CN111387059A - Method for regenerating plants from callus of schisandra chinensis - Google Patents
Method for regenerating plants from callus of schisandra chinensis Download PDFInfo
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- CN111387059A CN111387059A CN202010397711.2A CN202010397711A CN111387059A CN 111387059 A CN111387059 A CN 111387059A CN 202010397711 A CN202010397711 A CN 202010397711A CN 111387059 A CN111387059 A CN 111387059A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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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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- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a method for regenerating a plant from a callus of a schisandra chinensis, which relates to the technical field of plant tissue culture and comprises the following steps: induction of embryogenic callus, proliferation and propagation of embryogenic callus, embryogenesis, development of embryo, regeneration of plant, and transplantation of regenerated plant. The invention lays a foundation for the propagation of the good germplasm of the schisandra chinensis by using the leaves of the schisandra chinensis as callus regeneration plants, is beneficial to the preservation of the good germplasm and better and directly obtains the good characters of a parent.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for regenerating plants from callus of schisandra chinensis.
Background
The tissue culture of plants is a new asexual propagation technology developed in recent decades based on the theory that plant cells have totipotency. Tissue culture of plants, also called in vitro culture in a broad sense, refers to a technique of separating desired tissues, organs or cells, protoplasts, etc. from plant bodies, inoculating them on a culture medium containing various nutrients and plant hormones under aseptic conditions by aseptic manipulation to obtain regenerated whole plants or to produce other products having economic value. The narrow meaning refers to tissue culture, which refers to the culture of each part of plant tissue, such as cambium, parenchyma, mesophyll tissue, endosperm and the like to obtain regenerated plants, and also refers to the culture of callus generated on each organ in the culture process, and the callus is redifferentiated to form the regenerated plants.
The success or failure of plant tissue culture is closely related to the type of the explant, whether the selection of the explant is proper or not and whether the treatment method is proper or not, and is directly related to the difficulty degree and the culture direction of the tissue culture. As the material for inducing embryogenic callus, there are many kinds, such as seedling, hypocotyl, isolated root, stem, leaf, etc. The types, physiological states and material-taking parts of the explants directly influence the induction of the somatic embryogenic callus, and the explant materials used for inducing the somatic embryogenic callus by the schisandra chinensis comprise zygotic embryos and cotyledons after mature seeds germinate, hypocotyls, winter buds of the schisandra chinensis, tender stem segments, leaves, petioles and the like.
In the prior art, most researches are considered from the aspects of characteristics such as induction rate, induction days and the like, in the existing records, zygotic embryos of schisandra seeds are the best explants for inducing embryogenic callus, and then hypocotyls are used for researching the best explants with fresh leaves for inducing embryogenic callus. The zygotic embryo and the hypocotyl both belong to the component parts of the seeds, so that the heterozygous traits of the male parent and the female parent in the parent are kept, and the traits of the female parent are kept unfavorable.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for regenerating plants from the callus of the schisandra chinensis, which adopts the leaves of the schisandra chinensis as explants, induces embryogenic callus through vegetative propagation, forms regenerated plants through a somatic embryogenesis way, and can better and more directly obtain excellent parent properties.
A method for regenerating a plant from a callus of a schisandra chinensis, which comprises the following steps:
s1, induction of embryogenic callus,
inoculating sterile seedling leaves of fructus Schisandrae Bicoloris in culture medium No. 1, culturing for 28-32 days, transferring to new culture medium No. 1, culturing for 28-32 days, cutting off yellow brown hard part, transferring to new culture medium No. 1, and culturing for 28-32 days to obtain embryogenic callus;
s2, propagation and propagation of the embryogenic callus,
inoculating the embryogenic callus obtained in S1 into No. 2 culture medium, and culturing for 28-32 days to obtain late embryogenic callus;
the No. 2 culture medium is based on MS culture medium, and is added with 2, 4-D with the concentration of 0.1-02 mg/L and TDZ with the concentration of 0.1-0.2 mg/L;
s3, the generation of embryos,
cutting late embryogenic callus obtained in S2 into 0.4-0.6cm3Inoculating the block to No. 3 culture medium, and culturing for 28-32 days to obtain embryoid;
the No. 3 culture medium is based on an MS culture medium and is added with ABA with the concentration of 0.2-0.5 mg/L;
s4, development of embryo and plant regeneration,
inoculating the embryoid obtained in the step S3 into an MS culture medium for embryo development, obtaining cotyledonary embryos and embryos with radicles after 28-32 days, inoculating the cotyledonary embryos and the embryos with the radicles into a new MS culture medium, and continuing culturing for 28-30 days to form seedlings;
s5, transplanting the regeneration plant,
after the seedlings obtained in S4 become complete plants, hardening the seedlings for 2-4 days, taking out the complete plants from the bottles, washing the culture medium at the roots, and transplanting the complete plants into the mixed sterile sand and soil.
Preferably, in S2, the pH of the No. 2 medium is 5.6-6.0, and the culture conditions of the embryogenic callus are as follows: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8 h.
Preferably, in S3, the pH of the No. 3 medium is 5.6-6.0, and the culture conditions of the late embryogenic callus are as follows: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8 h.
Preferably, in S4, the pH of the MS medium is 5.6-6.0.
Preferably, in S4, the embryoid body, the cotyledonary embryo and the embryo with radicle are all cultured under the following conditions: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8 h.
Preferably, in S5, the mixing mass ratio of the sand to the soil is 1: 2.5-3.5.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention adopts the leaves of the schisandra chinensis, namely the safflower, as explants, performs embryogenic callus induction through vegetative propagation, and forms regenerated plants through a somatic embryogenesis way, thereby laying a foundation for the propagation of good germplasm of the schisandra chinensis, being beneficial to the preservation of the good germplasm and being capable of better and more directly obtaining the good characters of a parent;
2. the schisandra chinensis belongs to the magnolia family, the schisandra chinensis is in milky color, the material used by the invention is the 'safflower' schisandra chinensis with red flower quilt, the material is very rare, and good material is provided for researching scientific research experiments such as schisandra chinensis flower color and the like;
3. the ABA is added to improve the abnormal embryos, so that the abnormal embryos are greatly reduced after the ABA is added, and the embryo development and seedling formation are facilitated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram of spherical embryos developed from callus embryos of example 1 of the present invention;
FIG. 2 is a heart-shaped embryo diagram of callus embryo development in example 1 of the present invention;
FIG. 3 is a torpedo-shaped embryo diagram of callus embryo development in example 1 of the present invention;
FIG. 4 is a diagram of cotyledonary embryos for callus embryo development according to example 1 of the present invention;
FIG. 5 is a diagram of a plant regenerated by callus embryogenesis according to example 1 of the present invention;
FIG. 6 is a diagram of the root system of a plant regenerated by callus embryo development in example 1 of the present invention;
FIG. 7 shows a callus embryo before transplantation of a regenerated plant according to example 1 of the present invention;
FIG. 8 shows the example 1 of the present invention after transplanting the plants regenerated by callus embryo development.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.
Example 1:
a method for regenerating a plant from a callus of a schisandra chinensis, which comprises the following steps:
s1 Induction of embryogenic callus
Cutting off two ends and surface layer of leaves of fructus Schisandrae chinensis aseptic seedling to 0.4-0.6cm3The square of the strain is used as an explant and inoculated in a No. 1 culture medium, the culture is carried out for 28 days and then transferred to a new No. 1 culture medium for continuous culture 28, then a yellow brown hard part is cut off and then transferred to a new No. 1 culture medium for continuous culture for 28 days, and embryogenic callus is obtained;
the culture medium No. 1 is based on MS culture medium, 2, 4-D with the concentration of 1.0 mg/L and TDZ with the concentration of 0.2 mg/L are added, and the pH of the culture medium No. 1 is 5.6;
the temperature of the culture condition is 23 ℃, and the light-dark period is 16h/8 h;
s2, propagation and propagation of the embryogenic callus,
inoculating the embryogenic callus obtained in S1 into a No. 2 culture medium, and culturing for 28 days at 23 ℃ under the condition that the light-dark cycle is 16h/8h to obtain the embryogenic callus after proliferation and propagation;
the culture medium No. 2 is based on MS culture medium, 2, 4-D with the concentration of 0.1 mg/L and TDZ with the concentration of 0.1 mg/L are added, and the pH of the culture medium No. 2 is 5.6;
s3, the generation of embryos,
the embryogenic callus inoculated in the culture medium No. 2 in the S2 was cut into 0.4-0.6cm3Inoculating the small blocks into No. 3 culture medium, and culturing for 28 days to obtain embryoid;
the No. 3 culture medium is based on an MS culture medium and is added with ABA with the concentration of 0.5 mg/L;
the tissue culture temperature is 23 ℃, the light-dark period is 16h/8h, and the pH of the No. 3 culture medium is 5.6;
s4, development of embryo and plant regeneration,
inoculating embryoid generated in the culture medium No. 3 in the S3 into an MS culture medium for embryo development, forming cotyledonary embryos and embryos with radicles after 28 days, inoculating the cotyledonary embryos (figure 4) and the embryos with radicles into a new MS culture medium, and continuing to culture for 28 days to form seedlings (figures 5-6);
the tissue culture temperature is 23 ℃, the light-dark period is 16h/8h, and the pH of the MS culture medium is 5.6;
s5, transplanting of the regeneration plants (figure 7-8),
and (3) hardening the seedlings for 2 days after the seedlings form complete plants in the S4, taking out the seedlings from the bottles, cleaning culture medium at the roots, and transplanting the seedlings into mixed sterile sand and soil, wherein the mixing mass ratio of the sand to the soil is 1: 3.
the culture medium preparation steps in S1-S3 are as follows:
(1) weighing 30g of sucrose and 5.4g of agar powder (7 g of agar strips) in each 1L culture medium, and respectively weighing 20m L, 10m L, 10m L, 10m L, 10m L and 10m L mother solutions of No. 1, No. 2, No. 3, No. 4, No. 5 and No. 6 by using a pipette;
(2) dissolving, namely heating agar powder or agar strips and water in a pot until no blocks are transparent, adding cane sugar for dissolving, adding mother liquor, cleaning a conical flask containing the mother liquor for 3 times by using water, pouring the conical flask into the pot, continuously stirring by using a glass rod, pouring the conical flask into a large number of cups of 1L after the conical flask is boiled again, and adding distilled water to fix the volume to 1L;
(3) adjusting the pH value, measuring the pH value of the solution in a large quantity of cups by using pH test paper, and adjusting the pH value of the solution in the beakers to 5.8 by using sodium hydroxide or hydrochloric acid solution;
(4) adding hormone, when the hormone is added into the MS culture medium, firstly taking the hormone by using a pipette or a pipette according to the requirement, adding the hormone into a large number of cups, uniformly stirring, then measuring the pH value of the solution in the large number of cups by using pH test paper, and adjusting the pH value of the solution in the beakers to 5.8 by using sodium hydroxide or hydrochloric acid solution;
(5) subpackaging and sterilizing, pouring a large amount of the solution in the glass into 30-40 conical flasks, sealing the conical flasks, sterilizing in an autoclave at 121 deg.C for 20min, sounding 5 sound after sterilization, and cooling the culture medium.
Example 2:
a method for regenerating plants from the callus of schisandra chinensis, which is basically the same as the step of example 1, is characterized in that:
s1, cutting off the leaves of fructus Schisandrae chinensis (Hxatilis) aseptic seedling to 0.4-0.6cm3The square is used as an explant and inoculated in a No. 1 culture medium, the culture is carried out for 30 days, then the square is transferred to a new No. 1 culture medium for continuous culture for 30 days, then a tawny hard part is cut off, and then the square is transferred to the new No. 1 culture medium for continuous culture for 30 days to obtain embryogenic callus;
the culture medium No. 1 is based on MS culture medium, 2, 4-D with the concentration of 0.2 mg/L and TDZ with the concentration of 0.1 mg/L are added, the pH of the culture medium No. 1 is 5.8, and the culture condition is 25 ℃;
in S2, the embryogenic callus is inoculated into No. 2 culture medium and cultured for 30 days under the following culture conditions: obtaining the embryogenic callus after proliferation and propagation at the temperature of 25 ℃; ,
the culture medium No. 2 is based on MS culture medium, 2, 4-D with the concentration of 0.2 mg/L and TDZ with the concentration of 0.1 mg/L are added, and the pH of the culture medium No. 2 is 5.8;
in S3, cutting the embryogenic callus into small blocks, inoculating to No. 3 culture medium, and culturing at 25 deg.C for 30 days to obtain embryoid;
the culture medium No. 3 is based on MS culture medium, ABA with the concentration of 0.35 mg/L is added, and the pH of the culture medium No. 3 is 5.8;
s4, inoculating the cotyledonary embryo and the embryo with radicle to an MS culture medium, and continuing culturing for 30 days to form a seedling; the tissue culture temperature is 25 ℃, and the pH value of the MS culture medium is 5.8;
s5, hardening seedlings for 3 days after the seedlings form complete plants, wherein the mixing mass ratio of sand to soil is 1: 2.5.
example 3:
a method for regenerating plants from the callus of schisandra chinensis, which is basically the same as the step of example 1, is characterized in that:
s1, cutting off the leaves of fructus Schisandrae chinensis (Hxatilis) aseptic seedling to 0.4-0.6cm3The square of the strain is used as an explant and inoculated in a No. 1 culture medium, the culture is transferred to a new No. 1 culture medium for continuous culture for 32 days after 32 days of culture, then a tawny hard part is cut off, and the culture is transferred to the new No. 1 culture medium for continuous culture for 32 days to obtain embryogenic callus;
the culture medium No. 1 is based on MS culture medium, 2, 4-D with the concentration of 3.0 mg/L and TDZ with the concentration of 0.15 mg/L are added, the pH of the culture medium No. 1 is 6.0, and the culture condition is 27 ℃;
in S2, the embryogenic callus is inoculated into No. 2 culture medium and cultured for 32 days under the following culture conditions: obtaining the embryogenic callus after proliferation and propagation at the temperature of 27 ℃;
the culture medium No. 2 is based on MS culture medium, 2, 4-D with the concentration of 0.2 mg/L and TDZ with the concentration of 0.2 mg/L are added, and the pH of the culture medium No. 2 is 6.0;
in S3, cutting the embryogenic callus into small blocks, inoculating the small blocks into No. 3 culture medium, and continuously culturing for 32 days to obtain embryoid, wherein the tissue culture temperature is 27 ℃;
the No. 3 culture medium is based on an MS culture medium and is added with ABA with the concentration of 0.2 mg/L, and the pH of the No. 3 culture medium is 6.0;
s4, inoculating the cotyledonary embryo and the embryo with radicle to an MS culture medium, and continuously culturing for 32 days to form a seedling, wherein the tissue culture temperature is 27 ℃, and the pH value of the MS culture medium is 6.0;
s5, hardening seedlings for 3 days after the seedlings form complete plants, wherein the mixing mass ratio of sand to soil is 1: 3.5.
the results of the examples 1 to 3 are approximate, only the example 1 is taken as an example for relevant description, the leaf of the schisandra chinensis of the safflower in the example 1 is taken as an explant for inducing embryogenic callus, the growth condition is good, the structure is loose, and the transplanting survival rate reaches 100 percent; observing under a body type microscope to see various forms of embryo development, wherein the embryo development is carried out by a spherical embryo (figure 1), a heart-shaped embryo (figure 2), a torpedo-shaped embryo (figure 3) and a cotyledon-shaped embryo (figure 4); therefore, the five-spice leaf blade of safflower is used as the most suitable explant for inducing embryogenic callus;
the invention selects undeveloped leaves, stem segments and seed hypocotyls of dormant buds of schisandra chinensis as explants for inducing embryogenic callus, and uses the explants as comparative examples 1, 2 and 3 respectively, the influence of embryogenic callus induction when different parts of schisandra chinensis are used as the explants is researched, and culture steps which are not explicitly described in the comparative examples 1 to 3 are the same as the culture steps in the example 1.
Comparative example 1
Comparative example 1 provides a method for regenerating plants using unexpanded leaves of dormant buds of schisandra chinensis (schizandra sinensis) as explants, which is substantially the same as example 1 except that:
firstly, cleaning undeployed leaves of dormant buds with sterile water, soaking the leaves in 75% alcohol for 60s, washing with sterile water for 4 times, then placing the leaves into 0.1% mercuric chloride solution, standing for 10min, washing with sterile water for 5 times, and sucking water on the leaves for later use;
the rest of the cultivation steps and conditions were the same as in example 1.
Comparative example 2
Comparative example 2 provides a method for regenerating plants using stem segments of schisandra chinensis (schizandra sinensis) as explants, which is substantially the same as example 1 except that:
comparative example 2A stem section of Schisandra chinensis (Carthamus tinctorius) was used as an explant, the leaves of the young stem section of the upper half of the aseptic seedling were cut off, leaving only the stem section, the stem section was cut into 2-4cm pieces, and several cuts were made on the top with a scalpel, and the cut was sterilized and used.
Comparative example 3
Comparative example 3 provides a method for regenerating plants using hypocotyls of seeds of schisandra chinensis (schizandra androsaccensis) as explants, which is substantially the same as example 1 except that:
comparative example 3 the hypocotyl of the seed of schisandra chinensis (safflower) is used as the explant, and the early stage treatment is as follows: cutting the embryonic axis into 1-3cm segments, and cutting several knives thereon for inoculation.
As can be seen from the examples 1 and the comparative examples 1 to 3, the occurrence time sequence of the embryogenic callus is stem section > leaf section > undeployed leaf section, wherein the embryogenic callus which is visible to naked eyes appears at the incisions at the two ends after the stem section is inoculated for about 15 days, and then the embryogenic callus appears at the incision in the middle, the embryogenic callus is different in size, light yellow in color and loose in structure; the leaf blade is inoculated for about 15 days and begins to expand and curl, the embryogenic callus appears at the cut of the base part and the edge of the leaf blade for about 20 days, and then a thin layer of embryogenic callus appears on the surface part of the leaf blade, the color is light yellow green, and the structure is loose; the undeployed leaves begin to expand and curl about 20 days, embryogenic callus appears at the first edge cut in about 25 days, and the undeployed leaves are light green and green in color and loose in structure. As can be seen from Table 1, the embryogenic callus induced by the leaf and stem of the schisandra chinensis (Carthamus tinctorius L.) has good growth status, the embryogenic callus of the stem is early, the growth speed is high, and the embryogenic callus induced by the undeveloped leaf has relatively poor growth status;
TABLE 1 Effect of explant type on embryogenic Induction of embryogenic callus
The leaf and stem induced embryonic callus has good growth condition at the early stage, but in the late subculture process, a small part of the stem induced embryonic callus turns yellow brown, hard and compact.
Therefore, the leaf is the most suitable explant for inducing embryogenic callus, which is favorable for preserving excellent germplasm and better and directly obtaining excellent characteristics of a parent.
Compared with the invention, the comparison of different explants taken as comparative example regeneration plants shows that the growth conditions of the leaf and stem embryogenic callus induced by the leaf and stem are better and more, the embryogenic callus generation speed of the stem is high, but the browning is serious in the successive transfer process, the leaf browning is less, the number of leaf explants is more, the embryogenic callus generation speed of the unexpanded leaf is low, and the number of the embryogenic callus is less, so that the leaf is the most suitable explant for inducing the embryogenic callus.
The following comparative examples 4 to 6 were used to study the effect of exogenous plant growth regulator ABA on embryogenesis and development aberrations;
comparative example 4
Comparative example 4A method for regenerating plants from callus of Schisandra chinensis (Vaniot) is substantially the same as the step of example 1, except that:
in the step S3, the first step,
the No. 3 culture medium is based on an MS culture medium, is added with 0.2 mg/L2, 4-D +0.2 mg/L TDZ, and is not added with ABA;
comparative example 5
Comparative example 5A method for regenerating plants from callus of Schisandra chinensis (Vaniot) is basically the same as the comparative example 4, except that:
in the step S3, the first step,
the No. 3 culture medium is based on an MS culture medium, is added with 0.2 mg/L ABA +0.1 mg/L TDZ, and is not added with 2, 4-D;
comparative example 6
Comparative example 6A method for regenerating plants from the callus of Schisandra chinensis (Vaniot) is basically the same as the comparative example 4, except that:
in the step S3, the first step,
the No. 3 culture medium is based on an MS culture medium, is added with 0.2 mg/L2, 4-D +0.5 mg/L ABA and is not added with TDZ;
TABLE 2 Effect of exogenous plant growth regulator ABA concentration on embryogenesis and development
As can be seen from comparative examples 4-6, it is not easy to add TDZ and 2, 4-D to the MS medium during the embryogenesis, and the number of embryos is reduced; and the addition of ABA in the MS culture medium is beneficial to the improvement of the malformed embryos, greatly reduces the generation of the malformed embryos and is beneficial to the development of the embryos into seedlings (Table 2).
In conclusion, the invention discloses a method for regenerating plants from the callus of schisandra chinensis, which induces embryonic callus through vegetative propagation and forms regenerated plants through a somatic embryogenesis way, lays a foundation for the propagation of excellent germplasm of schisandra chinensis, is beneficial to the preservation of excellent germplasm, better and directly obtains excellent characters of a parent, greatly reduces the generation of abnormal embryos and is beneficial to the development of embryos into seedlings.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (6)
1. A method for regenerating plants from callus of schisandra chinensis, which is characterized by comprising the following steps:
s1, induction of embryogenic callus,
inoculating sterile seedling leaves of fructus Schisandrae Bicoloris in culture medium No. 1, culturing for 28-32 days, transferring to new culture medium No. 1, culturing for 28-32 days, cutting off yellow brown hard part, transferring to new culture medium No. 1, and culturing for 28-32 days to obtain embryogenic callus;
the No. 1 culture medium is based on MS culture medium, and is added with 2, 4-D with the concentration of 0.2-3.0 mg/L and TDZ with the concentration of 0.1-0.2 mg/L;
s2, propagation and propagation of the embryogenic callus,
inoculating the embryogenic callus obtained in S1 into No. 2 culture medium, and culturing for 28-32 days to obtain late embryogenic callus;
the No. 2 culture medium is based on MS culture medium, and is added with 2, 4-D with the concentration of 0.1-02 mg/L and TDZ with the concentration of 0.1-0.2 mg/L;
s3, the generation of embryos,
cutting late embryogenic callus obtained in S2 into 0.4-0.6cm3Inoculating the block to No. 3 culture medium, and culturing for 28-32 days to obtain embryoid;
the No. 3 culture medium is based on an MS culture medium and is added with ABA with the concentration of 0.2-0.5 mg/L;
s4, development of embryo and plant regeneration,
inoculating the embryoid obtained in the step S3 into an MS culture medium for embryo development, obtaining cotyledonary embryos and embryos with radicles after 28-32 days, inoculating the cotyledonary embryos and the embryos with the radicles into a new MS culture medium, and continuing culturing for 28-30 days to form seedlings;
s5, transplanting the regeneration plant,
after the seedlings obtained in S4 become complete plants, hardening the seedlings for 2-4 days, taking out the complete plants from the bottles, washing the culture medium at the roots, and transplanting the complete plants into the mixed sterile sand and soil.
2. The method for regenerating a plant from schisandra chinensis callus of claim 1, wherein in S2, the pH of the culture medium No. 2 is 5.6-6.0, and the culture conditions of said embryogenic callus are as follows: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8 h.
3. The method for regenerating a plant from schisandra chinensis callus of claim 1, wherein in S3, the pH of the culture medium No. 3 is 5.6-6.0, and the culture conditions of the embryogenic callus at the later stage are as follows: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8 h.
4. The method for regenerating a plant from the callus of schisandra chinensis as claimed in claim 1, wherein the pH of MS culture medium in S4 is 5.6-6.0.
5. The method for regenerating a plant from the callus of schisandra propinqua as claimed in claim 1, wherein in S4, said embryoid body and said cotyledonary embryo and radicle-growing embryo are cultured under the conditions: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8 h.
6. The method for regenerating plants from the callus of schisandra chinensis var safflower as claimed in claim 1, wherein in S5, the mixing mass ratio of sand and soil is 1: 2.5-3.5.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02154684A (en) * | 1988-12-05 | 1990-06-14 | Tsumura & Co | Medium for producing callus-inducing cell containing large amount of useful plant component and production of callus-inducing cell containing large amount of useful plant component |
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN1817106A (en) * | 2006-03-08 | 2006-08-16 | 东北林业大学 | Beiwuweizi cell embryo growth and strain regeneration |
| CN101904302A (en) * | 2010-07-13 | 2010-12-08 | 沈阳农业大学 | Method for somatic cell embryogeny and plant regeneration of medicinal plant schisandga chinensis baill |
| CN103299904A (en) * | 2013-05-30 | 2013-09-18 | 辽宁万亩五味子科技产业园区有限公司 | Artificial schisandra chinensis seed preparation and seedling culture method |
| CN104381129A (en) * | 2014-10-17 | 2015-03-04 | 南京帝道农业科技有限公司 | Rapid propagation method of green-leaf Schisandra chinensis plant regeneration |
| CN106171991A (en) * | 2016-07-15 | 2016-12-07 | 中国农业科学院特产研究所 | A kind of expanding propagation method of Radix Schisandrae Bicoloris |
| WO2019006466A1 (en) * | 2017-06-30 | 2019-01-03 | Booshoot Llc | Compositions and methods for large-scale in vitro plant bioculture |
-
2020
- 2020-05-12 CN CN202010397711.2A patent/CN111387059B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02154684A (en) * | 1988-12-05 | 1990-06-14 | Tsumura & Co | Medium for producing callus-inducing cell containing large amount of useful plant component and production of callus-inducing cell containing large amount of useful plant component |
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN1817106A (en) * | 2006-03-08 | 2006-08-16 | 东北林业大学 | Beiwuweizi cell embryo growth and strain regeneration |
| CN101904302A (en) * | 2010-07-13 | 2010-12-08 | 沈阳农业大学 | Method for somatic cell embryogeny and plant regeneration of medicinal plant schisandga chinensis baill |
| CN103299904A (en) * | 2013-05-30 | 2013-09-18 | 辽宁万亩五味子科技产业园区有限公司 | Artificial schisandra chinensis seed preparation and seedling culture method |
| CN104381129A (en) * | 2014-10-17 | 2015-03-04 | 南京帝道农业科技有限公司 | Rapid propagation method of green-leaf Schisandra chinensis plant regeneration |
| CN106171991A (en) * | 2016-07-15 | 2016-12-07 | 中国农业科学院特产研究所 | A kind of expanding propagation method of Radix Schisandrae Bicoloris |
| WO2019006466A1 (en) * | 2017-06-30 | 2019-01-03 | Booshoot Llc | Compositions and methods for large-scale in vitro plant bioculture |
Non-Patent Citations (6)
| Title |
|---|
| A.SMISKOVA等: "Somatic embryogenesis from zygotic embryos of Schisandra chinensis", 《BIOLOGIA PLANTARUM》 * |
| CHANG YEON YU等: "In vitro Callus formation and Plant Regeneration of Epimedium koreanum Nakai", 《KOREAN J. MEDICINAL CROP SCI.》 * |
| SANDRA CORREIA等: "Somatic embryogenesis in tamarillo (Cyphomandra betacea):approaches to increase efficiency of embryo formation and plant development", 《PLANT CELL TISS ORGAN CULT》 * |
| 刘茜等: "《高科技时代的植物组织培养新技术的研究应用》", 30 April 2018, 东北师范大学出版社 * |
| 孙丹等: "北五味子体细胞胚发生过程中内源IAA、ABA和GA3含量的动态变化", 《植物生理学报》 * |
| 武立丹等: "北五味子愈伤组织诱导研究", 《延边大学农学学报》 * |
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