CN112493126B - Method for induction of lagerstroemia indica somatic embryo and plant regeneration - Google Patents
Method for induction of lagerstroemia indica somatic embryo and plant regeneration Download PDFInfo
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Abstract
The invention relates to a method for induction of somatic embryos and plant regeneration of lagerstroemia indica, and belongs to the technical field of ornamental plant biology. The invention utilizes the cotyledon of the lagerstroemia indica to induce and generate a somatic embryo and establish a clone of a regeneration plant, and the main steps comprise: 1. culturing aseptic crape myrtle seedlings and selecting explants; 2. inducing the callus; 3. induction of embryonic callus; 4. somatic embryo development and development; 5. inducing and rooting somatic embryos; 6. transplanting to form seedlings. The somatic embryo induction and plant regeneration method of the ornamental plant lagerstroemia indica lays a foundation for the rapid propagation and accelerated industrialized production of lagerstroemia indica, the genetic transformation of lagerstroemia indica, the gene improvement and other researches.
Description
Technical Field
The invention relates to the technical field of ornamental plant biology, and particularly provides a method for induction of lagerstroemia indica somatic embryos and regeneration of plants.
Background
Lagerstroemia indica of lythraceae is an important ornamental horticultural plant. The traditional crape myrtle propagation modes mainly comprise seeding propagation and cutting propagation, but the seeding propagation is easy to generate variation, the propagation speed is low, and the survival rate of the cutting propagation is low.
In recent years, with the development of biotechnology, tissue culture rapid propagation technology has become an important approach to solve the above problems. The tender stem or seed of lagerstroemia indica with axillary buds is used as an explant to carry out lagerstroemia indica rapid propagation, and a lagerstroemia indica rapid propagation system is established. Researchers at home and abroad develop the production technology of artificial lagerstroemia indica seed capsule units and find that the explant of dwarf lagerstroemia indica has the highest survival rate of the stem segment with axillary buds.
With the development of theories and technologies such as cell biology, molecular biology and the like, establishment of clones is one of important ways for plant regeneration in cell engineering by utilizing a somatic embryogenesis technology. The somatic embryogenesis research of woody plants starts from the late 70 s and develops rapidly from the late 80 s to the early 90 s. In somatic embryogenesis studies of woody angiosperms, it has been observed that somatic embryos induce regeneration of plants from tissue cultures of more than 140 tree species of the 95 genus of the 46 family. Somatic embryogenesis has the characteristics of large quantity, high speed, complete structure, high regeneration rate and simple operation. But also provides a good foundation for researching differentiation, development, totipotency expression and genetic transformation of plant cells and screening mutants.
Previous studies on tissue culture and rapid propagation of lagerstroemia indica have been limited to the use of solid media to induce shoot or seed germination of multiple shoots. A method for establishing a lagerstroemia indica somatic embryogenesis, development and rooting system has no literature report at home and abroad. Therefore, a stable and controllable system for somatic embryogenesis, development and rooting of the lagerstroemia indica is developed by utilizing a cell engineering rapid propagation technology, and the system is particularly necessary for establishing a theoretical basis for realizing rapid propagation and accelerated industrialized production of the lagerstroemia indica and providing a feasible method; meanwhile, a good experimental system can be established for biotechnological breeding, germplasm resource preservation, rapid propagation and genetic transformation of the lagerstroemia indica.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for induction of lagerstroemia indica somatic embryos and regeneration of plants.
The invention provides a method for induction of somatic embryos and plant regeneration of lagerstroemia indica, which comprises the following steps:
step 1) cultivation of a crape myrtle aseptic seedling and selection of an explant; 2) inducing the callus; 3) induction of embryonic callus; 4) somatic embryo development and development; 5) inducing and rooting somatic embryos; 6) transplanting to form seedlings.
Further, aiming at the step 1), the method is characterized in that the culture of the aseptic seedlings is specifically as follows: soaking the seeds of the crape myrtle in tap water at room temperature for 24-28 hours; performing surface sterilization treatment before inoculation: soaking in 75% alcohol for 30 s, washing with sterile water for 2 times, and soaking in 0.1% HgCl2The solution is soaked for 10 minutes and then is washed for 5 to 6 times by sterile water; inoculating the seeds in MS0On a culture medium, the culture temperature is (25 +/-2) DEG C, the illumination intensity is 2000-3000 lx, and the illumination time is 12 h/d. The explant isCotyledon of Lagerstroemia indica Linn sterile seedling.
Further, aiming at step 2), the induction of callus is specifically: cutting cotyledons of the aseptic seedlings obtained in the step 1) into small pieces of 1.0-2.0mm, inoculating the small pieces of the aseptic seedlings to a culture medium for inducing callus, and inducing the callus under the dark condition at the constant temperature of 25 ℃.
Preferably, in the step 2), the callus induction medium is: MS +4mg/l 2,4-D +1.0mg/l BA.
Further, aiming at step 3), the induction of embryogenic callus is specifically: inoculating the callus obtained in the step 2) on an embryogenic callus induction culture medium, and inducing the embryogenic callus under the conditions of 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity. The embryogenic callus induction culture medium comprises: MS +4mg/l 2,4-D +0-1.0mg/l NAA +0-2.0mg/l BA or MS +4mg/l 2,4-D +0-1.0mg/l NAA +0-2.0mg/l KT.
Preferably, in step 3), the embryogenic callus induction medium is: MS +4mg/l 2,4-D +0.2mg/l NAA +1.0mg/l BA.
Further, with respect to step 4), it is characterized in that the somatic embryo development and development is specifically: inoculating the embryogenic callus obtained in the step 3) on a somatic embryogenesis development culture medium, and culturing at 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity. The somatic embryo development culture medium comprises: MS +0-4mg/l 2,4-D +0-0.2mg/l NAA +0-2.0mg/l BA +0-0.3mg/l IBA or MS +0-4mg/l 2,4-D +0.2mg/l NAA +0-2.0mg/l KT +0-0.3mg/l IBA or MS +0-4mg/l NAA0。
Preferably, the somatic embryo development medium is: MS +0.5mg/l BA +0.3mg/l IBA.
Further, aiming at the step 5), the induced rooting of the somatic embryos is specifically as follows: when the seedlings in the step 4) grow to be more than 3cm, the seedlings are inoculated into a culture medium for inducing rooting and cultured under the conditions of 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity. The induced rooting culture medium comprises: 1/2MS +1.0mg/l NAA, or 1/2MS +0.5mg/l IBA.
Further, aiming at the step 6), the method is characterized in that the transplanting seedling is specifically as follows: when the root length of the seedling in the step 5) reaches 1.5-2.0cm and 4 true leaves are unfolded, hardening the seedling and transplanting.
Preferably, the sucrose concentration is 30g/L, the agar is 9g/L, and the pH is 5.7-5.8.
In addition, the invention also discloses the application of any one of the induction methods in establishing a genetic transformation system of the lagerstroemia indica, culturing protoplasts, propagating excellent clones, genetic engineering and screening mutants.
The invention has the beneficial effects that:
the invention provides a method for induction of somatic embryos and plant regeneration of ornamental lagerstroemia indica, the induction rate and the embryo yield of the callus are both close to 100 percent, a convenient, quick and effective method is provided for lagerstroemia indica to obtain regenerated plants in a somatic embryogenesis mode, and the method has good social and economic benefits.
The method is in the leading position, is particularly necessary for establishing theoretical basis for realizing industrial seedling raising of the lagerstroemia indica somatic embryos and providing a feasible method, and also establishes basis for biotechnological breeding, rapid propagation and genetic transformation of the lagerstroemia indica.
Drawings
In order to more clearly illustrate the embodiments or the prior art solutions of the present invention, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive labor.
FIG. 1 shows the lagerstroemia indica callus obtained from callus induction medium No. 2;
FIG. 2 is a diagram showing proembryos obtained in somatic embryogenesis development medium (11) (12) after 10 days of culture;
FIG. 3 is a spherical embryo obtained in somatic embryogenesis development medium # 11 when cultured for 20 days;
FIG. 4 is a spherical embryo obtained in somatic embryogenesis development medium # 12 when cultured for 20 days;
FIG. 5 is a heart-shaped embryo obtained in somatic embryogenesis development medium No. (11) (12) after 30 days of culture;
FIG. 6 is a torpedo shaped embryo obtained in somatic embryogenesis development medium (11) (12) after 30 days of culture;
FIG. 7 is a cotyledonary embryo obtained in somatic embryogenesis development medium (11) (12) after 30 days of culture;
FIGS. 8-10 show whole somatic embryos obtained in somatic embryogenesis development medium # 11, when cultured for 30 days.
Detailed Description
The present invention will be described in further detail with reference to examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. Various substitutions and alterations made on the basis of the general knowledge and the conventional means in the art without departing from the technical idea of the invention as described above are intended to be included in the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 cultivation of aseptic seedlings of Lagerstroemia indica and explant selection
The crape myrtle seeds are collected from the institute of science and technology in Hebei province. Soaking the seeds of Lagerstroemia indica Linn in tap water at room temperature for 24-28 hr, and changing water every 5-6 hr. Performing surface sterilization treatment before inoculation, namely soaking in 75% alcohol for 30 s on an ultraclean workbench, washing with sterile water for 2 times, and soaking in HgCl with volume fraction of 0.1%2The solution was allowed to stand for 10 minutes and gently agitated with a glass rod to evenly sterilize the material. Washing with sterile water for 5-6 times, inoculating the seeds to MS0On a culture medium. The culture temperature is 25 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 12 h/d.
Selecting cotyledons of sterile seedlings as explants.
Example 2 callus Induction
Cotyledons of the aseptic seedlings in example 1 were cut into 1.0-2.0mm pieces perpendicular to the main leaf vein, and inoculated on callus-inducing media, 40 pieces of each media. Callus induction medium different kinds of hormones were added to the MS medium as follows:
(1)MS+4mg/l 2,4-D+1.0mg/l NAA
(2)MS+4mg/l 2,4-D+1.0mg/l BA
(3)MS+4mg/l 2,4-D+1.0mg/l KT
furthermore, the culture medium contained 3% sucrose, and the pH was adjusted to 5.7-5.8 before adding agar, followed by sterilization at 121 ℃ under a pressure of 101kp for 15 minutes under high temperature and high pressure conditions. Culturing at constant temperature of 25 deg.C for 10d, 20d, and 30d in dark condition to induce callus.
The 3 culture mediums enable the lagerstroemia indica cotyledon fragments to form callus, the induction rate is 100%, the induction rate is similar in the first 20 days, all the callus is formed in the last 30 days, but the induced callus is obviously different. The callus of culture medium (1) containing NAA is snowflake white, has villus on the surface, is non-embryogenic callus, and the callus of culture medium (2) containing BA is milky white, dense, granular, piled, mostly spherical under anatomical observation, has high water content, viscosity, and part of embryogenic callus (figure 1); the No. 3 culture medium containing KT is creamy yellow and soft, and the cytoplasm of the medium is thin and the vacuole is large when the medium is observed under a microscope. When the crape myrtle callus is induced, the BA effect is far better than KT.
Example 3 Induction of embryogenic callus
The calli induced in example 2 were inoculated on embryogenic callus induction medium, 40 pieces of each, which was supplemented with different kinds of hormones in MS medium as shown below:
(1)MS+4mg/l 2,4-D+1.0mg/l NAA
(2)MS+4mg/l 2,4-D+1.0mg/l BA
(3)MS+4mg/l 2,4-D+1.0mg/l KT
(4)MS+4mg/l 2,4-D+0.2mg/l NAA+0.5mg/l BA
(5)MS+4mg/l 2,4-D+0.2mg/l NAA+1.0mg/l BA
(6)MS+4mg/l 2,4-D+0.2mg/l NAA+1.5mg/l BA
(7)MS+4mg/l 2,4-D+0.2mg/l NAA+2.0mg/l BA
(8)MS+4mg/l 2,4-D+0.2mg/l NAA+0.5mg/l KT
(9)MS+4mg/l 2,4-D+0.2mg/l NAA+1.0mg/l KT
(10)MS+4mg/l 2,4-D+0.2mg/l NAA+1.5mg/l KT
(11)MS+4mg/l 2,4-D+0.2mg/l NAA+2.0mg/l KT
furthermore, the culture medium contained 3% sucrose, and the pH was adjusted to 5.7-5.8 before adding agar, followed by sterilization at 121 ℃ under a pressure of 101kp for 15 minutes under high temperature and high pressure conditions. Culturing at 26-28 deg.C under 12-14h/d illumination, 1800-2000lx illumination intensity, and 60-80% humidity for 20d to induce embryogenic callus.
The 11 culture mediums can induce embryogenic callus, but the induction rate is obviously different. (1) The induction rate of the embryogenic callus on the No. culture medium is only 25%; (2) the induction rates of the BA series treatments (4) to (7) on the embryogenic callus are all over 60 percent, the effect is obviously better than that of other treatments, wherein the best effect is achieved by matching the BA (4) to (7) with NAA, the induction rate is increased along with the increase of the BA concentration in the matched series, the induction rate is up to 100 percent when the BA concentration is up to 1.0mg/l, and then the induction rate is reduced along with the increase of the concentration (Table 1). In the induction of embryogenic callus, KT has much lower effect than BA, and the effect of inducing by matching cytokinin BA and 0.2ml/l NAA is better, and the induction rate reaches 100% when the concentration of BA is 1.0 mg/l.
TABLE 1 Effect of different treatments on embryogenic callus induction
Example 4 somatic embryogenesis and development
The embryogenic callus of example 3 was inoculated into somatic embryogenesis development medium, 40 pieces of each medium, and somatic embryos development medium was added with different kinds of hormones to the MS medium as shown below:
(1)MS+4mg/l 2,4-D+1.0mg/l BA
(2)MS+4mg/l 2,4-D+1.0mg/l KT
(3)MS+4mg/l 2,4-D+0.2mg/l NAA+0.5mg/l BA
(4)MS+4mg/l 2,4-D+0.2mg/l NAA+1.0mg/l BA
(5)MS+4mg/l 2,4-D+0.2mg/l NAA+1.5mg/l BA
(6)MS+4mg/l 2,4-D+0.2mg/l NAA+2.0mg/l BA
(7)MS+4mg/l 2,4-D+0.2mg/l NAA+0.5mg/l KT
(8)MS+4mg/l 2,4-D+0.2mg/l NAA+1.0mg/l KT
(9)MS+4mg/l 2,4-D+0.2mg/l NAA+1.5mg/l KT
(10)MS+4mg/l 2,4-D+0.2mg/l NAA+2.0mg/l KT
(11)MS+0.5mg/l BA+0.3mg/l IBA
(12)MS0
furthermore, the culture medium contained 3% sucrose, and the pH was adjusted to 5.7-5.8 before adding agar, followed by sterilization at 121 ℃ under a pressure of 101kp for 15 minutes under high temperature and high pressure conditions. Inducing the somatic embryo to germinate and develop at 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity. The culture was performed for 10d, 20d, and 30d, respectively, and observation was performed under a dissecting scope.
Microscopic examination was carried out at 10 days, while embryogenic calli remained in the same medium (1) - (10) as the embryogenic callus induction medium, proembryos appeared on medium (11) containing IBA and medium (12) without hormones (FIG. 2), and microscopic examination was carried out at 20 days, with proembryos appearing on medium (1) - (10) and green granular protrusions appearing on medium (11) and medium (12), and spherical embryos appeared by microscopic examination (FIG. 3-4). When the embryos were plated out on medium Nos. 1 to 10 for 30 days, spherical embryos and heart-shaped embryos appeared on the embryos, and the number of calli and somatic embryos increased greatly. (11) And (12) medium developed heart, torpedo and cotyledon shaped embryos (FIGS. 5-7), and the somatic embryos were easily separated from the surrounding callus at this time. After further culturing for 30 days, complete somatic embryos were formed in medium No. 11 (FIGS. 8-10), and the somatic embryos formed plantlets over time. While heart-shaped embryos, torpedo-shaped embryos and cotyledon-shaped embryos can be observed in the hormone-free medium No. 12. However, the continued culture produced abnormal plants with a large number of white root hairs.
(11) The number of somatic embryos induced by the medium is obviously the most, and the rate of abnormal embryos is the lowest. The rate of malformed embryos was lower on hormone-free medium, but the induction rate was relatively low. (1) The medium No. (10) has low induction rate of somatic embryos and high rate of abnormal embryos, and although the medium No. (4) can well induce embryogenic callus, the induction rate of the somatic embryos is obviously lower than that of the medium No. (11). This indicates that embryogenic callus induction medium is capable of somatic embryogenesis development, but less effective (Table 2).
TABLE 2 Effect of different treatments on somatic embryogenesis and development of Lagerstroemia indica
Example 5 Induction of rooting of somatic embryos
When the seedlings in example 4 grew to 3cm or more, the seedlings were cut out and inoculated into rooting induction media, 20 strains each, which were added with different kinds of hormones in 1/2MS medium, as shown below:
(1)1/2MS+0.1mg/l NAA
(2)1/2MS+0.5mg/l NAA
(3)1/2MS+1.0mg/l NAA
(4)1/2MS+1.5mg/l NAA
(5)1/2MS+2.0mg/l NAA
(6)1/2MS+0.1mg/l IBA
(7)1/2MS+0.5mg/l IBA
(8)1/2MS+1.0mg/l IBA
(9)1/2MS+1.5mg/l IBA
(10)1/2MS+2.0mg/l IBA
furthermore, the culture medium contained 3% sucrose, and the pH was adjusted to 5.7-5.8 before adding agar, followed by sterilization at 121 ℃ under a pressure of 101kp for 15 minutes under high temperature and high pressure conditions. Inducing the somatic embryo to root under the conditions of 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity. The rooting rate was investigated at 20 days of culture, and the root quality was observed.
After 20 days of culture, the rooting rate and the average number of roots per section are increased along with the increase of the concentration of the NAA, and the rooting rate is reduced along with the increase of the concentration of the NAA in the No. 3 culture medium, namely when the concentration of the NAA reaches 1.0mg/l, the rooting rate is highest; the average number of roots per stage was the highest in medium No. 4, i.e., at a NAA concentration of 1.5mg/l (Table 3).
TABLE 3 Effect of different treatments on rooting
From root quality analysis, the roots are slender and have more root hairs, the absorption capacity is strong, the roots are not easy to be damaged, and the transplanting survival is facilitated, and the roots on the culture medium No. 2 and the culture medium No. 3 are slender and have more root hairs. In general, the culture medium (3) has better rooting effect. The tendency of the series of treatments containing IBA on roots was similar to that of NAA, the highest rooting rate was observed at a concentration of 1.0mg/l, the average number of roots per stage was the highest at 0.5mg/l, and the quality of roots was almost the same as that of the roots treated in No. 10 except for callus. Compared with NAA and IBA treatment, the treatment rooting rate containing NAA is relatively higher, and the roots treated in (2) and (3) are better; the treatment with IBA showed less root callus, and although there were fewer root hairs, treatment No. 7 averaged 8 pieces per root.
Example 6 transplanting into seedlings
When the root length of the seedling on the rooting culture medium (2), (3) and (7) in example 5 reaches 1.5-2.0cm and 4 true leaves are unfolded, the seedling can be transplanted into sterilized vermiculite. And (3) washing the culture medium of the root part clean during transplanting, and damaging the root system as little as possible. After transplanting, watering thoroughly to prevent the seedlings from dying due to water loss, and covering a film to keep the water content between 80 and 90 percent at the temperature of 24 +/-1 ℃. Before the young seedlings develop new leaves, clear water is sprayed every day, and after the new leaves grow out, a proper amount of nutrient solution can be applied to promote the seedlings to be strong. The transplanting survival rate reaches more than 94 percent.
Claims (5)
1. A method for induction of somatic embryos and plant regeneration of lagerstroemia indica is characterized by comprising the following steps: 1) culturing aseptic crape myrtle seedlings and selecting explants; 2) inducing the callus; 3) induction of embryonic callus; 4) somatic embryo development and development; 5) inducing and rooting somatic embryos; 6) transplanting the seedlings into seedlings;
aiming at the step 1), the explant is a cotyledon of a lagerstroemia indica aseptic seedling;
aiming at the step 2), the induction of the callus is specifically as follows: shearing cotyledons of the aseptic seedlings obtained in the step 1) into small blocks with the diameter of 1.0-2.0mm, inoculating the small blocks on a culture medium for inducing callus, and inducing the callus under the dark condition at the constant temperature of 25 ℃;
the culture medium for inducing the callus is as follows: MS +4mg/l 2,4-D +1.0mg/l BA;
aiming at the step 3), the induction of the embryogenic callus specifically comprises the following steps: inoculating the callus obtained in the step 2) on an embryonic callus induction culture medium, and inducing the embryonic callus under the conditions of 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity;
the embryogenic callus induction culture medium comprises: MS +4mg/l 2,4-D +0.2mg/l NAA +1.0mg/l BA;
for step 4), the somatic embryo generation and development are specifically: inoculating the embryogenic callus obtained in the step 3) on a somatic embryogenesis development culture medium, and culturing at 26-28 ℃, 12-14h/d illumination, 1800-2000lx illumination intensity and 60-80% humidity;
the somatic embryogenesis development medium comprises: MS +0.5mg/l BA +0.3mg/l IBA;
aiming at the step 5), the induced rooting of the somatic embryo is specifically as follows: when the seedling in the step 4) grows to be more than 3cm, inoculating the seedling into a rooting induction culture medium, and culturing at 26-28 ℃, 12-14h/d illumination, 1800-;
the induced rooting culture medium comprises: 1/2MS +1.0mg/l NAA, or 1/2MS +0.5mg/l IBA.
2. The method of claim 1, directed to step 1), wherein the culturing of the sterile shoots is specifically: soaking the seeds of the crape myrtle in tap water at room temperature for 24-28 hours; performing surface sterilization treatment before inoculation: soaking in 75% alcohol for 30 s, washing with sterile water for 2 times, and soaking in 0.1% HgCl2The solution is soaked for 10 minutes and then is washed for 5 to 6 times by sterile water; inoculating the seeds in MS0On a culture medium, the culture temperature is 25 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 12 h/d.
3. The method of claim 1, directed to step 6), wherein transplanting a seedling is specifically: when the root length of the seedling in the step 5) reaches 1.5-2.0cm and 4 true leaves are unfolded, hardening the seedling and transplanting.
4. The method for induction of somatic embryos and regeneration of plants of lagerstroemia indica according to any one of claims 1 to 3, wherein sucrose is added to all the culture media at a concentration of 30g/L, agar is added at a concentration of 9g/L, and the pH is 5.7 to 5.8.
5. Use of the induction method according to any one of claims 1 to 4 in genetic transformation system establishment, protoplast culture, elite clonal propagation, genetic engineering and mutant screening of lagerstroemia indica.
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