CN109892225B - In-vitro preservation method for anthurium andraeanum germplasm resources - Google Patents
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Abstract
A method for in vitro preservation of anthurium andraeanum germplasm resources comprises the steps of taking stout and strong absorbing buds of anthurium andraeanum after leaves are removed as explants, washing the explants cleanly under tap water, disinfecting the explants for 45 to 60 seconds by using 72 to 75 percent alcohol, washing the explants for 1 time by using sterile water, treating the explants by using 0.1 percent mercury bichloride, washing the explants for 3 times by using the sterile water, and drying the explants by using sterile filter paper; cutting the explant into stem segments with nodes, inoculating the stem segments into a bud induction culture medium for culturing for 30 days, and then transferring the germinated axillary buds onto a proliferation culture medium for propagation for 40 days to obtain a cluster seedling; cutting the cluster seedlings into single plants and transferring the single plants into an in vitro preservation culture medium for preservation; in vitro preservation culture medium: 3g/L Huabao No. 1 + 1-3 g/L NH4NO3+ 0.5-1.0 g/L adult man and woman comprehensive mineral substance composite vitamin tablet +30g/L sucrose +4g/L agar powder. The method of the invention can preserve the 16-month anthurium andraeanum test-tube plantlet, the later-generation plant characters of the test-tube plantlet are not different from ISSR amplification bands, the preserved material is stable, the genetic variation can not occur, the sexual degeneration and the disease infection can be prevented, and the excellent characteristics of the germplasm can be ensured.
Description
Technical Field
The invention relates to the field of in vitro preservation of germplasm resources of plants, in particular to a method for in vitro preservation of Anthurium album germplasm resources.
Background
The white palm and the taro are perennial evergreen herbaceous plants of the taro genus of the Araceae family, can watch leaves and flowers, also have the function of absorbing toxic and harmful gases such as formaldehyde and the like, and are one of ten potted flowers popular in the current market. The white palm is distributed in tropical America, West Indian Islands and Malaysia Islands, and 41 original strains and more than 100 cultivated varieties are reported. The white palm is a heterozygote, the characters of offspring are easy to separate, the service life of seeds is extremely short, the germplasm resources are difficult to store through the seeds, the female parent is mainly stored through a facility cultivation means, and the conventional storage method has the defects of large consumption of manpower, material resources and financial resources, germplasm loss and degeneration caused by diseases and insect pests and the like.
In production, seedlings produced by the plant tissue culture rapid propagation industry are not only required to conform to the variation of market supply and demand, but also are restricted by the growth season, so that some mother bottle materials have no significance in propagation expansion at a certain stage, the mother bottle materials are required to be temporarily stored, and when actual production needs are met, the mother bottle materials can be rapidly restored to a normal rapid propagation state. Meanwhile, in high-temperature and high-humidity seasons, the subculture is easy to pollute, and if the mother bottle material can be stored for a short time, the production cost of the mother bottle material transfer is saved, and the variation rate of the tissue culture seedlings can be reduced.
In order to solve the defects of the traditional planting and preservation of the anthurium andraeanum and the practical problems in the production of the tissue culture and rapid propagation industry of the seedlings, the development of the research on the in vitro preservation of the anthurium andraeanum germplasm resources is increasingly urgent. The in vitro preservation has the advantages of time and labor saving, no germ plasm resource loss caused by diseases, maintenance of the excellent characteristics of the germ plasm and the like, and is convenient for virus-free germ plasm exchange. There are many reports on the in vitro preservation of plants. In Chinese patent literature, a method for in vitro preservation of chrysanthemum germplasm resources (application No. CN 200710019345.1) and a method for in vitro preservation of chrysanthemum by reducing macroelements in a culture medium (application No. CN 200710019346.6) respectively disclose a method for in vitro preservation of chrysanthemum by adding abscisic acid and reducing macroelements in the culture medium, and the preservation period of the chrysanthemum can reach 10 months; for another example, an in vitro conservation method of oriental lily germplasm resources (application number CN 201010138986.0) disclosed in Chinese patent literature discloses in vitro conservation of oriental lily germplasm by improving a culture medium formula and a culture step method, wherein the preservation period can reach 12-15 months; for another example, the Chinese patent document discloses an isolated preservation method of a color calla germ plasm resource (application number CN 200710164732.4), which is used for preserving the isolated germ plasm of the color calla by reducing macroelements in a culture medium and adding mannitol and penicillin, and the preservation period can reach 12 months.
However, the prior art does not disclose how to preserve the white palm test-tube plantlet, and the problem needs to be solved urgently.
Disclosure of Invention
The invention provides a method for in vitro preservation of Anthurium leucopoda germplasm resources, which mainly aims to overcome the defects that the greenhouse planting preservation cost of the existing Anthurium leucopoda germplasm resource facilities is high, the greenhouse planting preservation cost is easily influenced by diseases and insect pests, the normal subculture preservation and transfer frequency of tissue culture are high, and the cost is high.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for in vitro preservation of anthurium andraeanum germplasm resources comprises the following steps:
1) explant selection and sterilization: taking the stout and strong suckling buds of the white palms without the leaves as explants, firstly washing the explants cleanly under tap water, then disinfecting the surfaces of the explants for 45-60 seconds by using 72-75% alcohol, washing the explants for 1 time by using sterile water, then treating the explants for 10-12 minutes by using 0.1% mercuric chloride, washing the explants for 3 times by using the sterile water, and sucking the explants for later use by using sterile filter paper;
2) and (3) propagation of test-tube plantlets: cutting the explant treated in the step 1 into stem segments with nodes, and inoculating the stem segments into a bud induction culture medium with the pH value of 5.8-6.0 at the temperature of 27-29 ℃, the illumination intensity of 1200-1500 lux and the illuminationCulturing for 30 days under the condition of 12 hours/day, then transferring the germinated axillary buds to a proliferation culture medium with the pH value of 5.8-6.0, and performing propagation for 40 days under the conditions of the temperature of 27-29 ℃, the illumination intensity of 1200-1500 lux and the illumination time of 12 hours/day to obtain the plantlets; wherein, the bud induction culture medium: 4g/L Huabao No. 1 +30g/L cane sugar + 0.1-0.3 mg/LTDZ + 0.05-0.1 mg/LNAA +5g/L agar powder; proliferation culture medium: 3g/L Huabao No. 1 + 1-3 g/L NH4NO3+ 0.05-0.15 mg/LTDZ + 0.05-0.1 mg/LNAA +30g/L sucrose +5g/L agar powder;
3) and (3) storing test-tube plantlets: cutting the cluster seedlings obtained in the step 2 into single plants, and transferring the single plants into an in-vitro preservation culture medium with the pH value of 6.1-6.5 for preservation under the conditions of the temperature of 20-25 ℃, the illumination intensity of 1000-1200 lux and the illumination time of 8 hours/day; wherein the in vitro preservation medium: 3g/L Huabao No. 1 + 1-3 g/L NH4NO3+ 0.5-1.0 g/L adult man and woman comprehensive mineral substance composite vitamin tablet +30g/L sucrose +4g/L agar powder.
Further, the in vitro preservation culture medium is 3g/L Huabao No. 1 +1g/L NH4NO3+0.5 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder.
Further, the in vitro preservation culture medium is 3g/L Huabao No. 1 +1g/L NH4NO3+ 0.75 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder.
Further, the in vitro preservation culture medium is 3g/L Huabao No. 1 +1g/L NH4NO3+ 1.0 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder.
Further, the bud induction culture medium is 4g/L Huabao No. 1 +30g/L cane sugar +0.1mg/LTDZ +0.05mg/LNAA +5g/L agar powder.
Further, the proliferation culture medium is 3g/L Huabao No. 1 +1g/L NH4NO3+0.1mg/LTDZ +0.05mg/LNAA +30g/L sucrose +5g/L agar powder.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention preserves the white palm test-tube plantlet by adding the comprehensive mineral substance vitamin complex tablets which are good for preserving adults, men and women in an in vitro preservation culture medium, the survival rate of the test-tube plantlet after being preserved for 16 months is still 100 percent, the plant grows normally, and the test-tube plantlet on the conventional culture medium can only survive for 5 months.
2. The method of the invention can preserve the 16-month anthurium andraeanum test-tube plantlet, the later-generation plant characters of the test-tube plantlet are not different from ISSR amplification bands, the preserved material is stable, the genetic variation can not occur, the sexual degeneration and the disease infection can be prevented, and the excellent characteristics of the germplasm can be ensured.
3. The invention saves the test-tube plantlet of the white palm by adding the comprehensive mineral substance complex vitamin tablets capable of preserving adults, men and women in vitro into the in-vitro preservation culture medium, and can save the preservation space and reduce the workload compared with the traditional greenhouse planting and preservation.
4. The invention reduces the production relay inoculation work, reduces the unnecessary labor and material cost in the production operation process, and improves the economic benefit.
Drawings
FIG. 1 shows a test-tube plantlet preserved for 6 months in an in vitro preservation medium added with a comprehensive mineral multi-vitamin tablet for well-preserved adults and men.
FIG. 2 is a test-tube plantlet preserved for 16 months on an in vitro preservation culture medium added with a comprehensive mineral vitamin complex tablet for well-preserved adults and men.
FIG. 3 is a control plot of control tube plantlets and stored tube plantlets obtained by the method of the present invention after re-cultivation in a proliferation medium (left: control tube plantlet, right: stored tube plantlet of the present invention).
FIG. 4 is a comparison chart of a control plant and a regenerated plant obtained by transplanting a control test-tube plantlet and a stored test-tube plantlet obtained by the method of the present invention into a greenhouse, respectively (left: control plant, right: regenerated plant).
FIG. 5 shows ISSR maps of primer amplification (molecular weight markers of DNA of 1, 100 bpa; 2, white palm of "sunshine"; 3, control strain; 4, white palm of "Meijiu"; 5, white palm of "Vickers"; restored after preservation by the method of the present invention).
Detailed Description
The following describes embodiments of the present invention with reference to the drawings.
A method for in vitro preservation of anthurium andraeanum germplasm resources comprises the following steps:
1) explant selection and sterilization: taking the stout and strong suckling buds of the white palms without the leaves as explants, firstly washing the explants cleanly under tap water, then disinfecting the surfaces of the explants for 45-60 seconds by using 72-75% alcohol, washing the explants for 1 time by using sterile water, then treating the explants for 10-12 minutes by using 0.1% mercuric chloride, washing the explants for 3 times by using the sterile water, and sucking the explants for later use by using sterile filter paper; wherein, the 'good wine' white palm stout bud is used as an explant; and the best is 72 percent alcohol;
2) and (3) propagation of test-tube plantlets: cutting the explant treated in the step 1 into stem sections with nodes, inoculating the stem sections into a bud induction culture medium with the pH value of 5.8-6.0, culturing for 30 days under the conditions of the temperature of 27-29 ℃, the illumination intensity of 1200-1500 lux and the illumination time of 12 hours/day, and then transferring the germinated axillary buds to a proliferation culture medium with the pH value of 5.8-6.0, and performing propagation for 40 days under the conditions of the temperature of 27-29 ℃, the illumination intensity of 1200-1500 lux and the illumination time of 12 hours/day to obtain clump seedlings; wherein, the bud induction culture medium: 4g/L Huabao No. 1 +30g/L cane sugar + 0.1-0.3 mg/LTDZ + 0.05-0.1 mg/LNAA +5g/L agar powder; proliferation culture medium: 3g/L Huabao No. 1 + 1-3 g/L NH4NO3+ 0.05-0.15 mg/LTDZ + 0.05-0.1 mg/LNAA +30g/L sucrose +5g/L agar powder; specifically, the optimal composition of the bud induction culture medium is 4g/L Huabao No. 1 +30g/L cane sugar +0.1mg/LTDZ +0.05mg/LNAA +5g/L agar powder; the optimal composition of the proliferation culture medium is 3g/L Huabao No. 1 +1g/L NH4NO3+0.1mg/LTDZ +0.05mg/LNAA +30g/L sucrose +5g/L agar powder;
3) and (3) storing test-tube plantlets: cutting the cluster seedlings obtained in the step 2 into single plants, and transferring the single plants into an in-vitro preservation culture medium with the pH value of 6.1-6.5 for preservation under the conditions of the temperature of 20-25 ℃, the illumination intensity of 1000-1200 lux and the illumination time of 8 hours/day; wherein the in vitro preservation medium: 3g/L Huabao No. 1 + 1-3 g/L NH4NO3+ 0.5-1.0 g/L adult man and woman comprehensive mineral substance composite vitamin tablet +30g/L sucrose +4g/L agar powder. Specifically, the composition of the in vitro preservation medium has three implementationsExample (c): in vitro preservation of culture medium: 3g/L Huabao No. 1 +1g/L NH4NO3+0.5 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder; ② in vitro preservation culture medium: 3g/L Huabao No. 1 +1g/L NH4NO3+ 0.75 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder; ③ in vitro preservation culture medium: 3g/L Huabao No. 1 +1g/L NH4NO3+ 1.0 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder;
4) refer to fig. 1 and 2. Genetic stability identification of progeny of the preserved material: after 2 months of growth of the test-tube plantlet's wine on a control in vitro preservation culture medium without adding a comprehensive mineral substance complex vitamin tablet for well-preserved adults, the plant is full of the tissue culture bottle, most of the leaves turn yellow, and after 5 months, the plant is aged and withered. The test-tube plantlet of 'Mei wine' is slowly grown on an in vitro preservation culture medium added with a comprehensive mineral substance complex vitamin sheet which is good for storing adults, men and women, the plant grows normally after 6 months (as shown in figure 1), the top tip and the bottle mouth are shown after 16 months (as shown in figure 2), the stem of the plant is green, only a small amount of leaves at the lower part are withered and yellow, and the whole plant stem can be used; therefore, the subculture time of the test-tube plantlet is greatly prolonged after the in vitro preservation culture medium is added with the comprehensive mineral vitamin complex tablets for well-preserved adults, so that the subculture interval is changed from 1-time subculture in 1 month to 16-month subculture in ordinary times, the survival rate of the test-tube plantlet is 100% after 16 months, and the test-tube plantlet survives for 11 months after being prolonged compared with a control;
reference is made to fig. 3, 4 and 5. Transferring the test-tube plantlets stored for 16 months in the step 3 to a proliferation culture medium respectively with the traditional control test-tube plantlets which are subjected to 1-month subculture and 1-time normal culture, and after culturing for 20 days, differentiating adventitious buds with normal shapes from the stored test-tube plantlets, wherein the differentiation capacity is equivalent to that of the control test-tube plantlets, which indicates that the stored test-tube plantlets recover normal growth (shown in figure 3); transferring the regenerated adventitious bud to 1/2MS +0.3 mg/LNAA +1g/L activated carbon rooting culture medium after 30 days; transferring the seedlings to a greenhouse for hardening seedlings after 15 days, and transplanting the seedlings to peat soil after 7 days: culturing seedlings in a substrate with perlite =8: 2; when the height of the plantlet is 10 cm, cultivating the plantlets in cups with the specification of 120 mm; after 5 months, the growth was normal, with no difference in the vegetative traits from the control strain (as shown in FIG. 4); meanwhile, the total DNA of the white palm is extracted from the heart leaves, and no specific band is detected by ISSR molecular marker (as shown in figure 5). The results show that the culture medium is added with the compound vitamin tablets which are good for storing adult male and female comprehensive mineral matters, so that 16 carallula germplasms are stored, and good genetic stability is maintained.
Refer to fig. 5. The specific experimental method of ISSR molecular marker is as follows: extraction of materials and DNA: extracting genome DNA from plant heart leaves directly after quick freezing by liquid nitrogen, storing at-20 ℃ for later use, and adopting an improved CTAB method for the extraction method of the genome DNA of the anthurium andraeanum; ISSR marker amplification: and (3) PCR reaction system: 2 XPCR Reagent (Tiangen Biochemical KT 207) 10. mu.l, ISSR primer (10. mu.M) 1. mu.l, template DNA 1. mu.l, ddH2O is added to 20 μ l. Reaction procedure: the PCR reaction program is: 5min at 94 ℃ for 40 cycles (94 ℃ for 1min, 56 ℃ for 30s, 72 ℃ for 30 s); finally, extension is carried out for 10 min at 72 ℃. Taking 10. mu.l of amplification product to contain 0.5. mu.g.mL-1EB was run in a 1.2% agarose gel, examined on a gel imaging system and photographed. ISSR primers are synthesized from Shanghai; results and analysis: the bands amplified by the primer ISSR of the progeny plants of the isolated preservation material are consistent with the control, no specific band is generated (as shown in figure 5), and the result shows that the caralluma albiflora preserved for 16 months by adding the good-preservation piece into the culture medium has no genetic variation on the molecular level.
The invention preserves the white palm test-tube plantlet by adding the comprehensive mineral substance vitamin complex tablets which are good for preserving adults, men and women in an in vitro preservation culture medium, the survival rate of the test-tube plantlet after being preserved for 16 months is still 100 percent, the plant grows normally, and the test-tube plantlet on the conventional culture medium can only survive for 5 months. In addition, the method of the invention can preserve the 16-month test-tube plantlet of the white palm, the later-generation plant characters of the test-tube plantlet are not different from ISSR amplification bands, the preservation material is stable, genetic variation can not occur, the sexual degeneration and disease infection can be prevented, and the excellent characteristics of the germplasm can be ensured. Compared with the traditional greenhouse planting and preservation, the method can save the preservation space, reduce the workload, reduce the production relay inoculation work, reduce the unnecessary labor and material cost in the production operation process and improve the economic benefit.
The above description is only an embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing the present invention.
Claims (1)
1. A method for in vitro preservation of anthurium andraeanum germplasm resources is characterized by comprising the following steps:
1) explant selection and sterilization: taking the stout and strong suckling buds of the white palms without the leaves as explants, firstly washing the explants cleanly under tap water, then disinfecting the surfaces of the explants for 45-60 seconds by using 72-75% alcohol, washing the explants for 1 time by using sterile water, then treating the explants for 10-12 minutes by using 0.1% mercuric chloride, washing the explants for 3 times by using the sterile water, and sucking the explants for later use by using sterile filter paper;
2) and (3) propagation of test-tube plantlets: cutting the explant treated in the step 1 into stem sections with nodes, inoculating the stem sections into a bud induction culture medium with the pH value of 5.8-6.0, culturing for 30 days under the conditions of the temperature of 27-29 ℃, the illumination intensity of 1200-1500 lux and the illumination time of 12 hours/day, and then transferring the germinated axillary buds to a proliferation culture medium with the pH value of 5.8-6.0, and performing propagation for 40 days under the conditions of the temperature of 27-29 ℃, the illumination intensity of 1200-1500 lux and the illumination time of 12 hours/day to obtain clump seedlings; wherein, the bud induction culture medium: 4g/L Huabao No. 1 +30g/L cane sugar +0.1mg/LTDZ +0.05mg/LNAA +5g/L agar powder; proliferation culture medium: 3g/L Huabao No. 1 +1g/L NH4NO3+0.1mg/LTDZ +0.05mg/LNAA +30g/L sucrose +5g/L agar powder;
3) and (3) storing test-tube plantlets: cutting the cluster seedlings obtained in the step 2 into single plants, and transferring the single plants into an in-vitro preservation culture medium with the pH value of 6.1-6.5 for preservation under the conditions of the temperature of 20-25 ℃, the illumination intensity of 1000-1200 lux and the illumination time of 8 hours/day; wherein the in vitro preservation medium: 3g/L Huabao No. 1 +1g/L NH4NO3+0.5 g/L adult man and woman comprehensive mineral substance vitamin complex tablet +30g/L sucrose +4g/L agar powder.
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