CN110024693B - Tissue culture and rapid propagation method by utilizing stem tip meristem of coix lacryma-jobi - Google Patents
Tissue culture and rapid propagation method by utilizing stem tip meristem of coix lacryma-jobi Download PDFInfo
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Abstract
The invention relates to a tissue culture and rapid propagation method by utilizing a coix lacryma-jobi stem tip meristem. The method takes stem tips as materials, and comprises the following specific operation steps: (1) disinfecting and germinating seeds; (2) disinfecting the stem tip; (3) inducing shoot apical meristem cluster buds; (4) rooting cluster buds and transplanting tissue culture seedlings. The tissue culture rapid propagation method adopts the stem tip meristem of the coix lacryma-jobi as the material to carry out tissue culture rapid propagation, directly induces the cluster buds without a callus induction process, obtains the tissue culture seedlings with high propagation coefficient and stable heredity by using the method, wherein the seed germination rate, the cluster bud induction rate and the cluster bud rooting rate can respectively reach 45.3 percent, 76.9 percent and 100 percent, and can be popularized and planted in production.
Description
Technical Field
The invention belongs to the technical field of plant biotechnology, and particularly relates to a tissue culture and rapid propagation method by utilizing a coix lacryma-jobi stem tip meristem.
Background
Coix lachryma-jobi L is a medicinal and edible plant, contains abundant vitamins, polysaccharides, amino acids and phenols, and has high economic value. Guizhou is the largest province of China in coix production and accounts for 1/3 of the coix yield all over China. However, the breeding mode of the coix belongs to cross pollination, the filial generation is unstable, the character separation is serious, meanwhile, the coix planted in the open air is susceptible to diseases such as smut, leaf blight, leaf spot and the like, so that the yield and the quality of the coix are reduced, and the development of the coix industry is seriously restricted.
Tissue culture rapid propagation is carried out by using the stem tip meristem of the coix as a material, and cluster buds are directly induced without callus induction process, so that the tissue culture rapid propagation method has the advantages of high propagation coefficient, no influence of seasons, stable properties, low cost and the like. At present, no report related to tissue culture and rapid propagation technology by utilizing stem tip meristem of coix lacryma-jobi is seen.
Disclosure of Invention
The invention provides a tissue culture rapid propagation method for inducing cluster buds and roots by utilizing a stem tip meristem of coix, which can obtain a large amount of coix tissue culture seedlings with stable heredity in a short time and provide technical reference for developing a new coix variety propagation mode.
A tissue culture and rapid propagation method by utilizing a coix seed stem tip meristem comprises the following steps:
(1) tissue culture and disinfection of seeds and stem tips: selecting complete and full seeds without insect eyes for disinfection treatment, cutting stem tips after the seeds germinate, carrying out stem tip disinfection treatment in a super clean workbench, cutting off the stem tip meristem and parts of 0.3-0.5cm at the upper end and the lower end of the stem tip meristem, and then vertically inoculating the stem tip meristem in a cluster bud induction culture medium, wherein the vertical inoculation is that the morphological upper end of the stem tip meristem is upwards inoculated; (2) removing the superiority of the top of the stem tip: when the stem tip grows to 2-3cm, cutting off 0.3-0.5cm part of the upper and lower ends of the meristem of the stem tip, vertically inoculating in the cluster bud induction culture medium again, and repeating the step for 2-3 times.
(3) Rooting induction of cluster buds and transplanting of tissue culture seedlings: cutting off the cluster buds when the cluster buds grow to about 3-5cm, inoculating the cluster buds into a cluster bud rooting culture medium, and hardening and transplanting after the root system is strong;
the cluster bud induction culture medium comprises: MS +9.0mg.L_16-BA+0.5mg.L-1NAA, pH 5.80; the rooting culture medium of the cluster buds comprises: MS +0.5mg.L-1NAA，pH＝5.80。
The MS minimal medium formula comprises 4.4443g/L MS powder, 30g/L sucrose and 7g/L agar.
The seed disinfection treatment method in the step (1) comprises the following steps: selecting complete and plump seeds without wormholes, putting the seeds into a 1% detergent solution for rubbing and washing to whiten, washing the detergent with tap water, carrying out boiling water bath for 3-5s, immediately putting the seeds into a 50% carbendazim solution for soaking for 7-8h, taking out the seeds, and then washing the seeds with tap water.
The stem tip disinfection treatment method in the step (1) comprises the following steps: soaking the stem tip in 1% detergent solution for 10min, washing with tap water for 30min, sterilizing with 75% alcohol for 30-40s in a super clean bench, washing with sterile water for 4 times, sterilizing with 0.2% mercuric chloride for 7-8min, washing with sterile water for 4 times, and finally sucking surface water with sterile absorbent paper for inoculation;
in the step (3), the seedling hardening time is 2-3 days, and the matrix used for transplanting is vermiculite and nutrient soil which are uniformly mixed in a ratio of 1: 4.
The seed germination condition is dark culture at 28 ℃ for 96 h.
In the culture processes of the steps (2) and (3), the illumination intensity is 2000lx, the illumination is 12h every day, and the temperature is 24-28 ℃.
The invention takes the shoot apical meristem as the material and contains 9.0mg.L-16-BA and 0.5mg.L-1Shoot apical meristem culture was performed in MS medium of NAA. Cutting off the upper 0.3-0.5cm part of the meristem of the stem tip for multiple times to remove the top advantage of the stem tip and promote the growth of lateral buds; meanwhile, the part of 0.3-0.5cm below the meristem of the stem tip is cut off to maintain the physiological state and the development age of the stem tip, so that the higher morphogenetic capacity of the tissue culture is ensured in the tissue culture process, a large number of cluster buds are differentiated, and the cluster buds are induced to root to obtain the tissue culture seedling. Compared with the open-air culture, the tissue culture seedling obtained by the tissue culture rapid propagation method has stable heredity and is not easy to be infected by germs such as smut, leaf blight, leaf spot and the like during the seedling period; compared with tissue culture regeneration, the method has the advantages of direct induction to generate cluster buds without callus induction, shortened seedling time by about 30 days, high propagation coefficient and transplanting survival rate, no limitation of seasons and the like. By using the method, the seed germination rate, the cluster bud induction rate and the cluster bud rooting rate can respectively reach 45.3 percent, 76.9 percent and 100 percent.
The tissue culture rapid propagation method utilizes the stem tip meristem of the coix as a material to carry out tissue culture rapid propagation, directly induces cluster buds without a callus induction process, and has the advantages of high propagation coefficient, no influence of seasons, stable properties, low cost and the like. The invention successfully establishes an efficient stem tip meristem tissue culture rapid propagation system, and can be popularized and planted in production.
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FIG. 1 is a diagram of tissue culture and rapid propagation of a stem tip meristem of Coix lacryma-jobi.
Wherein, A: selecting a part of a shoot apical meristem; b: cutting off 0.3-0.5cm stem tip at upper and lower ends of meristem part of stem tip for the first time; c: cutting off 0.3-0.5cm stem tip at the upper and lower ends of the meristem part of the stem tip for the second time; d: cutting off 0.3-0.5cm stem tip at the upper and lower ends of the meristem part of the stem tip for the third time; e: after the top advantages of the stem tip are removed, inducing cluster buds for 60 days; f: inducing the cluster buds to root for 20 days; g: transplanting the seedlings for 40 days.
FIG. 2 shows the 1-6 generation tissue culture seedlings of Job's tears.
FIG. 3 detection of SSR molecular marker genetic stability of Coix lacryma-jobi subculture.
Wherein, A-D is the verification of the PCR electrophoretogram of the subculture by four primers (GBssrJT32, GBssrJT136, GBssrJT149 and GBssrJT 198); 1-6 is the number of subcultures; a to c are the number of repetitions.
Detailed Description
The present invention will be described in further detail with reference to examples.
Culture medium and culture conditions: the culture medium for inducing the cluster buds comprises: MS +9.0mg.L_16-BA+0.5mg.L-1NAA; the rooting culture medium of the cluster buds comprises: MS +0.5mg.L-1NAA. The illumination intensity is 2000lx at each stage of the culture, the illumination is carried out for 12h every day, and the temperature is 24-28 ℃.
Example 1 establishment of tissue culture and Rapid propagation System Using Stem tip meristem of Coix lacryma-jobi
1.1 seed disinfection: selecting complete and plump seeds without insect eyes, putting the seeds into a 1% detergent solution for rubbing and washing to whiten, washing the detergent with tap water, carrying out boiling water bath for 3-5s, immediately putting the seeds into a 50% carbendazim solution for soaking for 6h, taking out the seeds, washing the carbendazim with the tap water, and putting the seeds into a seedling raising tray for dark culture at 28 ℃ for 96 h.
As can be seen from Table 1, the effect of the coix seed treatment by the boiling water bath is better than that of the coix seed treatment at normal temperature, the fungal contamination of the coix seed is reduced along with the increase of 50 percent carbendazim treatment time, the fungal contamination is not seen in 6h and 9h, the survival rate is reduced along with the increase of 50 percent carbendazim treatment time, the best combination is that the coix seed treatment is carried out by the boiling water bath for 3 to 5s, the 50 percent carbendazim treatment is carried out for 6h, the contamination rate is 0, and the survival rate is 45.32 percent.
TABLE 1 results of Coix seed treatment with different combinations of disinfectants
1.2 tissue culture and disinfection of stem tips: soaking the stem tip in 1% detergent solution for 10min, washing with tap water for 30min, sterilizing with 75% alcohol for 30-40s in a super clean bench, washing with sterile water for 4 times, sterilizing with 0.2% mercuric chloride for 7-8min, washing with sterile water for 4 times, and drying with sterile absorbent paper for subsequent experiments. As shown in fig. 1 at a.
As can be seen from Table 2, the stem tip contamination rate and the survival rate of the coix seed are gradually reduced as the disinfection time of 75% alcohol and 0.2% mercury bichloride is increased. The optimal disinfection conditions are 3-3 groups, namely 75% alcohol treatment for 30s, 0.2% mercury bichloride treatment for 7min, the pollution rate is 8%, and the survival rate is 92%.
TABLE 2 treatment results of stem tips of Coix lacryma-jobi with different combinations of disinfectants
1.3 cluster bud induction: cutting off the stem tip meristem and the upper and lower 0.3-0.5cm parts, vertically inoculating the cut stem tip meristem in the multiple shoot induction culture medium (the upper part of the stem tip meristem is upward morphologically), cutting off the upper and lower 0.3-0.5cm parts of the stem tip meristem when the shoot grows to 2-3cm, and vertically inoculating the cut stem tip meristem in the multiple shoot induction culture medium (the upper part of the stem tip meristem is upward morphologically). This step was repeated 2-3 times. The culture medium for inducing cluster buds contains 9.0mg.L_16-BA and 0.5mg.L-1MS medium of NAA. As shown in figure 1B, C, D, E.
As can be seen from Table 3, when the upper and lower ends of the stem apex meristem of Coix lacryma-jobi were cut at 0.3-0.5cm for the 4 th time, the meristem growth coefficient of the treated stem apex increased with the increase of the concentration of 6-BA, which was most significant in 2-3 groups, and when 0.5mg/L NAA and 9mg/L6-BA were added, the stem apex growth coefficient reached 2.9, and the germination induction rate was 76.9%, which was the best in combination.
TABLE 3 Cluster bud Induction results with different combinations of hormone concentrations
1.4 subculture and rooting induction of cluster buds: cutting when the cluster bud grows to about 3-5cm, inoculating 0.5mg.L cluster bud-1Inducing rooting in MS culture medium of NAA and subculturing in MS culture medium of 0.5mg/LNAA +9 mg/L6-BA. As shown in fig. 1F and 2.
The rooting of the secondary buds is induced by utilizing auxin, and the induction result is shown in Table 4, the rooting rate, the root hair density, the root length and the root thickness of the secondary buds in an MS + NAA culture medium are better than those of 1/2MS, MS and MS + NAA, the rooting number of the MS culture medium containing 0.5mg/L NAA reaches 3.9, the rooting rate is 100%, and the combined rooting is optimal.
TABLE 4 Cluster bud rooting induction results with different auxin concentration combinations
1.5 transplanting tissue culture seedlings: after the tissue culture seedlings growing into thick and strong root systems are uncapped and water is added to harden the seedlings for 2 to 3 days, the seedlings are transplanted, the matrix used for transplanting is vermiculite and nutrient soil (1: 4) are uniformly mixed, and the survival rate is 95 percent. As shown at G in fig. 1.
1.6 detecting the genetic stability of the subculture seedlings: the selected detection material is a subculture seedling generated by tissue culture and rapid propagation of a stem tip tissue, and whether the strains of 6 generations of subcultures are stable in heredity is detected by using a molecular marking method as follows:
(1) the DNA extraction method comprises the following steps: adding 50mg of subculture seedling powder ground by liquid nitrogen into 1.5mL centrifuge tube of 20uL mercaptoethanol and 1000uL 2% CTAB, incubating in 65 ℃ water bath for 1h, reversing each 10min for mixing, centrifuging at 13200rpm for 7min, transferring supernatant into a new centrifuge tube, adding phenol-chloroform with equal volume, shaking, centrifuging at 13200rpm for 10min, transferring supernatant into a new centrifuge tubeAdding equal volume of chloroform into a centrifuge tube, shaking up continuously, centrifuging at 13200rpm for 5min, transferring the supernatant into a new centrifuge tube, repeating for 1 time, adding 0.6 times volume of isopropanol, shaking up continuously, standing in a refrigerator at-20 deg.C for 20min, centrifuging at 13200rpm for 10min, discarding the supernatant, adding 1ml of 75% ethanol, centrifuging at 13200rpm for 3min, discarding the supernatant, adding 1ml of 75% ethanol, centrifuging at 13200rpm for 3min, discarding the supernatant, drying the centrifuge tube in an oven at 37 deg.C, taking out after ethanol is completely volatilized, adding 45-50uL of Rnaase H containing RNAase H2O, storing at-20 ℃.
(2) SSR primer information:
and (3) PCR reaction system: 10 μ L of PCR system contained 1.0 μ L of 100ng/uL DNA, 0.2 μ L of primer, 1 μ L of 2.5mM dNTP, 1 μ L of 10 XTaq buffer, 5mM Mg2+0.8 μ L, 5U/. mu.L Taq enzyme 0.05 μ L, ddH2O 5.75μL。
And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30S, denaturation at 56-60 deg.C for 30S, and denaturation at 72 deg.C for 30S, and circulation for 35 times, electrophoresis at 72 deg.C for 7min, at 12 deg.C for infinity, 2.0% agarose gel, and 90V.
GBssrJT32, GBssrJT136, GBssrJT149 and GBssrJT 1984 with good polymorphism and strong detectability are used for carrying out PCR polymorphism detection on the DNA of the tissue culture seedlings of the 6 generations of successive generations by SSR primers, and gel electrophoresis detection results show that (figure 3) bands amplified by the DNA of successive generations of seedling strains are all at the same position, which indicates that the polymorphism of the tissue culture seedlings of the 6 generations of successive generations is consistent with that of parent strains, and simultaneously indicates that the tissue culture rapid propagation method of the meristematic tissue of the coix lacryma can be applied to production.
Claims (7)
1. A tissue culture and rapid propagation method by utilizing a coix seed stem tip meristem comprises the following steps:
(1) tissue culture and disinfection of seeds and stem tips: selecting complete and full seeds without insect eyes for seed disinfection treatment, cutting stem tips after the seeds germinate, carrying out stem tip disinfection treatment in a super-clean workbench, cutting off the stem tip meristem and parts of 0.3-0.5cm at the upper end and the lower end of the stem tip meristem, and then vertically inoculating the stem tip meristem in a cluster bud induction culture medium, wherein the vertical inoculation is that the morphological upper end of the stem tip meristem is upwards inoculated;
(2) removing the superiority of the top of the stem tip: when the stem tip grows to 2-3cm, cutting off 0.3-0.5cm part of the upper and lower ends of the meristem of the stem tip, vertically inoculating in the cluster bud induction culture medium again, and repeating the step for 2-3 times;
(3) rooting induction of cluster buds and transplanting of tissue culture seedlings: cutting off the cluster buds when the cluster buds grow to about 3-5cm, inoculating the cluster buds into a cluster bud rooting culture medium, hardening and transplanting after the root system is strong;
the cluster bud induction culture medium comprises: MS +9.0mg. L-1 6-BA+0.5mg·L-1NAA, pH 5.80; the rooting culture medium of the cluster buds comprises: MS +0.5mg. L-1NAA，pH＝5.80。
2. The method of claim 1, wherein the MS minimal medium formulation is 4.4443g/L MS powder, 30g/L sucrose, and 7g/L agar.
3. The method of claim 1, wherein the step (1) seed disinfection treatment method comprises: selecting complete and plump seeds without wormholes, putting the seeds into a 1% detergent solution for rubbing and washing to whiten, washing the detergent with tap water, carrying out boiling water bath for 3-5s, immediately putting the seeds into a 50% carbendazim solution for soaking for 7-8h, taking out the seeds, and then washing the seeds with tap water.
4. The method according to claim 1, wherein the step (1) stem tip disinfection treatment method comprises: soaking the stem tip in 1% detergent solution for 10min, washing with tap water for 30min, sterilizing with 75% alcohol for 30-40s in a super clean bench, washing with sterile water for 4 times, sterilizing with 0.2% mercuric chloride for 7-8min, washing with sterile water for 4 times, and absorbing surface water with sterile absorbent paper for inoculation.
5. The method according to claim 1, wherein in the step (3), the seedling hardening time is 2-3 days, and the matrix used for transplanting is vermiculite and nutrient soil which are mixed uniformly in a ratio of 1: 4.
6. The method of claim 1, wherein the seed is germinated in a dark culture at 28 ℃ for 96 hours.
7. The method according to claim 1, wherein the cultivation process of the steps (2) and (3) comprises the illumination intensity of 2000lx, the daily illumination time of 12h and the temperature of 24-28 ℃.
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