CN113100068B - In-vitro culture and regeneration method of cinnamomum camphora - Google Patents
In-vitro culture and regeneration method of cinnamomum camphora Download PDFInfo
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a camphor in-vitro culture and regeneration method, which specifically comprises the following steps: (1) selecting immature fruits of healthy disease-free adult cinnamomum camphora, and sterilizing and inducing to obtain cotyledonary somatic embryos; (2) dark multiplication and subculture are carried out to obtain a clone cotyledon-shaped embryo; (3) culturing in dark/weak light, inoculating and healing to obtain germinated embryos; (4) performing illumination culture to obtain a somatic embryogenesis related culture material and clumped seedlings; (5) selecting single small branches with terminal buds in the cluster seedlings to carry out illumination culture and rooting to obtain complete plants. The cinnamomum camphora leaf-shaped somatic embryo is used as an induction starting material and a proliferation intermediate material for somatic embryo germination, the embryo of the germinated somatic embryo is used as a key material for obtaining the cinnamomum camphora somatic embryogenesis and organogenesis (seedling clumping), the single branch with the apical bud is used as an induction material for forming a complete plant, the operation is simple, various sterile materials are obtained and easily proliferated, and strong cell totipotency can be shown.
Description
Technical Field
The invention relates to the technical field of plant cell engineering, in particular to an in vitro culture and regeneration method of cinnamomum camphora.
Background
Cinnamomum camphora (Cinnamomum camphora L.) is a plant of Lauraceae and belongs to the genus Cinnamomum, and is a kind of evergreen arbor mainly distributed in Yangtze river basin and in the south area, and is called four famous trees in south of Yangtze river together with nan, catalpa and tung. With the development of urban garden construction, cinnamomum camphora is widely applied to urban street trees, shade trees, landscape landscaping and the like. Moreover, many garden workers try to introduce the cinnamomum camphora in the north, but the cinnamomum camphora is warm in preference and cannot adapt to the cold climate in the north, the low-temperature freezing injury prevents the transplantation and planting of the cinnamomum camphora in the north, and the agenda schedule is provided for cultivating the new germplasm of the cinnamomum camphora with cold resistance.
With the development of plant cell engineering and plant transgenic technology, theoretical basis and technical possibility are provided for obtaining the cinnamomum camphora with cold resistance in molecular breeding. The high-frequency plant regeneration system is a prerequisite foundation for applying a transgenic technology, however, the cinnamomum camphora is a woody plant, the genetic background is complex, the isolated culture is difficult, a proper isolated culture material is selected, a set of plant regeneration system with stable material source and high-efficiency plant regeneration is established, and the high-frequency plant regeneration system is particularly important for improving the cold resistance of the cinnamomum camphora of the woody plant by a molecular means; especially, the steps of identifying and screening the transgenic chimera can be greatly reduced through a plant regeneration way (single cell origin) of somatic embryogenesis.
Therefore, the technical problem to be solved by the technical personnel in the field is how to provide a cinnamomum camphora somatic embryo germination (the process of simultaneously forming stem segments and root tips on a culture material in the process of in vitro culture), in vitro culture and plant regeneration method.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for in vitro culture and regeneration of cinnamomum camphora, so as to solve the deficiencies in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
an in vitro culture and regeneration method of cinnamomum camphora comprises the following steps:
(1) selecting immature fruits of healthy disease-free adult cinnamomum camphora, cutting and stripping cotyledon embryos after disinfection treatment, and then inoculating the cotyledon embryos on a somatic embryo induction culture medium for induction to obtain cotyledon-like somatic embryos;
(2) inoculating the cotyledon somatic embryos on the somatic embryo induction culture medium again for dark multiplication and subculture to obtain clone cotyledon somatic embryos;
(3) inoculating the clone cotyledon somatic embryos on a somatic embryo induction culture medium again for dark/weak light culture, and then inoculating the small-particle cotyledon somatic embryos for callus formation to obtain germinating somatic embryos;
(4) cutting off the embryo of the germinated embryo and switching the embryo into an embryo inducing culture medium for illumination culture to obtain embryo generation related culture materials and clustered seedlings;
(5) and (4) selecting single small branches with terminal buds in the cluster seedlings, transferring the single small branches with the terminal buds into an MS culture medium, and performing illumination culture for rooting to obtain complete plants.
The invention has the advantages that the cinnamomum camphora cotyledon somatic embryo is used as an induction starting material and a proliferation intermediate material for somatic embryo germination, the embryo of the germinated somatic embryo is used as a key material for obtaining the induction of cinnamomum camphora somatic embryogenesis and organogenesis (clump seedling), the single-plant twig with the terminal bud is used as an induction material for forming a complete plant, the operation is simple, various sterile materials are obtained and easily proliferated, and strong cell totipotency can be shown.
Wherein, the somatic embryo germination is used for realizing the material state preservation (before and after transformation) of the high-frequency cell totipotency; the somatic embryo bud cluster seedling is used for the quick propagation of the transgenic clone and the regeneration of the complete plant (after transformation); cotyledonary somatic embryos can be used for donor material multiplication (before transformation), transgenic recipient material (during transformation) and recipient material somatic embryo germination (after transformation); embryogenic callus can be used for donor material propagation (pre-transformation) and transgenic recipient material (in transformation); although non-embryogenic callus is a redundant material, it is an essential induction link for the germination of single-cell somatic embryos.
Further, in the step (1), the sterilization treatment specifically comprises: firstly, putting immature fruits of cinnamomum camphora into an alcohol solution with the volume concentration of 70-75% for soaking for 30-45 s, taking out the fruits, and then putting the fruits into a mercuric chloride solution with the mass concentration of 0.1% for soaking for 7 min.
The beneficial effect of adopting the further technical scheme is that the cotyledon embryo in the fruit is taken out after the fruit is disinfected, so that the poison of the disinfectant to the explant can be reduced, and the survival rate of the explant and the induction rate of the somatic embryo can be improved.
Further, in the step (1), the somatic embryo induction culture medium comprises 1.2-2.0 mg/L of 6-BA and 0.2-0.5 mg/L of IBA.
The adoption of the further technical scheme has the beneficial effects that the somatic embryo induction culture medium is a combination of high-concentration cytokinin and low-concentration auxin, is beneficial to the cell division of the cotyledon embryo, promotes the explant to induce the cotyledon-shaped somatic embryo, and is used for the induction and proliferation of the cotyledon embryo, the proliferation of embryogenic callus and the induction and proliferation of clump seedlings.
Further, in the step (1), the induced illumination period is 16h illumination/8 h dark, the illumination intensity is 2000Lx, and the time is 15 days.
The beneficial effect of adopting the further technical scheme is that the multiplication activity of the cotyledonary somatic embryo can be ensured by observing that the cotyledonary somatic embryo is taken down from the explant in time.
Further, in the step (2), the subculture period is 4 to 6 weeks.
Further, in the step (3), the dark/low light culture period is 4 to 6 weeks.
Further, in the step (3), the particle size of the small particle leaf-like embryo is 3-5 mm.
Further, in the step (4), the somatic embryogenesis related culture material comprises embryogenic callus, granular somatic embryos and germinated somatic embryos.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. as the plant regeneration mode established by the invention is derived from the germination of the somatic embryo originated from single cells, and the subsequent proliferation and induction system is established by using the embryo of the germinated somatic embryo, when the plant regeneration mode is used as a transgenic technology receptor material, on one hand, the high-frequency realization of the cell totipotency of the material can be maintained, on the other hand, the identification and screening steps of transgenic chimeras are not needed, and the important effect on improving the cinnamomum camphora by molecular means can be played.
2. The invention organically integrates two different in vitro plant regeneration ways of organogenesis and somatic embryogenesis for the first time, and can be applied to selection, preservation and proliferation of good regeneration capacity materials (transgenic receptor materials) of woody plants and plant regeneration and rapid propagation of transgenic positive materials.
3. The camphor tree cotyledon embryo clone obtained by screening and the established camphor tree somatic embryo germination related material proliferation and induction method provide sufficient receptor materials and plant regeneration systems for improving the camphor tree cold resistance by the agrobacterium-mediated biotechnology.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The in vitro culture and regeneration method of cinnamomum camphora specifically comprises the following steps:
(1) selecting healthy disease-free adult cinnamomum camphora (in campus of south Yang academy of academic Press, Henan province), selecting immature fruits of cinnamomum camphora with the diameter of about 1cm, firstly soaking the immature fruits in 70% alcohol solution for 30s, taking out the immature fruits, then soaking the immature fruits in 0.1% mercury chloride solution for 7min, then placing the immature fruits on sterile filter paper, cutting the immature fruits at a position which is perpendicular to a fruit base and is away from the fruit base 2/3 by using a scalpel, peeling cotyledon embryos by using a dissecting needle, inoculating the dissected embryos onto a somatic embryo induction culture medium (6-BA with the concentration of 1.2mg/L and IBA with the concentration of 0.2 mg/L), wherein the illumination period is 16h illumination/8 h, the illumination intensity is 2000Lx, and after inoculating and culturing for 15 days, milky white cotyledon embryos can be observed to be generated on the cotyledon of cinnamomum camphora;
(2) stripping cotyledon-shaped somatic embryos by using forceps, inoculating the cotyledon-shaped somatic embryos on a somatic embryo induction culture medium again, placing the somatic embryos in a dark environment for proliferation, and carrying out subculture once every 4-6 weeks to obtain clone cotyledon-shaped somatic embryos;
(3) placing the clone cotyledon embryo on sterile filter paper, cutting with a scalpel, inoculating on a somatic embryo induction culture medium again for dark/weak light culture, culturing large-particle cotyledon embryo with the size of 5-7mm after inoculating for 4-6 weeks, and observing proliferation of the cotyledon embryo; culturing small-particle leaf-shaped somatic embryos with the size of 3-5mm, observing callus formation of the somatic embryos, and inducing the surface of the obtained callus to see somatic embryo germination, namely simultaneously forming stem sections and root tips of the cultured material;
(4) selecting embryo of a germinating embryo as a key material for germinating cinnamomum camphora embryo, cutting off a transformant embryo induction culture medium in time for illumination culture, observing cluster bud proliferation after 10-15 days, continuously culturing, and sequentially observing cluster bud growth (cluster seedlings) and generation of white and transparent non-embryogenic callus, wherein after 4-6 weeks, the cinnamomum camphora embryo generation related culture material can be observed on the non-embryogenic callus: yellow embryogenic callus, granular embryos and germinated embryos;
when the induction of the material is observed, immediately inoculating the yellow embryonic callus into a somatic embryo induction culture medium for dark culture; then, if subculture is carried out once every 15 days, yellow embryonic callus proliferation can be obtained; if subculture is carried out once every 4-6 weeks, granular somatic embryos can be obtained by induction on the yellow embryogenic callus, cotyledonary somatic embryos can be observed to form after the granular somatic embryos are inoculated in a somatic embryo induction culture medium for 4 weeks, and at the moment, cotyledonary somatic embryo proliferation or somatic embryo germination can reappear when the cotyledonary somatic embryos are treated according to the step (3);
(5) selecting single small branches with terminal buds about 1.5-2cm higher in the cluster seedlings, transferring the single small branches into an MS culture medium, and culturing for about 10 days under illumination to observe rooting to gradually form a complete plant; and cutting off all terminal buds of the clustered seedlings with the length of less than 1cm, cutting the base parts of the clustered seedlings into blocks, placing the blocks in a culture medium with the same formula, and observing the multiplication of the clustered seedlings after the seedlings are cultured for 15 days by illumination.
Example 2
The camphor in-vitro culture and regeneration method specifically comprises the following steps:
(1) selecting healthy disease-free adult cinnamomum camphora (in campus of south Yang academy of academic Press, Henan province), selecting immature fruits of cinnamomum camphora with the diameter of about 1cm, firstly soaking the immature fruits in 72% alcohol solution for 40s, taking out the immature fruits, then soaking the immature fruits in 0.1% mercury chloride solution for 7min, then placing the immature fruits on sterile filter paper, cutting the immature fruits at a position which is perpendicular to a fruit base and is away from the fruit base 2/3 by using a scalpel, peeling cotyledon embryos by using a dissecting needle, inoculating the dissected embryos onto a somatic embryo induction culture medium (6-BA with the concentration of 1.5mg/L and IBA with the concentration of 0.4 mg/L), wherein the illumination period is 16h illumination/8 h, the illumination intensity is 2000Lx, and after inoculating and culturing for 15 days, milky white cotyledon embryos can be observed to be generated on the cotyledon of cinnamomum camphora;
(2) stripping cotyledon-shaped somatic embryos by using forceps, inoculating the cotyledon-shaped somatic embryos on a somatic embryo induction culture medium again, placing the somatic embryos in a dark environment for proliferation, and carrying out subculture once every 4-6 weeks to obtain clone cotyledon-shaped somatic embryos;
(3) placing the clone cotyledon embryo on sterile filter paper, cutting with a scalpel, inoculating on a somatic embryo induction culture medium again for dark/weak light culture, culturing large-particle cotyledon embryo with the size of 5-7mm after inoculating for 4-6 weeks, and observing proliferation of the cotyledon embryo; culturing small-particle leaf-shaped somatic embryos with the size of 3-5mm, observing callus formation of the somatic embryos, and inducing the surface of the obtained callus to see somatic embryo germination, namely simultaneously forming stem sections and root tips of the cultured material;
(4) selecting embryo of the germinating embryo as a key material for germinating the cinnamomum camphora somatic embryo, cutting off a transformant embryo induction culture medium in time for illumination culture, observing cluster bud proliferation after 10-15 days, continuously culturing, observing cluster bud growth (cluster seedlings) and generation of white and transparent non-embryogenic callus, and observing a cinnamomum camphora somatic embryogenesis related culture material on the non-embryogenic callus after 4-6 weeks: yellow embryogenic callus, granular somatic embryos and germinated somatic embryos;
when the induction of the material is observed, immediately inoculating the yellow embryonic callus into a somatic embryo induction culture medium for dark culture; then, if subculture is carried out once every 15 days, yellow embryonic callus proliferation can be obtained; if subculture is carried out once every 4-6 weeks, granular somatic embryos can be obtained by induction on the yellow embryogenic callus, cotyledonary somatic embryos can be observed to form after the granular somatic embryos are inoculated in a somatic embryo induction culture medium for 4 weeks, and at the moment, cotyledonary somatic embryo proliferation or somatic embryo germination can reappear when the cotyledonary somatic embryos are treated according to the step (3);
(5) selecting single small branches with terminal buds about 1.5-2cm higher in the cluster seedlings, transferring the single small branches into an MS culture medium, and culturing for about 10 days under illumination to observe rooting to gradually form a complete plant; and cutting off all terminal buds of the clustered seedlings with the length of less than 1cm, cutting the base parts of the clustered seedlings into blocks, placing the blocks in a culture medium with the same formula, and observing the multiplication of the clustered seedlings after the seedlings are cultured for 15 days by illumination.
Example 3
The camphor in-vitro culture and regeneration method specifically comprises the following steps:
(1) selecting healthy disease-free adult cinnamomum camphora (in campus of south Yang academy of academic Press, Henan province), selecting immature fruits of cinnamomum camphora with the diameter of about 1cm, firstly soaking the immature fruits in 75% alcohol solution by volume for 45s, taking out the immature fruits, then soaking the immature fruits in 0.1% mercury chloride solution by mass concentration for 7min, then placing the immature fruits on sterile filter paper, cutting the immature fruits at a position which is perpendicular to a fruit base and is away from the fruit base 2/3 by using a scalpel, peeling cotyledon embryos by using a dissecting needle, inoculating the dissected embryos onto a somatic embryo induction culture medium (6-BA of 2.0mg/L and IBA of 0.5 mg/L), wherein the illumination period is 16h of illumination/8 h, the illumination intensity is 2000Lx, and after inoculating and culturing for 15 days, milky white cotyledon embryos can be observed to be generated on the cotyledon of cinnamomum camphora;
(2) peeling off cotyledonary somatic embryos by using forceps, inoculating the cotyledonary somatic embryos on a somatic embryo induction culture medium again, placing the somatic embryos in a dark environment for proliferation, and carrying out subculture once every 4 to 6 weeks to obtain clone cotyledonary somatic embryos;
(3) placing the clone cotyledon somatic embryos on sterile filter paper, cutting up the cotyledon somatic embryos by using a scalpel, inoculating the cotyledon somatic embryos on a somatic embryo induction culture medium again for dark/low light culture, and culturing large-particle cotyledon somatic embryos with the size of 5-7mm in a cutting block after inoculating for 4-6 weeks, wherein the multiplication of the cotyledon somatic embryos can be observed; culturing small particle leaf-shaped somatic embryos with the size of 3-5mm, observing callus organization of the somatic embryos, and inducing the surface of the obtained callus to see somatic embryo germination, namely forming stem sections and root tips of the cultured material at the same time;
(4) selecting embryo of a germinating embryo as a key material for germinating cinnamomum camphora embryo, cutting off a transformant embryo induction culture medium in time for illumination culture, observing cluster bud proliferation after 10-15 days, continuously culturing, and sequentially observing cluster bud growth (cluster seedlings) and generation of white and transparent non-embryogenic callus, wherein after 4-6 weeks, the cinnamomum camphora embryo generation related culture material can be observed on the non-embryogenic callus: yellow embryogenic callus, granular somatic embryos and germinated somatic embryos;
when the induction of the material is observed, immediately inoculating the yellow embryonic callus into a somatic embryo induction culture medium for dark culture; then, if subculture is carried out once every 15 days, yellow embryonic callus proliferation can be obtained; if subculture is carried out once every 4-6 weeks, granular somatic embryos can be obtained by induction on the yellow embryogenic callus, cotyledonary somatic embryos can be observed to form after the granular somatic embryos are inoculated in a somatic embryo induction culture medium for 4 weeks, and at the moment, cotyledonary somatic embryo proliferation or somatic embryo germination can reappear when the cotyledonary somatic embryos are treated according to the step (3);
(5) selecting single small branches with terminal buds of about 1.5-2cm higher in the cluster seedlings, transferring the single small branches with the terminal buds into an MS culture medium, and culturing the single small branches with the terminal buds in light for about 10 days to observe rooting and gradually form complete plants; and cutting off all terminal buds of the clustered seedlings with the length of less than 1cm, cutting the base parts of the clustered seedlings into blocks, placing the blocks in a culture medium with the same formula, and observing the multiplication of the clustered seedlings after the seedlings are cultured for 15 days by illumination.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. An in vitro culture and regeneration method of cinnamomum camphora is characterized by comprising the following steps:
(1) selecting immature fruits of healthy disease-free adult cinnamomum camphora, cutting and stripping cotyledon embryos after disinfection treatment, and then inoculating the cotyledon embryos on an embryo induction culture medium for illumination induction to obtain cotyledon-like embryos; the somatic embryo induction culture medium comprises 1.2-2.0 mg/L of 6-BA and 0.2-0.5 mg/L of IBA; the induced illumination period is 16h illumination/8 h darkness, the illumination intensity is 2000Lx, and the time is 15 days;
(2) inoculating the cotyledon somatic embryos on the somatic embryo induction culture medium again for dark multiplication and subculture to obtain clone cotyledon somatic embryos; the subculture time is 4-6 weeks;
(3) placing the clone cotyledon somatic embryos on sterile filter paper, cutting up the cotyledon somatic embryos by using a scalpel, inoculating the cotyledon somatic embryos on a somatic embryo induction culture medium again for dark/low light culture, and culturing large-particle cotyledon somatic embryos with the size of 5-7mm in a cutting block after inoculating for 4-6 weeks, wherein the multiplication of the cotyledon somatic embryos can be observed; culturing small-particle leaf-shaped somatic embryos with the size of 3-5mm, observing callus formation of the somatic embryos, and inducing the surface of the obtained callus to see somatic embryo germination, namely simultaneously forming stem sections and root tips of the cultured material;
(4) selecting embryo of the germinating embryo as a key material for germinating cinnamomum camphora embryo, cutting off a transformant embryo induction culture medium in time for illumination culture, observing cluster bud proliferation after 10-15 days, continuing culture, observing cluster bud growth and generation of white and transparent non-embryogenic callus, and observing cinnamomum camphora embryogenic related culture materials on the non-embryogenic callus after 4-6 weeks: yellow embryogenic callus, granular somatic embryos and germinated somatic embryos;
when the induction of the material is observed, immediately inoculating the yellow embryonic callus into a somatic embryo induction culture medium for dark culture; then, if subculture is carried out once every 15 days, yellow embryonic callus proliferation can be obtained; if subculture is carried out once every 4-6 weeks, granular somatic embryos can be obtained by induction on the yellow embryogenic callus, cotyledonary somatic embryos can be observed to form after the granular somatic embryos are inoculated in a somatic embryo induction culture medium for 4 weeks, and at the moment, cotyledonary somatic embryo proliferation or somatic embryo germination can reappear when the cotyledonary somatic embryos are treated according to the step (3);
(5) and (4) selecting single small branches with terminal buds in the cluster seedlings, transferring the single small branches with the terminal buds into an MS culture medium, and performing illumination culture for rooting to obtain complete plants.
2. The in vitro culture and regeneration method of cinnamomum camphora according to claim 1, wherein in step (1), the disinfection treatment specifically comprises: firstly, putting immature fruits of cinnamomum camphora into an alcohol solution with the volume concentration of 70-75% for soaking for 30-45 s, taking out the fruits, and then putting the fruits into a mercuric chloride solution with the mass concentration of 0.1% for soaking for 7 min.
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