CN111557244B - Method for inducing somatic embryos of gingkoes and regenerating plants - Google Patents
Method for inducing somatic embryos of gingkoes and regenerating plants Download PDFInfo
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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
- 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention relates to a method for inducing somatic embryos of ginkgo biloba and regenerating plants, belonging to the technical field of plant propagation. The method comprises the following steps: step 1: selecting young ginkgo leaves and performing disinfection and sterilization treatment; step 2: induction of embryonic callus; and step 3: propagation of embryogenic callus: cutting the induced callus into 5X 5mm2Sequentially inoculating the blocks into a subculture medium with the hormone concentration from high to low for culture; and 4, step 4: maintaining embryonic callus; and 5: development of somatic embryos; step 6: maturation of somatic embryos; and 7: germination of somatic embryos. The invention successfully induces embryoid and germinates and develops into seedlings by a somatic embryo induction method which is independently developed.
Description
Technical Field
The invention relates to the technical field of plant propagation, and particularly provides a method for inducing gingko somatic embryos and regenerating plants.
Background
Ginkgo biloba is one of the oldest 'wiggles', is called 'activated stone' in the plant kingdom and is regarded as 'treasure tree' in China. The tree appearance, leaf shape and leaf color are unique and aesthetic, and the tree is an indispensable ornamental tree species in urban greening. The wood has excellent material quality, is suitable for processing, has special medicinal fragrance, and becomes a representative of high-grade wood in the wood market. The active ingredients of ginkgo, such as flavonoid, terpene lactone and the like, have the effects of resisting aging, improving the immune function and preventing and treating cardiovascular diseases, and are widely applied to medical preparations, health-care foods, cosmetics and the like. The market demands for ginkgo leaves, fruits and nursery stocks are increasing day by day, however, ginkgo leaves as a gymnosperm of a male and female heteroplant have long growth period and low propagation efficiency, and the traditional propagation modes of seeds, grafting, cuttage and the like are limited by time, environment, materials and the like, so that the rapid and stable production of excellent nursery stocks is difficult to realize, and the ginkgo market is always in a state of short supply and short demand. Therefore, the application of biotechnology to variety improvement and the rapid propagation prospect of excellent varieties are very wide.
Somatic embryogenesis of plants refers to a process of morphogenesis in which haploid or diploid somatic cells develop new individuals under specific conditions through a similar pathway as zygote embryogenesis without sexual cell fusion. Compared with organogenesis, somatic embryogenesis has remarkable characteristics: the first two polarities: in the early stage of somatic embryogenesis, there are two poles of radicle and germ, while adventitious bud and/or adventitious root are of unidirectional polarity. ② there is physiological isolation: after the somatic embryo is formed, the vascular bundle system of the parent plant or the explant is less connected, and the somatic embryo is easily separated from the original tissue. The somatic embryo is in relatively independent state, and can absorb nutrients from the explant or callus via root end or structure similar to embryonic stem and grow into one independent plant under proper condition. ③ stability of inheritance: the embryoid is originated from a single cell or a cell mass, and in the process of generation and development of the embryoid, once the embryoid is formed, the embryoid passes through a differentiation process, the structure is stable, long-time dedifferentiation and redifferentiation processes are not needed, the frequency of cell variation is low, the seedling rate is high, and the genetic stability is good. Large occurrence quantity: after the plant embryoid with high multiplication rate is formed, the embryoid can be regenerated under proper conditions, namely a large amount of secondary embryoid is formed. Reengineering the characteristic of fertilized egg morphogenesis. Although the several modes of morphogenesis in plant tissue culture are concrete expressions of totipotency of plant cells, the somatic embryogenesis way is the most complete mode of totipotency expression of cells, which not only shows that plant somatic cells have complete sets of genetic information, but also recapitulates the process of zygotic embryo morphogenesis.
When gingko is propagated rapidly through callus, except that adventitious buds can be induced by callus of gingko embryos, other organs are difficult to differentiate. In the research aspect of ginkgo embryos, more reports are reported about the induction of embryo seedling and mature embryo callus, while the research on the induction and differentiation of embryo callus in different growth periods is rarely reported. In the 90 th 20 th century, scholars at home and abroad successively adopted different explants, such as pollen, female gametophytes, immature embryos and mature embryos to successfully induce embryoids, but the embryoids cannot continuously germinate, only stay in cotyledon type embryos on a microstructure at most, and only stay for 1mm, do not grow any more and die due to browning.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for inducing gingko somatic embryos and regenerating plants.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for inducing somatic embryos of ginkgo biloba and regenerating plants, which comprises the following steps:
step 1: selecting young ginkgo leaves and performing disinfection and sterilization treatment: taking ginkgo disease-free young leaves as explant materials;
step 2: induction of embryogenic callus: cutting the explant into 3 x 3cm pieces, and inoculating the pieces to a callus induction culture medium;
the callus induction culture medium is MK +6-10 mg/L2, 4-D +3-6mg/L BA +400mg/L CH +400mg/L glutamine;
and step 3: propagation of embryogenic callus: selecting embryogenic culture with vigorous growth, slightly yellow color and surface protrusion from induced callus, cutting into 5 × 5mm2Sequentially inoculating the blocks into a subculture medium with the hormone concentration from high to low for culture;
the subculture medium is MK +4 mg/L2, 4-D +2mg/L BA +400mg/L CH +400mg/L glutamine, MK +2 mg/L2, 4-D +1mg/L BA +400mg/L CH +400mg/L glutamine and MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine;
and 4, step 4: maintenance of embryogenic callus: removing brown dead non-embryogenic callus after subculture, and maintaining embryogenic property of the obtained embryogenic callus on an embryogenic property maintaining culture medium;
the embryogenic maintenance medium is MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine;
and 5: development of somatic embryos: transferring the stable embryogenic callus with the granules in the step 4 into a development culture medium for culturing;
step 6: maturation of somatic embryos: selecting embryogenic callus which grows vigorously and is compact in structure and obvious in granular surface in the step 5, and transferring the embryogenic callus into a mature culture medium;
and 7: germination of somatic embryos: and (4) separating the embryoid subjected to maturation culture in the step 6 from the callus, and transferring the embryoid into an MK germination culture medium without hormone for germination.
Preferably, in the step 1, the sterilization treatment specifically includes: soaking the explant for 1h by using a detergent, and washing with running water to remove surface impurities and strains; performing 75% alcohol treatment on a sterile operating table for 20s, and washing with sterile water for 3-4 times; and finally, performing disinfection treatment by using 2% sodium hypochlorite for 10-30min, and washing by using sterile water for 3-4 times.
Preferably, in the step 2, the callus induction medium is MK +8 mg/L2, 4-D +4mg/L BA +400mg/L CH +400mg/L glutamine.
Preferably, in the step 3, 0.05-0.lmg/L ABA is added to the culture medium at least once every two subcultures.
Further, in the step 5, the development medium is MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine +8g/L inositol.
Further, in the step 6, the maturation culture medium is MK +3-6mg/L ABA +60-100g/L PEG +3-6g/L activated carbon.
Preferably, the maturation medium is MK +4mg/L ABA +75g/L PEG +5g/L activated carbon.
Further, in the step 7, the germination culture medium is MK + 5% -20% coconut juice +0.2-1g/L hydrolyzed casein +0.2-0.5g/L glutamine
Preferably, the germination medium is MK +10% coconut milk +0.5g/L hydrolyzed casein +0.5g/L glutamine.
Preferably, the concentration of the added sucrose in all the culture media is 30g/L, the concentration of the added agar is 7g/L, and the pH value is 5.85;
the step 2-4 is dark culture at the temperature of 21 +/-1 ℃; the steps 5-7 are illumination culture, the illumination is 14h/d, the light intensity is 1500Lx, and the temperature is 23 +/-1 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the induction of stable embryogenic callus with embryogenesis capacity is the basis for the research of callus morphogenesis, and the obtaining of embryogenic callus is the first step of plant somatic embryogenesis. The invention utilizes different culture mediums in specific steps to obtain stable embryogenic callus. In order to solve the problem that the differentiation capacity of the embryogenic callus is reduced in the long-term culture process, so that the embryogenic callus keeps vigorous differentiation capacity and is always in a high-frequency somatic embryogenesis period, the subculture medium with sequentially reduced hormone concentration is adopted in the subculture process, so that the callus can be continuously proliferated, the embryogenic property of the callus can be continuously maintained without loss, and the stable embryogenic callus is obtained. The non-embryogenic callus gradually browned and mostly dead as the hormone concentration decreased during the process.
In order to solve the problem that the embryogenic callus subculture medium inhibits the maturation of somatic embryos, after the subculture medium is cultured for a period of time, the culture needs to be transferred into a new culture medium for the development induction of the somatic embryos and the maturation and germination of the somatic embryos, the seedling rate of the embryoids can reach 40 percent, the germination rate is greatly improved compared with the prior art, and the induction of the gingko somatic embryos and the construction of a plant regeneration system are completed.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.
The reagents and materials used in the examples and comparative examples were commercially available unless otherwise specified.
The invention provides a method for inducing somatic embryos of ginkgo biloba and regenerating plants, and the specific embodiment is as follows.
Example 1
The induction of stable embryogenic callus with embryogenesis capacity is the basis for the research of callus morphogenesis, and the obtaining of embryogenic callus is the first step of plant somatic embryogenesis. In order to solve the above technical problem, the present invention provides step 1.
The step 1 comprises the following steps: selecting materials: taking disease-free young leaves of basic medicinal ginkgo Y1 young seedlings in 3-4 months as explant materials.
And (3) explant disinfection treatment: soaking the detergent for 2 hours, and washing with running water to remove surface impurities and strains; performing 75% alcohol treatment on a sterile operating table for 20s, and washing with sterile water for 3-4 times; and finally, sterilizing with 4% sodium hypochlorite for 5-15min, and washing with sterile water for 3-4 times.
The selected explant material was subjected to induction treatment, step 2, as follows.
Explant inoculation induction: drying the surface water of the young leaves with sterile filter paper on a sterile operating table, and cutting with a scalpel into pieces of about 3 × 3cm2And (5) small pieces, and inoculating to a callus induction culture medium.
The induction culture medium is MK +6-10 mg/L2, 4-D +3-6mg/L BA +400mg/L CH +400mg/L glutamine.
After the young ginkgo leaves are cultured on the induction culture medium for 10 days, the explants can obviously expand, and callus can appear in about 15 days. The young ginkgo leaves can easily induce callus, the callus growth rate is 92-100%, wherein the induction culture medium is MK +8 mg/L2, 4-D +4mg/L BA +400mg/L CH +400mg/L glutamine is the most preferable, and the callus growth rate can reach 100%.
The callus induced by the young ginkgo leaves in the invention mainly comprises four types: the adhesive, milky white and slightly yellow, smooth surface and hilly bulge are embryonic callus; viscous, white, brilliant, some with filamentous structure, embryonic callus; soft, beige and granular, the callus can be proliferated quickly; the callus is a non-embryogenic callus with compact, hard, brown and large granular structure, and the callus gradually browns and dies after being subcultured for many times. The callus obtained in the experiment is mostly the last two types, and the callus grows faster. Only part of the embryogenic callus was obtained in the experiment, and only 198 out of 300 explants processed produced embryogenic callus, with an embryogenic callus induction rate of about 66%.
In order to solve the problem that the differentiation capacity of the embryogenic callus is reduced in the long-term culture process, so that the embryogenic callus keeps the vigorous differentiation capacity and is always in the period of high-frequency somatic embryogenesis, the invention provides the steps 3 and 4.
The step 3 comprises the following steps: selecting embryo culture with vigorous growth, slightly yellow color and surface protrusion from the induced callus, cutting into pieces of 5 × 5mm2Clumps, inoculated in subculture medium, subcultured every 3 weeks.
The subculture medium comprises: selecting an induction culture medium with the best induction effect, wherein the type of the plant growth regulator is unchanged, the concentration is gradually reduced to the original concentrations of 1/2, 1/4 and 1/8, and other components are unchanged. Namely, the subculture medium MK +4 mg/L2, 4-D +2mg/L BA +400mg/L CH +400mg/L glutamine, MK +2 mg/L2, 4-D +1mg/L BA +400mg/L CH +400mg/L glutamine, MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine.
Subculturing on subculture medium with 1/2, 1/4 and 1/8 induction medium hormone concentration for 1 time respectively, removing browning dead non-embryogenic callus, and maintaining embryogenic callus on embryogenic maintenance medium MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine.
In the process of subculture, firstly, the callus is transferred to a subculture medium with 1/2 hormone concentration, then the embryogenic culture is transferred to the subculture medium, the culture is rapidly divided and proliferated, and the culture volume is obviously increased. At this time, the embryogenic state of the callus is still unstable, and a few of the embryogenic cells lose embryogenic property, while some of the non-embryogenic cells are induced to develop embryogenic property. The hormone concentration is gradually reduced to 1/4 and 1/8, and the embryogenic callus can be considered as more stable embryogenic callus when the callus is continuously proliferated and the embryogenic property of the callus can be continuously maintained without loss after the embryogenic callus is subcultured for 2 and 3 generations. While the hormone concentration is gradually reduced from 1/2 to 1/8, 0.05-0.lmg/L ABA is added into the subculture medium at least once every two subcultures, so that the multiplication speed can be increased, and the embryogenic state can be maintained. The non-embryogenic callus gradually browned and mostly dead as the 2,4-D concentration decreased during the subculture. While white semitransparent filamentous callus has the advantages that the concentration of 2,4-D is reduced along with the increase of the subculture frequency, the osmotic pressure in the culture medium is adjusted, ABA and the like are added, the callus is changed into beige or brown-white from white, the structure is dense, and the surface is slightly granular.
In order to solve the problem that the culture medium for the embryogenic callus subculture inhibits the somatic embryo maturation, after the culture medium is cultured for a period of time, the culture needs to be transferred into a new culture medium for the development induction of the somatic embryo, and the invention provides a step 5.
The step 5 comprises the following steps: transferring the stable embryogenic callus with granules obtained by subculture into a development culture medium.
Most preferably, the development medium is MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine +8g/L inositol.
After the embryogenic callus is transferred into a development culture medium for illumination culture, the color of the callus gradually turns green from beige and brown white, the structure is more compact, and the surface presents obvious particles.
In order for the callus to form embryoid bodies, it needs to be transferred to maturation medium for further culture, and the present invention provides step 6.
The step 6 comprises the following steps: selecting embryogenic callus with vigorous growth, compact structure and obvious granular surface, and transferring into mature culture medium.
The maturation culture medium is MK +3-6mg/L ABA +60-100g/L PEG +3-6g/L activated carbon.
After the embryogenic callus was inoculated on the ABA and PEG-containing somatic embryo induction medium for 5d, the callus surface turned pale green and formed smooth dome-shaped protrusions. After 20 days of inoculation, the color of the callus deepens, the callus is dark green, and green bud points begin to appear; after 35 days of inoculation, the green buds gradually developed into embryoid bodies. Embryoid bodies grow on the callus until a certain stage, and then do not grow any more. In order to successfully induce the embryoid to successfully develop into the seedling, the invention provides a step 7.
The providing step 7 includes:
the embryoid cultured by ABA maturation is separated from the callus and transferred into MK germination medium without hormone.
The germination culture medium comprises: MK + coconut juice + other ingredients. The coconut juice has a concentration of 5-20%, 4 concentration gradients are set, and other components are hydrolyzed casein 0.2-1g/L and glutamine 0.2-0.5 g/L.
30 embryoids are inoculated in each group, and after a period of culture, the development and seedling condition of the embryoids is counted. After the embryos are transferred into a germination culture medium for 15 days, the embryoids further grow and curled leaflets begin to appear; inoculating 18d, 2-3 young leaves and unfolding; after 25 days of inoculation, the stem was further elongated and more leaves appeared; after 30 days of inoculation, tissue culture seedlings with good growth state are formed. The results of the invention show that when the concentration of the coconut juice is 5%, 10%, 15% and 20%, the seedling rate of the embryoid is 23.3%, 40%, 26.6% and 13.3%, respectively.
Meanwhile, the concentration of the added sucrose in all the culture media in the application is 30g/L, the concentration of the added agar is 7g/L, and the pH value is 5.85;
the step 2-4 is dark culture at the temperature of 21 +/-1 ℃; the steps 5-7 are illumination culture, the illumination is 14h/d, the light intensity is 1500Lx, and the temperature is 23 +/-1 ℃.
To further illustrate the advantageous effects of the present invention, comparative examples were constructed as follows.
Comparative example 1
The MK medium in steps 2-7 was replaced with MS medium, and the other conditions were the same as in example 1.
In comparative example 1, after the MK medium was replaced with the MS medium, callus growth rate, callus state, and embryoid induction rate were greatly different from callus induction to final embryoid germination and seedling formation in steps 2 to 7, as shown in Table 1. Meanwhile, the callus cultured by the MS culture medium has loose texture and part of the callus is in a water stain shape. The callus cultured by the MK culture medium has more compact texture and is easy to agglomerate. In addition, although some embryoid bodies grew on MS medium, they eventually broke and died, whereas MK medium embryoid bodies developed into seedlings, see Table 2.
TABLE 1
TABLE 2
Comparative example 2
The subculture medium in step 3 was MK +4 mg/L2, 4-D +2mg/L BA +400mg/L CH +400mg/L glutamine, and the hormone concentration was not decreased, except for the same conditions as in example 1.
Embryogenic cultures which grow vigorously, are slightly yellow in color and have surface protrusions are selected from the induced calli and inoculated into the subculture medium. The following are found: after inoculation, the callus continuously grows, white callus is newly generated around the original callus, and the texture is loose. Along with the increase of the subculture time, the water stain of the callus is serious, and no particles appear on the surface of the callus. The long-term action of 2,4-D is not beneficial to inducing adventitious buds from callus and the later-stage morphogenesis of somatic embryos, and after the 2,4-D is gradually removed from a culture medium and osmotic pressure is adjusted, the continuous effect of the 2,4-D can be gradually eliminated, the embryogenic callus proliferation is regulated and controlled, and the morphogenesis of the somatic embryos is perfected.
Comparative example 3
The subculture medium in step 3 was MK +2 mg/L2, 4-D +1mg/L BA +400mg/L CH +400mg/L glutamine, and the hormone concentration was not decreased, except for the same conditions as in example 1.
Embryogenic cultures which grow vigorously, are slightly yellow in color and have surface protrusions are selected from the induced calli and inoculated into the subculture medium. The following are found: the volume of the inoculated callus is increased, the texture is gradually densified from loose, the texture of the callus after multiple subcultures is unstable, compact callus and loose callus alternately appear, and the embryogenic property of the callus is unstable. With the increase of the subculture time, the inside of the callus gradually becomes brown and dead.
Comparative example 4
The subculture medium in step 3 was MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine, and the hormone concentration was not decreased, except for the same conditions as in example 1.
Embryogenic cultures which grow vigorously, are slightly yellow in color and have surface protrusions are selected from the induced calli and inoculated into the subculture medium. The following are found: the plant hormone is greatly reduced, the inoculated callus grows slowly, the callus proliferation amount is low, the texture is compact and dry, and the maintenance of the embryogenic property of the callus and the formation of an embryoid are not facilitated.
Comparative example 5
Embryogenic calli which grew vigorously and were granular were selected and inoculated into a medium containing MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine + myo-inositol, wherein myo-inositol was 0g/L, 4g/L, 8g/L, and 12g/L, respectively, and the same conditions as in example 1 were followed. Approximately 15 flasks were treated with each medium, 3-4 clumps per flask. The amount of callus growth was counted and is shown in Table 3.
TABLE 3
Test results show that the inositol dosage has very obvious influence on the proliferation rate of the callus. When the dosage of inositol is increased within the range of 4-8g/L, the proliferation rate is gradually increased, and when the dosage of inositol reaches 8g/L, the proliferation rate reaches 201%, and the proliferation effect is best. However, when the concentration of inositol is increased, the proliferation rate is reduced sharply, and the callus is browned seriously. The inositol is used as an osmotic regulator, and when the concentration is proper, the inositol can promote the absorption of nutrients in a culture medium by the callus, provide enough nutrients for cell division and proliferation, and easily cause the cells to lose water and brown and die due to overhigh concentration. Meanwhile, researches show that the inositol concentration, namely the osmotic pressure is increased, the formation and the development of the proembryo at the later stage can be promoted, and the granular embryogenic callus is obviously increased.
Comparative example 6
The maturation medium in step 6 was MK +4mg/L BA +80g/L PEG +3-6g/L activated carbon, and the rest of the conditions were the same as in example 1.
The ABA is replaced by BA, smooth dome-shaped bulges can be formed on the callus, the callus is dark green, green bud points appear, but the bud points cannot further develop into embryoids and gradually become brown and die.
Comparative example 7
The germination medium was freed of coconut juice in step 7, and the rest of the conditions were the same as in example 1.
The germination medium without coconut juice can enable embryoid to grow slightly, but the embryoid can be browned and died soon, and the seedling rate of the embryoid is very low, about 5%. The development of embryoid is obviously improved by adding coconut juice into MK culture medium. When the concentration of the coconut juice is 10%, the seedling rate of the embryoid is 40%.
In conclusion, in order to solve the problem that the differentiation capacity of the embryogenic callus is reduced in the long-term culture process, so that the embryogenic callus keeps a relatively vigorous differentiation capacity and is always in a high-frequency somatic embryogenesis period, the subculture medium with sequentially reduced hormone concentration is adopted in the subculture process, so that the callus can be continuously proliferated, the embryogenic property of the callus can be continuously maintained without loss, and the relatively stable embryogenic callus is obtained; meanwhile, the seedling rate of the embryoid can reach more than 40 percent, and the induction of the gingko somatic embryo and the construction of a plant regeneration system are completed.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (4)
1. A method for inducing somatic embryos of ginkgo biloba and regenerating plants, which is characterized by comprising the following steps:
step 1: selecting young ginkgo leaves and performing disinfection and sterilization treatment: taking ginkgo disease-free young leaves as explant materials;
step 2: induction of embryogenic callus: cutting the explant into 3cm multiplied by 3cm slices, and inoculating the slices to a callus induction culture medium for culture;
the callus induction culture medium is MK +8 mg/L2, 4-D +4mg/L BA +400mg/L CH +400mg/L glutamine +30g/L cane sugar +7g/L agar, and the pH value is 5.85;
and step 3: propagation of embryogenic callus: selecting embryogenic culture with vigorous growth, slightly yellow color and surface protrusion from the induced callus, cutting into 5mm × 5mm blocks, sequentially inoculating into subculture medium with hormone concentration from high to low, and culturing;
the subculture medium is MK +4 mg/L2, 4-D +2mg/L BA +400mg/L CH +400mg/L glutamine +0.05-0.L mg/L ABA +30g/L sucrose +7g/L agar, pH 5.85, MK +2 mg/L2, 4-D +1mg/L BA +400mg/L CH +400mg/L glutamine +0.05-0.L mg/L ABA +30g/L sucrose +7g/L agar, pH 5.85 and MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine +0.05-0.L mg/L ABA +30g/L sucrose +7g/L agar, pH 5.85;
and 4, step 4: maintenance of embryogenic callus: removing browning dead non-embryogenic callus after subculture, and maintaining the embryogenic callus on an embryogenic maintenance culture medium;
the embryogenic maintenance medium is MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine +30g/L sucrose +7g/L agar, and the pH is 5.85;
and 5: development of somatic embryos: transferring the stable embryogenic callus with the granules in the step 4 into a development culture medium for culturing; the development culture medium is MK +1 mg/L2, 4-D +0.5mg/L BA +400mg/L CH +400mg/L glutamine +8g/L inositol +30g/L sucrose +7g/L agar, and the pH value is 5.85;
step 6: maturation of somatic embryos: selecting the embryogenic callus which grows vigorously and has a compact structure and obvious granular surface in the step 5, and transferring the embryogenic callus into a mature culture medium for culture; the mature culture medium is MK +4mg/L ABA +75g/L PEG +5g/L active carbon +30g/L sucrose +7g/L agar, and the pH value is 5.85;
and 7: germination of somatic embryos: separating the embryoid subjected to maturation culture in the step 6 from the callus, and transferring the embryoid into an MK germination culture medium without hormone for germination; the germination medium is MK, 10% coconut juice, 0.2-1g/L hydrolyzed casein, 0.2-0.5g/L glutamine, 30g/L sucrose and 7g/L agar, and the pH value is 5.85.
2. The method for inducing somatic embryos of ginkgo biloba according to claim 1 and regenerating plants, wherein the sterilization treatment in the step 1 is specifically: soaking the explant for 1h by using a detergent, and washing with running water to remove surface impurities and strains; performing 75% alcohol treatment on a sterile operating table for 20s, and washing with sterile water for 3-4 times; and finally, carrying out disinfection treatment by using 2wt% of sodium hypochlorite for 10-30min, and washing with sterile water for 3-4 times.
3. The method for inducing somatic embryos of ginkgo biloba according to claim 1, wherein the germination medium is MK +10% coconut juice +0.5g/L hydrolyzed casein +0.5g/L glutamine +30g/L sucrose +7g/L agar and pH 5.85.
4. The method for inducing somatic embryos of ginkgo biloba according to any one of claims 1 to 3, wherein the step 2 to 4 is dark culture at a temperature of 21 ± 1 ℃; the steps 5-7 are illumination culture, the illumination is 14h/d, the light intensity is 1500Lx, and the temperature is 23 +/-1 ℃.
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| CN108849529A (en) * | 2018-08-29 | 2018-11-23 | 南京林业大学 | A kind of method of ginkgo endosperm induced embryonic callus |
| CN110291989A (en) * | 2019-07-29 | 2019-10-01 | 南京林业大学 | A kind of method for building up in the sterile leaf source for gingkgo callus induction |
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| CN108849529A (en) * | 2018-08-29 | 2018-11-23 | 南京林业大学 | A kind of method of ginkgo endosperm induced embryonic callus |
| CN110291989A (en) * | 2019-07-29 | 2019-10-01 | 南京林业大学 | A kind of method for building up in the sterile leaf source for gingkgo callus induction |
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