nutrient components, selects picloram, NAA and ZT as growth regulators, and also adds gamma-aminobutyric acid, spermidine, sodium nitroprusside, furathiocarbamide and pea leaching liquor which are special components, can promote the protoplast to grow cell walls again, starts a splitting procedure, enables the protoplast to realize continuous splitting and proliferation, finally forms callus, and has the cell splitting frequency of 26.55 percent and the plant plate rate of 8.03 percent, thereby obviously improving the callus compared with the prior art.
2) On the basis of the previous research, the invention determines the culture medium and method for the pepper protoplast culture to plant regeneration through years of practice, establishes a complete pepper protoplast culture system, and lays a good foundation for the research of pepper somatic cell hybridization, genetic transformation, germplasm innovation and the like.
Drawings
FIG. 1 protoplast cell division;
FIG. 2 protoplast culture to form a cell mass;
FIG. 3 protoplast culture to form small calli;
FIG. 4 callus proliferation;
FIG. 5 differentiation of adventitious buds from callus;
FIG. 6 forms a complete regenerated plant.
Detailed Description
Example 1 isolation, purification and culture of protoplasts of Zanthoxylum bungeanum
The test material is sterile test-tube plantlet of Zanthoxylum piperitum of Zanthoxylum bungeanum L.var.bungeanum L.var.cultivation.
1. Pretreatment of
Taking the test-tube seedling leaves growing for 4-5 weeks, and pretreating at the low temperature of 4 ℃ for 1 h.
2. Protoplast isolation and purification
Cutting the pretreated leaves into 1-2 mm thin strips, adding an enzymolysis solution according to the proportion of 1: 10, and carrying out oscillatory enzymolysis for 8 hours at the rotating speed of 40r/min at the temperature of 28 ℃ under the dark condition. After enzymolysis, filtering the mixed solution by a stainless steel mesh sieve of 45 mu m to remove non-enzymolysis materials and large cell masses, transferring the filtrate into a 10mL centrifuge tube, centrifuging at 800r/min for 6min, removing supernatant, and suspending the precipitate by using a CPW-13% mannitol solution; adding CPW-26% sucrose solution into another clean centrifuge tube, slowly adding suspended protoplast, centrifuging at 800r/min for 3min to obtain a clear zone at the middle interface, sucking out the protoplast zone with a suction tube, and washing with liquid protoplast culture medium for 1 time.
The components of the enzymolysis solution are CPW solution, 1.0 percent of cellulase, 0.5 percent of pectinase, 0.2 percent of eductase, 0.7mol/L of mannitol and 5mmol/LMES, and the pH value is 5.8.
3. Protoplast culture
Suspending the separated and purified pepper protoplast in a protoplast culture medium, and adjusting the density to 1 × 105Sucking the protoplast suspension by using a rubber-tipped dropper, dripping the protoplast suspension into culture dishes (the diameter is 3 cm), dripping about 15-20 drops into each dish to form a liquid shallow layer, sealing by using a sealing film, and culturing at 25 ℃ in the dark; and supplementing the protoplast culture medium without the osmotic pressure regulator once after culturing for 5-8 days, and supplementing the protoplast culture medium without the osmotic pressure regulator once every 10 days, wherein the supplemented protoplast culture medium without the osmotic pressure regulator is about 30% of the protoplast culture medium.
The protoplast culture medium consists of: k2SO4700mg/L，NH4NO3200mg/L，Ca(NO3)2·4H2O435mg/L，CaCl2·2H2O 170mg/L， MgSO4·7H2O 340mg/L， KH2PO4180mg/L，Fe-EDDHA28.5mg/L，MnSO4·4H2O 17.8mg/L，ZnSO4·7H2O 8.4mg/L，H3BO33.5mg/L，KI 0.81mg/L，Na2MoO4·2H2O 0.25mg/L，CoCl2·6H2O 0.025mg/L，CuSO4·5H2O0.025 mg/L, thiamine hydrochloride 1.0mg/L, pyridoxine hydrochloride 1.0mg/L, nicotinic acid 1.0mg/L, choline chloride 0.4mg/L, biotin 0.02mg/L, ascorbic acid 1.8mg/L, sodium pyruvate 22.5mg/L, inositol 125mg/L, proline 140mg/L, bovine serum albumin 225mg/L, rhamnose 75mg/L, fructose 75mg/L, xylose 75mg/L, ribose 75mg/L, cellobiose 250mg/L, gamma-aminobutyric acid 5.0mmol/L, picloram 2.0mg/L, NAA0.5mg/L, ZT1.0mg/L, spermidine 9.0mg/L, sodium nitroprusside 100. mu. mol/L, furbenuron 1.6mg/L, pea extract 20 mL/L; osmotic pressure regulator: 0.65mol/L of sucrose; pH 5.8.
4. Optimization of protoplast culture media
1) Influence of concentration of gamma-aminobutyric acid on splitting capacity of pepper protoplast
Adding 0, 1.0, 3.0, 5.0 and 7.0mmol/L gamma-aminobutyric acid into the protoplast culture medium respectively, keeping the other components unchanged, suspending the separated and purified pepper protoplast in the protoplast culture medium, and adjusting the density to 1 × 105And performing liquid shallow layer static culture, counting the cell division frequency of the protoplast after culturing for 15 days, and counting the plating rate after culturing for 30 days, wherein the results are shown in the following table 1.
As can be seen from Table 1, the addition of gamma-aminobutyric acid to the protoplast culture medium can improve the division capability of pepper protoplasts, and as the concentration of gamma-aminobutyric acid increases, the cell division frequency and the plating rate gradually increase and reach the highest value at 5.0mmol/L and then start to decrease, so that the optimal concentration of gamma-aminobutyric acid is 5.0 mmol/L.
2) Influence of sodium nitroprusside concentration on division capability of pepper protoplast
Adding sodium nitroprusside 0, 20, 40, 60 and 80 μmol/L into protoplast culture medium respectively, keeping other components unchanged, suspending the separated and purified pepper protoplast in the original suspensionIn a biomass culture medium, and adjusting the density to 1 × 105And performing liquid shallow layer static culture, counting the cell division frequency of the protoplast after culturing for 15 days, and counting the plating rate after culturing for 30 days, wherein the results are shown in the following table 2.
As can be seen from Table 2, with the increase of the sodium nitroprusside concentration, the cell division frequency and the plate planting rate of the pepper protoplast show a trend of increasing firstly and then decreasing, when the sodium nitroprusside concentration is 100 mu mol/L, the division frequency reaches 26.70%, the plate planting rate reaches 8.04%, and the difference with the control is obvious; however, the high concentration of sodium nitroprusside is not favorable for the continuous division of the pepper protoplast, so the optimal concentration of the sodium nitroprusside is 100 mu mol/L.
3) Influence of different culture media on division capability of pepper protoplast
The pepper protoplast culture medium of the invention, the pepper protoplast culture medium WPM +2,4-D0.5mg/L +6-BA 1.0mg/L + NAA 1.0mg/L which is researched by the predecessor, the osmotic pressure regulator mannitol 0.6mol/L, the pH value adjusted to 5.8 (the separation and culture research of the pepper protoplast in Linan, Yang Xiuping, Zhongzheng Jun, Dengpeng. pepper protoplast) are used, the separated and purified pepper protoplast is respectively suspended in the two protoplast culture media, and the density is adjusted to 1 × 105Performing liquid shallow layer static culture, counting the cell division frequency of the protoplast after culturing for 15 days, counting the plate planting rate after culturing for 30 days, and performing liquid shallow layer culture on the pepper protoplast respectively, wherein the results are shown in the following table 3.
The type of medium directly affects the frequency of protoplast division, plating rate, and formation of small cell masses. As can be seen from Table 3, the division capacity of the pepper protoplast is greatly improved by using the protoplast culture medium of the invention, the cell division frequency can reach 26.55%, the plating rate can reach 8.03%, and the culture medium is obviously improved compared with the protoplast culture medium studied by the predecessor. The protoplast culture medium disclosed by the invention has the advantages that the proportioning of major elements and trace elements is adjusted, a plurality of nutrient components such as choline chloride, biotin, ascorbic acid, sodium pyruvate, proline, bovine serum albumin and cellobiose are added, picloram, NAA and ZT are selected as growth regulators, and special components such as gamma-aminobutyric acid, spermidine, sodium nitroprusside, furathiocarbamide and pea leaching liquor are additionally added, so that the protoplast can be promoted to grow cell walls again, the division program is started, the protoplast can be continuously divided and proliferated, and finally callus is formed.
Example 2 callus culture and plant regeneration
1. Callus proliferation culture
Picking out small callus (figure 3) with the diameter of about 1-2 mm formed by culturing the protoplast in example 1, inoculating the small callus on a callus proliferation culture medium (WPM basic culture medium +2, 4-D1.0 mg/L + ZT 0.5mg/L + coconut milk 20ml/L + sucrose 50g/L + agar 7 g/L), culturing at 25 ℃ under the condition of scattered light, and proliferating the callus to 4-5 mm after 30-40 days (figure 4); and cutting the callus by using a scalpel at intervals to form a small tissue block, transferring the tissue block to a fresh multiplication culture medium for subculture, and providing more basic materials for later-stage experiments.
2. Differentiation culture of callus
Selecting callus with diameter of about 5mm, inoculating to differentiation medium (WPM minimal medium + ZT1.0mg/L +6-BA 1.0mg/L + NAA0.1mg/L + H)2O280 mu mol/L, 200mg/L of asparagine, 1g/L of polyvinyl alcohol, 20ml/L of coconut milk, 30g/L of cane sugar and 7g/L of agar, wherein the culture temperature is 25 ℃, the coconut milk is cultured for 3-4 d under the condition of weak light (illumination intensity of 400lx and illumination time of 13 h/d), then cultured for 30-35 d under the condition of normal light (illumination intensity of 1750lx and illumination time of 13 h/d), and adventitious buds are differentiated from the callus (figure 5).
3. Rooting culture
When the adventitious bud differentiated from the callus grows to about 1.0-2.0 cm, cutting and transferring the adventitious bud to a rooting culture medium (1/4 MS basic culture medium + IBA2.0mg/L + NAA0.2mg/L + activated carbon 1.0g/L + sucrose 20g/L + agar 6 g/L), and culturing for 20-30 days under the conditions of 25 ℃ of temperature, 1750lx of illumination intensity and 15h/d of illumination time to grow into a complete plant (figure 6).
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (8)
1. The protoplast culture medium special for the pepper is characterized by comprising the following components: k2SO4650~750mg/L，NH4NO3180~220mg/L，Ca(NO3)2·4H2O 415~455mg/L，CaCl2·2H2O 150~190mg/L， MgSO4·7H2O 320~360mg/L， KH2PO4160~200mg/L，Fe-EDDHA 28.3~28.7mg/L，MnSO4·4H2O 17.6~18.0mg/L，ZnSO4·7H2O 8.1~8.5mg/L，H3BO33.3~3.7mg/L，KI 0.79~0.83mg/L，Na2MoO4·2H2O 0.23~0.27mg/L，CoCl2·6H2O 0.02~0.03mg/L，CuSO4·5H20.02-0.03 mg/L of O, 0.8-1.2 mg/L of thiamine hydrochloride, 0.8-1.2 mg/L of pyridoxine hydrochloride, 0.8-1.2 mg/L of nicotinic acid, 0.3-0.5 mg/L of choline chloride, 0.01-0.03 mg/L of biotin, 1.6-2.0 mg/L of ascorbic acid, 20-25 mg/L of sodium pyruvate, 100-150 mg/L of inositol, 120-160 mg/L of proline, 200-250 mg/L of bovine serum albumin, 50-100 mg/L of rhamnose, 50-100 mg/L of fructose, 50-100 mg/L of xylose, 50-100 mg/L of ribose, 225-275 mg/L of cellobiose, 4.5-5.5 mmol/L of gamma-aminobutyric acid, 1.9-2.1 mg/L of picloram, 0.4mg/L of NAA, 0.9-1.1 mg/L of NANA, 0.8-0.8 mg/L of spermidine, 90-110 mu mol/L of sodium nitroprusside, 1.5-1.7 mg/L of furbenuron and 15-25 mL/L of pea leaching liquor; osmotic pressure regulator: 0.6-0.7 mol/L of sucrose.
2. The protoplast culture medium for pepper according to claim 1, wherein the pH value of the culture medium is adjusted to 5.6-6.0.
3. A method for culturing regenerated plants by pepper protoplasts is characterized by comprising the following steps:
1) protoplast culture: suspending pepper protoplasts in a protoplast culture medium, adjusting the density to a proper value, sucking the protoplast suspension by using a rubber-tipped dropper, dripping the protoplast suspension into a culture dish (the diameter is 3 cm), dripping about 15-20 drops into each dish to form a liquid shallow layer, sealing by using a sealing film, and standing and culturing at the temperature of 25 +/-2 ℃ in the dark; supplementing a protoplast culture medium without an osmotic pressure regulator once after culturing for 5-8 days, and supplementing the protoplast culture medium once every 10 days;
2) and (3) callus proliferation culture: when protoplasts are cultured to form small callus with the diameter of about 1-2 mm, picking out and inoculating the callus to a callus proliferation culture medium (WPM basic culture medium +2, 4-D1.0 mg/L + ZT 0.5mg/L + coconut milk 20ml/L + sucrose 50g/L + agar 7 g/L), and culturing at the temperature of 25 +/-2 ℃ under the condition of scattered light; cutting the callus with a scalpel at intervals to form smaller tissue blocks, and transferring the tissue blocks to a fresh multiplication culture medium for subculture;
3) differentiation culture of callus: selecting callus with diameter of about 5mm, inoculating to differentiation medium (WPM minimal medium + ZT1.0mg/L +6-BA 1.0mg/L + NAA0.1mg/L + H)2O280 mu mol/L + asparagine 200mg/L + polyvinyl alcohol 1g/L + coconut milk 20ml/L + sucrose 30g/L + agar 7 g/L), culturing for 3-4 days under the condition of weak light, and culturing under the condition of normal light, wherein the culture temperature is 25 +/-2 ℃;
4) rooting culture: when the adventitious bud differentiated from the callus grows to about 1-2 cm, cutting and transferring the adventitious bud to a rooting culture medium (1/4 MS minimal medium + IBA2.0mg/L + NAA0.2mg/L + activated carbon 1.0g/L + sucrose 20g/L + agar 6 g/L) for culturing to form a complete plant.
4. The method for culturing regenerated plants by using pepper protoplasts as claimed in claim 3, wherein the pepper protoplasts in step 1) are isolated and purified from the leaves of the pepper sterile test tube plantlet.
5. The method for culturing regenerated plants by using pepper protoplasts as claimed in claim 3, wherein the density of the protoplasts in step 1) is adjusted to 1 × 105~2×105one/mL.
6. The method for culturing regenerated plants by using pepper protoplasts as claimed in claim 3, wherein the supplemented protoplast culture medium without osmo-regulator in step 1) is about 30% of the protoplast culture medium.
7. The method for culturing regenerated plants by using protoplasts of pepper according to claim 3, wherein the illumination intensity in step 3) is 300-500 lx and the illumination time is 12-14 h/d, and the normal illumination condition is 1500-2000 lx and the illumination time is 12-14 h/d.
8. The method for culturing regenerated plants by pepper protoplasts according to claim 3, wherein the culturing conditions in step 4) are as follows: the temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 14-16 h/d.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146638A (en) * | 2013-03-14 | 2013-06-12 | 青岛农业大学 | Induced proliferation method for glehnia littoralis hairy roots |
| CN105145355A (en) * | 2015-09-08 | 2015-12-16 | 中国林业科学研究院亚热带林业研究所 | Phyllostachys edulis protoplast culture method |
| EP3115456A1 (en) * | 2014-03-06 | 2017-01-11 | Riken | Plant transformation method |
| CN107156828A (en) * | 2017-01-18 | 2017-09-15 | 恩施硒域传奇健康管理有限公司 | A kind of full nutrition formula food suitable for patients with inflammatory bowel |
| CN109645429A (en) * | 2018-12-10 | 2019-04-19 | 山东天博食品配料有限公司 | A kind of rattan green pepper chicken cream and preparation method thereof |
| CN110214699A (en) * | 2019-07-16 | 2019-09-10 | 郭根霞 | A kind of sainfoin anther callus differential medium |
| CN110214700A (en) * | 2019-07-16 | 2019-09-10 | 郭根霞 | A kind of abductive approach and its special culture media of sainfoin anther callus |
-
2020
- 2020-06-08 CN CN202010509943.2A patent/CN111713405A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146638A (en) * | 2013-03-14 | 2013-06-12 | 青岛农业大学 | Induced proliferation method for glehnia littoralis hairy roots |
| EP3115456A1 (en) * | 2014-03-06 | 2017-01-11 | Riken | Plant transformation method |
| CN105145355A (en) * | 2015-09-08 | 2015-12-16 | 中国林业科学研究院亚热带林业研究所 | Phyllostachys edulis protoplast culture method |
| CN107156828A (en) * | 2017-01-18 | 2017-09-15 | 恩施硒域传奇健康管理有限公司 | A kind of full nutrition formula food suitable for patients with inflammatory bowel |
| CN109645429A (en) * | 2018-12-10 | 2019-04-19 | 山东天博食品配料有限公司 | A kind of rattan green pepper chicken cream and preparation method thereof |
| CN110214699A (en) * | 2019-07-16 | 2019-09-10 | 郭根霞 | A kind of sainfoin anther callus differential medium |
| CN110214700A (en) * | 2019-07-16 | 2019-09-10 | 郭根霞 | A kind of abductive approach and its special culture media of sainfoin anther callus |
Non-Patent Citations (2)
| Title |
|---|
| 李南: "花椒原生质体分离与培养", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 李南等: "花椒原生质体分离与培养研究", 《西北林学院学报》 * |
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