CN111041044A - Genetic transformation and transgenic plant regeneration method for triploid Chinese white poplar - Google Patents
Genetic transformation and transgenic plant regeneration method for triploid Chinese white poplar Download PDFInfo
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Abstract
The invention discloses a genetic transformation and transgenic plant regeneration method of triploid Chinese white poplar, which uses the tender leaf of triploid Chinese white poplar as gene receptor material to make agrobacterium-mediated transformation so as to obtain transgenic plant. The method mainly comprises the following steps: obtaining young leaves of triploid Chinese white poplar, culturing agrobacterium, infecting agrobacterium, screening and differentiating adventitious buds of triploid Chinese white poplar, regenerating plants and hardening seedlings and transplanting. The method is simple to operate and high in reliability, and opens up a new way for applying the modern biotechnology to the variety improvement of the triploid Chinese white poplar.
Description
Technical Field
The invention belongs to the technical field of biotechnology and modern agriculture, and relates to a plant tissue culture and genetic transformation method, in particular to a method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa.
Background
The triploid Chinese white poplar is a scientific research team leading in Wittig professor of Chinese academy of engineering, adopts the technologies of partial replacement of cell chromosomes, chromosome doubling and the like, is a new variety obtained through hardy assault for many years, and has the characteristics of fast growth, high quality, high efficiency and the like. The variety can be grown into trees in 1 year, forest in 3 years and wood in 5 years. The diameter at breast height of a single plant can reach 20 cm after 5 years of growth, the volume of the single plant is 0.1-0.2 cubic meter, and the accumulation amount per mu can reach 10-20 cubic meters. The propagation speed is high, the disease resistance is strong, the dry shape is straight, and the plant can be used as a preferred tree species for high-yield woods, farmland protection forest nets and highway greening, and is an rare raw material for paper pulp construction. The ecological adaptability is strong: the rust-resistant and aphid-resistant ecological greening base is mainly characterized by wide distribution range, rust resistance, longicorn resistance and aphid resistance, and is suitable for manufacturing high-yield forests, farmland forest nets, plain greening, channel greening and urban greening, and has high ecological protection benefit. Is suitable for yellow river basin and the three north area, and can be planted in river mudflat and Sichuan lands with the altitude of 2-2000 m. The triploid populus industry has become recognized by various levels of governments, both national and local.
Disclosure of Invention
The invention aims to provide a genetic transformation and transgenic plant regeneration method of triploid Chinese white poplar, which takes tender leaves of triploid Chinese white poplar as a gene receptor material to mediate agrobacterium to obtain a transgenic plant.
The invention is realized by the following technical scheme:
a triploid Chinese white poplar genetic transformation and transgenic plant regeneration method comprises the following steps:
1) culturing triploid Chinese white poplar seedling with sterile seedling culture medium at 25 deg.c for 16 hr each day;
2) the agrobacterium tumefaciens EHA105 containing a binary plant expression vector PCAMBIA2300-PtCLE is singly cloned on a YM culture medium containing kanamycin and rifampicin for streak culture, the agrobacterium tumefaciens thallus is placed in a liquid culture medium, and acetosyringone is added to prepare an invasion solution;
3) transversely shearing the leaves for 2-3 times, wherein the shearing amplitude is two thirds of the width of the leaves, adding an infection solution for dip dyeing, sucking residual infection solution on the leaves by using filter paper, and transferring the leaves to a co-culture medium for culture;
4) transferring the leaves into a screening culture medium for culture, and transferring the generated adventitious buds into a strong seedling culture medium for continuous culture;
5) selecting strong adventitious buds, transplanting into a rooting culture medium to form a root system, transplanting the seedlings into a growth culture medium, and transplanting when the root system is developed and the seedlings grow to be more than 6 cm.
Further, the sterile seedling culture medium is as follows: MS + sucrose 30g/L + agar 7g/L, pH 5.8.
Further, the concentration of kanamycin and rifampicin is 50mg/L and 25mg/L respectively.
Further, the liquid culture medium is: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L, pH5.8.
Further, the OD600 of the thalli in the dip dyeing solution is 0.3-0.6.
Furthermore, the addition concentration of the acetosyringone is 100 mu mol/L.
Further, the co-culture medium is: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L + agar 7g/L + acetosyringone 100 mu mol/L, pH5.8, the culture condition is at 25 ℃, and the illumination is 16 hours per day.
Further, the screening medium is: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L + agar 7g/L + kanamycin 20mg/L + cefamycin 300mg/L, pH5.8, the culture condition is that the culture temperature is 25 ℃, and the illumination is 16 hours per day.
Further, the strong seedling culture medium is as follows: MS +6-BA0.2mg/L + TDZ0.001mg/L + kanamycin 20mg/L + cefamycin 300mg/L + sucrose 30g/L + agar 7g/L, p H5.8.8.
Further, the rooting medium is as follows: 1/2MS + IBA0.05mg/L + NAA0.05mg/L + kanamycin 20mg/L + cefamycin 300mg/L + sucrose 30g/L + agar 7g/L, pH 5.8.
Further, the growth medium is: MS + sucrose 20g/L + agar 7g/L, p H5.8.8.
The invention has the beneficial effects that:
the invention utilizes the tissue culture technology, the tender leaves of the in vitro poplar can form adventitious buds, and the adventitious buds grow into regeneration plants after induced rooting. The triploid Chinese white poplar young leaves are used as gene receptor materials, and an agrobacterium-mediated genetic transformation regeneration system has the characteristics of simple method, high reliability and easiness in operation, and opens up a new way for applying modern biotechnology to triploid Chinese white poplar variety improvement.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
The invention provides a triploid Chinese white poplar genetic transformation and transgenic plant regeneration method, which mainly comprises the following steps: obtaining young leaves of triploid Chinese white poplar, culturing agrobacterium, infecting agrobacterium, screening and differentiating adventitious buds of triploid Chinese white poplar, regenerating plants and hardening seedlings and transplanting.
The basic process is as follows:
step 1, propagating sterile triploid Chinese white poplar seedlings, and collecting tender leaves to obtain a gene receptor material;
step 2, culturing the constructed plasmid and the monoclonal agrobacterium;
step 3, agrobacterium infection;
step 4, screening and differentiating adventitious buds of triploid Chinese white poplar;
and 5, regenerating and hardening seedlings and transplanting the triploid Chinese white poplar.
The operation method of the step 1 is as follows:
culturing triploid Chinese white poplar seedling with sterile seedling culture medium at 25 deg.c for 16 hr each day.
Selecting 2 nd to 4 th leaves of the tremula alba young leaves, wherein the length of the leaves is 27-40 mm.
The culture medium of the triploid Chinese white poplar aseptic seedling is as follows: MS + sucrose 30g/L + agar 7g/L, pH5.8.
The operation method of the step 2 is as follows:
agrobacterium EHA105 containing binary plant expression vector PCAMBIA2300-PtCLE was single-cloned on YM medium containing kanamycin 50mg/L and rifampicin 25mg/L, and cultured at 28 ℃ for 3 days. And (3) collecting agrobacterium tumefaciens thalli, suspending the agrobacterium tumefaciens thalli in a liquid culture medium, and continuously shaking for 30min until the OD600 of the thalli is 0.3-0.6, namely, the infection liquid is used for transformation. The acetosyringone is added into the staining solution with the concentration of 100 mu mol/L.
The liquid culture medium is: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L, pH5.8.
The operation method of the step 3 is as follows:
the specific parameters of infection are as follows: transversely shearing 2-3 blades according to the size of the leaves, wherein the shearing width is two thirds of the width of the leaves, adding 100 ml of dye-soaking solution into 40 leaves, and infecting for 15 minutes; sucking the residual infection liquid on the leaves with filter paper, and transferring the leaves to a co-culture medium for culturing for 2-4 days; co-culture conditions: the temperature was 25 ℃ and the light was irradiated for 16 hours per day.
The co-culture medium is as follows: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L + agar 7g/L + acetosyringone 100 mu mol/L, pH5.8.
The operation method of the step 4 is as follows:
and (3) transferring the co-cultured leaves into a screening culture medium, wherein the culture temperature is 25 ℃, the illumination is carried out for 16 hours every day, the culture medium is changed every 2 weeks, and a small amount of adventitious buds are gradually generated after the four weeks. Transplanting the adventitious bud into a strong seedling culture medium for culturing for about 3 weeks, and selecting a healthy and strong bud with the height of about 20 mm for rooting culture. The strong seedling culture condition is that the temperature is 25 ℃, and the illumination is 16 hours per day.
The screening culture medium is as follows: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L + agar 7g/L + kanamycin 20mg/L + cefamycin 300mg/L, pH 5.8.
The strong seedling culture medium comprises: MS +6-BA0.2mg/L + TDZ0.001mg/L + kanamycin 20mg/L + cefamycin 300mg/L + sucrose 30g/L + agar 7g/L, and pH is 5.8.
The operation method of the step 5 is as follows:
and (4) selecting a strong adventitious bud, and transferring the bud into a rooting culture medium. The culture temperature was 25 ℃ and the light irradiation was carried out for 16 hours per day. After one week, forming root system, transplanting the seedling into growth culture medium at 25 deg.C under illumination for 16 hr, transplanting when the root system is developed and the seedling grows to more than 6cm, wherein the culture medium is peat soil: 1:1(v/v) of vermiculite, culturing in an illumination incubator for 1 month, transferring to a shading greenhouse for culturing, and culturing in a field forest land after 2 months.
The rooting medium comprises the following components: 1/2MS + IBA0.05mg/L + NAA0.05mg/L + kanamycin 20mg/L + cefamycin 300mg/L + sucrose 30g/L + agar 7g/L, pH 5.8.
The growth medium consists of: MS + sucrose 20g/L + agar 7g/L, pH 5.8.
Example 1
1) Culturing triploid Chinese white poplar seedling with sterile seedling culture medium at 25 deg.c for 16 hr each day. Selecting the 2 nd leaf of the tremule poplar young leaf, wherein the length of the leaf is 27 mm.
2) Agrobacterium E HA105 containing binary plant expression vector PCAMBIA2300-PtCLE was streaked on YM medium containing kanamycin 50mg/L and rifampicin 25mg/L, and cultured at 28 ℃ for 3 days. And (3) collecting agrobacterium tumefaciens thalli, suspending the agrobacterium tumefaciens thalli in a liquid culture medium, and continuously shaking for 30min until the OD600 of the thalli is 0.3, namely, the agrobacterium tumefaciens thalli is used for transformation. The acetosyringone is added into the staining solution with the concentration of 100 mu mol/L.
3) The specific parameters of infection are as follows: transversely shearing 2 blades according to the size of the leaves, wherein the shearing amplitude is two thirds of the width of the leaves, adding 100 ml of dye-soaking solution into 40 blades, and infecting for 15 minutes; sucking the residual infection liquid on the leaves by using filter paper, and transferring the leaves to a co-culture medium for culturing for 2 days; co-culture conditions: the temperature was 25 ℃ and the light was irradiated for 16 hours per day.
4) And (3) transferring the co-cultured leaves into a screening culture medium, wherein the culture temperature is 25 ℃, the illumination is carried out for 16 hours every day, the culture medium is changed every 2 weeks, and a small amount of adventitious buds are gradually generated after the four weeks. Transplanting the adventitious bud into a strong seedling culture medium for culturing for about 3 weeks, and selecting a healthy and strong bud with the height of about 20 mm for rooting culture. The strong seedling culture condition is that the temperature is 25 ℃, and the illumination is 16 hours per day.
5) And (4) selecting a strong adventitious bud, and transferring the bud into a rooting culture medium. The culture temperature was 25 ℃ and the light irradiation was carried out for 16 hours per day. After one week, forming root system, transplanting the seedling into growth culture medium at 25 deg.C under illumination for 16 hr, transplanting when the root system is developed and the seedling grows to more than 6cm, wherein the culture medium is peat soil: 1:1(v/v) of vermiculite, culturing in an illumination incubator for 1 month, transferring to a shading greenhouse for culturing, and culturing in a field forest land after 2 months.
Example 2
1) Culturing triploid Chinese white poplar seedling with sterile seedling culture medium at 25 deg.c for 16 hr each day. Selecting the 4 th leaf of the tremule poplar young leaf, wherein the length of the leaf is 40 mm.
2) Agrobacterium E HA105 containing binary plant expression vector PCAMBIA2300-PtCLE was streaked on YM medium containing kanamycin 50mg/L and rifampicin 25mg/L, and cultured at 28 ℃ for 3 days. And (3) collecting agrobacterium tumefaciens thalli, suspending the agrobacterium tumefaciens thalli in a liquid culture medium, and continuously shaking for 30min until the OD600 of the thalli is 0.6, namely, the agrobacterium tumefaciens thalli is used for transformation. The acetosyringone is added into the staining solution with the concentration of 100 mu mol/L.
3) The specific parameters of infection are as follows: transversely shearing 3 blades according to the size of the leaves, wherein the shearing amplitude is two thirds of the width of the leaves, adding 100 ml of dye-soaking solution into 40 leaves, and infecting for 15 minutes; sucking the residual infection liquid on the leaves by using filter paper, and transferring the leaves to a co-culture medium for culture for 4 days; co-culture conditions: the temperature was 25 ℃ and the light was irradiated for 16 hours per day.
4) And (3) transferring the co-cultured leaves into a screening culture medium, wherein the culture temperature is 25 ℃, the illumination is carried out for 16 hours every day, the culture medium is changed every 2 weeks, and a small amount of adventitious buds are gradually generated after the four weeks. Transplanting the adventitious bud into a strong seedling culture medium for culturing for about 3 weeks, and selecting a healthy and strong bud with the height of about 20 mm for rooting culture. The strong seedling culture condition is that the temperature is 25 ℃, and the illumination is 16 hours per day.
5) And (4) selecting a strong adventitious bud, and transferring the bud into a rooting culture medium. The culture temperature was 25 ℃ and the light irradiation was carried out for 16 hours per day. After one week, forming root system, transplanting the seedling into growth culture medium at 25 deg.C under illumination for 16 hr, transplanting when the root system is developed and the seedling grows to more than 6cm, wherein the culture medium is peat soil: 1:1(v/v) of vermiculite, culturing in an illumination incubator for 1 month, transferring to a shading greenhouse for culturing, and culturing in a field forest land after 2 months.
Example 3
1) Culturing triploid Chinese white poplar seedling with sterile seedling culture medium at 25 deg.c for 16 hr each day. Selecting the No. 3 leaf of the tremule poplar young leaf, wherein the length of the leaf is 35 mm.
2) Agrobacterium E HA105 containing binary plant expression vector PCAMBIA2300-PtCLE was streaked on YM medium containing kanamycin 50mg/L and rifampicin 25mg/L, and cultured at 28 ℃ for 3 days. And (3) collecting agrobacterium tumefaciens thalli, suspending the agrobacterium tumefaciens thalli in a liquid culture medium, and continuously shaking for 30min until the OD600 of the thalli is 0.4, namely, the agrobacterium tumefaciens thalli is used for transformation. The acetosyringone is added into the staining solution with the concentration of 100 mu mol/L.
3) The specific parameters of infection are as follows: transversely shearing 2 blades according to the size of the leaves, wherein the shearing amplitude is two thirds of the width of the leaves, adding 100 ml of dye-soaking solution into 40 blades, and infecting for 15 minutes; sucking the residual infection liquid on the leaves by using filter paper, and transferring the leaves to a co-culture medium for 3 days; co-culture conditions: the temperature was 25 ℃ and the light was irradiated for 16 hours per day.
4) And (3) transferring the co-cultured leaves into a screening culture medium, wherein the culture temperature is 25 ℃, the illumination is carried out for 16 hours every day, the culture medium is changed every 2 weeks, and a small amount of adventitious buds are gradually generated after the four weeks. Transplanting the adventitious bud into a strong seedling culture medium for culturing for about 3 weeks, and selecting a healthy and strong bud with the height of about 20 mm for rooting culture. The strong seedling culture condition is that the temperature is 25 ℃, and the illumination is 16 hours per day.
5) And (4) selecting a strong adventitious bud, and transferring the bud into a rooting culture medium. The culture temperature was 25 ℃ and the light irradiation was carried out for 16 hours per day. After one week, forming root system, transplanting the seedling into growth culture medium at 25 deg.C under illumination for 16 hr, transplanting when the root system is developed and the seedling grows to more than 6cm, wherein the culture medium is peat soil: 1:1(v/v) of vermiculite, culturing in an illumination incubator for 1 month, transferring to a shading greenhouse for culturing, and culturing in a field forest land after 2 months.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A triploid Chinese white poplar genetic transformation and transgenic plant regeneration method is characterized by comprising the following steps:
1) culturing triploid Chinese white poplar seedling with sterile seedling culture medium at 25 deg.c for 16 hr each day;
2) the agrobacterium tumefaciens EHA105 containing a binary plant expression vector PCAMBIA2300-PtCLE is singly cloned on a YM culture medium containing kanamycin and rifampicin for streak culture, the agrobacterium tumefaciens thallus is placed in a liquid culture medium, and acetosyringone is added to prepare an invasion solution;
3) transversely shearing the leaves for 2-3 times, wherein the shearing amplitude is two thirds of the width of the leaves, adding an infection solution for dip dyeing, sucking residual infection solution on the leaves by using filter paper, and transferring the leaves to a co-culture medium for culture;
4) transferring the leaves into a screening culture medium for culture, and transferring the generated adventitious buds into a strong seedling culture medium for continuous culture;
5) selecting strong adventitious buds, transferring into a rooting culture medium to form a root system, transferring the seedlings into a growth culture medium, and transplanting when the root system is developed and the seedlings grow to be more than 6 cm;
the liquid culture medium is as follows: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L, pH5.8;
the co-culture medium is as follows: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L + agar 7g/L + acetosyringone 100 mu mol/L, pH5.8;
the screening culture medium comprises: MS +6-BA0.5mg/L + NAA0.05mg/L + KT1.0mg/L + sucrose 30g/L + agar 7g/L + kanamycin 20mg/L + cefamycin 300mg/L, pH 5.8.
2. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa as claimed in claim 1, wherein the sterile seedling medium is: MS + sucrose 30g/L + agar 7g/L, pH 5.8.
3. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa as claimed in claim 1, wherein the concentrations of kanamycin and rifampicin are 50mg/L and 25mg/L, respectively.
4. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa according to claim 1, wherein the OD600 of the thalli in the staining solution is 0.3-0.6.
5. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa as claimed in claim 1, wherein the addition concentration of acetosyringone is 100 μmol/L.
6. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa as claimed in claim 1, wherein the strong seedling culture medium is: MS +6-BA0.2mg/L + TDZ0.001mg/L + kanamycin 20mg/L + cefamycin 300mg/L + sucrose 30g/L + agar 7g/L, and pH is 5.8.
7. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa as claimed in claim 1, wherein the rooting medium is: 1/2MS + IBA0.05mg/L + NAA0.05mg/L + kanamycin 20mg/L + cefamycin 300mg/L + sucrose 30g/L + agar 7g/L, pH 5.8.
8. The method for genetic transformation and transgenic plant regeneration of triploid populus tomentosa as claimed in claim 1, wherein the growth medium is: MS + sucrose 20g/L + agar 7g/L, pH 5.8.
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| CN110295191A (en) * | 2019-07-15 | 2019-10-01 | 南京林业大学 | A kind of genetic transforming method of diploid Chinese white poplar |
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