CN111387059B - Method for regenerating plants from callus of schisandra chinensis - Google Patents
Method for regenerating plants from callus of schisandra chinensis Download PDFInfo
- Publication number
- CN111387059B CN111387059B CN202010397711.2A CN202010397711A CN111387059B CN 111387059 B CN111387059 B CN 111387059B CN 202010397711 A CN202010397711 A CN 202010397711A CN 111387059 B CN111387059 B CN 111387059B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- callus
- days
- culture
- schisandra chinensis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for regenerating a plant from a safflower schisandra callus, which relates to the technical field of plant tissue culture and comprises the following steps: induction of embryonic callus, proliferation and propagation of embryonic callus, embryogenesis, development and plant regeneration of embryo, 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 embryogenic callus of the explants, and the explants used for inducing the embryogenic callus of the schisandra chinensis have zygotic embryos and cotyledons after the germination of mature seeds, hypocotyls, winter buds of schisandra chinensis, tender stem sections, 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 parents can be 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, inducing the embryogenic callus,
inoculating sterile seedling leaves of fructus Schisandrae chinensis (safflower) in culture medium No. 1, culturing for 28-32 days, transferring to new culture medium No. 1, culturing for 28-32 days, cutting off 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 the S1 into a No. 2 culture medium to be cultured for 28-32 days to obtain later embryogenic callus;
the culture medium No. 2 is based on MS culture medium, and is added with 2,4-D with the concentration of 0.1-02mg/L and TDZ with the concentration of 0.1-0.2 mg/L;
s3, generating an embryo,
cutting the late embryogenic callus obtained in S2 into 0.4-0.6cm 3 Inoculating 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, the development of embryo and the regeneration of plant,
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 radicles into a new MS culture medium, and continuing culturing for 28-30 days to form seedlings;
s5, transplanting the regeneration plants,
and (4) after the seedlings obtained in the step (S4) form complete plants, hardening the seedlings for 2-4 days, taking out the complete plants from the bottles, cleaning culture media 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/8h.
Preferably, in S3, the pH of the No. 3 culture 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/8h.
Preferably, in S4, the pH of the MS culture medium is 5.6-6.0.
Preferably, in S4, the culture conditions of the embryoid body, the cotyledonary embryo and the embryo with radicle are as follows: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8h.
Preferably, in S5, the mixing mass ratio of sand to 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 magnolia schisandra chinensis, the corolla is milky, and the material used by the invention is the 'safflower' schisandra chinensis with red flower cover, the material is rare, and provides better material for researching scientific research experiments such as schisandra chinensis flower color and the like;
3. the invention improves the abnormal embryos by adding the ABA, greatly reduces the generation of the abnormal embryos after adding the ABA and is beneficial to the development of the embryos into seedlings.
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 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 plants from the callus of 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 0.4-0.6cm 3 The 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 an MS culture medium, 2,4-D with the concentration of 1.0mg/L and TDZ with the concentration of 0.2mg/L are added, and the pH value of the culture medium No. 1 is 5.6;
the temperature of the culture condition is 23 ℃, and the light-dark period is 16h/8h;
s2, propagation and propagation of the embryogenic callus,
inoculating the embryogenic callus obtained in the S1 into a No. 2 culture medium, and culturing for 28 days under the conditions that the temperature is 23 ℃ and the light-dark cycle is 16h/8h to obtain the embryogenic callus after proliferation and propagation;
the culture medium No. 2 is based on an MS culture medium, 2,4-D with the concentration of 0.1mg/L and TDZ with the concentration of 0.1mg/L are added, and the pH value of the culture medium No. 2 is 5.6;
s3, generating an embryo,
the embryogenic callus inoculated in the No. 2 culture medium in the S2 is cut into 0.4-0.6cm 3 Inoculating 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, the development of the embryo and the 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 the regeneration plants (figures 7-8),
and (5) hardening the seedlings for 2 days after the seedlings form complete plants in the S4, taking out the seedlings from the bottles, cleaning a culture medium at the root, and transplanting the seedlings into mixed sterile sand and soil, wherein the mixed mass ratio of the sand to the soil is 1:3.
the preparation steps of the culture medium in the S1-S3 are as follows:
(1) Weighing 30g of sucrose and 5.4g of agar powder (7 g of agar strips) in each 1L of culture medium, and respectively weighing 20mL, 10mL and 10mL of No. 1, 2, 3, 4, 5 and 6 mother liquor 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 1L cups 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 the large measuring cup by using pH test paper, and adjusting the pH value of the solution in the beaker 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 a 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 schisandra chinensis callus tissues is basically the same as the step of example 1, and the difference is that:
s1, cutting off two ends and the surface layer of leaves of the sterile seedlings of the schisandra chinensis (Carthamus tinctorius L.) and then shearing the leaves into 0.4-0.6cm 3 The 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 an MS culture medium, 2,4-D with the concentration of 0.2mg/L and TDZ with the concentration of 0.1mg/L are added, the pH of the culture medium No. 1 is 5.8, and the culture condition is 25 ℃;
s2, inoculating the embryogenic callus into a No. 2 culture medium for culturing for 30 days, wherein the culture conditions are as follows: 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 0.2mg/L and TDZ with the concentration of 0.1mg/L are added, and the pH value of the culture medium No. 2 is 5.8;
s3, cutting the embryogenic callus into small blocks, inoculating the small blocks to a No. 3 culture medium, and continuously culturing for 30 days at the culture temperature of 25 ℃ to obtain embryoids;
the No. 3 culture medium is based on an MS culture medium, and is added with ABA with the concentration of 0.35mg/L, and the pH of the No. 3 culture medium is 5.8;
s4, inoculating the cotyledon embryo and the embryo with radicle to an MS culture medium to continue 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 two ends and the surface layer of leaves of the sterile seedlings of the schisandra chinensis (Carthamus tinctorius L.) and then shearing the leaves into 0.4-0.6cm 3 Taking the square as an explant, inoculating the explant in a No. 1 culture medium, transferring the explant to a new No. 1 culture medium after culturing for 32 days, continuously culturing for 32 days, cutting off a yellow brown hard part, and transferring the explant to the new No. 1 culture medium to continuously 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 3.0mg/L and TDZ with the concentration of 0.15mg/L are added, the pH of the culture medium No. 1 is 6.0, and the culture condition is 27 ℃;
s2, inoculating the embryogenic callus into a No. 2 culture medium for 32 days, wherein the culture conditions are as follows: obtaining the embryogenic callus after proliferation and propagation at the temperature of 27 ℃;
the culture medium No. 2 is based on an MS culture medium, 2,4-D with the concentration of 0.2mg/L and TDZ with the concentration of 0.2mg/L are added, and the pH value of the culture medium No. 2 is 6.0;
s3, cutting the embryogenic callus into small blocks, inoculating the small blocks into a 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 0.2mg/L, and the pH value of the No. 3 culture medium is 6.0;
s4, inoculating the cotyledon-shaped embryos and the embryos with radicles to an MS culture medium, and continuously culturing for 32 days to form seedlings, 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; the embryo development can be seen in various forms under a body type microscope, and 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 undeveloped leaves of dormant buds with sterile water, soaking the leaves in 75% alcohol for 60s, washing the leaves with the sterile water for 4 times, then putting the leaves into 0.1% mercuric chloride solution, standing for 10min, washing the leaves with the sterile water for 5 times, and sucking water on the leaves for later use;
the rest of the culturing 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 the schisandra chinensis is taken as the explant, and the early 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 stage 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 different explants taken as comparative example regeneration plants are found to have better growth conditions and more amount of the leaf and stem induced embryogenic callus, the stem embryogenic callus has high speed, but the browning is more serious in the successive transfer process, the leaf browning is less, the number of the leaf explants is more, the embryogenic callus speed of the unexpanded leaves is low, and the amount 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 influence of exogenous plant growth regulator ABA on the development and development of embryos;
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 step of,
the No. 3 culture medium is based on an MS culture medium, is added with 0.2 mg/L2, 4-D +0.2mg/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 0.2mg/L ABA +0.1mg/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 step of,
the No. 3 culture medium is based on an MS culture medium, is added with ABA with the concentration of 0.2 mg/L2, 4-D +0.5mg/L, 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 was not easy to add TDZ and 2,4-D to the MS medium during the embryogenesis, and the number of embryos decreased; and the addition of ABA in the MS culture medium is beneficial to improving the abnormal embryos, greatly reduces the generation of the abnormal 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, inducing the embryogenic callus,
inoculating sterile seedling leaves of fructus Schisandrae chinensis (Carthami flos) in culture medium No. 1, culturing for 28-32 days, transferring to new culture medium No. 1, culturing for 28-32 days, cutting off 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 an MS culture medium and is added with 2,4-D with the concentration of 0.2-3.0mg/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 the S1 into a No. 2 culture medium to be cultured for 28-32 days to obtain later embryogenic callus;
the No. 2 culture medium is based on an MS culture medium and is added with 2,4-D with the concentration of 0.1-0.2mg/L and TDZ with the concentration of 0.1-0.2 mg/L;
s3, generating an embryo,
cutting the late embryogenic callus obtained in S2 into 0.4-0.6cm 3 Inoculating 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, the development of embryo and the regeneration of plant,
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 radicles into a new MS culture medium, and continuing culturing for 28-30 days to form seedlings;
s5, transplanting the regeneration plants,
and (5) hardening the seedlings for 2-4 days after the seedlings obtained in the step (S4) form complete plants, taking out the complete plants from the bottle, cleaning 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 the embryogenic callus are as follows: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8h.
3. The method for regenerating a plant from schisandra chinensis callus of claim 1, wherein in S3, the pH of medium 3 is 5.6-6.0, and the culture conditions of said later embryogenic callus are: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8h.
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 schisandra chinensis callus derived from claim 1, wherein in S4, said embryoid body, said cotyledonary embryo and radicular embryo are cultured under the conditions that: the culture temperature is 23-27 deg.C, and the light-dark period is 16h/8h.
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010397711.2A CN111387059B (en) | 2020-05-12 | 2020-05-12 | Method for regenerating plants from callus of schisandra chinensis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010397711.2A CN111387059B (en) | 2020-05-12 | 2020-05-12 | Method for regenerating plants from callus of schisandra chinensis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111387059A CN111387059A (en) | 2020-07-10 |
| CN111387059B true CN111387059B (en) | 2022-11-22 |
Family
ID=71412006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010397711.2A Active CN111387059B (en) | 2020-05-12 | 2020-05-12 | Method for regenerating plants from callus of schisandra chinensis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111387059B (en) |
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 |
-
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 (5)
| Title |
|---|
| In vitro Callus formation and Plant Regeneration of Epimedium koreanum Nakai;Chang Yeon Yu等;《Korean J. Medicinal Crop Sci.》;20021231;第10卷(第4期);第259-262页 * |
| Somatic embryogenesis from zygotic embryos of Schisandra chinensis;A.SMISKOVA等;《BIOLOGIA PLANTARUM》;20051231;第49卷(第3期);第451-454页 * |
| Somatic embryogenesis in tamarillo (Cyphomandra betacea):approaches to increase efficiency of embryo formation and plant development;Sandra Correia等;《Plant Cell Tiss Organ Cult》;20121231;第109卷;第143–152页 * |
| 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;孙丹等;《植物生理学报》;20131231;第49卷(第1期);第70-74页 * |
| 北五味子愈伤组织诱导研究;武立丹等;《延边大学农学学报》;20080930;第30卷(第3期);第167-171、187页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111387059A (en) | 2020-07-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Litz et al. | In vitro somatic embryogenesis and plant regeneration from Carica papaya L. ovular callus | |
| Cheema | Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature Himalayan poplar (Populus ciliata) | |
| CN102499080B (en) | Plant fast propagating method using fagopyrum tataricum leaf stalks as explants | |
| CN105815213A (en) | Establishing method for in-vitro regeneration system of Kiwi berry | |
| Sö ndahl et al. | Cacao somatic embryogenesis | |
| Liu | Factors affecting induction, somatic embryogenesis and plant regeneration of callus from cultured immature inflorescences of sugarcane | |
| CN112931198B (en) | Preparation method of pineapple cold-resistant germplasm | |
| CN102907318A (en) | Rapid propagation of notoginseng regenerated plant by using bioreactor to cultivate notoginseng somatic embryos | |
| CN106613997A (en) | In-vitro regeneration culture method of tree peonies | |
| CN105028212A (en) | Establishment method of efficient regeneration system of mniochloa abersend | |
| CN103053423B (en) | Method for establishing high-efficiency regeneration system by using broccoli microspore embryo as explant | |
| CN104737912A (en) | Tissue culture and rapid propagation culture medium for gerbera jamesonii and culture method thereof | |
| CN109392721A (en) | A method of induction sweet potato plant regeneration | |
| CN108064699A (en) | A kind of tissue Propagation In Vitro method of pueraria lobata plant | |
| CN109717080B (en) | Method for improving successive transfer effect of agapanthus embryonic cells | |
| CN111387059B (en) | Method for regenerating plants from callus of schisandra chinensis | |
| CN114532227B (en) | Method for inducing and proliferating calluses of agapanthus radicis roots and tips | |
| CN107022519B (en) | Separation culture method of tea suspension single cells | |
| CN112493126B (en) | Method for induction of lagerstroemia indica somatic embryo and plant regeneration | |
| CN104115751A (en) | Culture method for obtaining regenerated plantlet by utilizing Chinese cabbage bulb leaves | |
| WO1993012645A1 (en) | Somatic embryogenesis and plant regeneration of cacao | |
| CN112189562B (en) | Method for inducing callus to regenerate plant by bamboo joint | |
| CN110896853B (en) | Liquid suspension culture method for promoting maturation of somatic embryos of liquidambar plants | |
| Huang et al. | Chinese gooseberry, kiwifruit (Actinidia spp.) | |
| CN109729979B (en) | Method for promoting agapanthus somatic embryo germination synchronization rate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |