CN110050696B - Method for obtaining high-polysaccharide high-yield dendrobium officinale through ion beam mutagenesis and application - Google Patents
Method for obtaining high-polysaccharide high-yield dendrobium officinale through ion beam mutagenesis and application Download PDFInfo
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Abstract
The invention discloses a method for obtaining high-polysaccharide high-yield dendrobium officinale by ion beam mutagenesis and application thereof, wherein the method comprises the following steps: selecting full seeds without insect pests and sterilizing; carrying out ion beam mutagenesis treatment on the disinfected dendrobium officinale seeds; carrying out tissue culture seedling cultivation on the mutagenized seeds; and (3) cultivating the obtained tissue culture seedling, selecting a plant of which the seed is not subjected to mutagenesis treatment as a control, screening to obtain a high-yield plant with character variation, independently collecting the seed of the plant with character variation, continuously subculturing, and screening to obtain the dendrobium officinale with stable character. The method for obtaining the dendrobium officinale with high polysaccharide and high yield through ion beam mutagenesis provided by the invention can generate non-directional mutation after ion beam injection mutagenesis, and can carry out screening according to a set screening target in a large number of mutagenesis screening experiments, so that excellent dendrobium officinale germplasm with required characters is screened, and the polysaccharide content of the bred dendrobium officinale is improved by 13.35 percent on the basis of improving the yield.
Description
Technical Field
The invention relates to the technical field of dendrobium officinale breeding, in particular to a method for obtaining high-polysaccharide and high-yield dendrobium officinale through ion beam mutagenesis and application.
Background
Dendrobium officinale (Dendrobium officinale Kimura et Migo) is also called Equisetum nigrum, is a traditional and rare Chinese medicinal material in China, has the effects of nourishing yin, clearing heat, tonifying stomach, promoting fluid production, moistening lung, relieving cough and the like, and has a long history of food and medicine. As early as in the book Shen nong Ben Cao Jing, the records of 'treating injury, removing arthralgia, descending qi, tonifying five internal organs, consumptive disease, emaciation, yin strengthening and stomach thickening after long-term administration' exist.
The dendrobium officinale has special growing environment, extremely difficult self-propagation and high economic value, leads to less and less wild resources due to long-term over-excavation and unreasonable development and utilization of people, and is listed in the famous rare or endangered protected plant directory of China at present. In order to solve the problem of shortage of wild dendrobium officinale resources, people continuously research and domesticate to realize artificial cultivation, and meanwhile, the application of a tissue culture technology enables the dendrobium officinale industry to develop rapidly. In recent years, the dendrobium officinale cultivation area is expanded from the traditional Zhejiang and Yunnan to Guangxi, Guizhou, Guangdong, Fujian, Anhui, Jiangsu and other places, the demand of excellent dendrobium officinale germplasm is rapidly increased due to the rapid expansion of the dendrobium officinale cultivation area, and the dendrobium officinale germplasm with excellent properties and adapting to different regional environments needs to be cultivated to meet the market demand and promote the development of the dendrobium officinale industry.
At present, the breeding of the dendrobium officinale takes hybridization as the main part, a few reports of radiation mutagenesis exist, Zhanzhonggen and the like are published in 2009 nuclear agriculture newspaper, and different doses are researched137The Cs gamma ray irradiation influences the growth and differentiation effect of protocorm of the dendrobium officinale seed embryo, and the phenomena of stem bifurcation, blade green deficiency, white-green interphase and the like of variant seedlings generated by irradiation appear; xiwavelet et al published a paper in Zhejiang agricultural science 2016, and in order to obtain a proper dose of gamma-ray irradiation to protocorm of Dendrobium officinale, Dendrobium officinale Kimura et Migo No. 1 was used as a material and its protocorm was treated137The irradiation research of Cs gamma rays and the analysis of the offspring variation thereof, the obtained result is that the dosage near the half lethal dosage of 86.4 Gy can be used for the irradiation mutagenesis treatment of the dendrobium officinale protocorm.
However, no reports related to ion beam mutagenesis of dendrobium officinale seeds have been found so far.
Disclosure of Invention
The invention aims to provide a method for obtaining high-polysaccharide and high-yield dendrobium officinale by ion beam mutagenesis and application aiming at short plates and defects in the breeding of the dendrobium officinale in the prior art.
The invention adopts the following technical scheme:
a method for obtaining high-polysaccharide high-yield dendrobium officinale by ion beam mutagenesis comprises the following steps:
s1, selecting plump and insect-pest-free dendrobium officinale seeds and disinfecting;
s2, performing ion beam mutagenesis treatment on the disinfected dendrobium officinale seeds to obtain mutagenized seeds;
wherein the mutagenic ion beam is6+C, the mutagenesis dose is 10-80Gy, and the dosage rate is 5-50 Gy/min;
s3, carrying out tissue culture seedling cultivation on the mutagenic seeds obtained in the step S2;
s4, cultivating the tissue culture seedling obtained in the step S3, selecting a plant of which the seed is not subjected to mutagenesis treatment as a control, screening a high-yield plant with character variation, separately collecting the seed of the plant with character variation, carrying out multi-generation continuous purification culture on the seed, and screening to obtain the dendrobium officinale with stable character.
In the above technical solution, in step S4, the method for screening the high-yield plants with characteristic variation includes screening the cultivated seedlings with stem number, length and diameter larger than those of the control plants, and then screening the cultivated seedlings with fresh-strip polysaccharide content higher than that of the control plants by 5% as the high-yield plants with characteristic variation.
Further, in the above technical solution, in step S4, the method for measuring the content of fresh striped polysaccharide is a phenol-sulfuric acid method.
Still further, in the above technical solution, in step S4, the culture medium for culturing the tissue culture seedling is a mixture of pine bark and coconut coir in a mass ratio of 1.8-2.4: 1.
In the above technical solution, in step S2, the mutagenesis dose of the ion beam mutagenesis treatment is 40 to 60Gy, and the dose rate is 30 Gy/min.
In the above technical solution, in step S3, the tissue culture seedling cultivation of the mutagenized seed sequentially includes induced germination, differentiation culture, and rooting culture of the seed.
In detail, the illumination time for inducing germination of the seeds is 12h/d, the temperature is 21-25 ℃, the illumination intensity is 1000-.
In detail, the illumination time of the differentiation culture is 12h/d, the temperature is 21-25 ℃, the illumination intensity is 1000-.
In detail, the illumination time of the rooting culture is 12h/d, the temperature is 21-25 ℃, the illumination intensity is 2000-3000lx, and the culture time is 45-55 d.
Further, in the above technical solution, in step S3, the culture medium for inducing germination of the seeds is 1/2MS culture medium + sucrose 20 g/L + activated carbon 1 g/L + agar 5 g/L.
Further, in the above technical solution, in step S3, the differentiation culture medium is 1/2MS medium + sucrose 20 g/L + α -naphthylacetic acid (NAA)0.5 mg/L + banana 100 g/L + activated carbon 5 g/L + agar 5 g/L;.
Further, in the above technical solution, in step S3, the culture medium for rooting culture is 1/2MS culture medium + sucrose 20 g/L + α -naphthylacetic acid (NAA)0.5 mg/L + banana 200 g/L + activated carbon 5 g/L + agar 5 g/L.
Further, in the above technical scheme, in step S3, the tissue culture seedling cultivation of the mutagenized seed further comprises hardening off after rooting cultivation, the hardening off needs to be covered with a sunshade net, and the hardening off time is 25-32 days.
In the above technical solution, in step S1, the sterilizing specifically includes sterilizing with 70-80 wt% alcohol for 20-60S, washing with sterile water for 2-5 times, sterilizing with 0.1-0.2 wt% mercuric chloride for 15-30min, and finally rinsing with sterile water for 4-6 times.
According to another aspect of the invention, the invention also provides application of the method in cultivation and screening of the dendrobium officinale variety with high polysaccharide and high yield.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method for obtaining the excellent new germplasm of the dendrobium officinale by ion beam mutagenesis is an effective method for obtaining a mutant plant of the dendrobium officinale, seeds of the dendrobium officinale can generate non-directional mutation after being subjected to ion beam injection mutagenesis, and purposeful screening can be carried out according to the set screening target, namely the quantity, the plant height, the diameter, the polysaccharide content and the like of the dendrobium officinale stems in a large amount of mutagenesis screening experiments, so that the excellent dendrobium officinale germplasm with required properties can be screened;
(2) the method for obtaining the excellent new germplasm of the dendrobium officinale by ion beam mutagenesis provided by the invention is not only suitable for the dendrobium officinale, but also can be applied to the breeding research work of other dendrobium nobile and other excellent plant germplasm through proper improvement.
Drawings
FIG. 1 is a graph showing the relationship between the survival rate of Dendrobium officinale seed and the mutagenesis dose in example 1 of the present invention.
Detailed Description
In order that the invention may be more readily understood, reference will now be made in detail to the present embodiments of the invention.
The following examples are intended to illustrate the present invention, but not to limit the scope of the invention, which is defined by the claims.
Unless otherwise specified, experimental reagents and materials used in the examples of the present invention are commercially available, and unless otherwise specified, technical means used in the examples of the present invention are conventional means well known to those skilled in the art.
The dendrobium officinale seeds used in the embodiment of the invention are provided by Yixing biotechnology limited company in Beijing.
The method for obtaining the excellent and new germplasm of dendrobium officinale by ion beam mutagenesis in the embodiment of the invention specifically comprises the following steps:
1. mutation of dendrobium officinale seeds and statistics of survival rate
Selecting mature and uncracked fruits to obtain seeds, sterilizing the surfaces of the fruits in an ultra-clean workbench of an aseptic inoculation room by using 70-80% of alcohol for 20-60 seconds, washing the fruits for 2-5 times by using sterile water, sterilizing the fruits in 0.1-0.2% of mercuric chloride for 15-30 minutes, rinsing the fruits for 4-6 times by using the sterile water, cutting the fruits in an aseptic culture dish, putting the seeds into an aseptic EP (ethylene propylene) tube, sealing the seeds by using a sealing film and storing the seeds in a refrigerator at 4 ℃ for later use.
Performing ion beam mutagenesis on seeds stored in an EP tube by using a heavy ion research device (HIRF L) of Lanzhou heavy ion accelerator national laboratory of recent physical research institute of Chinese academy of sciences, wherein the mutagenized ions are6+C ion beam, the mutagenesis dosage is 10Gy, 20Gy, 40Gy, 60Gy, 80Gy and 100Gy, the dosage rate is 30Gy/min, and the mutagenized seeds are taken back to the laboratory for tissue culture.
A seed germination culture medium comprising 1/2MS culture medium, 20 g/L of sucrose, 1 g/L of activated carbon and 5 g/L of agar.
Transferring the ion beam-mutagenized dendrobium officinale seeds to a seed germination plate culture medium for culture, counting seed germination rates, taking the seed germination rate without ion beam mutagenesis as a blank control, and calculating the survival rate of the ion beam-mutagenized seeds, wherein the calculation formula is as follows:
survival (%) < mutagenized seed germination/blank control seed germination × 100%
The relationship between the mutation dosage and the survival rate of the dendrobium officinale seeds obtained according to the survival rate calculation formula is shown in fig. 1, and the survival rate of the blank control which is not subjected to ion beam mutagenesis is taken as 100%.
From the data in fig. 1, it can be concluded that the survival rate of the seeds without ion beam implantation mutagenesis is 100% when the mutagenesis dose is 0Gy, and the survival rate is reduced from 100% to the lowest when the mutagenesis dose is from 0Gy to 20Gy, and then the survival rate of the dendrobium officinale seeds is gradually increased and then reduced to 0 when the mutagenesis dose is increased. This is a "saddle" curve of survival rate for ion beam mutagenesis and is also a reference for selecting the optimal mutagenesis dose.
2. Culture of dendrobium officinale tissue culture seedling
(1) Seed germination culture:
an induced germination culture medium comprises 1/2MS culture medium, 20 g/L of sucrose, 1 g/L of activated carbon and 5 g/L of agar;
inoculating the mutagenized seeds into an induced germination culture medium in a clean bench of a sterile room, then carrying out seed germination culture, placing the seeds on a culture frame of a tissue culture laboratory, setting the illumination time to be 12h/d, setting the temperature of the tissue culture room to be 21-25 ℃, setting the illumination intensity to be 1000-2000lx, and culturing for 60-70 days to form protocorms.
(2) Differentiation culture:
a differentiation culture medium which is 1/2MS culture medium, sucrose 20 g/L + α -naphthylacetic acid (NAA)0.5 mg/L + banana 100 g/L + active carbon 5 g/L + agar 5 g/L;
transferring the protocorm into a differentiation culture medium for proliferation culture, placing the protocorm on a culture frame of a tissue culture laboratory, setting the illumination time to be 12h/d, setting the temperature of the tissue culture laboratory to be 21-25 ℃, setting the illumination intensity to be 1000-2000lx, culturing for 60-70 days, growing buds of the protocorm and differentiating to grow leaves, and growing to about 1-2cm with 2-4 leaves to obtain differentiated tissue culture seedlings.
(3) Rooting culture:
a rooting culture medium comprising 1/2MS culture medium, sucrose 20 g/L + α -naphthylacetic acid (NAA)0.5 mg/L + banana 200 g/L + active carbon 5 g/L + agar 5 g/L;
transferring the differentiated tissue culture seedlings into a rooting culture medium for rooting culture, placing the tissue culture seedlings on a culture frame of a tissue culture laboratory, setting the illumination time to be 12h/d, setting the temperature of the tissue culture room to be 21-25 ℃, setting the illumination intensity to be 2000-3000lx, culturing for 20-25 days, enabling 2-3 white roots to grow out from the base part and grow for about 1-2cm, then continuing to grow, enabling the roots to grow, enabling the plant to continue to grow high, increasing the leaves, enabling the rooting rate to reach 90% in 30-35 days, continuing to culture for 40-50 days, enabling the roots of the dendrobium officinale seedlings to be fully distributed in the culture medium, and enabling the tissue culture seedlings to be about 3-6cm high, thereby obtaining the rooted tissue culture seedlings.
(4) Hardening seedlings:
placing the rooting tissue culture seedling band bottle obtained in the tissue culture laboratory into a greenhouse for continuous culture, covering a sunshade net in the greenhouse to prevent over-sufficient sunshine, hardening the seedling for about 30 days, and obtaining the tissue culture seedling which can be transplanted, wherein the height of the tissue culture seedling is about 4-8 cm.
3. Cultivation of dendrobium officinale
Directly smashing a tissue culture seedling bottle after seedling hardening, taking out the tissue culture seedling, putting the tissue culture seedling into clear water for cleaning, and mainly aiming at removing culture medium components at the root of the tissue culture seedling, grouping the tissue culture seedlings with the same mutagenesis dose according to the size, then taking 6-8 tissue culture seedlings with similar sizes as 1 clump to be planted into a pot with the diameter of 9cm, wherein the culture medium is formed by mixing pine bark and coconut chaff according to the proportion of 2: 1. And putting the planted dendrobium officinale plantlets into a tray, putting the seedlings in order in a seedbed, watering, paying attention to shade, and watering and maintaining at regular intervals.
4. Screening of Dendrobium officinale
(1) Method for measuring dendrobium officinale fresh strip polysaccharide
Accurately weighing glucose with a constant dry weight at 105 ℃, adding water to prepare a glucose standard solution with a concentration of 100 mu g/ml, respectively sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0ml of the glucose standard solution, placing the glucose standard solution into a 10ml test tube with a plug, respectively adding water to supplement the glucose standard solution to 1.0ml, accurately adding 1.0ml of 5% phenol solution, shaking up, accurately adding 5ml of concentrated sulfuric acid, shaking up, placing in a boiling water bath to heat for 20min, taking out, and rapidly placing in an ice water bath to cool for 5 min. The absorbance value was measured at 490nm wavelength using the reagent as a blank, and a glucose standard curve was drawn using the glucose content (μ g) as abscissa and the absorbance value as ordinate. The standard curve equation is obtained as follows: y is 0.0068X-0.0121, R2＝0.9992。
(2) Dendrobium officinale screening method
And comparing and screening the dendrobium officinale seedlings cultivated for 12 months, wherein the aim is to screen the dendrobium officinale seedlings with larger appearance change.
Firstly, selecting the dendrobium officinale with vigorous growth, selecting the dendrobium officinale pot plants with the indexes such as the total quantity, the length and the like of the stems of each pot of the dendrobium officinale higher than those of the control, then measuring the growth conditions of the dendrobium officinale pot one by one, measuring the length and the diameter of the maximum 5 stems in each pot, and aiming at counting the yield of the dendrobium officinale pot plants and screening to obtain the dendrobium officinale pot plants with the yield possibly improved. And screening the dendrobium officinale with the improved average length or diameter of each pot of stems, continuously culturing, flowering and pollinating, and collecting seeds for later use.
The method comprises the steps of taking the yield of fresh dendrobium officinale strips and the polysaccharide content as screening indexes, screening the dendrobium officinale with improved yield, measuring the polysaccharide content of the fresh dendrobium officinale strips with improved yield, screening the dendrobium officinale with improved polysaccharide content, and obtaining the dendrobium officinale germplasm with improved yield and polysaccharide content. Culturing the dendrobium officinale with collected seeds for 24 months, collecting partial fresh strips to measure the characters of the dendrobium officinale, measuring the plant height, the stem thickness and the single stem weight, taking an average value for each treatment, screening the dendrobium officinale with improved yield, then measuring the polysaccharide content of the fresh strips of the dendrobium officinale, taking 1.0g of the fresh strips, shearing the fresh strips, grinding the fresh strips into a mortar, washing the mortar by 50ml of pure water for times, combining the washing liquids, putting the washing liquids into a ground triangular flask, adding a reflux device, heating and refluxing in a boiling water bath for 2 hours, taking the refluxed supernatant, diluting different times according to the sample concentration, measuring the polysaccharide content by adopting a phenol-sulfuric acid method according to a standard curve manufacturing method, then calculating the polysaccharide content according to the dilution times, and screening the dendrobium officinale with the polysaccharide content improved by 5% or more. And (4) obtaining the excellent dendrobium officinale germplasm through the screening.
5. Propagation of dendrobium officinale
And continuously carrying out tissue culture propagation on the seeds corresponding to the dendrobium officinale with the screened yield and the polysaccharide content, then cultivating the tissue culture seedlings, and screening excellent germplasm resources to obtain the dendrobium officinale with stable genetic characters.
6. Determination of dendrobium officinale traits
Randomly screening 100 pots of the control and screened dendrobium candidum germplasm, measuring the plant height, the stem thickness, the single stem weight and the polysaccharide content of the dendrobium candidum, and calculating to obtain an average value, wherein the specific data are shown in the following table 1.
TABLE 1 determination of Dendrobium officinale traits
As can be seen from Table 1, compared with a control, the dendrobium officinale obtained by mutation breeding has greatly improved plant height, thick stem, single stem weight and polysaccharide content, and the germplasm characteristics of the obtained dendrobium officinale are obviously superior to the control, so that the ion beam mutation is an effective method for breeding excellent germplasm of the dendrobium officinale.
In conclusion, ion beams mutate the seeds of the dendrobium officinale, although mutation is uncertain, germplasm with excellent characters can be obtained through screening a large number of mutations as long as a clear screening target is provided, the research takes plant height and diameter as the screening target, firstly, the dendrobium officinale with possibly improved yield is screened, and then, the dendrobium officinale with improved polysaccharide content is continuously screened on the basis, so that the germplasm with excellent characters can be finally obtained.
Finally, the above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for obtaining high-polysaccharide high-yield dendrobium officinale by ion beam mutagenesis is characterized by comprising the following steps:
s1, selecting plump and insect-pest-free dendrobium officinale seeds and disinfecting;
s2, performing ion beam mutagenesis treatment on the disinfected dendrobium officinale seeds to obtain mutagenized seeds;
wherein the mutagenic ion beam is6+C, the mutagenesis dose is 10-80Gy, and the dosage rate is 5-50 Gy/min;
s3, carrying out tissue culture seedling cultivation on the mutagenic seeds obtained in the step S2;
s4, cultivating the tissue culture seedling obtained in the step S3, selecting a plant of which the seed is not subjected to mutagenesis treatment as a control, screening a high-yield plant with character variation, separately collecting the seed of the plant with character variation, carrying out multi-generation continuous purification culture on the seed, and screening to obtain the dendrobium officinale with stable character;
in step S3, the germination induction culture medium of the seeds is 1/2MS culture medium + sucrose 20 g/L + activated carbon 1 g/L + agar 5 g/L1, the differentiation culture medium is 1/2MS culture medium + sucrose 20 g/L2 + L0-naphthylacetic acid 0.5 mg/L3 + banana 100 g/L4 + activated carbon 5 g/L + agar 5 g/L, and the rooting culture medium is 1/2MS culture medium + sucrose 20 g/L + α -naphthylacetic acid 0.5 mg/L + banana 200 g/L + activated carbon 5 g/L + agar 5 g/L;
in step S4, the culture medium for culturing the tissue culture seedling is a mixture of pine bark and coconut coir in a mass ratio of 1.8-2.4: 1.
2. The method of claim 1, wherein in step S4, the method for selecting the plants with high yield of variation in traits comprises selecting the seedlings with stem number, length and diameter larger than those of the control plants, and selecting the seedlings with fresh-shoot polysaccharide content higher than 5% of that of the control plants as the plants with high yield of variation in traits.
3. The method according to claim 2, wherein in step S4, the fresh streak polysaccharide content is determined by a phenol-sulfuric acid method.
4. The method according to claim 1, wherein in step S2, the ion beam mutagenesis treatment is performed at a mutagenesis dose of 40-60Gy and a dose rate of 30 Gy/min.
5. The method according to claim 1, wherein in step S3, the tissue culture seedling cultivation of the mutagenized seed comprises induced germination, differentiation culture and rooting culture of the seed in sequence; wherein:
the illumination time for inducing germination of the seeds is 12h/d, the temperature is 21-25 ℃, the illumination intensity is 1000-;
the illumination time of the differentiation culture is 12h/d, the temperature is 21-25 ℃, the illumination intensity is 1000-;
the illumination time of the rooting culture is 12h/d, the temperature is 21-25 ℃, the illumination intensity is 2000-3000lx, and the culture time is 45-55 d.
6. The method according to claim 5, wherein in step S3, the tissue culture seedling cultivation of the mutagenized seeds further comprises seedling exercising after rooting cultivation, the exercising needs to be covered with a sunshade net, and the seedling exercising time is 25-32 days.
7. The method as claimed in claim 1, wherein the sterilizing step S1 comprises sterilizing with 70-80 wt% ethanol for 20-60S, washing with sterile water for 2-5 times, sterilizing with 0.1-0.2 wt% mercuric chloride for 15-30min, and rinsing with sterile water for 4-6 times.
8. The use of the method of any one of claims 1-7 for breeding and screening high-polysaccharide high-yield Dendrobium officinale varieties.
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