CN111616049B - Tissue culture seedling method for amomum tsao-ko - Google Patents
Tissue culture seedling method for amomum tsao-ko Download PDFInfo
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Classifications
-
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of medicinal plant tissue culture, and discloses a tissue culture seedling method for amomum tsao-ko, which comprises the following steps: selection of explants: selecting tender stem sections at the base of the tsaoko stems; sterilizing explants; primary induction culture; subculture multiplication culture; the invention discloses a tissue culture seedling method of grass fruits, which adopts explants of tender stem sections at the stem base, and compared with tender shoots at the near soil surface adopted at the beginning of the experiment, the pollution rate is reduced from 95 percent of the tender shoots at the near soil surface to 12 percent of the tender stem sections, while the induction rate of the tender stem sections at the stem base is 80 percent and is only slightly lower than the induction rate of 84 percent of the tender shoots at the near soil surface.
Description
Technical Field
The invention belongs to the field of medicinal plant tissue culture, and particularly relates to a tissue culture seedling method for amomum tsao-ko.
Background
The Amomum tsao-koCrevost et Lemaire is a perennial herbaceous plant of the genus Amomum in the family Zingiberaceae, grows in tropical and subtropical shade-wet sparse forest with the altitude of 1100-1800 m, and is mainly distributed in Yunnan, Guangxi, Guizhou and the like. It is prepared from dry mature fruit, has effects of eliminating dampness, warming middle warmer, eliminating phlegm, and stopping malaria, and is mainly used for treating cold-dampness obstruction, abdominal pain, distention and fullness, emesis, malaria, and chills and fever. Meanwhile, the tsaoko belongs to medicinal and edible medicinal materials, is widely used as a flavoring raw material of food in folk, and the whole plant (the most common fruits) of the tsaoko contains aromatic substances such as 1, 8-cineole, trans-2-decaolefine aldehyde, citral-b and the like, and is usually used for extracting volatile essential oil as essence and spice.
The tsaoko is introduced from southeast Asia countries such as Vietnam, the planting area of the tsaoko in China is nearly 200 ten thousand mu, wherein the planting area of Yunnan is the largest and accounts for 90% of the planting area in China, and the tsaoko is the largest tsaoko industry base in China. Although the tsaoko is a foreign introduced variety, with the continuous research and development of the Chinese tsaoko planting technology, the yield and quality of the Chinese tsaoko are far higher than those of the original seed land at present. At present, from the national germplasm resource survey results, because seedling raising is adopted all year round, the hybridization among population plants is formed, the variation of the tsaoko amomum fruits is caused to a large extent, the variety is various among the production regions of the tsaoko amomum fruits and even in the same production region, the quality and the yield of the tsaoko amomum fruits are uneven, and the tsaoko amomum fruits are propagated and experimentally planted in partial regions by adopting a plant division propagation mode, although the female parent characters can be kept to the maximum extent, the degradation of the tsaoko amomum fruits is found to be serious because the planting years are long in the planting process, and meanwhile, the disease and insect pest are very easy to cause the popularization of the disease.
In recent years, although there are reports of tissue culture seedling raising methods of tsaoko amomum fruits, relatively few studies are made, and there are reports of tissue culture (callosa and the like) by using tsaoko amomum fruits, but the embryos are obtained by using tsaoko amomum fruits seeds for culture, the possibility of hybridization variation also exists, the embryos of the tsaoko amomum fruits are used as explants for tissue culture in the operation process, aseptic seedlings of the tsaoko amomum fruits are cultured, stem sections of the aseptic seedlings of the tsaoko amomum fruits are taken for adventitious bud induction, then rooting culture is carried out, the period of the whole culture process is long, if large-scale production is carried out, the cost is increased, the existing tissue culture method adopting the stem tips of the tsaoko amomum fruits (Hongdong and the like) is adopted, but the multiplication times of the method is only up to 2.34, the vitrification phenomenon of the adventitious buds occurs, and various variations. In addition, the culture medium generates browning phenomena during the induction and proliferation culture processes, and the browning phenomena causes death of tissues of the culture, thereby influencing the growth and development of the culture and even causing death of the whole culture.
In conclusion, the screened high-quality tsaoko germplasm with high aromatic oil content and large yield is subjected to asexual propagation by using the modern biotechnology, so that the female parent characters can be maintained to the greatest extent, a large number of high-quality seedlings can be rapidly obtained, and the popularization of the excellent variety of the tsaoko in a short period becomes very urgent.
Disclosure of Invention
The invention mainly aims to provide a tissue culture seedling method for tsaoko amomum fruits, which aims to overcome the defects of the traditional propagation technology and the defects of the existing tissue culture technology, improve the multiplication coefficient and keep the excellent characters of female parents to the maximum extent.
In order to achieve the purpose, the invention provides the following technical scheme:
a tissue culture seedling method for tsaoko includes the following steps:
(1) selection of explants: selecting tender stem sections at the base of the tsaoko stems;
(2) sterilizing explants;
(3) performing primary induction culture, wherein the used primary induction culture medium is WPM + NAA 0.01-0.05 mg/L +6-BA 8.0-10.0 mg/L + active carbon 0.5 g/L;
(4) subculture proliferation, wherein the subculture proliferation medium is MS +6-BA 2.0-3.0 mg/L + KT 5.0-6.0 mg/L + coconut water 50-100 ml/L;
(5) rooting culture, wherein the used rooting culture medium is 1/2WPM, 0.5-1.0 mg/L NAA, 0.2-0.5 mg/L IBA and 0.2-0.5 mg/L paclobutrazol.
Further, after the explant is selected, cleaning of the explant is also included, the explant is cut into 2-3 cm long, the explant is placed under tap water and is washed for 1 hour, then the explant is washed for 10min by soap liquid, then the explant is washed for 2-3 times by clear water until no soap liquid remains, and finally the explant is washed for 1-2 hours under the tap water.
Further, the explant sterilization method comprises the steps of firstly sterilizing with 75% alcohol for 20s, washing with sterile water for 3 times, then sterilizing with 8% hydrogen peroxide for 12min, washing with sterile water for 3-4 times, finally sterilizing with 0.1% mercuric chloride and 2 drops of Tween-80 for 10min, and washing with sterile water for 8 times.
Further, the primary induction culture also comprises the step of placing the culture in an environment with the temperature of 25 +/-2 ℃, wherein the dark culture is carried out for the first 15 days, and the light-dark alternate culture is carried out for the last 45 days at the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 h.
Further, the subculture for root growth and rooting culture also comprises the step of culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours.
Further, the primary induction culture medium is WPM + NAA0.02mg/L +6-BA8.0 mg/L + active carbon 0.5g/L.
Furthermore, the subculture multiplication medium is MS +6-BA2mg/L + KT5.0mg/L + coconut water 100 ml/L.
Furthermore, the rooting culture medium is 1/2WPM, NAA0.5mg/L, IBA0.2 mg/L and paclobutrazol 0.2 mg/L.
The invention has the following beneficial effects:
1. according to the tissue culture seedling method for the tsaoko, the adopted explants are tender stem sections at the stem base, compared with tender shoots at the near soil surface adopted at the beginning of the experiment, the pollution rate is reduced from 95% of the tender shoots at the near soil surface to 12% of the tender stem sections, and the induction rate of the tender stem sections at the stem base is 80% and is only slightly lower than the induction rate of 84% of the tender shoots at the near soil surface.
2. The invention relates to a tissue culture seedling method of tsaoko amomum fruits, which adopts explants which are tender stem sections of stem bases, belongs to maternal vegetative organs, has the same gene as a maternal parent completely, and keeps the female parent characters to the maximum extent.
3. According to the method for tissue culture seedling of the amomum tsao-ko, in the induction culture and proliferation culture processes, NAA with extremely low concentration is adopted, so that cluster buds are directly induced and proliferated without callus, the generation of callus is effectively avoided, the callus stage which is most likely to have variation in the tissue culture process is reduced to the maximum extent, meanwhile, the tissue culture link is simplified and the production cost of the tissue culture is reduced because the callus is not passed.
4. According to the tissue culture seedling method for the tsaoko amomum fruits, 50-100 ml/L of coconut water is added in the proliferation process, so that the proliferation coefficient of the coconut water is increased from 3.0 without adding the coconut water to 6.0, the propagation efficiency is improved, the production time is shortened, and the production cost is reduced.
5. According to the method for tissue culture seedling of the amomum tsao-ko, 0.2-0.5 mg/L of paclobutrazol is added in the rooting culture process, compared with a rooting culture medium without the paclobutrazol, the rooting rate is 10% higher, seedlings are robust, the color is bright green, strong seedling culture is not needed between proliferation and rooting culture, the culture link is simplified, the production cost is reduced, and the occurrence rate of high stiff seedlings after excessive paclobutrazol addition is avoided.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. To avoid unnecessary detail, the following examples will not describe the known art in detail. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified; in the following examples,% is by mass unless otherwise specified.
The invention provides a tissue culture seedling method for tsaoko amomum fruits, which comprises the following steps:
(1) selecting tender stem segments at the base of the tsaoko stems as explants;
(2) sterilizing the cut explants;
(3) absorbing surface moisture of the sterilized explant by using sterile filter paper, cutting wounds at two ends, and carrying out primary induction culture to obtain cluster buds, wherein the used primary induction culture medium is WPM + NAA 0.01-0.05 mg/L +6-BA 8.0-10.0 mg/L + active carbon 0.5 g/L;
(4) carrying out induction culture to obtain cluster buds, cutting the cluster buds into single buds, inoculating the single buds into a subculture multiplication medium according to polarity, and carrying out subculture multiplication, wherein the subculture multiplication medium is MS +6-BA 2.0-3.0 mg/L + KT 5.0-6.0 mg/L + coconut water 50-100 ml/L;
(5) and (3) carrying out induction culture or propagation culture to obtain cluster buds, cutting the cluster buds into single buds, inoculating the single buds into a rooting culture medium according to polarity, and carrying out rooting culture, wherein the rooting culture medium is 1/2WPM, 0.5-1.0 mg/L NAA, 0.2-0.5 mg/L IBA and 0.2-0.5 mg/L paclobutrazol.
The explant adopted by the invention is a tender stem section of the stem base, the pollution rate is reduced to 12% of the tender stem section from 95% of the tender bud of the near soil surface, and the induction rate of the tender stem section of the stem base is 80% and is only slightly lower than the induction rate of 84% of the tender bud of the near soil surface.
The source of the explant is an important factor for determining the success or failure of tissue culture, but the cleanliness of the explant needs to be considered, explants selected from the outside or indoors carry various microorganisms to different degrees, and once the contamination sources are brought into a culture medium, the contamination of the culture can be caused, so the cleanliness of the explant needs to be considered simultaneously when being selected. Generally, cleaning is directly carried out by adopting underwater rinsing, and further, after the explant is selected, the explant is cut into 2-3 cm long, is rinsed for 1 hour under tap water, is rinsed for 10min by using soap liquid, is rinsed for 2-3 times by using clear water until no soap liquid remains, and is finally rinsed for 1-2 hours under tap water.
The disinfectant has various types, but the basic selection principle is to remove microorganisms on materials urgently and cannot damage the materials, and various reagents have advantages and limitations, so one reagent or a plurality of reagents are selected to be combined for use, the maximum effect can be obtained at the lowest cost, and further, the explant disinfection method comprises the steps of disinfecting with 75% alcohol for 20s, washing with sterile water for 3 times, disinfecting with 8% hydrogen peroxide for 12min, washing with sterile water for 3-4 times, disinfecting with 0.1% mercuric chloride and 2 drops of tween-80 for 10min, and washing with sterile water for 8 times.
Further, the primary induction culture also comprises the step of placing the culture in an environment with the temperature of 25 +/-2 ℃, wherein the dark culture is carried out for the first 15 days, and the light-dark alternate culture is carried out for the last 45 days at the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 h. Further, the subculture for root growth and rooting culture also comprises the step of culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours. The initial division and proliferation of explant cells and the differentiation of organs have important influence on the illumination intensity, generally speaking, the illumination intensity is strong, the seedlings grow robustly, but the illumination intensity is weak, the seedlings are easy to grow in vain, the instant cumquat and the like in the research of the culture technology of the tsaoko embryo, the initial generation illumination intensity is 1600-2100 lx, the secondary generation illumination intensity is 2000 lx, the rooting is 1500-2000 lx, and the like in the research of the rapid propagation and seedling culture of the tissue culture of the tsaoko, the initial generation secondary illumination intensity is 1000-2000 lx, the rooting is 1500-2000 lx, which are all greatly different from the illumination intensity adopted by the invention, but by using the technical scheme of the invention, the seedlings are comprehensively in the states of being sturdy, uniform and high in transplanting survival rate.
The physiological activity of the plants in the growth and development process is not only influenced by a single factor but controlled by the interaction of a plurality of hormones, and the plant growth regulator has dual properties, namely, the growth is promoted by lower concentration, the growth is inhibited by higher concentration, the requirements of different plants and different parts of the same plant on the variety, the concentration and the proportion of the plant growth regulator are different, so most of the plants can meet the specific requirements by reasonable matching of different plant growth regulators, the primary induction culture medium of the invention is WPM + NAA 0.01-0.05 mg/L +6-BA 8.0-10.0 mg/L + active carbon 0.5g/L, preferably, the WPM + NAA0.02mg/L +6-BA8.0 mg/L + active carbon 0.5g/L, in the induction culture process, the invention adopts A with extremely low concentration, the tissue culture method has the advantages that the tissue culture method is directly induced and proliferated without callus, callus is effectively avoided, the callus stage which is most likely to have variation in the tissue culture process is reduced to the maximum extent, meanwhile, the tissue culture link is simplified and the production cost of the tissue culture is reduced because callus is not used, the subculture proliferation medium selected by the method is MS +6-BA 2.0-3.0 mg/L + KT 5.0-6.0 mg/L + coconut water 50-100 ml/L, preferably MS +6-BA2mg/L + KT5.0mg/L + coconut water 100ml/L, in the proliferation process, 50-100 ml/L of coconut water is added, the proliferation coefficient of the coconut water is increased from 3.0 without adding coconut water to 6.0, the proliferation efficiency is improved, the production time is shortened, the production cost is reduced, and the rooting medium selected by the method is 1/2M + A0.5-1.0 mg/L + A0.2-0.5 mg 0.2-0.5 mg/L of/L + paclobutrazol, preferably 1/2WPM + NAA 0.5-1.0 mg/L + IBA 0.2-0.5 mg/L + paclobutrazol 0.2-0.5 mg/L, wherein in the rooting culture process, 0.2-0.5 mg/L paclobutrazol is added, compared with the rooting culture medium without paclobutrazol, the rooting rate is 10% higher, seedlings are robust, the color is bright green, strong seedling culture is not needed between proliferation and rooting culture, the culture link is simplified, the production cost is reduced, after the paclobutrazol is excessively added, the incidence rate of the stiff seedlings is high, and the coconut water is fresh coconut purchased in the market and internal juice obtained by splitting.
Coconut water is mainly derived from tender fruit of coconut, also called coconut green. The research on coconut water by some coconut research institutions in countries such as India shows that the coconut water contains abundant nutrient substances, the content of arginine, alanine, cystine and serine in coconut water protein is higher than that of milk, more than 17 kinds of free amino acids are contained, and the coconut water protein contains VC and VB, such as nicotinic acid, pantothenic acid, folic acid, VH, VB1, VB2 and the like. In addition, the mineral substances in coconut water include Na, Ca, Fe, Mg, Cu, S, P, Cl and other elements; several active substances which cause rapid and irregular division of mature cells of higher plants and thus stimulate plant growth, of which 2 have been identified as 1, 3-diphenylurea and indolylhexylase. The coconut water contains the mineral elements with the highest content of K + and Fe2 +. According to the invention, coconut water is specifically added into the subculture medium, so that nutrient elements are added to the culture medium for plant growth to a certain extent, the growth environment is optimized, and various amino acids and trace elements in the coconut water naturally exist, so that the absorption and adaptability of plants are enhanced.
The tissue culture seedling raising method for tsaoko provided by the invention is described in detail below with reference to specific examples, but the method is not to be construed as limiting the scope of the invention.
Example 1
1. Explant selection and cleaning
Selecting germplasm with large fruits and high aromatic oil content as female parent, shearing young stem segments of the base parts of healthy and disease-and-insect-free grass fruits into 2-3 cm long, flushing the young stem segments under tap water for 1 hour, rinsing the young stem segments with soap liquid for 10min, flushing the young stem segments with clear water for 2-3 times until no soap liquid remains, and finally flushing the young stem segments under tap water for 1-2 hours.
2. Disinfection of explants
And (2) placing the clean explant in a super clean bench, sterilizing the explant by using 75% alcohol for 20s, washing the explant by using sterile water for 3 times, then sterilizing the explant by using 8% hydrogen peroxide for 12min, washing the explant by using the sterile water for 3-4 times, finally sterilizing the explant by using 0.1% mercuric chloride and 2 drops of Tween-80 for 10min, washing the explant by using the sterile water for 8 times, wherein the pollution rate in 7 days is 13.2%, and the disinfection death rate of the explant is 2.7%.
3. Induction of explants
The sterilized explants are dried by using sterile filter paper, wounds at two ends are cut off, the explants are flatly paved in an induction culture medium of WPM + NAA0.01 mg/L +6-BA8.0 mg/L + active carbon 0.5g/L, and are cultured in an environment with the temperature of 25 +/-2 ℃, wherein the first 15 days of culture are dark culture, the last 45 days of culture are alternately cultured in light and dark with the illumination intensity of 2000-3000 lx and the single-day illumination time of 12h, the induction rate of the 60-day cluster buds is 78.6%, and no callus is generated.
4. Subculture multiplication
And (2) obtaining cluster buds through induction culture, cutting the cluster buds into single buds, inoculating the single buds into an enrichment culture medium of MS +6-BA 2.0 mg/L + KT5.0mg/L + coconut water 100ml/L according to polarity, and culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours, wherein the enrichment coefficient is 6.2 after culturing for 40 days, and no callus is generated.
5. Rooting culture
And (2) carrying out induction culture or propagation culture to obtain cluster buds, cutting the cluster buds into single buds, inoculating the single buds into a rooting culture medium of 1/2WPM, NAA0.5mg/L, IBA0.2 mg/L and paclobutrazol 0.2mg/L according to polarity, and culturing for 40 days under the environment of temperature of 25 +/-2 ℃, illumination intensity of 2000-3000 lx and single-day illumination time of 12h, wherein the rooting rate is 98.9%, most of the seedlings have 3-5 robust roots, the seedling height is 4-8 cm, and the leaves are thick and fresh green.
Example 2
1. Explant selection and cleaning
The explant adopted by the invention is a tender stem section of the base part of a straw fruit stem, is cut into 2-3 cm long, is rinsed under tap water for 1 hour, is rinsed for 10min with soap solution, is rinsed with clear water for 2-3 times until no soap solution remains, and is finally rinsed under tap water for 1-2 hours.
2. Disinfection of explants
The clean explant is placed in a super clean bench and is firstly sterilized by 75% alcohol for 30s, sterilized by sterile water for 3 times, sterilized by 8% hydrogen peroxide for 11min, and then sterilized by sterile water for 3 times, and finally sterilized by 0.1% mercuric chloride and 2 drops of Tween-80 for 15min, and is washed by sterile water for 8 times, the pollution rate in 7 days is 10.7%, and the death rate of the sterilized explant is 5.2%.
3. Induction of explants
The sterilized explants are dried by using sterile filter paper, wounds at two ends are cut off, the explants are flatly paved in an induction culture medium of WPM + NAA0.02mg/L +6-BA8.0 mg/L + active carbon 0.5g/L, and are cultured in an environment with the temperature of 25 +/-2 ℃, wherein the first 15 days of culture are dark culture, the last 45 days of culture are alternately cultured in light and dark with the illumination intensity of 2000-3000 lx and the single-day illumination time of 12h, the induction rate of the 60-day cluster buds is 79.5%, and no callus is generated.
4. Subculture multiplication
And (2) obtaining cluster buds through induction culture, cutting the cluster buds into single buds, inoculating the single buds into an enrichment culture medium of MS +6-BA 3.0 mg/L + KT5.0mg/L + coconut water 50ml/L according to polarity, and culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours, wherein the enrichment coefficient is 5.5 after culturing for 40 days, and no callus is generated.
5. Rooting culture
And (2) carrying out induced culture or propagation culture to obtain cluster buds, cutting the cluster buds into single buds, inoculating the single buds into a rooting culture medium of 1/2WPM, 1.0mg/L NAA, 0.2mg/L IBA and 0.4mg/L paclobutrazol according to polarity, culturing for 40 days under the environment of temperature of 25 +/-2 ℃, illumination intensity of 2000-3000 lx and single-day illumination time of 12h, wherein the rooting rate is 96.0%, 3-5 strong roots are provided for most of the healthy, the seedling height is 4-7 cm, and the leaves are thick and fresh green.
Example 3
1. Explant selection and cleaning
Selecting germplasm with large fruits and high aromatic oil content as female parent, shearing young stem segments of the base parts of healthy and disease-and-insect-free grass fruits into 2-3 cm long, flushing the young stem segments under tap water for 1 hour, rinsing the young stem segments with soap liquid for 10min, flushing the young stem segments with clear water for 2-3 times until no soap liquid remains, and finally flushing the young stem segments under tap water for 1-2 hours.
2. Disinfection of explants
And (2) placing the clean explant in a super clean bench, sterilizing the explant by using 75% alcohol for 15s, washing the explant by using sterile water for 3 times, then sterilizing the explant by using 8% hydrogen peroxide for 10min, washing the explant by using the sterile water for 3-4 times, finally sterilizing the explant by using 0.1% mercuric chloride and 2 drops of Tween-80 for 15min, washing the explant by using the sterile water for 8 times, wherein the pollution rate in 7 days is 13.2%, and the disinfection death rate of the explant is 4.7%.
3. Induction of explants
The sterilized explants are dried by using sterile filter paper, wounds at two ends are cut off, the explants are flatly paved in an induction culture medium of WPM + NAA0.01 mg/L +6-BA 10.0mg/L + active carbon 0.5g/L, and are cultured in an environment with the temperature of 25 +/-2 ℃, wherein the first 15 days of culture are dark culture, the last 45 days of culture are alternately cultured in light and dark with the illumination intensity of 2000-3000 lx and the single-day illumination time of 12h, the induction rate of the 60-day cluster buds is 78.4%, and no callus is generated.
4. Subculture multiplication
And (2) obtaining cluster buds through induction culture, cutting the cluster buds into single buds, inoculating the single buds into a proliferation culture medium of MS +6-BA 2.5mg/L + KT 6.0 mg/L + coconut water 80ml/L according to polarity, and culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours, wherein the proliferation coefficient is 5.4 after culturing for 40 days, and no callus is generated.
5. Rooting culture
And (2) carrying out induction culture or propagation culture to obtain cluster buds, cutting the cluster buds into single buds, inoculating the single buds into a rooting culture medium of 1/2WPM, NAA 0.6mg/L, IBA0.5 mg/L and paclobutrazol 0.5mg/L according to polarity, and culturing for 40 days under the environment of 25 +/-2 ℃ of temperature, 2000-3000 lx of illumination intensity and single-day illumination time of 12h, wherein the rooting rate is 97.2%, most of the seedlings have 3-5 robust roots, the seedling height is 4-8 cm, and the leaves are thick and fresh green.
Example 4
1. Explant selection and cleaning
Selecting germplasm with large fruits and high aromatic oil content as female parent, shearing young stem segments of the base parts of healthy and disease-and-insect-free grass fruits into 2-3 cm long, flushing the young stem segments under tap water for 1 hour, rinsing the young stem segments with soap liquid for 10min, flushing the young stem segments with clear water for 2-3 times until no soap liquid remains, and finally flushing the young stem segments under tap water for 1-2 hours.
2. Disinfection of explants
And (2) placing the clean explant in a super clean bench, sterilizing the explant by using 75% alcohol for 20s, washing the explant by using sterile water for 3 times, then sterilizing the explant by using 8% hydrogen peroxide for 12min, washing the explant by using the sterile water for 3-4 times, finally sterilizing the explant by using 0.1% mercuric chloride and 2 drops of Tween-80 for 10min, washing the explant by using the sterile water for 8 times, wherein the pollution rate in 7 days is 13.2%, and the disinfection death rate of the explant is 2.7%.
3. Induction of explants
The sterilized explants are dried by using sterile filter paper, wounds at two ends are cut off, the explants are flatly paved in an induction culture medium of WPM + NAA 0.05 mg/L +6-BA 9.0mg/L + active carbon 0.5g/L, and are cultured in an environment with the temperature of 25 +/-2 ℃, wherein the first 15 days of culture are dark culture, the last 45 days of culture are alternately cultured in light and dark with the illumination intensity of 2000-3000 lx and the single-day illumination time of 12h, the induction rate of the 60-day cluster buds is 79.1%, and no callus is generated.
4. Subculture multiplication
And (2) obtaining cluster buds through induction culture, cutting the cluster buds into single buds, inoculating the single buds into an enrichment culture medium of MS +6-BA 2.0 mg/L + KT 5.5 mg/L + coconut water 90ml/L according to polarity, and culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours, wherein the enrichment coefficient is 4.9 after culturing for 40 days, and no callus is generated.
5. Rooting culture
And (2) carrying out induction culture or propagation culture to obtain cluster buds, cutting the cluster buds into single buds, inoculating the single buds into a rooting culture medium of 1/2WPM, NAA 0.8mg/L, IBA0.3 mg/L and paclobutrazol 0.3 mg/L according to polarity, and culturing for 40 days under the environment of 25 +/-2 ℃ of temperature, 2000-3000 lx of illumination intensity and single-day illumination time of 12h, wherein the rooting rate is 97.9%, most of the seedlings have 3-5 robust roots, the seedling height is 4-8 cm, and the leaves are thick and fresh green.
Comparative example 1
The other steps are identical to those of example 1, except for the secondary propagation step:
and (3) obtaining cluster buds through induction culture, cutting the cluster buds into single buds, inoculating the single buds into a proliferation culture medium of MS +6-BA 2.0 mg/L + KT5.0mg/L according to polarity, and culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12h, wherein the proliferation coefficient is 3.0 after 40 days of culture, and no callus is generated.
Comparative example 2
The other steps are the same as example 1, except for the rooting culture step:
and (3) obtaining cluster buds through induction culture or proliferation culture, cutting the cluster buds into single buds, inoculating the single buds into a rooting culture medium of 1/2WPM + NAA0.5mg/L + IBA0.2 mg/L according to polarity, and culturing for 40 days in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours, wherein the rooting rate is 88.3%, the number of robust roots is small, the seedling height is 6-9 cm, and the leaves are thin and slightly green.
Explants refer to the various inoculated materials in plant tissue culture. Theoretically, plant cells all have totipotency, new plants can be regenerated, and any organ, any tissue, single cell and protoplast can be used as explants. However, in practice, different kinds of plants and different organs of the same plant are inconsistent to the induction condition reaction chamber, the induced differentiation rate of some parts is high, some parts are difficult to dedifferentiate, or the redifferentiation frequency is very low, even only bud is differentiated and does not grow roots or only bud but not grow roots, in order to ensure that the tissue culture of the plant is successful, the selection of a proper explant is very important, for most plants, the stem tip is a better part, the shape of the stem tip is basically built, the growth speed is high, the inheritance is stable, but the restriction of material sources is easy to be caused, and the explant selects young stem segments at the base part of the tsaoko stem, increases the material sources and ensures the inheritance. Compared with the tender shoots near the soil surface adopted in the initial test, the explants adopted by the invention are tender stem sections at the stem base, the pollution rate is reduced from 95 percent of the tender shoots near the soil surface to 12 percent of the tender stem sections, and the induction rate of the tender stem sections at the stem base is 80 percent and is only slightly lower than the induction rate of 84 percent of the tender shoots near the soil surface.
Claims (6)
1. A tissue culture seedling method of tsaoko amomum fruits is characterized by comprising the following steps:
(1) selection of explants: selecting tender stem sections at the base of the tsaoko stems;
(2) sterilizing explants;
(3) performing primary induction culture, wherein the used primary induction culture medium is WPM + NAA 0.01-0.05 mg/L +6-BA 8.0-10.0 mg/L + active carbon 0.5 g/L;
(4) subculture proliferation, wherein the subculture proliferation medium is MS +6-BA 2.0-3.0 mg/L + KT 5.0-6.0 mg/L + coconut water 50-100 ml/L;
(5) rooting culture, wherein the used rooting culture medium is 1/2WPM, 0.5-1.0 mg/L NAA, 0.2-0.5 mg/L IBA and 0.2-0.5 mg/L paclobutrazol;
in the step (3), the primary induction culture further comprises culturing in an environment with the temperature of 25 +/-2 ℃, dark culture is carried out for the first 15 days, and light-dark alternate culture is carried out for the last 45 days at the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 hours;
the subculture of proliferation and rooting in the steps (4) and (5) further comprises culturing in an environment with the temperature of 25 +/-2 ℃, the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 h.
2. The tissue culture seedling raising method for the tsaoko amomum fruits according to claim 1, characterized in that after the explant is selected in the step (1), the explant is cleaned, the explant is cut into 2-3 cm in length, the explant is placed under tap water to be washed for 1 hour, then the explant is rinsed for 10min by using soap liquid, then the explant is washed for 2-3 times by using clear water until no soap liquid remains, and finally the explant is washed under the tap water for 1-2 hours.
3. The tissue culture seedling raising method for the tsaoko amomum fruits according to claim 1, wherein the explant sterilization method in the step (2) comprises the steps of firstly sterilizing with 75% alcohol for 20s, washing with sterile water for 3 times, then sterilizing with 8% hydrogen peroxide for 12min, washing with the sterile water for 3-4 times, finally sterilizing with 0.1% mercuric chloride and 2 drops of tween-80 for 10min, and washing with the sterile water for 8 times.
4. The tissue culture seedling raising method for the tsaoko amomum fruits according to claim 1, wherein the primary induction culture medium used in the step (3) is WPM + NAA0.02mg/L +6-BA8.0 mg/L + activated carbon 0.5g/L.
5. The tissue culture seedling raising method for tsaoko amomum fruits according to claim 1, wherein the subculture multiplication medium used in the step (4) is MS +6-BA2mg/L + KT5.0mg/L + coconut water 100 ml/L.
6. The tissue culture seedling raising method for the tsaoko amomum fruits according to claim 1, wherein the rooting culture medium used in the step (5) is 1/2WPM + NAA0.5mg/L + IBA0.2 mg/L + paclobutrazol 0.2 mg/L.
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