CN109673519B - Method for virus-free rapid propagation of lily bulbels - Google Patents
Method for virus-free rapid propagation of lily bulbels Download PDFInfo
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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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 provides a lily bulbil detoxification and rapid propagation method, which takes lily bulbil as a material, adopts a method of combining low-temperature and high-temperature alternate temperature treatment with 2 times of stem tip detoxification culture, can effectively remove lily virus diseases such as lily mottle virus, lily symptomless virus and the like, has the detoxification rate of more than 85 percent, has the stripped stem tip length of about 1.0-1.5 mm, has low requirements on instruments, is easy to operate, is easier to survive on the stem tip, has the survival rate of more than 90 percent, can obtain strong seedlings in a short time, and has higher efficiency. Further, the outer partial scale of the non-toxic seedling is utilized for tissue culture and rapid propagation, the adventitious bud induction coefficient and the proliferation rate are improved, the bud induction coefficient is 6.35, the adventitious bud proliferation rate is 3.37, a large number of high-quality virus-free seedlings can be effectively obtained, the cost is low, the efficiency is high, and a foundation is laid for industrial seedling culture.
Description
Technical Field
The invention belongs to the technical field of horticultural plant propagation, and particularly relates to a lily bulbil detoxification and rapid propagation method.
Background
Lilium tigrinum (Lilium lancifolium) belongs to Lilium of liliaceae, 8 essential amino acid contents in Lilium tigrinum are 7.05% higher than that of Lilium davidii of Lanzhou, and the total amino acid content is 11.24% higher than that of Lilium davidii of Lanzhou, so that the Lilium tigrinum has potential values of developing low fat, high nutrition and good health care functions, is one of main medicinal and edible plants in China, and reaches 4666.7 hm only in planting area of Longshan county in Hunan province in 20132The yield is over 10 million yuan, which is one of the agricultural support industries in this county. Seed balls or scales are mostly adopted for carrying out asexual propagation in lilium tigrinum production, but the asexual propagation has the defects of small propagation coefficient, continuous accumulation of viruses and the like, so that virus diseases are easy to occur to plants, and the virus diseases mainly comprise Lily Symptomless Virus (LSV), Lily mottle virus (LMoV), Cucumber Mosaic Virus (CMV) and the like. After the lily is infected with the virus, the lily is cultured,the plants are infected for the whole life and are damaged for a long time, the normal physiological functions of the plants are damaged, the yield is reduced, the quality is deteriorated, the development of the lilium tigrinum industry is severely restricted, the unit yield and the product quality of the medicinal lily are improved, and the method is a precondition for the development conception of the whole lily industry. Therefore, the urgent need is to obtain lily virus-free seedlings through virus-free treatment, cut off virus infection from the source and establish a corresponding rapid propagation system to achieve maximum benefit.
The ultra-low temperature detoxification technology is to dehydrate the pre-cultured material, then place the material in liquid nitrogen for 24 hours, then unfreeze the material, and finally culture the material. Although the ultra-low temperature detoxification time is short, the survival rate and the detoxification rate are both about 50% (agrimony, oriental lily virus detection and stem tip ultra-low temperature detoxification research [ D ]. fujian agriculture and forestry university, 2009), and the operation requirement is high, which is not favorable for large-scale production.
At present, the main method for overcoming the harm of virus diseases is to use stem tip culture to produce virus-free seedlings, because the virus is easy to move through a vascular system, the vascular system of the stem tip is not developed completely, the virus can only transfer from cell to cell through a small amount of plasmodesmata just formed, and most viruses also need to modify the plasmodesmata by means of motor proteins so as to allow the virus nucleic acid to pass through. Therefore, the virus transmission rate in the shoot apical meristematic region is very slow, and the probability of obtaining a non-toxic shoot after tissue culture is also high (moweing. botany [ M ]. higher education Press, 2015). Simple stem tip culture is difficult to remove viruses, and multiple methods are generally adopted for combined detoxification, for example, after 65 ℃ high-temperature pretreatment is carried out on a tissue culture seedling for 30 min by using the radix cynanchi bungei and the like, stem tips of 0.2-0.5 mm are stripped for detoxification culture (application research of stem tip culture and heat treatment technology in lily detoxification, university academy of Shanxi agriculture, 2010, 30(6):528 and 532), but the operation of the treatment method of the tissue culture seedling is complicated, the detoxification effect is not ideal, the survival rate of the detoxification material is low, and the detoxification material is difficult to obtain in a short time; liu Yan Ni and the like use perfume Bai to treat for 4 weeks at the high temperature of 32 ℃, then cut 0.3-0.6 mm stem tips and culture the stem tips on the virus oxazole with the concentration of 15mg/L (study on lily detoxification technology [ J ]. Ningxia agriculture and forestry science 2015,56(11): 12-14); after high-temperature heat treatment at 38 ℃ for 20 days such as Zhongxiao, the stem tips of 0.1-0.3 mm are stripped and cultured, and the seedling rate is 36.7% (study on the detoxification method of 3 major viruses of lilium tigrinum, Hunan agricultural science, 2016 (10): 7-10). However, the stem tips stripped by the method are small and are smaller than 0.8mm, the stem tips are not easy to strip, instruments and equipment such as a dissecting mirror are needed, the operation is complex, and the regeneration rate is low. Because the smaller the stripped stem tip is, the higher the detoxification rate is, but the higher the death rate is, the lower the seedling rate is; on the contrary, the death rate is greatly reduced, but the detoxification rate is lower or the aim of detoxification cannot be achieved. Therefore, the method overcomes the contradiction between the two methods, and the method becomes the bottleneck of plant detoxification production by improving the regeneration rate and the detoxification rate.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a lily bulbil detoxification and rapid propagation method, which solves the problems of complex operation, low regeneration rate, low seedling rate or poor detoxification effect of the existing detoxification method.
In order to achieve the purpose, the invention adopts the following scheme: a lily bulbil detoxification and rapid propagation method specifically comprises the following steps:
1) cleaning lily bulbels, soaking the lily bulbels in a washing powder solution for 5-20 min, washing the lily bulbels under running water for 1-2 h, and airing the lily bulbels for later use;
2) placing the pretreated lily bulbels at a low temperature of 4 ℃ for 3-4 days, and then placing the lily bulbels at a high temperature of 38 ℃ for 3-4 days, so that the temperature is alternately treated and the lily bulbels are continuously treated for 24-32 days; then, removing the scale on the outermost layer of the bulbil and then disinfecting; the illumination intensity during the high-temperature treatment is 1500lx-2000lx, the illumination time is 12h/d, and sterile water at 38 ℃ is sprayed to keep the humidity;
3) taking the sterilized bulbils obtained in the step 2), stripping visible stem tips, then inoculating the bulbils to an MS +1.5 g/L6-BA +0.2g/L NAA +10 mg/L ribavirin culture medium, carrying out dark culture for 7 days, then transferring the bulbils to an illumination environment with the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500lx-2000lx and the illumination time of 12h/d, culturing for 20-30 d, and inducing adventitious buds; continuously stripping the stem tip visible to the naked eye from the adventitious bud, inoculating the stem tip to an MS +1.5 g/L6-BA +0.2g/L NAA +10 mg/L ribavirin culture medium, carrying out dark culture for 7 d, and then transferring to an illumination environment with the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500lx-2000lx and the illumination time of 12h/d for culturing for 20-30 d to obtain a virus-free seedling;
4) and (3) continuously culturing the virus-free seedlings in the step 3) for 15-30 days, inoculating outer-layer scales of the virus-free seedlings to an adventitious bud induction culture medium, performing primary culture induction to generate adventitious buds, inoculating the adventitious buds to a subculture multiplication culture medium, performing subculture to obtain proliferated seedlings, and transferring the proliferated seedlings to a rooting culture medium to culture the proliferated seedlings to obtain rooted seedlings, namely the virus-free tissue culture seedlings.
Preferably, the sterilization is to soak the bulbils for 5-15 min by using a washing powder solution, wash the bulbils under running water for 1-2 h, place the bulbils on a sterile operating platform for ultraviolet irradiation for 20min, soak the bulbils in 75% ethanol for 30 s, treat the bulbils for 15min by using 0.2% mercuric chloride solution, and finally wash the bulbils for 3-5 times by using sterile water.
Preferably, the length of the stem tip stripped in the step 3) is 1.0-1.5 mm.
Preferably, the outer layer scale in the step 4) is an outer layer lower half scale.
Preferably, the adventitious bud induction culture medium is MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L +6-7g/L agar, and the pH value of the culture medium is 5.8-6.2.
Preferably, the subculture multiplication medium is MS + 2.5 mg/L6-BA + 0.3 mg/L NAA +30 g/L sucrose +6-7g/L agar, and the pH value of the medium is 5.8-6.2.
Preferably, the rooting medium is 1/2MS +0.5 mg/L6-BA +0.2mg/L NAA +30 g/L sucrose +6-7g/L agar, and the pH value of the medium is 5.8-6.2.
Preferably, the conditions of the adventitious bud induction culture, the subculture proliferation culture and the rooting culture are that the adventitious bud is cultured for 30-45 days under the environment conditions that the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 1500lx-2000lx and the sunshine time is 12-14 h/d.
The mechanism of the invention is as follows: based on the fact that no virus exists at the growth point of the plant or the concentration of the virus is very low, the plant growth point and the vicinity of the growth point are alternately treated at low temperature (4 ℃) and high temperature (38 ℃), wherein the low temperature of 4 ℃ can enable the virus to be in a dormant state, and the high temperature of 38 ℃ can passivate and inhibit the virus growth, so that after the temperature change treatment is repeated for 3-4 weeks, the virus content at the stem tip of the bulbus sprout is remarkably reduced and controlled, and the stem tip is continuously stripped for 2 times for virus removal culture, so that the virus removal efficiency of the stem tip is greatly improved, and the stem tip with the length of 1.0-1.5 mm or even longer can be directly stripped by naked eyes; meanwhile, the bulbil stem tip can be protected by low-temperature treatment at 4 ℃ after treatment at 38 ℃, so that the growth and development of the bulbil stem tip are facilitated, and the seedling rate is improved.
Compared with the prior art, the invention has the following beneficial effects:
1. the Lily bulblets are used as materials, and a method of low-temperature-high-temperature alternative temperature treatment and 2-time stem tip detoxification culture is adopted, so Lily mottle virus (LMoV), Lily Symptomless Virus (LSV) and other Lily virus diseases can be effectively removed, and the detoxification rate reaches more than 85%; the length of the stripped stem tip is about 1.0-1.5 mm, instrument equipment such as a dissecting mirror and the like is not needed, the stem tip with the length can be directly stripped by naked eyes, the operation is simple and convenient, and the cost is low; and the stem tip is easier to survive, the survival rate is up to more than 90 percent, strong seedlings can be obtained in a short time, and the efficiency is higher. The invention solves the problems of high difficulty in stem tip peeling, difficulty in survival and slow growth in the prior art.
2. According to the invention, the lower part of the outer-layer scale of the non-toxic seedling is utilized for tissue culture and rapid propagation, so that the adventitious bud induction coefficient and the multiplication rate are improved, the bud induction coefficient is 6.35, the adventitious bud multiplication rate is 3.37, high-quality virus-free seedlings can be effectively obtained in a large quantity, reliable seedling guarantee is brought for large-area planting, the yield increase and the quality improvement are ensured, the virus-free rapid propagation method is low in cost and high in efficiency, has a good application prospect, and lays a foundation for industrial seedling culture.
Drawings
FIG. 1 is an electrophoresis chart of LMoV detoxification validation after the first shoot tip culture; m is 5000 Marker, and 1-12 are respectively detection samples;
FIG. 2 is a LMoV detoxification validation electropherogram after a second shoot tip culture; m is 5000 Marker, and 1-12 are respectively detection samples;
FIG. 3 is an electrophoresis chart of LSV detoxification validation after the first stem tip culture; m is 2000 Marker, and 1-12 are respectively detection samples;
FIG. 4 is an electrophoresis chart of LSV detoxification validation after the second stem tip culture; m is 2000 Marker, and 1-12 are respectively detection samples.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. The experimental material used in the following examples was lilium tigrinum bulbil planted in the southern Sichuan area of Chongqing.
Example 1 detection of viruses in Lilium tigrinum materials
Extracting lily RNA: extracting multiple sample bulbels as material, extracting genome RNA by TIANGEN plant RNA miniprep kit (R4151), and storing at-20 deg.C.
And (3) cDNA synthesis: cDNA Synthesis Total volume was 20. mu.l per reaction, first strand cDNA was synthesized using TIANGEN plant cDNA Rapid reverse transcription kit (KR 116), and the reaction reagents included 2. mu.l total RNA, 2. mu.l 5 Xg DNA Buffer, RNase-freeddH2O6. mu.l, incubating at 42 ℃ for 5min, and standing on ice; 10 XKing RT Buffer 2, FastKing RT Enzyme Mix 1. mu.l, FQ-RT Primer Mix 2. mu.l, RNase-Free ddH were added2O5. mu.l, reaction program 42 ℃ incubation for 30 min, 70 ℃ inactivation for 5 min.
And (3) PCR amplification: PCR reagent selects and uses Dalianbao biological products (A)RR001A) The PCR reaction total volume is 20 mu l, and the method specifically comprises the following steps: mu.l of reverse transcribed synthetic cDNA, 10 XBuffer (20 mM Mg)2+) Mu.l of 2. mu.l, 2.5 mM/l dNTP 2. mu.l, 0.5. mu.l of each of 10. mu.M/l forward and reverse primers, 0.2. mu.l of 5U/. mu.l Taq DNA polymerase, 13.8. mu.l ddH2O; the amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 52-54 ℃ for 30 s, and extension at 72 ℃ for 30 s for 38 cycles; extending for 10min at 72 ℃; storing at 12 deg.C.
Specific primer pairs of LMoV, LSV and CMV are designed through a lily virus CP (coat protein) gene sequence, and the sizes of PCR products amplified respectively are 623bp, 439bp and 255 bp. Wherein the specific sequence is shown as follows:
LMoV-F：5’-GTTCCAGGCAAATGAGACACTC-3’
LMoV-R：5’-GTTCCAGGCAAATGAGACACTC-3’
LSV -F：5’- ATGAAGGTTGGCGTCGTAT -3’
LSV -R：5’- CCTCAGCAGAAGTGGGTC -3’
CMV -F：5’- TTGCGTTTCGTCTACTGGATCT -3’
CMV -R：5’- CAAAGGTTGGGTGGTTAATGG -3’
PCR analysis shows that only LMoV and LSV viruses are detected in the experimental material, and CMV virus is not contained in the experimental material.
Example 2 detoxification of Lilium tigrinum bulblets
1) Pretreatment of lilium tigrinum bulbil
Selecting healthy and plump lily bulblets in the current year, cleaning dirt on the surfaces of the lilium tigrinum bulblets in tap water, soaking for 10min by using washing powder, washing for 1-2 hours in running water, and drying in the air.
2) Detoxification treatment of bulbil of lilium tigrinum
And (3) treating the dried bulbils at a low temperature of 4 ℃ for 3-4 days, then treating the bulbils at a high temperature of 38 ℃ for 3-4 days, wherein the illumination intensity is 1500lx-2000lx, the illumination time is 12h/d, spraying sterile water at 38 ℃ to keep the humidity of the bulbils, and carrying out alternate treatment at the temperature for 24-32 days continuously.
3) Sterilizing bulbil of Lilium tigrinum and Bulbus Lilii
Removing the outermost layer of scales from the treated bulbule, soaking with washing powder for 10min, washing under running water for 1-2 h, placing in a sterile operating table, irradiating with ultraviolet rays for 20min, soaking with 75% ethanol for 30 s, treating with 0.2% mercuric chloride solution for 15min (submerging and shaking), and washing with sterile water for 3-5 times.
4) First shoot tip culture of Plumbum Preparatium bulblet
The sterilized bulbils are taken and dried by using a sterilized filter paper, and then the stem tips of about 1.2mm are stripped by naked eyes by using a scalpel, a pair of tweezers and a dissecting needle. Inoculating the seeds into a solid culture medium (MS +1.5 g/L6-BA +0.2g/L NAA +10 mg/L ribavirin) according to a bottle-by-bottle specification, culturing for 7 d in dark, transferring to a light environment with the culture temperature of 25 +/-2 ℃, the light intensity of 1500lx-2000lx and the sunshine time of 12h/d, culturing for 30d, and inducing adventitious buds. After 30d, the stem tip seedling rate was counted, and the stem tip seedling rate = the number of seedlings generated/(total number-number of contaminations), and the results were found to be shown in table 1. The stem tip seedlings thus cultured were used as a material, and 50 plants were randomly picked and tested for viruses according to the method described in example 1, and the results are shown in FIGS. 1 and 3.
TABLE 1 Stem tip seedling Rate
| Total number of stem tips | Number of contaminations | Number of emerged seedlings | Stem tip seedling rate |
| 50 | 6 | 40 | 90.9% |
| 50 | 9 | 37 | 90.2% |
| 50 | 8 | 38 | 90.5% |
| 50 | 5 | 40 | 88.9% |
As can be seen from Table 1, the seedling rate of the stem tip of about 1.2mm for induction culture is as high as more than 90%, and the seedling rate is greatly improved.
TABLE 2 detoxification rate of first shoot tip culture
| Virus | Total number of detections | Number of undetected | Rate of detoxification |
| LSV | 50 | 26 | 52% |
| LMOV | 50 | 22 | 44% |
As can be seen from fig. 1 and fig. 3, the target bands of LSV or LMOV could not be amplified, i.e. LSV or LMOV was detoxified, and the statistics are shown in table 2, after the first shoot tip culture, the detoxication rate of LSV was 52%, and the detoxication rate of LMOV was 44%.
5) Second shoot tip culture of the Plumbum Preparatium bulblet
Taking the adventitious bud induced in the step 4), and peeling off the stem tip of about 1.0-1.5 mm by using a scalpel, a forceps and a dissecting needle under naked eyes. Inoculating the seeds into a solid culture medium (MS +1.5 g/L6-BA +0.2g/L NAA +10 mg/L ribavirin) according to a bottle-by-bottle specification, culturing in dark for 7 d, and culturing in an illumination environment with the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500lx-2000lx and the illumination time of 12h/d for 30 d. The stem tip seedlings thus cultured were used as the material, and 50 plants were randomly picked and tested for viruses according to the method described in example 1, and the results are shown in FIGS. 2 and 4.
TABLE 3 detoxification rate of second shoot tip culture
| Virus | Total number of detections | Number of undetected | Rate of detoxification |
| LSV | 50 | 44 | 88% |
| LMOV | 50 | 43 | 86% |
As can be seen from fig. 2 and 4, the LSV or LMoV target band is not amplified, i.e. the LSV or LMoV is detoxified, the statistics is shown in table 3, after the second stem tip culture, the LSV detoxification rate is 88%, the LMoV detoxification rate is 86%, and the stem tip seedling without virus is the detoxified seedling.
Example 3 tissue culture Rapid propagation of detoxified Plumbum Preparatium seedlings
1) Adventitious bud induction:
continuously culturing the virus-free seedlings in the embodiment 2 for 15-30 days, transversely cutting an outer layer scale into an upper part and a lower part, and respectively inoculating the upper part, the lower part and an integral scale into an adventitious bud induction culture medium of MS +6-BA + NAA + sucrose (30 g/L), wherein the concentration of 6-BA is 0.5mg/L, 1 mg/L, 1.5mg/L and 2 mg/L; the concentration of NAA was 0.05 mg/L and 0.2mg/L, respectively. 3 lower scales are placed on each bottle of culture medium, and 10 bottles of culture medium are placed on each concentration gradient. The induction culture conditions are that the culture temperature is 25 +/-2 ℃, the humidity is 60% -70%, the illumination intensity is 1500lx-2000lx, and the illumination time is 12-14 h/d. The total number of shoots, rate of shoots, induction coefficient (average induced scale) of the material were counted at 45d, induction coefficient = total number of adventitious shoots/total number of germinated scales (slice). The results are shown in tables 4 to 6.
TABLE 4 Medium optimization of partial scale on outer layer
| 6-BA concentration (mg/L) | NAA concentration (mg/L) | Total number of scales (sheet) | Total number of budding scales (pieces) | Rate of induction | Number of adventitious buds | Coefficient of |
| 1 | 0.05 | 30 | 10 | 33.3% | 13 | 1.33 |
| 1 | 0.2 | 30 | 7 | 23.3% | 16 | 2.24 |
| 1.5 | 0.05 | 30 | 9 | 30.0% | 25 | 2.78 |
| 1.5 | 0.2 | 30 | 12 | 40.0% | 35 | 2.91 |
| 2 | 0.05 | 30 | 7 | 23.3% | 19 | 2.71 |
| 2 | 0.2 | 30 | 14 | 46.7% | 28 | 2.02 |
TABLE 5 Medium optimization of the lower outer partial scale
| 6-BA concentration (mg/L) | NAA concentration (mg/L) | Total number of scales (sheet) | Total number of budding scales (pieces) | Rate of induction | Number of adventitious buds | Coefficient of |
| 1 | 0.05 | 30 | 19 | 63.3% | 43 | 2.26 |
| 1 | 0.2 | 30 | 21 | 70.0% | 66 | 3.14 |
| 1.5 | 0.05 | 30 | 22 | 73.3% | 70 | 3.18 |
| 1.5 | 0.2 | 30 | 26 | 86.7% | 165 | 6.35 |
| 2 | 0.05 | 30 | 25 | 83.3% | 80 | 3.20 |
| 2 | 0.2 | 30 | 27 | 90.0% | 69 | 2.56 |
TABLE 6 Medium optimization of outer intact scales
| 6-BA concentration (mg/L) | NAA concentration (mg/L) | Total number of scales (sheet) | Total number of budding scales (pieces) | Rate of induction | Number of adventitious buds | Coefficient of |
| 1 | 0.05 | 30 | 21 | 70.0% | 55 | 2.62 |
| 1 | 0.2 | 30 | 17 | 56.7% | 43 | 2.53 |
| 1.5 | 0.05 | 30 | 23 | 76.7% | 68 | 2.95 |
| 1.5 | 0.2 | 30 | 28 | 93.3% | 106 | 3.79 |
| 2 | 0.05 | 30 | 20 | 66.7% | 71 | 3.55 |
| 2 | 0.2 | 30 | 21 | 70% | 41 | 1.95 |
And (4) analyzing results: the result of adventitious bud induction by different hormone ratios and different inoculation parts shows that the total rate of adventitious bud induction of the outer lower half-layer scale is higher than that of the outer whole-flap (complete) scale and the outer upper half-layer scale. Wherein the adventitious bud induced by the lower half scale of the outer layer in the culture medium of MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L is the most, the adventitious bud induction coefficient is 6.35, and the induction rate is 86.7%.
2) Multiplication subculture by using adventitious buds of virus-free seedlings
Respectively inoculating the adventitious buds induced in the step 1) into secondary multiplication culture media of MS (1/2 MS) +6-BA + NAA 0.3 mg/L + sucrose 30g/L, wherein the concentrations of 6-BA are 0.5mg/L, 1.5mg/L and 2.5 mg/L respectively; 5 adventitious buds are placed on each bottle of culture medium, and 10 bottles of culture medium are placed on each concentration gradient. Induction culture conditions: culturing for 30-45 d under the illumination conditions that the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 1500lx-2000lx and the sunshine time is 12-14 h/d to obtain the proliferated seedlings. And counting the total number of the proliferated buds and the proliferation coefficient of the material at 45 d. Proliferation factor = total number of proliferating shoots/total number of inoculated bulbs. The results are shown in Table 7.
TABLE 7 multiplication subculture medium optimization of adventitious buds
| Minimal medium | Concentration of NAA | 6-BA concentration | Number of inoculated buds | Total number of proliferated shoots | Coefficient of proliferation |
| MS | 0.3 | 0.5 | 50 | 92 | 1.84 |
| MS | 0.3 | 1.5 | 50 | 127 | 2.54 |
| MS | 0.3 | 2.5 | 50 | 169 | 3.37 |
| 1/2MS | 0.3 | 0.5 | 50 | 53 | 1.05 |
| 1/2MS | 0.3 | 1.5 | 50 | 88 | 1.76 |
| 1/2MS | 0.3 | 2.5 | 50 | 84 | 1.68 |
And (4) analyzing results: the analysis of the multiplication and subculture results of the adventitious buds by the MS and NAA proportioning concentration shows that the total number of the multiplication buds is the most in MS +6-BA2.5 mg/L + NAA 0.3 mg/L + sucrose 30g/L, and the multiplication coefficient reaches 3.37; when MS is halved, the number of proliferated shoots is significantly reduced.
3) Inducing rooting by using proliferated seedling
Inoculating the proliferated seedling obtained in the step 2) into 1/2MS +0.5 mg/L6-BA +0.2mg/L NAA +30 g/L sucrose rooting culture medium for culture, and inducing the culture conditions: culturing for 30-45 days under the illumination conditions that the culture temperature is 25 +/-2 ℃, the humidity is 60-70%, the illumination intensity is 1500lx-2000lx and the illumination time is 12-14 h/d until rooting, and obtaining the virus-free tissue culture seedling.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, although the applicant has described the present invention 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 to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention and shall be covered by the claims of the present invention.
Claims (7)
1. A lily bulbil detoxification and rapid propagation method is characterized by comprising the following steps:
1) cleaning lily bulbels, soaking the lily bulbels in a washing powder solution for 5-20 min, washing the lily bulbels under running water for 1-2 h, and airing the lily bulbels for later use;
2) placing the pretreated lily bulbels at a low temperature of 4 ℃ for 3-4 days, then placing the pretreated lily bulbels at a high temperature of 38 ℃ for 3-4 days, carrying out temperature alternate treatment for 24-32 days continuously, and then removing the outermost layer scales of the bulbels for disinfection; the illumination intensity during the high-temperature treatment is 1500lx-2000lx, the illumination time is 12h/d, and sterile water with the temperature of 38 ℃ is sprayed to keep the humidity;
3) taking the sterilized bulbils obtained in the step 2), stripping visible stem tips, then inoculating the bulbils to an MS +1.5 g/L6-BA +0.2g/L NAA +10 mg/L ribavirin culture medium, carrying out dark culture for 7 days, then transferring the bulbils to an illumination environment with the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500lx-2000lx and the illumination time of 12h/d, culturing for 20-30 d, and inducing adventitious buds; continuously stripping the stem tip visible to the naked eye from the adventitious bud, inoculating the stem tip to an MS +1.5 g/L6-BA +0.2g/L NAA +10 mg/L ribavirin culture medium, carrying out dark culture for 7 d, and then transferring to an illumination environment with the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500lx-2000lx and the illumination time of 12h/d for culturing for 20-30 d to obtain a virus-free seedling; the length of the stripped stem tip is 1.0-1.5 mm;
4) and (3) continuously culturing the virus-free seedlings in the step 3) for 15-30 days, inoculating outer-layer scales of the virus-free seedlings to an adventitious bud induction culture medium, performing primary culture induction to generate adventitious buds, inoculating the adventitious buds to a subculture multiplication culture medium, performing subculture to obtain proliferated seedlings, and transferring the proliferated seedlings to a rooting culture medium to culture the proliferated seedlings to obtain rooted seedlings, namely the virus-free tissue culture seedlings.
2. A lily bulbil detoxification and rapid propagation method according to claim 1, wherein the disinfection is to soak the bulbil with a washing powder solution for 5-15 min, wash the bulbil under running water for 1-2 h, place the bulbil on an aseptic operation table for ultraviolet irradiation for 20min, then soak the bulbil with 75% ethanol by volume for 30 s, then treat the bulbil with 0.2% mercuric chloride solution by mass for 15min, and finally wash the bulbil with sterile water for 3-5 times.
3. The lily bulbil detoxification and rapid propagation method according to claim 1, wherein the outer layer scale of step 4) is the outer lower half scale.
4. The lily bulbil detoxification and rapid propagation method according to claim 1, wherein the adventitious bud induction culture medium is MS +6-BA 1.5mg/L + NAA 0.2mg/L + sucrose 30g/L +6-7g/L agar, and the pH value of the culture medium is 5.8-6.2.
5. The lily bulbil detoxification and rapid propagation method according to claim 1, wherein the subculture multiplication medium is MS + 2.5 mg/L6-BA + 0.3 mg/L NAA +30 g/L sucrose +6-7g/L agar, and the pH value of the medium is 5.8-6.2.
6. The lily bulbil detoxification and rapid propagation method according to claim 1, wherein the rooting medium is 1/2MS +0.5 mg/L6-BA +0.2mg/L NAA +30 g/L sucrose +6-7g/L agar, and the pH value of the medium is 5.8-6.2.
7. The lily bulbil detoxification and rapid propagation method according to claim 1, wherein the conditions of adventitious bud induction culture, subculture proliferation culture and rooting culture are culture for 30-45 days under the environment conditions of culture temperature of 25 +/-2 ℃, humidity of 60-70%, illumination intensity of 1500lx-2000lx and sunshine time of 12-14 h/d.
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