CN109937886B - Method for observing explant generating way - Google Patents
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- CN109937886B CN109937886B CN201910383496.8A CN201910383496A CN109937886B CN 109937886 B CN109937886 B CN 109937886B CN 201910383496 A CN201910383496 A CN 201910383496A CN 109937886 B CN109937886 B CN 109937886B
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Abstract
The invention discloses a method for observing explant generating ways, which is characterized in that leaves and stem segments of crassula argentea are used as explants to be inoculated into MS culture media containing plant growth regulators with different concentrations, and four different explant generating ways of sterile short shoot type, cluster bud multiplication type, embryoid generation type and organ generation type can be observed after 30 days. The teaching process is optimized, and the teaching effect is improved; is beneficial to the cultivation of the practical ability and the observation ability of students. The method comprises the following steps: (1) obtaining a material; (2) performing aseptic treatment; (3) sterile short-shoot type; (4) cluster bud proliferative type; (5) embryoid generation type; (6) organogenesis type i; (7) organotypic type II.
Description
Technical Field
The invention relates to the field of experimental teaching of plant tissue culture courses, in particular to a tissue culture method of crassula argentea.
Background
In plant tissue culture courses, the routes of explant generation are classified into sterile brachytic, cluster bud-proliferating, organogenic, embryoid-proliferating and orchid-specific protocorm-proliferating routes according to the theory of cell totipotency. Students are often confused about how to quickly and efficiently distinguish between these several approaches.
The crassula argentea is perennial evergreen succulent plant of genus Cynomorium of family Crassulaceae. The fleshy leaves are opposite, and the color is emerald; the stem is round, thick, rich and has multiple branches, and the plant shape is full and round. In recent years, stem sections and leaves of crassula argentea are selected as explants and inoculated to MS culture media containing different plant growth regulators, and other four explant generation ways except the original bulb proliferation way specific to orchidaceae plants can be realized on the plants; the change of the explants is continuously observed after inoculation, the cultivation of the practical ability and the observation ability of students is facilitated, and good teaching effect is achieved.
Disclosure of Invention
The invention aims to provide a method for observing the generation path of an explant, which is characterized in that leaves and stem segments of crassula argentea are used as the explant to be inoculated into MS culture media containing plant growth regulators with different concentrations, and four different explant generation paths can be observed after 30 days.
The invention relates to a method for observing an explant generating path, which comprises the following steps:
(1) obtaining of materials: picking fresh healthy leaves and stem segments of crassula argentea, and washing the surface with tap water for 20 min;
(2) and (3) sterile treatment: soaking the washed leaves and stem segments on an ultra-clean workbench for 15s by using 75% alcohol, washing the leaves and stem segments with sterile water for 1 time after soaking, then soaking the leaves and stem segments in 0.1% mercuric chloride solution for disinfection for 6min, washing the leaves and stem segments with sterile water for 3-5 times after disinfection, and finally sucking the water on the surfaces of the leaves and stem segments by using disinfection filter paper;
(3) sterile brachytic generation pathway: cutting the stem section obtained in the step (2) into small stem sections with a pair of axillary buds, inoculating the small stem sections on an MS minimal medium, and inducing the stem sections to grow;
(4) the proliferation pathway of the cluster buds: cutting the stem section obtained in the step (2) into small stem sections with a pair of axillary buds, inoculating the small stem sections on a cluster bud multiplication culture medium, and inducing to generate cluster buds;
(5) the embryogenic pathway: cutting the leaves obtained in the step (2) into the size of about 1.0cm multiplied by 1.0cm, inoculating the cut leaves onto an MS minimal medium, and inducing the adventitious buds;
(6) organotypic pathway i: cutting the leaf obtained in the step (2) into about 1.0cm multiplied by 1.0cm, inoculating the leaf to a callus induction culture medium, and inducing to generate callus;
(7) organogenic pathway ii: cutting the callus obtained in the step (6) into 0.5cm multiplied by 0.5cm, inoculating the cut callus to an MS basic culture medium, and inducing the callus to redifferentiate to form a seedling;
2. the invention provides a method for observing an explant generating path, which is characterized by comprising the following steps: and (3) before the sterile operating platform in the step (2) is used, an ultraviolet sterilizing lamp and a fan are required to be started for sterilizing for 30 min.
3. The invention provides a method for observing an explant generation path through tissue culture of crassula argentea in plant tissue culture teaching, which is characterized by comprising the following steps: the 0.15% mercuric chloride solution in the step (2) contains 1-2 drops of 20mg/L Tween.
4. The invention provides a method for observing the generation path of explants, which is characterized in that: the culture medium of the sterile brachytic pathway in the step (3) is MS + sucrose 20g/L + agar 6g/L, and the pH value is 5.8-6.0.
5. The invention provides a method for observing an explant generating path, which is characterized by comprising the following steps: the cluster bud multiplication medium in the step (4) is prepared from MS +6-BA0.5mg/L + NAA0.5 mg/L + sucrose 20g/L + agar 6g/L, and the pH value is 5.8-6.0.
6. The invention provides a method for observing an explant generating path, which is characterized by comprising the following steps: the culture medium of the embryoid generation pathway in the step (5) is MS + sucrose 20g/L + agar 6g/L, and the pH value is 5.8-6.0.
7. The invention provides a method for observing an explant generating path, which is characterized by comprising the following steps: the culture medium of the step (6) is prepared from MS +6-BA2mg/L + NAA0.2mg/L + sucrose 20g/L + agar 6g/L, and the pH value is 5.8-6.0.
8. The invention provides a method for observing an explant generating path, which is characterized by comprising the following steps: the culture medium of the step (7) is prepared from MS, sucrose 20g/L and agar 6g/L, and the pH value is 5.8-6.0.
9. The invention provides a method for observing an explant generating path, which is characterized by comprising the following steps: the culture conditions from the step (3) to the step (7) are 24-26 ℃, the illumination intensity is 2000-3000lux, and the illumination time is 10-12 h/d.
Compared with the prior art, the invention has the beneficial effects that:
1. the material drawing is convenient: the crassula argentea has strong asexual reproduction capability, is generally cutting reproduction, is inserted into a sand bed after being dried by thick tender stems or leaves in a growing season, and can take roots and grow buds after 3-4 weeks; the cultivation management is simple and extensive, the watering and fertilizing are less, and the foliage plant is a popular foliage plant.
2. The operation is simple, the observation effect is good: the leaves and stem segments of the crassula argentea are thick and large, and are convenient to clamp during inoculation and clear during observation.
3. The sterilization effect is good: the stem and leaf surfaces of the crassula argentea are smooth and have no epidermal hair, the outer surface of the leaf epidermal cell is covered with a thicker wax layer, the surface disinfection effect of the explant is good, and the success rate of the inoculated contamination rate is low.
4. The plant material of the crassula argentea can realize four different explant generation ways, and simplify the propagation, so that the experimental teaching of the course of 'plant tissue culture' is more vivid and intuitive, and students can systematically master the explant generation ways in the plant tissue culture.
5. 4 different explant generation ways can be observed 30 days after inoculation, the time is short, the effect is good, and great convenience is brought to the experimental teaching of plant tissue culture.
6. Through continuous observation after explant inoculation, the detailed observation and analysis capability of students can be improved, the learning interest of students is stimulated, the teaching efficiency is improved, and the teaching process is optimized.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The invention discloses a method for observing an explant generating path, which comprises the following steps:
obtaining of materials: picking fresh healthy leaves and stem sections of crassula argentea, and washing the surface of the picked leaves for 20min under tap water;
and (3) sterile treatment: opening an ultraviolet sterilizing lamp and a fan of the sterile operating table 30min before operation, closing the fan 10min, fully sterilizing the sterile operating table by the ultraviolet sterilizing lamp, soaking the washed blades on the sterile operating table for 15s by using 75% alcohol after sterilization, washing the blades with sterile water for 1 time after soaking, then soaking the blades in 0.1% mercuric chloride solution for sterilization for 6min, washing the blades with sterile water for 3-5 times after sterilization, and finally absorbing the moisture on the surfaces of the blades and the stems by using sterile filter paper;
sterile short shoot type: firstly, mixing the components of an MS culture medium together to prepare a mixed solution, adding 2% of sucrose and 0.65% of agar powder into the mixed solution to prepare an MS basic culture medium, subpackaging the prepared MS basic culture medium into conical flasks, sterilizing at 98kPa and 121 ℃ for 20min, taking out the conical flasks after the sterilization is finished, naturally cooling and solidifying the culture medium in the conical flasks, cutting stem sections after the sterilization treatment into small stem sections containing a pair of axillary buds, inoculating the small stem sections onto the MS basic culture medium, providing illumination for 12-14h each day by using a fluorescent lamp at the temperature of 24-26 ℃, wherein the illumination intensity is 2000-3000lx, the axillary buds can be observed to grow out after 30 days, and the stem sections are high in length;
cluster bud multiplication type: firstly mixing an MS culture medium, 0.5 mg/L6-BA and 0.5mg/L NAA together to prepare a mixed solution, adding 2% of sucrose and 0.65% of agar powder into the mixed solution to prepare a cluster bud multiplication culture medium, subpackaging the prepared cluster bud multiplication culture medium into conical flasks, sterilizing the conical flasks at 98kPa and 121 ℃ for 20min, taking out the conical flasks after the sterilization is finished, naturally cooling and solidifying the cluster bud multiplication culture medium in the conical flasks, cutting stem sections after the sterilization treatment into small stem sections containing a pair of axillary buds, transferring the small stem sections to the cluster bud multiplication culture medium, inducing the cluster buds at the temperature of 26 ℃, providing illumination for 12-14h every day by adopting a fluorescent lamp, wherein the illumination intensity is 2000 + 3000lx, and generating a large number of fleshy and green cluster buds at the bottoms of leaf axillary and stem sections after 30 days;
embryoid generation type: mixing the components of an MS culture medium together to prepare a mixed solution, adding 2% of sucrose and 0.65% of agar powder into the mixed solution to prepare an MS basic culture medium, subpackaging the prepared MS basic culture medium into conical flasks, sterilizing at 98kPa and 121 ℃ for 20min, taking out the conical flasks after the sterilization is finished, naturally cooling and solidifying the MS basic culture medium in the conical flasks, cutting the leaves subjected to the sterilization treatment into pieces with the size of 1.0cm multiplied by 1.0cm, inoculating the pieces into the MS basic culture medium, inducing at the temperature of 26 ℃ to generate adventitious buds, providing illumination for 12-14h each day by using a fluorescent lamp, wherein the illumination intensity is 2000 and 3000lx, a green bulge is formed at the cut of the leaves after 30 days, the small bulge grows up to form a first leaf of a crassula argentea plantlet, and then a pair of opposite leaves are grown to form roots at the same time;
organogenic type i: mixing an MS culture medium, 2 mg/L6-BA and 0.2mg/L NAA together to prepare a mixed solution, adding 2% of cane sugar and 0.65% of agar powder into the mixed solution to prepare a callus induction basic culture medium, subpackaging the prepared callus induction basic culture medium into conical flasks, sterilizing the conical flasks at 98kPa and 121 ℃ for 20min, taking out the conical flasks after the sterilization is finished, naturally cooling and solidifying the callus induction basic culture medium, cutting the sterile-treated leaves into 1.0cm multiplied by 1.0cm, inoculating the cut leaves into the callus induction basic culture medium, performing callus induction at the temperature of 26 ℃, providing illumination for 12-14h each day by adopting a fluorescent lamp, wherein the illumination intensity is 2000 and 3000lx, and generating compact yellow-green spherical callus at the cut of the leaves after 30 days;
organogenesis type ii: mixing the components of an MS culture medium together to prepare a mixed solution, adding 2% of sucrose and 0.65% of agar powder into the mixed solution to prepare an MS basic culture medium, subpackaging the prepared MS basic culture medium into conical flasks, sterilizing at 98kPa and 121 ℃ for 20min, taking out the conical flasks after the sterilization is finished, naturally cooling and solidifying the MS basic culture medium in the conical flasks, cutting the callus generated in the organogenesis type I into the size of 0.5cm multiplied by 0.5cm, inoculating the cut callus onto the MS basic culture medium, inducing the callus to redifferentiate at the temperature of 26 ℃, providing 12-14h of illumination by adopting a fluorescent lamp every day, wherein the illumination intensity is 2000 and 3000lx, and the callus redifferentiate generates green protrusions after 30 days, and the green protrusions grow into fleshy adventitious buds with fresh green color.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for observing the route of explant occurrence comprising the steps of:
(1) obtaining of materials: picking fresh healthy leaves and stem segments of crassula argentea, and washing the surface with tap water for 20 min;
(2) and (3) sterile treatment: soaking the washed leaves and stem segments on an ultra-clean workbench for 15s by using 75% alcohol, washing the leaves and stem segments with sterile water for 1 time after soaking, then soaking the leaves and stem segments in 0.1% mercuric chloride solution for disinfection for 6min, washing the leaves and stem segments with sterile water for 3-5 times after disinfection, and finally sucking the water on the surfaces of the leaves and stem segments by using disinfection filter paper;
(3) sterile short shoot type: cutting the stem section obtained in the step (2) into small stem sections with a pair of axillary buds, inoculating the small stem sections on an MS minimal medium, and inducing the stem sections to grow; the MS minimal medium is MS + sucrose 20g/L + agar 6g/L, and the pH is 5.8-6.0;
(4) cluster bud multiplication type: cutting the stem section obtained in the step (2) into small stem sections with a pair of axillary buds, inoculating the small stem sections on a cluster bud multiplication culture medium, and inducing to generate cluster buds; the cluster bud multiplication culture medium is MS +6-BA0.5mg/L + NAA0.5 mg/L + sucrose 20g/L + agar 6g/L, and the pH value is 5.8-6.0;
(5) embryoid generation type: cutting the leaves obtained in the step (2) into the size of about 1.0cm multiplied by 1.0cm, inoculating the cut leaves onto an MS minimal medium, and inducing the adventitious buds; the MS minimal medium is MS, sucrose 20g/L and agar 6g/L, and the pH value is 5.8-6.0;
(6) organogenic type i: cutting the leaf obtained in the step (2) into about 1.0cm multiplied by 1.0cm, inoculating the leaf to a callus induction culture medium, and inducing to generate callus; the callus induction culture medium is MS +6-BA2mg/L + NAA0.2mg/L + sucrose 20g/L + agar 6g/L, and the pH value is 5.8-6.0;
(7) organogenesis type ii: cutting the callus obtained in the step (6) into 0.5cm multiplied by 0.5cm, inoculating the cut callus to an MS basic culture medium, and inducing the callus to redifferentiate to form a seedling; the MS minimal medium is composed of MS, cane sugar 20g/L and agar 6g/L, and the pH value is 5.8-6.0.
2. A method of observing the route of explant occurrence according to claim 1, wherein: and (3) before the sterile operating platform in the step (2) is used, an ultraviolet sterilizing lamp and a fan are required to be started for sterilizing for 30 min.
3. A method of observing the route of explant occurrence according to claim 1, wherein: the 0.1% mercuric chloride solution in the step (2) contains 1-2 drops of 20mg/L Tween.
4. A method of observing the route of explant occurrence according to claim 1, wherein: the culture conditions from the step (3) to the step (7) are that the temperature is 24-26 ℃, the illumination intensity is 2000-3000lux, and the illumination time is 10-12 h/d.
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| CN105359981A (en) * | 2015-12-07 | 2016-03-02 | 宋宏婷 | Tissue culture method for crassula agenten thunb seedlings |
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| CN105359981A (en) * | 2015-12-07 | 2016-03-02 | 宋宏婷 | Tissue culture method for crassula agenten thunb seedlings |
Non-Patent Citations (6)
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| CALLUS FORMATION AND ORGANOGENESIS FROM CULTURED LEAF SEGMENTS OF CRASSULA ARGENTEA: CYTOKININ-INDUCED DEVELOPMENTAL PATTERN CHANGES;Paterson et al.;《Amer. J. Bot.》;19811231;第68卷(第7期);第965-972页 * |
| EFFECTS OF LIGHT AND HORMONES ON REGENERATION OF CRASSULA ARGENTEA FROM LEAVES;Paterson et al.;《Amer. J. Bot.》;19791231;第66卷(第4期);第463-470页 * |
| Indirect Regeneration and Somatic Embryogenesis from Leaf and Stem Explants of Crassula ovata (Mill.) Druce – An Ornamental Medicinal Plant;Ahmed et al.;《International Scholarly and Scientific Research & Innovation》;20141231;第8卷(第12期);第1158-1161页 * |
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| 燕子掌的组织培养与植株再生;蒲仕明等;《北方园艺》;20121231(第23期);第125-128页 * |
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