CN110669783B - Genetic transformation method for kokstroemia indica - Google Patents
Genetic transformation method for kokstroemia indica Download PDFInfo
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Abstract
The invention provides a method for genetic transformation of hevea brasiliensis, which comprises (1) cutting young leaves of the germplasm of the hevea brasiliensis, and culturing in an inducing culture medium containing MS + 1.5-1.6 mg/L6-BA +0.1mg/LNAA +20g/L sucrose and pH5.8 bud; after 50 days, selecting excellent germplasm of the rubber grass suitable for tissue culture; transferring to 1/2MS +20g/L sucrose and a rooting culture medium with pH of 5.8 to obtain regenerated hevea brasiliensis tissue culture seedlings; (2) cutting seedling leaves, and pre-culturing on a bud induction culture medium for 2 days; (3) adding agrobacterium infection solution for dip dyeing, transferring to a bud induction culture medium, and culturing in the dark for 3 days; transferring to a bud induction culture medium containing 400mg/L Car for culturing for one week; transferring to a screening culture medium of MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg at pH5.8 to induce the regeneration of resistant buds; transferring to a new rooting culture medium of 1/2MS +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, and pH5.8 to obtain a hevea brasiliensis resistance regeneration plant; the genetic transformation method of the hevea brasiliensis has the advantages that the inductivity of the resistant regeneration buds is more than 30%, the plant aberration rate of the transgenic regeneration seedlings is low, and the positive detection rate of the transgenic hevea brasiliensis is more than 90%.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a genetic transformation method of hevea brasiliensis.
Background
The Hevea brasiliensis is also known as Taraxacum kok saghyz Rodin, is a plant of Taraxacum of Cichorium of Compositae, and is native to Hassakestan, Europe, Xinjiang, etc. The roots of the rubber grasses contain high-quality rubber with the content of more than 20 percent, and the rubber grasses are gradually used as a second natural rubber resource.
In recent years, scholars at home and abroad have conducted some researches on the tissue culture technology of the kochia scoparia. For example, the leaf of kochia scoparia is used as an explant to induce adventitious buds or callus, but the strain of the kochia scoparia is seriously mixed due to the self-incompatible breeding characteristics of the kochia scoparia. Due to the difference of tissue culture conditions and methods among different strains, the problems of low bud induction rate, low regeneration capacity of regenerated seedlings and the like are easily caused, the genetic transformation of the hevea brasiliensis is difficult to carry out, and in the process of the genetic transformation, the induction rate of the resistant regenerated buds is extremely low, the genetic transformation period is long, the aberration rate is high, and the hevea brasiliensis transgenic strains are difficult to effectively obtain.
Disclosure of Invention
In view of the above, the invention provides a high-transformation-efficiency genetic transformation method for the rubber grass, the inductivity of the resistant regeneration buds is more than 30%, the plant aberration rate of the transgenic regeneration seedlings is low, the growth speed is high, and the positive detection rate of the transgenic rubber grass is more than 90%.
The technical scheme of the invention is realized as follows:
the invention provides a genetic transformation method of kokstroemia indica, which comprises the following steps:
(1) screening and obtaining of plant material genetically transformed with kokstroemia indica
a. Collecting and selecting Xinjiang rubber grass germplasm, cutting complete young and tender leaves, sterilizing and cutting, and placing in a formula comprising: MS + 1.5-1.6 mg/L6-BA +0.1mg/LNAA +20g/L sucrose, and culturing in a bud induction culture medium with pH of 5.8; transferring the culture medium to a new same culture medium after 28-32 days of culture, and continuing to grow for 20-30 days; selecting excellent germplasm of the Xinjiang rubber grasses suitable for tissue culture according to the regeneration rate and the growth state of the regenerated buds cultured on a bud induction culture medium for 48-52 days;
b. the selected excellent germplasm of the Xinjiang rubber plant is cut off when the plant height reaches 4-6 cm, and the process is transferred to a formula comprising the following steps: 1/2MS +20g/L sucrose, and in the rooting culture medium with pH5.8 to culture roots, becoming complete regeneration rubber grass tissue culture seedlings;
(2) pre-culture of explants of Hedychium plant
Cutting the leaves of the tissue culture seedlings of the hevea brasiliensis and pre-culturing the cut leaves on a bud induction culture medium for 3 days;
(3) acquisition of resistant regenerated rubber grass plants
a. Collecting pre-cultured rubber grass explants, putting the rubber grass explants into a sterile culture bottle, adding a pre-prepared agrobacterium infection solution until the rubber grass explants are completely immersed, and performing dip dyeing;
b. after the explant is subjected to bacterium liquid sucking-drying, transferring the explant to a bud induction culture medium, carrying out co-culture for 3 days at the temperature of 22-23 ℃ in the dark, wherein the pH is 5.2-5.3; taking out and washing with sterile water, and then washing with an MS liquid culture medium added with 395-405 mg/L Car; transferring the culture medium to a bud induction culture medium added with 395-405 mg/L Car for culturing for one week;
c. transferring the explant to the formula comprises: MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, inducing the regeneration of resistant buds on a screening culture medium with pH of 5.8, and subculturing once every 2 weeks;
d. when the resistant regenerated shoots grew to 3-5cm, the transfer to the formula included: 1/2MS +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, and culturing on new rooting culture medium with pH of 5.8 to root until becoming complete resistant regenerated rubber grass plant.
Further, the cutting treatment method for the blade in the step (1) and the step (2) comprises the following steps: cutting the leaf into 2-3cm length, scratching the leaf 1/2-3/4 width across the vein every 0.8-1.2 cm, and cutting the petiole into 1-1.5cm length.
Further explaining, in the step (1), the root system of the tissue culture seedling with the diameter of more than or equal to 1mm is selected from the rubberella seedlings after 30 days, the length of 2.8-3.2 cm is cut, and the formula is transferred as follows: 1/2MS, germinating adventitious buds on the root system until rooting to form complete tissue culture seedling, and taking tender leaf for the second time as the plant material for genetic transformation of Hevea brasiliensis. Is beneficial to keeping the young state of the rubber grass for a long time, keeping the regeneration capacity of the rubber grass and reducing the distortion rate.
Further, in the step (3), the preparation method of the agrobacterium infection solution comprises the following steps:
(1) selecting a single agrobacterium colony of a target gene to be cultured in 10mL of YEP culture medium with corresponding resistance at 28 ℃ and 200rpm overnight;
(2) adding 1mL of overnight culture liquid into 100mL of YEP culture medium with the same resistance, and culturing at 28 ℃ and 200rpm for 10-12 h;
(3) centrifuging with 50mL sterile centrifuge tube at 4 deg.C and 6000rpm for 10min to collect thallus; resuspending the Agrobacterium tumefaciens precipitate with MS liquid culture medium, centrifuging at 4 deg.C and 6000rpm for 10min, and collecting thallus; re-suspending the agrobacterium tumefaciens precipitate by using an MS liquid culture medium, and adjusting OD600 to 0.6 to prepare an agrobacterium tumefaciens infection liquid; the MS liquid culture medium comprises the following components in percentage by weight: MS +20g/L sucrose, pH 5.8.
Further explaining, in the step (3), acetosyringone with the final concentration of 100mg/L is added into the agrobacterium tumefaciens dip dyeing solution before dip dyeing.
Further, in the step (1), the culture conditions for bud induction and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.
Further, in the steps (2) and (3), the conditions for preculture, induction of resistant bud and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.
Further, in step (1), the formulation of the shoot induction medium comprises: MS +1.5mg/L6-BA +0.1mg/LNAA +20g/L sucrose, pH 5.8; the formula of the rooting culture medium comprises: 1/2MS +20g/L sucrose, pH 5.8.
Further, in step (3), the formulation of the screening medium comprises: MS +1.5mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.
Further, in step (3), the formulation of the new rooting medium comprises: 1/2MS +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, through screening of excellent germplasms of the Xinjiang rubber grass, the rubber grass regenerated seedling with strong regeneration capacity is obtained for genetic transformation, and hygromycin resistance screening of different concentrations of the rubber grass is carried out, so that the induction rate of the resistant regenerated bud is improved, and the rubber grass genetic transformation with high transformation rate and low deformity rate is realized. Has the following characteristics:
1. the screened excellent germplasm of the Xinjiang rubber plant has high seed germination rate, and the germination rate in the same batch of seeds reaches 100 percent; the growth speed is high, the continuous growth activity is strong, and the leaves are not easy to age; high bud induction rate, strong regeneration capability and high regeneration seedling forming efficiency, and is favorable for genetic transformation.
2. In the process of genetic transformation, the transformation efficiency is high, and the induction rate of the resistance regeneration bud is more than 30 percent; the genetic transformation period is short, and only about 3 months are needed from the infection of agrobacterium to the acquisition of the complete transgenic regeneration seedlings; the transgenic regenerated seedling has complete plant shape and low aberration rate; the transgenic regeneration seedlings have vigorous activity and high growth speed; the transgenic regenerated seedling has high propagation efficiency and is easy to obtain transgenic strains.
3. In the shoot induction medium for selecting resistant regenerated shoots, callus and shoots induced to be regenerated by explants which have not been successfully genetically transformed have a plurality of manifestation symptoms: (1) growth is severely inhibited; (2) easy vitrification; (3) the tip of the seedling leaf is easy to brown and necrose. According to the characteristics, the regeneration buds which are not successfully genetically transformed can be directly removed, and the positive detection rate of the transgenic rubber grass is over 90 percent finally.
Drawings
FIG. 1 shows the excellent germplasm of Xinjiang rubber grass suitable for tissue culture screened by the embodiment of the invention;
FIG. 2 is an explant of genetically transformed Columba glabra according to an embodiment of the present invention;
FIG. 3 is a graph of a first week of growth of a RUBENCH PLANT on a selection medium in accordance with an embodiment of the present invention;
FIG. 4 is a third week of growth of a RUBENCH PLANT on screening media in accordance with an embodiment of the present invention;
FIG. 5 shows the growth of a Hevea brasiliensis explant on a selection medium for 50 days according to an embodiment of the present invention;
FIG. 6 is a 75 day growth of a Hevea brasiliensis explant on selection medium according to an embodiment of the present invention;
FIG. 7 shows the expanding propagation of transgenic Hevea brasiliensis line according to an embodiment of the present invention;
FIG. 8 is the electrophoresis diagram of the PCR detection of the exogenous DNA of the transgenic hevea brasiliensis according to the embodiment of the present invention;
FIG. 9 shows the regeneration of multiple shoots after 30 days of treatment of RUBENCAO with hygromycin at different concentrations in accordance with the present invention.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1-a method for genetic transformation of hevea brasiliensis, comprising the steps of:
(1) screening and obtaining of plant material genetically transformed with kokstroemia indica
a. Collecting and selecting Xinjiang rubber grass germplasm, cutting complete and tender leaves, cutting the leaves into 2-3cm lengths after sterilization, scratching the leaves 1/2-3/4 width across veins every 0.8-1.2 cm, cutting petioles into 1-1.5cm lengths, and placing the leaves in a formula, wherein the cut length comprises the following steps: MS +1.6mg/L6-BA +0.1mg/LNAA +20g/L sucrose, and culturing in a bud induction culture medium with pH of 5.8; transferring to a new same culture medium after culturing for 30 days, and continuing to grow for 20-30 days; selecting excellent germplasm of the Xinjiang rubber grasses suitable for tissue culture according to the regeneration rate and the growth state of the regenerated buds after the Xinjiang rubber grasses are cultured on a bud induction culture medium for 50 days;
b. the selected excellent germplasm of the Xinjiang rubber plant is cut off when the plant height reaches 4-6 cm, and the process is transferred to a formula comprising the following steps: 1/2MS +20g/L sucrose, and in the rooting culture medium with pH5.8 to culture roots, becoming complete regeneration rubber grass tissue culture seedlings;
c. selecting a root system of a tissue culture seedling with the diameter of more than or equal to 1mm from the rubber grass seedlings after 30 days, cutting the root system to the length of 2.8-3.2 cm, and transferring to a formula as follows: 1/2MS, germinating adventitious buds on the root system until rooting to form complete tissue culture seedling, and taking tender leaf for the second time as the plant material for genetic transformation of Hevea brasiliensis.
(2) Pre-culture of explants of Hedychium plant
Cutting off leaves of the tissue culture seedlings of the hevea brasiliensis to be 2-3cm in length, scratching leaves 1/2-3/4 across veins every 0.8-1.2 cm, cutting off petioles to be 1-1.5cm in length, and pre-culturing on a bud induction culture medium for 2 days;
(3) acquisition of resistant regenerated rubber grass plants
a. Collecting pre-cultured rubber grass explants, putting the rubber grass explants into a sterile culture bottle, adding a pre-prepared agrobacterium infection solution until the rubber grass explants are completely immersed, and carrying out dip dyeing for 30 minutes;
b. after the explant is sucked to dry bacterial liquid, the explant is transferred to a bud induction culture medium, the pH value is 5.2, and the explant is cultured for 3 days in the dark at the temperature of 22-23 ℃; taking out, washing with sterile water, and washing with MS liquid culture medium with 400mg/L Car added; transferring to a bud induction culture medium added with 400mg/L Car for culturing for one week;
c. transferring the explant to the formula comprises: MS +1.6mg/L6-BA +0.1mg/LNAA +20g/L sucrose +400mg/L Car and 8mg/L Hyg, inducing the regeneration of resistant buds on a pH5.8 screening culture medium, and subculturing once every 2 weeks;
d. when the resistant regenerated shoots grew to 3-5cm, the transfer to the formula included: 1/2MS +20g/L sucrose +400mg/L Car and 8mg/L Hyg, and culturing on new rooting culture medium (pH5.9) to root until becoming complete resistant regenerated rubber grass plant.
According to the genetic transformation method, the germination rate of the screened rubber grass germplasm is 100%, the induction rate of the resistant regeneration bud is 32.6% in the genetic transformation process, the positive detection rate of the transgenic rubber grass is 92.3%, the transgenic regeneration seedling is complete in plant form, low in aberration rate, high in growth speed and high in propagation expansion efficiency, and a transgenic plant line can be quickly obtained.
Example 2-a method for genetic transformation of hevea brasiliensis, according to the transformation method of example 1, with the difference that in step (1), the culture conditions for bud induction and rooting culture are: at 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 plus 2000 lux; in the steps (2) and (3), the conditions of pre-culture, induction of resistant buds and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.
According to the genetic transformation method, the germination rate of the screened rubber grass germplasm is 100%, the induction rate of the resistant regeneration bud is 33.8% in the genetic transformation process, the positive detection rate of the transgenic rubber grass is 93.4%, the transgenic regeneration seedling is complete in plant form, low in aberration rate, high in growth speed and high in propagation expansion efficiency, and a transgenic plant line can be quickly obtained.
Example 3-a method for genetic transformation of hevea brasiliensis, according to the transformation method of example 1, with the difference that, in step (1), the formulation of the shoot induction medium comprises: MS +1.5mg/L6-BA +0.1mg/LNAA +20g/L sucrose, pH 5.8; the formula of the rooting culture medium comprises: 1/2MS +20g/L sucrose, pH 5.8;
in the step (3), the formula of the screening medium comprises: MS +1.5mg/L6-BA +0.1mg/LNAA +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH5.8; the formula of the new rooting culture medium comprises: 1/2MS +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH5.8.
According to the genetic transformation method, the germination rate of the screened rubber grass germplasm is 100%, the induction rate of the resistant regeneration bud is 35.1% in the genetic transformation process, the positive detection rate of the transgenic rubber grass is 96%, the transgenic regeneration seedling is complete in plant form, low in aberration rate, high in growth speed and high in propagation expansion efficiency, and the transgenic plant line can be quickly obtained.
Example 4-a method for genetic transformation of hevea brasiliensis, comprising the steps of:
(1) screening and obtaining of plant material genetically transformed with kokstroemia indica
a. Collecting and selecting Xinjiang rubber grass germplasm with luxuriant growth state, cutting complete and tender leaves of the rubber grass, placing the leaves in a beaker, washing the leaves clean by running water, treating the leaves for 30 seconds by 75% ethanol, and washing the leaves for 1 time by using sterile water; treating with 1% sodium hypochlorite solution on a shaker at 60rpm for 20 min, and washing with sterile water for 5 times, each for 5 min;
b. cutting off the leaf blade on sterile filter paper to 2-3cm length, scratching the leaf blade 2/3 width across vein every 1cm, and cutting off the petiole to 1-1.5cm length;
c. placing the plant material on a bud induction medium (MS +1.5mg/L6-BA +0.1mg/L NAA +20g/L sucrose, pH5.8) to grow; the culture condition is 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800-; transferring to a new same culture medium after culturing for 30 days, and continuing to grow for 20-30 days;
d. selecting excellent Xinjiang rubber grass germplasm 12-16 suitable for tissue culture according to the regeneration rate and the growth state of the regeneration bud after culturing for 50 days on a bud induction culture medium, wherein the regeneration rate and the growth state comprise the plant length, the leaf green degree, the vitrification degree and the induction rate of the regeneration bud;
the screening standard of the excellent germplasm of the hevea brasiliensis tissue culture is as follows:
grade I regeneration bud: the color is light green-green, the leaf length is more than 3cm, and no vitrification exists;
grade II regeneration bud: good, light green-green color, 3cm >1.5cm leaf length, no vitrification;
grade III regenerated bud: the color is light green-green, the leaf length is more than 0.5cm and is 1.5cm, and vitrification is avoided;
grade IV regeneration bud: poor, yellow or brown in color, leaf length <0.5cm, or vitrified;
the observation time is 20-25 days after the second subculture, wherein the total number of explants of 12-16 of the Sinkiang rubber grass germplasm is 54, the proportion of the I-grade regenerated buds is 80%, and the proportion of the IV-grade regenerated buds is 0; the induction rate of the regenerated buds is 89.36 percent;
e. when the plant height reaches 5cm, the plant is cut off and transferred to a rooting medium (1/2MS +20g/L sucrose, pH5.8) to be cultured and rooted (roots are usually germinated in about 10 days) to form a complete regeneration seedling which is used as the plant material for the genetic transformation of the hevea brasiliensis grass, as shown in figure 1.
f. Selecting a root system of a vigorously growing tissue culture seedling with the diameter of more than or equal to 1mm from the rubberella seedlings after 30 days, cutting the root system by a blade to a length of 2.8-3.2 cm, and transferring to a formula as follows: 1/2MS, after 10 days, sprouting adventitious buds on the root system until rooting to form complete tissue culture seedlings; the buds are not easy to distort, can keep the young state of the rubber grass for a long time, keeps the regeneration capacity of the rubber grass, is used for carrying out secondary tender leaf taking and is used as a plant material for genetic transformation of the rubber grass.
(2) Pre-culture of Hedychium plant explants
As shown in figure 2, the leaf of the tissue culture seedling of the Hevea brasiliensis is taken, cut to 2-3cm length by a blade on sterile filter paper, the leaf is scratched 2/3 width across veins every 1cm, and the petiole is cut to 1-1.5cm length; placing the mixture on a bud induction culture medium for pre-culture for 2 days, wherein the culture condition is 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 plus 2000 lux.
(3) Preparation of agrobacterium infection liquid
Selecting a single agrobacterium colony of a target gene to be cultured in 10mL of YEP culture medium with corresponding resistance at 28 ℃ and 200rpm overnight;
adding 1mL of the bacterial liquid into 100mL of YEP culture medium with corresponding resistance, and culturing at 28 ℃ and 200rpm for 10-12 h;
the cells were collected by centrifugation at 6000rpm in a 50mL sterile centrifuge tube at 4 ℃ for 10 minutes. Resuspending Agrobacterium precipitation with MS liquid culture medium (MS +20g/L sucrose, pH5.8), centrifuging at 4 deg.C and 6000rpm for 10min, and collecting thallus; resuspending the Agrobacterium pellet with MS liquid culture medium and adjusting OD600 to 0.6 to prepare Agrobacterium infection solution.
(4) Acquisition of resistant regenerated rubber grass plants
a. Collecting pre-cultured Hedychium notatum explants, placing into a sterile culture flask, adding Agrobacterium tumefaciens staining solution (acetosyringone with final concentration of 100 mg/L) until completely immersed, staining at room temperature for 30min, and slightly shaking to ensure the explants to fully contact with the staining solution;
b. placing the explant on sterile filter paper, sucking the bacterial liquid to the greatest extent, transferring the explant to a culture dish filled with a bud induction culture medium, carrying out co-culture for 3 days at the temperature of 22 ℃ in the dark; taking out explant, rapidly cleaning with sterile water for 3-5 times, sufficiently cleaning with MS liquid culture medium supplemented with 400mg/L carbenicillin (Car) for 5min, and placing on sterile filter paper to absorb water; then transferring the culture medium to a bud induction culture medium added with 400mg/L Car for culturing for one week, wherein the culture condition is 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 plus 2000 lux;
c. transferring the explant to a screening culture medium (MS +1.5mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH5.8), inducing the regeneration of the resistant bud, subculturing once every 2 weeks under the culture condition of 22 ℃, the illumination period of 16 hours/day and the illumination intensity of 1800 plus 2000 lux; as shown in FIGS. 3-6; the rubberella plants grow on the screening culture medium for the first week, and part of the explants begin browning and necrosis; growing on the screening culture medium for the third week, wherein most area of the explant undergoes browning necrosis, and the length of the induced bud is about 1-3 mm; growing on a screening culture medium for 50 days, and inducing buds to be about 5-10mm long; growing on a screening culture medium for 75 days, and inducing buds to be about 3-5cm long;
d. when the resistant regeneration bud grows to 3-5 cm; transferring to a new rooting culture medium (1/2MS +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH5.8) for rooting culture; the culture condition is 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800-; until they became intact resistant regenerated rubber grass plants, as shown in FIG. 7.
As shown in fig. 8, the PCR detection electropherogram of the exogenous DNA of transgenic hevea brasiliensis; m: marker; 1-34: transgenic rubber grass; +: positive plasmid control; -: wild type kokstroemia indica;
according to the genetic transformation method, the germination rate of the screened rubber grass germplasm is 100%, the induction rate of the resistant regeneration bud is 37.2% in the genetic transformation process, the positive detection rate of the transgenic rubber grass is 98%, the transgenic regeneration seedling is complete in plant form, low in aberration rate, high in growth speed and high in propagation expansion efficiency, and the transgenic plant line can be quickly obtained.
Comparative experiment group 1-according to the transformation method of example 4, during the screening of the plant material genetically transformed with kochia scoparia, the screening of the formulations of different kochia scoparia bud induction media was performed and the germination rates of different explants were counted with the following results:
the formula of the rubber grass bud induction culture medium is as follows:
the formula I is as follows: MS +1.5mg/L6-BA +0.1mg/L NAA, and 20g/L sucrose; agar 7-8 g/L; the pH value is 5.8;
and a second formula: MS +1.0 mg/L6-BA +0.2mg/L NAA, and 20g/L sucrose; agar 7-8 g/L; the pH value is 5.8;
and the formula III: MS +1.0 mg/L6-BA +0.1mg/L IAA, and 20g/L sucrose; agar 7-8 g/L; the pH value is 5.8; each group was set with 3 replicates;
as can be seen from the above table, the formula of the rubber grass bud induction medium is as follows: MS +1.5mg/L6-BA +0.1mg/L NAA for bud induction culture, the average budding rate is obviously improved, and the germplasm screening is facilitated, namely, excellent germplasm of the Xinjiang rubber plant suitable for tissue culture is selected according to the growth state of the regeneration bud, including the plant length, the green degree of leaves, the vitrification degree and the induction rate of the regeneration bud.
Comparative experiment group 2-transformation method according to example 4, during the course of genetic transformation, hygromycin resistance screening of the plants of hevea brasiliensis was carried out at different concentrations in the screening medium, respectively: 8mg/L, 10mg/L, 12mg/L, 14mg/L, 16mg/L and 18mg/L, and counting the growth condition of the explants after the treatment of the rubber grass explants with hygromycin for 30 days, wherein the results are as follows:
as can be seen from the above table, all explants in the group treated with 16-18mg/L hygromycin died due to browning; in the group treated with 8-10mg/L hygromycin, a small number of cluster buds were regenerated, but the growth of the cluster buds was significantly inhibited, and the average length of the cluster buds was <5 mm. The regeneration of the cluster buds in the hygromycin treatment group of 12-14mg/L is obviously reduced, the growth of the cluster buds is more seriously inhibited, the cluster buds in the hygromycin treatment group of 12mg/L can not be normally regenerated basically, as shown in figure 9, the regeneration condition of the cluster buds after the hygromycin treatment of the hevea brasiliensis for 30 days at different concentrations is that the hygromycin concentration A is 8 mg/L; b, 10 mg/L; c, 12 mg/L; d, 14 mg/L; e, 16 mg/L; f is 18mg/L, when the hygromycin concentration is lower than 8mg/L, the regeneration seedlings with successful genetic transformation and unsuccessful genetic transformation cannot be effectively distinguished, and the positive detection rate of the transgenic rubber seedlings is low finally.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A method for genetic transformation of kokstroemia indica, which is characterized in that: the method comprises the following steps:
(1) screening and obtaining of plant material genetically transformed with kokstroemia indica
a. Collecting and selecting Xinjiang rubber grass germplasm, cutting complete young and tender leaves, sterilizing and cutting, and placing in a formula comprising: MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L cane sugar, and culturing in a bud induction culture medium with pH of 5.8; transferring the culture medium to a new same culture medium after 28-32 days of culture, and continuing to grow for 20-30 days; selecting excellent germplasm of the Xinjiang rubber grasses suitable for tissue culture according to the regeneration rate and the growth state of the regenerated buds cultured on a bud induction culture medium for 48-52 days;
b. the selected excellent germplasm of the Xinjiang rubber plant is cut off when the plant height reaches 4-6 cm, and the process is transferred to a formula comprising the following steps: 1/2MS +20g/L sucrose, and in the rooting culture medium with pH5.8 to culture roots, becoming complete regeneration rubber grass tissue culture seedlings;
selecting a root system of a tissue culture seedling with the diameter of more than or equal to 1mm from the rubber grass seedlings after 30 days, cutting the root system to the length of 2.8-3.2 cm, and transferring to a formula as follows: 1/2MS, germinating adventitious buds on the root system until rooting to form complete tissue culture seedling, and taking tender leaf for the second time to serve as plant material for genetic transformation of Hevea brasiliensis;
(2) pre-culture of Hedychium plant explants
Cutting the leaves of the tissue culture seedlings of the hevea brasiliensis and pre-culturing the cut leaves on a bud induction culture medium for 2 days;
(3) acquisition of a resistant regenerated plant of RUBENCAO
a. Collecting pre-cultured rubber grass explants, putting the rubber grass explants into a sterile culture bottle, adding a pre-prepared agrobacterium infection solution until the rubber grass explants are completely immersed, and carrying out dip dyeing for 30 minutes;
b. after the explant is subjected to bacterium liquid sucking-drying, transferring the explant to a bud induction culture medium, carrying out co-culture for 3 days at the temperature of 22-23 ℃ in the dark, wherein the pH is 5.2-5.3; taking out and washing with sterile water, and then washing with an MS liquid culture medium added with 395-405 mg/L Car; transferring the culture medium to a bud induction culture medium added with 395-405 mg/L Car for culturing for one week;
c. transferring the explant to the formula comprises: MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, inducing the regeneration of resistant buds on a screening culture medium with pH of 5.8, and subculturing once every 2 weeks;
d. when the resistant regenerated shoots grew to 3-5cm, the transfer to the formula included: 1/2MS +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, and culturing on new rooting culture medium with pH of 5.8 to root until becoming complete resistant regenerated rubber grass plant.
2. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: the cutting processing method for the blade in the steps (1) and (2) comprises the following steps: cutting the leaf into 2-3cm length, scratching the leaf 1/2-3/4 width across the vein every 0.8-1.2 cm, and cutting the petiole into 1-1.5cm length.
3. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), the preparation method of the agrobacterium infection liquid comprises the following steps:
(1) selecting a single agrobacterium colony of a target gene to be cultured in 10mL of YEP culture medium with corresponding resistance at 28 ℃ and 200rpm overnight;
(2) adding 1mL of overnight culture liquid into 100mL of YEP culture medium with the same resistance, and culturing at 28 ℃ and 200rpm for 10-12 h;
(3) centrifuging with 50mL sterile centrifuge tube at 4 deg.C and 6000rpm for 10min to collect thallus; resuspending the Agrobacterium tumefaciens precipitate with MS liquid culture medium, centrifuging at 4 deg.C and 6000rpm for 10min, and collecting thallus; re-suspending the agrobacterium tumefaciens precipitate by using an MS liquid culture medium, and adjusting OD600 to 0.6 to prepare an agrobacterium tumefaciens infection liquid; the MS liquid culture medium comprises the following components in percentage by weight: MS +20g/L sucrose, pH 5.8.
4. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), acetosyringone with the final concentration of 100mg/L is added into the agrobacterium infection liquid before dip dyeing.
5. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (1), the culture conditions of bud induction and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.
6. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the steps (2) and (3), the conditions of pre-culture, induction of resistant buds and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.
7. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (1), the formula of the bud induction medium comprises: MS +1.5mg/L6-BA +0.1mg/L NAA +20g/L sucrose, pH 5.8; the formula of the rooting culture medium comprises: 1/2MS +20g/L sucrose, pH 5.8.
8. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), the formula of the screening medium comprises: MS +1.5mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.
9. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), the formula of the new rooting medium comprises: 1/2MS +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.
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