CN113711918B - Tissue culture rapid propagation method of Chinese pine - Google Patents
Tissue culture rapid propagation method of Chinese pine Download PDFInfo
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Abstract
The invention provides a tissue culture rapid propagation method of Pinus pumila, which comprises the following steps: obtaining yellow mountain pine seeds; and removing the testa; carrying out combined disinfection by adopting 75% alcohol and 1% sodium hypochlorite solution; then, sucking water, and picking out a seed embryo; inoculating the explant to a callus induction culture medium for callus induction; selecting callus, transferring the callus to an adventitious bud induction culture medium for adventitious bud induction; transferring the adventitious bud to an elongation culture medium for adventitious bud elongation culture; selecting an adventitious bud of the yellow mountain pine with the height of 1-2 cm, removing a needle leaf at the base of the bud, and transferring the bud to a rooting culture medium for direct rooting culture. The invention has the advantages that: firstly, embryo of the seed of the yellow mountain pine is taken as an experimental material, and a yellow mountain pine tissue culture and rapid propagation technology is established; and the method has the advantages of simple operation, low cultivation cost, high propagation efficiency, small influence of natural environment, and good market and industrial application prospects.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method of Pinus pumila.
Background
The yellow mountain pine is a special conifer species in China, and is an important species for mountain greening, forestation and ecological restoration in the subtropical high-altitude area of the east of China. The yellow mountain pine has the advantages of strong ecological succession function, strong adaptability, high ecological environmental protection function and unique natural landscape effect. Pine is a harmful host of pine wood nematodes, pine wood nematode disease is introduced into China, and the pine wood nematode disease has spread to 726 county-level administrative districts of 18 provinces (autonomous regions, direct prefectures) in China by 12-31 months in 2020, and seriously threatens the ecological safety, biological safety and economic development of China. The yellow mountain pine is one of hosts of pine wood nematode diseases, and in 2004, in the elevation forest land near 700m high in the Anqing Dalong mountain forest land, it is found for the first time that the pine wood nematode infects the yellow mountain pine in a natural state, and causes a plurality of yellow mountain pine plants to die, which causes great threat to the germplasm resources of the yellow mountain pine in the yellow mountain scenic region. However, no report is found on the research on the germplasm resource preservation and tissue culture rapid propagation system of the Pinus fulva.
Plant tissue culture is an important way for plant germplasm resources to be preserved. The plant tissue culture has the advantages of short growth cycle, high reproduction rate, convenient management and the like, and is favorable for industrial production and automatic control. By utilizing the plant tissue culture technology, the rare endangered plants can be rapidly propagated in a short time, so that the rare species can be preserved. Meanwhile, the technology can rapidly propagate a famous and excellent new variety to obtain a plant with excellent characteristics of the female parent.
Therefore, if the tissue culture and rapid propagation technology of the yellow mountain pine can be carried out by taking the embryo of the yellow mountain pine seed as an explant, the method has important significance in the aspects of yellow mountain pine germplasm resource preservation, genetic improvement, new variety cultivation, high-quality seedling propagation and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a tissue culture rapid propagation method of the Pinus pumila, which has the advantages of simple operation, low cultivation cost, high propagation efficiency, small influence by natural environment and good market and industrialized application prospects.
The invention adopts the following technical scheme to solve the technical problems:
a tissue culture and rapid propagation method of Pinus pumila comprises the following steps:
(1) pretreatment of explants:
drying the cones of the yellow mountain pine under natural illumination to obtain yellow mountain pine seeds; then, removing the testa of the Pinus massoniana seed;
(2) and (3) disinfection of explants:
sterilizing the seed of the Pinus sylvestris without the testa by combining 75% alcohol and 1% sodium hypochlorite solution; then, sucking water on sterile filter paper, and picking out a seed embryo to obtain a sterile explant required by inoculation;
(3) callus induction:
under the aseptic condition, inoculating the aseptic explant obtained in the step (2) on a callus induction culture medium to induce the callus; wherein, the formula of the callus induction culture medium is as follows: MS +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.2mg/L NAA, pH 5.8;
(4) induction and elongation of adventitious buds:
selecting callus, transferring the callus to an adventitious bud induction culture medium, and inducing adventitious buds; wherein the formula of the adventitious bud induction culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.05mg/L NAA, pH 5.8;
transferring the induced adventitious bud to an elongation culture medium after the adventitious bud grows out, and performing elongation culture on the adventitious bud; wherein, the formula of the elongation culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +0.1 mg/L6-BA +0.05mg/L NAA, pH 5.8;
(5) rooting culture:
selecting 1-2 cm-high Huangshan pine adventitious buds, removing coniferous leaves of bud bases, and transferring the buds to a rooting culture medium for direct rooting culture; wherein, the formula of the rooting culture medium is as follows: 1/2DCR +30g/L sucrose +7g/L agar powder +2mg/L IBA +0.05mg/L NAA, pH 5.8.
In a preferred embodiment of the present invention, in the step (1), the pinus sylvestris is obtained from a scenic spot of the yellow mountain of anhui.
In a preferred embodiment of the present invention, in the step (2), the specific process of performing combined disinfection by using 75% alcohol and 1% sodium hypochlorite solution is as follows: taking out the Pinus pumila seeds without the seed coats for later use; and then, in a superclean bench, cleaning the seeds with sterile water for 3-5 times, rinsing with 75% alcohol for 60s, soaking with 1% sodium hypochlorite solution for 10min, and finally washing with sterile water for 3-5 times.
In a preferred embodiment of the present invention, in the step (2), the seed is sterilized by a combination of 75% alcohol and 1% sodium hypochlorite solution, and the contamination rate of the seeds is 2%.
In a preferred embodiment of the present invention, in the step (3), when the induction culture is performed for 12-16 days, the callus formation of the explant is started, and the callus formation rate is 93.65-96.35%.
In a preferred embodiment of the present invention, in the step (4), the callus starts to grow adventitious buds after the induction culture for 25 to 35 days, and the average number of adventitious buds is 2.42 to 3.72.
In a preferred embodiment of the present invention, in the step (5), when the rooting culture is performed for 35-45 days, rooting is started, and the rooting rate is 28%.
As one of the preferable modes of the invention, the MS, DCR and 1/2DCR minimal medium are all the conventional mediums in the field.
In a preferred embodiment of the present invention, the callus induction, adventitious bud induction and elongation, and rooting culture are performed under the following conditions: the culture temperature is 25 +/-2 ℃, the relative humidity is 70-80%, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
As one of the preferable modes of the invention, the method further comprises the hardening-seedling transplanting step of the step (6); the hardening and transplanting process comprises the following steps:
after the rooting seedlings of the yellow mountain pine grow to develop root systems, opening a tissue culture seedling bottle cap, hardening the seedlings for 3-5 days, and keeping sufficient water in the culture medium in the period; then, mashing the culture medium in the tissue culture bottle by using a glass rod, and taking out the tissue culture seedling from the tissue culture bottle; then, washing the root culture medium with tap water, and transplanting the root culture medium into a flowerpot; and finally, placing the transplanted yellow mountain pine seedlings in a culture room for culture.
Compared with the prior art, the invention has the advantages that:
(1) the invention firstly uses the seed embryo of the yellow mountain pine seed as an experimental material to carry out the construction of a yellow mountain seed embryo disinfection system and the induction test of callus, adventitious bud and rooting, and finally establishes the tissue culture and rapid propagation technology of the yellow mountain pine; the method has the advantages of simple operation, low cultivation cost, high propagation efficiency, small influence of natural environment and good market and industrialization application prospect;
(2) the invention fills the blank of the in vitro rapid propagation technology of the yellow mountain pine, solves the bottlenecks of difficult asexual propagation and low seedling survival rate of the yellow mountain pine, greatly reduces the cost of purchasing seedlings, reduces the risks of poor afforestation effect and the like caused by uneven seed quality, realizes the large-scale planting of the yellow mountain pine and increases the forest farmer income.
Drawings
FIG. 1 is a graph of a seed of Pinus sylvestris with the testa removed in example 3;
FIG. 2 is a culture diagram of callus induction culture of the sterile seed embryos transferred to the induction medium in example 3;
FIG. 3 is the state diagram of the callus induced by the explants in example 3;
FIG. 4 is a culture diagram of callus transferred to adventitious bud induction medium for adventitious bud induction culture in example 3;
FIG. 5 is a diagram showing the state of adventitious buds outgrowth from callus in example 3;
FIG. 6 is a culture diagram of adventitious bud elongation culture in example 3 in which adventitious buds are transferred to an elongation medium;
FIG. 7 is a graph showing the rooting of adventitious buds in example 3;
FIG. 8 is a diagram of uncapping seedlings for tissue culture seedlings in example 3;
FIG. 9 is a state diagram after transplanting the rooted seedling to a flowerpot in example 3;
FIG. 10 is a graph showing the sterilization effect of different sterilization combinations on the seed embryo of Pinus sylvestris in example 4;
FIG. 11 is a graph showing the effect of different media on the rooting rate of adventitious buds in example 4.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The tissue culture rapid propagation method of the Pinus pumila comprises the following steps:
(1) pretreatment of explants:
drying the Huangshan pine cones collected from the Huangshan scenic spot of Anhui province under natural illumination to obtain the seeds of the Huangshan pine; then, the testa of the Pinus massoniana seed was removed and placed in a refrigerator at 4 ℃ for further use.
(2) And (3) disinfection of explants:
taking out the yellow mountain pine seeds with the seed coats removed from the refrigerator; then, in a superclean bench, cleaning the seeds with sterile water for 3 times, rinsing with 75% alcohol for 60s, soaking with 1% sodium hypochlorite solution for 10min, and finally rinsing with sterile water for 3 times; subsequently, water is sucked dry on sterile filter paper, and the embryo of the seed is picked out by a scalpel, thus obtaining the sterile explant required by inoculation.
(3) Callus induction:
under the aseptic condition, inoculating the aseptic explant obtained in the step (2) on a callus induction culture medium to induce the callus; when induction culture reached 12d, the explants began to callus.
Wherein, the formula of the callus induction culture medium is as follows: MS +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.2mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 23 ℃, the relative humidity is 70%, the illumination time is 14h/d, and the illumination intensity is 2000 Lx.
(4) Induction and elongation of adventitious buds:
selecting callus, transferring the callus to an adventitious bud induction culture medium, and inducing adventitious buds; when the induction culture is carried out for 25 days, adventitious buds begin to grow on the callus. Wherein the formula of the adventitious bud induction culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 23 ℃, the relative humidity is 70%, the illumination time is 14h/d, and the illumination intensity is 2000 Lx.
And transferring the induced adventitious bud to an elongation culture medium after the adventitious bud grows out, and performing elongation culture on the adventitious bud. Wherein, the formula of the elongation culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +0.1 mg/L6-BA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 23 ℃, the relative humidity is 70%, the illumination time is 14h/d, and the illumination intensity is 2000 Lx.
(5) Rooting culture:
selecting adventitious buds of the Pinus fulva with the height of 1cm, removing coniferous leaves at the base parts of the buds, and transferring the buds to a rooting culture medium for direct rooting culture; and when the rooting culture is carried out for 35d, rooting is started.
Wherein, the formula of the rooting culture medium is as follows: 1/2DCR +30g/L sucrose +7g/L agar powder +2mg/L IBA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 23 ℃, the relative humidity is 70%, the illumination time is 14h/d, and the illumination intensity is 2000 Lx.
(6) Hardening and transplanting seedlings:
gradually opening the cap of the tissue culture seedling after the rooting seedling of the yellow mountain pine grows to have a developed root system, hardening the seedling for 3 days, and keeping sufficient water in the culture medium in the period; then, mashing the culture medium in the tissue culture bottle by using a glass rod, and taking out the tissue culture seedling from the tissue culture bottle; then, washing the root culture medium with tap water, and transplanting the root culture medium into a flowerpot; and finally, placing the transplanted yellow mountain pine seedlings in a culture room for culture, and keeping soil moisture sufficient to ensure that the transplanted yellow mountain pine seedlings survive and start to grow.
Example 2
The tissue culture rapid propagation method of the Pinus pumila comprises the following steps:
(1) pretreatment of explants:
drying the Huangshan pine cones collected from the Huangshan scenic spot of Anhui province under natural illumination to obtain the seeds of the Huangshan pine; then, the testa of the Pinus massoniana seed was removed and placed in a refrigerator at 4 ℃ for further use.
(2) And (3) disinfection of explants:
taking out the yellow mountain pine seeds with the seed coats removed from the refrigerator; then, in a superclean bench, cleaning the seeds with sterile water for 5 times, rinsing with 75% alcohol for 60s, soaking with 1% sodium hypochlorite solution for 10min, and finally rinsing with sterile water for 5 times; subsequently, water is sucked dry on sterile filter paper, and the embryo of the seed is picked out by a scalpel, thus obtaining the sterile explant required by inoculation.
(3) Callus induction:
under the aseptic condition, inoculating the aseptic explant obtained in the step (2) on a callus induction culture medium to induce the callus; when induction culture reached 16d, the explants began to callus.
Wherein, the formula of the callus induction culture medium is as follows: MS, 30g/L of sucrose, 7g/L of agar powder, 1mg/L of 6-BA and 0.2mg/L of NAA, and the pH value is 5.8; the culture conditions were: the culture temperature is 27 ℃, the relative humidity is 80%, the illumination time is 16h/d, and the illumination intensity is 2500 Lx.
(4) Induction and elongation of adventitious buds:
selecting callus, transferring the callus to an adventitious bud induction culture medium, and inducing adventitious buds; when the induction culture is carried out for 35d, adventitious buds begin to grow on the callus. Wherein the formula of the adventitious bud induction culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 27 ℃, the relative humidity is 80%, the illumination time is 16h/d, and the illumination intensity is 2500 Lx.
And transferring the induced adventitious bud to an elongation culture medium after the adventitious bud grows out, and performing elongation culture on the adventitious bud. Wherein, the formula of the elongation culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +0.1 mg/L6-BA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 27 ℃, the relative humidity is 80%, the illumination time is 16h/d, and the illumination intensity is 2500 Lx.
(5) Rooting culture:
selecting 2 cm-high adventitious buds of Pinus densiflora, removing coniferous leaves at the bud base, and transferring to rooting culture medium for direct rooting culture; and when the rooting culture is carried out for 45 days, rooting is started.
Wherein, the formula of the rooting culture medium is as follows: 1/2DCR +30g/L sucrose +7g/L agar powder +2mg/L IBA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 27 ℃, the relative humidity is 80%, the illumination time is 16h/d, and the illumination intensity is 2500 Lx.
(6) Hardening and transplanting seedlings:
after the rooting seedlings of the yellow mountain pine grow to develop root systems, gradually opening the caps of the tissue culture seedlings, hardening the seedlings for 5 days, and keeping sufficient water in the culture medium in the period; then, mashing the culture medium in the tissue culture bottle by using a glass rod, and taking out the tissue culture seedling from the tissue culture bottle; then, washing the root culture medium with tap water, and transplanting the root culture medium into a flowerpot; and finally, placing the transplanted yellow mountain pine seedlings in a culture room for culture, and keeping sufficient soil moisture so that the transplanted yellow mountain pine seedlings survive and start to grow.
Example 3
The tissue culture rapid propagation method of the Pinus pumila comprises the following steps:
(1) pretreatment of explants:
drying the Huangshan pine cones collected from the Huangshan scenic spot of Anhui province under natural illumination to obtain the seeds of the Huangshan pine; next, the testa of the Pinus pumila seeds was removed (as shown in FIG. 1) and placed in a refrigerator at 4 ℃ for further use.
(2) And (3) disinfection of explants:
taking out the yellow mountain pine seeds with the seed coats removed from the refrigerator; then, in a superclean bench, cleaning the seeds with sterile water for 4 times, rinsing with 75% alcohol for 60s, soaking with 1% sodium hypochlorite solution for 10min, and finally rinsing with sterile water for 4 times; subsequently, water is sucked dry on sterile filter paper, and the embryo of the seed is picked out by a scalpel, thus obtaining the sterile explant required by inoculation.
(3) Callus induction:
inoculating the sterile explant obtained in the step (2) to a callus induction culture medium under a sterile condition, and inducing the callus (shown in figure 2); at induction culture until 14d, the explants began to callus (as shown in FIG. 3).
Wherein, the formula of the callus induction culture medium is as follows: MS +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.2mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the relative humidity is 75%, the illumination time is 15h/d, and the illumination intensity is 2250 Lx.
(4) Induction and elongation of adventitious buds:
selecting callus, transferring to adventitious bud induction culture medium, and inducing adventitious bud (shown in FIG. 4); when the induction culture is carried out for 30 days, adventitious buds begin to grow on the callus (as shown in FIG. 5). Wherein the formula of the adventitious bud induction culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the relative humidity is 75%, the illumination time is 15h/d, and the illumination intensity is 2250 Lx.
After the adventitious bud grows, the induced adventitious bud is transferred to an elongation medium, and elongation culture of the adventitious bud is performed (as shown in FIG. 6). Wherein, the formula of the elongation culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +0.1 mg/L6-BA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the relative humidity is 75%, the illumination time is 15h/d, and the illumination intensity is 2250 Lx.
(5) Rooting culture:
selecting an adventitious bud of the yellow mountain pine with the height of 1.5cm, removing a needle leaf at the base part of the bud, and transferring the bud to a rooting culture medium for direct rooting culture; rooting culture was started for 40 days (see FIG. 7).
Wherein, the formula of the rooting culture medium is as follows: 1/2DCR +30g/L sucrose +7g/L agar powder +2mg/L IBA +0.05mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the relative humidity is 75%, the illumination time is 15h/d, and the illumination intensity is 2250 Lx.
(6) Hardening and transplanting seedlings:
after the rooting seedlings of the yellow mountain pine grow to develop a developed root system, gradually opening the caps of the tissue culture seedlings, hardening the seedlings for 4 days, and keeping sufficient water in the culture medium (as shown in figure 8); then, mashing the culture medium in the tissue culture bottle by using a glass rod, and taking out the tissue culture seedling from the tissue culture bottle; then, the root culture medium is washed clean by tap water and transplanted into a flowerpot (as shown in fig. 9); and finally, placing the transplanted yellow mountain pine seedlings in a culture room for culture, and keeping sufficient soil moisture so that the transplanted yellow mountain pine seedlings survive and start to grow.
Example 4
This example is an analysis of the parameter selection in examples 1-2 above:
influence of different disinfection combinations on seed contamination rate
The seed from which the seed coat was removed was sterilized with 75% alcohol prepared in advance and 3% sodium hypochlorite, and different combinations of sterilization times were set (see table 1). Three gradients (30s, 60s and 90s) are respectively set for the disinfection time of 75% alcohol, and the disinfection time of 3% sodium hypochlorite is respectively set for 5min, 10min and 15 min. Subsequently, the water was blotted on sterile filter paper, and the seed embryos were picked with a scalpel and inoculated in MS medium without any hormone added.
TABLE 1 Disinfection of explants by different treatment combinations
After 1 week, the contamination rate of the embryos of each sterilization combination was counted, and the results are shown in FIG. 10. The results show that: under the treatment of the disinfection mode A, D, G, the seed embryo pollution rate of the yellow mountain pine is very significantly lower than that of other disinfection combinations, and the disinfection combination D (75% alcohol disinfection for 60 seconds and 3% sodium hypochlorite disinfection for 10 minutes) is the combination with the best disinfection effect, and the seed pollution rate is only 2%.
Second, influence of different hormone ratios on callus induction
MS and DCR are used as basic culture media, and auxin and cytokinin with different types and concentrations are added to perform callus induction on the seed embryo of the Pinus sylvestris. As shown in Table 2, a total of 21 callus induction media were prepared in this experiment. The sterile yellow pine embryos are inoculated onto culture media of different formulations, 10 bottles of each culture medium are inoculated, 3 embryos are inoculated per bottle, and 3 repetitions are carried out. After 28 days of culture in the tissue culture chamber, the induction rate of callus on each medium was observed and counted. .
TABLE 2 results of callus induction of Pinus sylvestris seed embryos by different culture media
As can be seen from Table 2, the induction rate of the embryo callus is different under the induction of the culture medium with different formulations. Data ofStatistical analysis shows that in the MS basic culture medium, the callus induction rate of the seed embryo of the Pinus sylvestris is generally higher than that of the DCR culture medium. Wherein MS is taken as a culture medium, 1mg/L of 6-BA and 0.2mg/L of NAA are added, and the induction rate of the embryo callus is as high as about 95 percent. Relevant studies showed NH4 in minimal medium+And NO3+The concentration has obvious effect on the dedifferentiation of mature zygotic embryo of conifer and the induction of callus, and the high concentration of NH4+And NO3+The content can effectively promote the induction of the callus. Our experiments show that the MS culture medium with high salt concentration has more obvious induction effect on the callus of the seed embryo of the yellow mountain pine compared with the DCR culture medium.
Further comparative analysis shows that the concentration change of 6-BA in the MS culture medium has extremely obvious influence on the induction effect of the callus. Lower or higher concentrations of 6-BA reduced the induction of callus (see Table 3). When the addition amount of 6-BA is 1mg/L, the callus induction effect of the embryo is the best. Similarly, the difference of the efficiencies of the auxin 2,4-D and NAA on the callus is large, and the effect of adding the auxin NAA to induce the callus is higher than that of 2, 4-D. When the addition amount of NAA is 0.2mg/L, the callus induction rate is the highest (see Table 4). In addition, the induction effect of the embryo callus can be influenced by too high or too low concentration of NAA.
TABLE 3 Duncan multiple comparison of the Effect of different concentrations of 6-BA on the callus induction rate of embryos
TABLE 4 Duncan multiple comparison of the Effect of different concentrations of 2,4-D, NAA on the callus induction rate of embryos
Influence of different hormone ratios on adventitious bud induction
In the experiment, a DCR culture medium suitable for the growth of conifers is used as a basic culture medium, and different types of hormone combinations are added for inducing the adventitious buds of the larix olgensis. As shown in Table 5, adventitious bud induction experiments were performed using DCR media containing 6-BA at concentrations of 0.5 to 2.0mg/L and NAA at concentrations of 0.05 to 0.45mg/L, 10 flasks were inoculated with each medium formulation, 3 embryos were inoculated per flask, and 3 replicates were performed. Meanwhile, changes of the differentiation medium at various stages are observed and recorded, and the influence of different concentrations of the plant hormone on the induction of adventitious bud formation is compared. After a period of cultivation, the induction rate of adventitious buds was counted, and changes in the adventitious bud induction process were observed, and the results are shown in Table 5.
As can be seen from table 5, DCR media containing different concentrations of auxin in combination with cytokinin had significant differences in the effect of inducing adventitious buds of the yellow pine. Wherein, in the DCR culture medium with the addition of 6-BA of 1mg/L and the NAA of 0.05mg/L, the induced quantity of the adventitious buds of the yellow mountain pine is high and the growth condition of the adventitious buds is better.
TABLE 5 results of adventitious bud Induction in different differentiation media
Multiple comparative analysis shows that (Table 6), when the concentration of 6-BA is 1mg/L, the induction rate of the adventitious bud of the seed embryo of the yellow mountain pine is the highest. When the concentration of 6-BA is too high or too low, the induction rate of adventitious buds is remarkably reduced. Multiple comparative analysis of auxin with different concentrations on the adventitious bud induction rate of the seed embryo of the Pinus sylvestris shows that NAA with low concentration has obvious effect on the induction of the adventitious bud of the seed embryo of the Pinus sylvestris, the effect is obviously reduced along with the increase of the NAA concentration, and the effect is best when the NAA is 0.05 mg/L.
TABLE 6 Duncan multiple comparison of the Effect of different concentrations of 6-BA on adventitious bud Induction Rate
TABLE 7 Duncan multiple comparison of the Effect of NAA at different concentrations on adventitious bud Induction Rate
Influence of different hormone ratios on adventitious bud elongation
In the experiment, a DCR culture medium is used as a basic culture medium, the concentrations of cytokinin 6-BA and auxin NAA in the culture medium are reduced in time, and the proportion of hormones is adjusted to ensure that adventitious buds grow in a rapid and elongated manner. The induced adventitious buds were transferred to DCR media containing 0.1mg/L, 0.3mg/L, 0.5 mg/L6-BA in combination with 0.05mg/L NAA, respectively, and the elongation effect of the adventitious buds was observed. By culturing for 30 days, we found that the effect of adventitious bud elongation is best in DCR medium with 0.1mg/L, NAA of 6-BA being 0.05 mg/L.
Fifth, the influence of different hormone proportions on the rooting of adventitious buds
1/2DCR is used as a basic culture medium (the number of major elements is reduced by half), IBA and NAA with different concentrations are added for combination (shown in a table 8), and a proper rooting system is explored. Transferring the adventitious buds of the Pinus fulva with the height of 1-2 cm into different rooting culture media, removing the needle leaves at the base of the buds by using a scalpel before transferring, and counting the rooting condition of the adventitious buds of the Pinus fulva about 40 days. The results are shown in FIG. 11.
TABLE 8 rooting media in different combinations
As can be seen from FIG. 11, the rooting induction efficiency of the adventitious buds of Pinus pumila was different for the hormone combinations of different concentrations, and the rooting rate of the adventitious buds of Pinus pumila was the highest when the IBA content was 2mg/L and the NAA content was 0.05mg/L (combination 4). In 1/2DCR with the addition of 2mg/L of auxin IBA, the rooting rate of adventitious buds is obviously higher than that of other concentrations, and the rooting rate of the adventitious buds is reduced by over-high or over-low concentration of the IBA, which indicates that 2mg/L is the optimal concentration of the IBA. In addition, the NAA which is most suitable for rooting the adventitious bud of the Pinus pumila is 0.05mg/L, and the induction effect of the NAA on the adventitious bud is obviously reduced along with the increase of the NAA concentration.
Sixth, discuss
The research takes the seed embryo of the yellow mountain pine as an experimental material to construct the tissue culture and rapid propagation technology of the yellow mountain pine. The result shows that the optimal disinfection system of the seed embryo of the yellow mountain pine is that 75 percent alcohol is disinfected for 1 minute, and 3 percent sodium hypochlorite is disinfected for 10 minutes; the optimal induction culture medium of the callus is MS +1 mg/L6-BA +0.2mg/L NAA; the optimal induction culture medium of the adventitious bud is DCR +1 mg/L6-BA +0.05mg/L NAA; the best inducing condition for adventitious bud elongation is DCR +0.1 mg/L6-BA +0.05mg/L NAA; the best culture medium for rooting is 1/2DCR +2mg/L IBA +0.05mg/L NAA. The research result has important significance for the preservation, genetic improvement, new variety cultivation and high-quality seedling propagation of the germplasm resources of the Pinus pinaster.
In addition, it should be noted that the MS, DCR and 1/2DCR adopted in the invention are all the culture media conventional in the field, the formula of the MS culture medium is shown in Table 9, and the formula of the DCR culture medium is shown in Table 10.
TABLE 9 MS Medium formulation
TABLE 10 DCR Medium formulation
Note: 1/2 the DCR culture medium is obtained by halving the major elements of DCR culture medium.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (5)
1. A tissue culture rapid propagation method of Pinus pumila is characterized by comprising the following steps:
(1) pretreatment of explants:
drying the cones of the yellow mountain pine under natural illumination to obtain yellow mountain pine seeds; then, removing the testa of the Pinus massoniana seed;
(2) and (3) disinfection of explants:
sterilizing the seed of the Pinus sylvestris without the testa by combining 75% alcohol and 1% sodium hypochlorite solution; then, sucking water on sterile filter paper, and picking out a seed embryo to obtain a sterile explant required by inoculation;
(3) callus induction:
under the aseptic condition, inoculating the aseptic explant obtained in the step (2) on a callus induction culture medium to induce the callus; when the induction culture is carried out for 12-16 days, the explants begin to be callus; wherein, the formula of the callus induction culture medium is as follows: MS +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.2mg/L NAA, pH 5.8;
(4) induction and elongation of adventitious buds:
selecting callus, transferring the callus to an adventitious bud induction culture medium, and inducing adventitious buds; when the induction culture is carried out for 25-35 d, the callus begins to grow adventitious buds; wherein the formula of the adventitious bud induction culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +1 mg/L6-BA +0.05mg/L NAA, pH 5.8;
transferring the induced adventitious bud to an elongation culture medium after the adventitious bud grows out, and performing elongation culture on the adventitious bud; wherein, the formula of the elongation culture medium is as follows: DCR +30g/L sucrose +7g/L agar powder +0.1 mg/L6-BA +0.05mg/L NAA, pH 5.8;
(5) rooting culture:
selecting 1-2 cm-high Huangshan pine adventitious buds, removing coniferous leaves of bud bases, and transferring the buds to a rooting culture medium for direct rooting culture; when the rooting culture is carried out for 35-45 days, rooting is started; wherein, the formula of the rooting culture medium is as follows: 1/2DCR +30g/L sucrose +7g/L agar powder +2mg/L IBA +0.05mg/L NAA, pH 5.8.
2. The tissue culture rapid propagation method of the Pinus pumila as claimed in claim 1, wherein in the step (1), the Pinus pumila cones are collected from the scenic spot of the Huangshan mountain in Anhui province.
3. The tissue culture and rapid propagation method of Pinus pumila according to claim 1, wherein the specific process of performing combined disinfection by using 75% alcohol and 1% sodium hypochlorite solution in step (2) comprises: taking out the yellow mountain pine seeds without the seed coats for later use; and then, in a superclean bench, cleaning the seeds with sterile water for 3-5 times, rinsing with 75% alcohol for 60s, soaking with 1% sodium hypochlorite solution for 10min, and finally washing with sterile water for 3-5 times.
4. The tissue culture rapid propagation method of the Pinus huangshanensis according to claim 1, characterized in that the callus induction, the adventitious bud induction and the elongation, and the rooting culture process, the culture conditions are: the culture temperature is 25 +/-2 ℃, the relative humidity is 70% -80%, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
5. The tissue culture rapid propagation method of the Pinus pumila as claimed in any one of claims 1 to 4, further comprising a seedling hardening and transplanting step of the step (6); the hardening and transplanting process comprises the following steps:
after the rooting seedlings of the yellow mountain pine grow to develop root systems, opening a tissue culture seedling bottle cap, hardening the seedlings for 3-5 days, and keeping sufficient water in the culture medium in the period; then, mashing the culture medium in the tissue culture bottle by using a glass rod, and taking out the tissue culture seedling from the tissue culture bottle; then, washing the root culture medium with tap water, and transplanting the root culture medium into a flowerpot; and finally, placing the transplanted yellow mountain pine seedlings in a culture room for culture.
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