CN106755079A - Diamond dust aids in the Efficient Conversion method of Agrobacterium-mediated Transformation green bristlegrass seed - Google Patents
Diamond dust aids in the Efficient Conversion method of Agrobacterium-mediated Transformation green bristlegrass seed Download PDFInfo
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Abstract
The invention belongs to biological technical field, it is related to the processes such as a kind of Efficient Conversion method of diamond dust auxiliary Agrobacterium-mediated Transformation green bristlegrass seed, treatment and activation, diamond dust wound treatment and During Agrobacterium, co-cultivation, antibacterial and screening and culturing, the culture of kanamycin-resistant callus tissue seedling, culture of rootage including explant.Step of the present invention is simple, it is easy to operate, it is explant with ripe green bristlegrass seed, after seed is through activation process of germinateing, is aided in causing wound with diamond dust, carry out Agrobacterium-mediated Transformation, the transformation efficiency of green bristlegrass is substantially increased, the transformation period is shortened, Transformation Program is simplified, the scope of conversion strain is expanded, is that green bristlegrass establishes a kind of flexible, cycle is short, efficient transgenic technology system.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of green bristlegrass Agrobacterium Efficient Conversion method, specifically a kind of Buddha's warrior attendant
Sand aids in the Efficient Conversion method of Agrobacterium-mediated Transformation green bristlegrass seed.
Background technology
C4 photosynthesis are the productivity for driving several staple food crops and bioenergy grass class, including corn (Zea
Mays), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor), Chinese silvergrass (Miscanthus
) and switchgrass (Panicum virgatum) etc. giganteus.Green bristlegrass (Setaria viridis) is one real two
Times body, the characteristic of genetic analysis, including relatively small genome (510Mb), of short and small stature, life cycle are suitable to many
Short and grain weight is big etc., therefore, green bristlegrass is the excellent model for studying C4 photosynthesis, while being also research abiotic stress
The model of tolerance and bioenergy raw material.Green bristlegrass is morphologically similar to most of gramineous grasses, including primary biological energy
Source crop switchgrass (Panicum virgatum) and Chinese silvergrass (Miscanthus giganteus) etc., geographically divide extensively
Cloth, and occupy diversified ecological niche.
At present, after the transgenic technology of green bristlegrass is mainly germinateed using mature seed, lured from young shoot node separate living tissue
Leading embryo callus carries out Agrobacterium-mediated Transformation, and the greatest problem of this method is low frequency of embryonic callus induction, because of strain
Different with seed situation, the ratio of general embryonic callus induction number and seed number is both less than 10%；Next to that embryo callus subculture
Tissue induction time is more long, when a small amount of embryo callus for obtaining the first stage to culture medium from inoculation need about one month
Between, need just to have after subculture 2 times () enough embryo callus afterwards for Agrobacterium-mediated Transformation, entirely per first quarter moon subculture once
Transformation period needs about 4 months；Meanwhile, this embryo callus are intolerant to subculture, it is impossible to which long-term subculture is preserved for turning
Change, the embryo callus transformation efficiency that subculture is more than 3 times is substantially reduced, be not suitable for continuing on for conversion；Additionally, embryo callus subculture
Selecting for tissue needs certain experience.These flexibilities for all reducing this set transformation technology and simple and practical property.As
Important model plant, be green bristlegrass set up it is simpler, flexibly, cycle is short, efficient transgenic technology turned into association area
Research direction.
The content of the invention
The purpose of the present invention is directed to the deficiencies in the prior art and provides a kind of diamond dust auxiliary Agrobacterium-mediated Transformation green bristlegrass
The Efficient Conversion method of seed, with green bristlegrass mature seed as explant, wound treatment is carried out with diamond dust after induction dew bud, is located
Contaminated with Agrobacterium after reason, transfer-gen plant is obtained with plant regeneration through co-cultivation, antibacterial, screening, with simple, spirit
Living, quick, efficient the features such as.
The technical solution adopted in the present invention：
A kind of diamond dust aids in the Efficient Conversion method of Agrobacterium-mediated Transformation green bristlegrass seed, and it is comprised the following steps that：
1st, the treatment and activation of explant
The green bristlegrass dry mature seed processed through dormancy is chosen, the seed after kind of skin will be removed and loaded centrifuge tube, with having
Effect concentration is 1~3% liquor natrii hypochloritis's sterilization treatment, after sterile water wash, by mass volume ratio：Seed：Bud is lured
Lead fluid nutrient medium=1:6~8 ratio adds bud induced fluid culture medium, shaken cultivation 1~7 day, culture in centrifuge tube
Condition：Optical culture, 24~26 DEG C of cultivation temperature.The bud induced fluid culture medium is the culture medium based on MS culture mediums, its
Middle micronutrient levels is doubled, and pH value is 5.0~6.0, and adds hormone；The hormone is 2,4-D, Dicamba, KT, 6-BA
In one or more, its addition is respectively：Below 10mg/L.
2nd, the treatment of diamond dust wound and During Agrobacterium
The diamond dust of medium mesh degree of equivalent is weighed according to seed volume (i.e. by volume：Seed：Diamond dust=1:1),
High-temperature sterilization treatment, outwells the bud induced fluid culture medium in centrifuge tube, adds diamond dust, be subsequently adding it is a small amount of containing 0.6~
1.0OD600nmThe dip-dye culture medium of Agrobacterium, high speed processing 2~3 minutes under turbula shaker, adds the leaching containing Agrobacterium
Dye culture medium, make the cumulative volume of the dip-dye culture medium containing Agrobacterium for 8~12 times of seed volume (i.e. by volume：Seed：Leaching
Dye agrobacterium liquid=1:8~12), low-speed oscillation is contaminated 0.5~1 hour, condition of culture：Optical culture, 24~26 DEG C of cultivation temperature.
The dip-dye culture medium is the culture medium based on MS culture mediums, and wherein a great number of elements content halves, and micronutrient levels is doubled,
PH value is 5.0~6.0, addition hormone, acetosyringone (Acetosyringone) 20~60mg/L, surfactant 0.1~
1% (such as Synperonic, mass volume ratio)；The hormone is 2, one or more in 4-D, Dicamba, KT, 6-BA, its
Addition is respectively：Below 10mg/L.
3rd, co-culture
Seed after dip-dye is connected on the co-cultivation culture medium with filter paper, light culture 3~9 days, cultivation temperature 20~
(can be adjusted according to the different agrobacterium strains for using) between 28 DEG C.The co-cultivation culture medium is with MS culture mediums as base
Basal culture medium, wherein a great number of elements content halves, and micronutrient levels is doubled, and pH value is 5.0~6.0, addition hormone, acetyl fourth
Ketone musk (Acetosyringone) 20~60mg/L；The hormone is 2, one or more in 4-D, Dicamba, KT, 6-BA,
Its addition is respectively：Below 10mg/L.
4th, antibacterial and screening and culturing
Seed after co-cultivation is connected on antibacterial and screening and culturing medium and is screened, screening is in two stages：First rank
Section, first adds the semilethal metering of selective agent in antibacterial and screening and culturing medium, screens 1 week；Second stage, will be by the first rank
Section screening seed subculture to addition the full lethal metering of selective agent antibacterial and screening and culturing medium in, screening 3~4 weeks.Culture bar
Part：First light culture, last 1~2 week optical culture, 24~26 DEG C of cultivation temperature.Described antibacterial and screening and culturing medium is cultivated with MS
Culture medium based on base, wherein micronutrient levels are doubled, and pH value is 5.0~6.0, addition hormone, the antibiosis of suppression Agrobacterium
100~500mg/L of element (such as Timentin)；The hormone is 2, and one or more in 4-D, Dicamba, KT, 6-BA, it adds
Dosage is respectively：Below 10mg/L.
5th, kanamycin-resistant callus tissue seedling culture
The embryo callus subculture that will be induced by screening stage on seedling culture medium, condition of culture：Optical culture, training
Support 24~26 DEG C of temperature, incubation time 2~4 weeks.The seedling culture medium is the culture medium based on MS culture mediums, wherein organic
Element and micronutrient levels are doubled, sugarcane, 15~20g/L, and pH value is 5.0~6.0, addition hormone, the antibiosis of suppression Agrobacterium
100~500mg/L of element (such as Timentin), without or addition selective agent semilethal metering.The hormone is 2,4-D, KT, 6-
One or more in BA, its addition is respectively：Below 10mg/L.
6th, culture of rootage
The bud for reaching more than 1cm that will be regenerated on seedling culture medium individually takes out, and is connected on root media and is given birth to
Root culture, condition of culture：Optical culture, 24~26 DEG C of cultivation temperature, incubation time 1~2 week.The root media is trained with MS
Culture medium based on base is supported, wherein a great number of elements content halves, and cane sugar content halves, pH value is 5.0~6.0, adds selective agent
Semilethal is measured, and is added or without IBA, and its addition is：Below 10mg/L.
Step of the present invention is simple, easy to operate, is explant with ripe green bristlegrass seed, and seed is through activation process of germinateing
Afterwards, aided in causing wound with diamond dust, carry out Agrobacterium-mediated Transformation, substantially increase the transformation efficiency of green bristlegrass, shorten conversion
In the cycle, simplify Transformation Program, expand the scope of conversion strain, be green bristlegrass establish it is a kind of flexibly, it is cycle is short, efficient
Transgenic technology system.
Brief description of the drawings
Fig. 1 is pZmUbi-GUS (ZmUbis-HPH) the plasmid vector figure for converting.
Not same order is co-cultured when Fig. 2 is green bristlegrass different lines (A10, ME34) seed diamond dust (emery) aid in treatment
Section (3 days, 5 days, 7 days, 9 days) gus gene transient expression disparity map.A10, ME34 strain left side are all not use diamond dust auxiliary
The changing effect for helping, the right is all to use diamond dust auxiliaring effect；Using diamond dust aid in seed convert efficiency apparently higher than
Do not use：When co-culturing 3 days, seed young shoot all GUS dyeing, the transient expression effect of transformed gene of diamond dust treatment
Very strong, the seed young shoot without diamond dust treatment is not almost colored, transient expression poor effect, during with co-culturing
Between extension, the seed transient expression situation of non-process has increase, but is all nothing like the expression effect of diamond dust treatment.
Fig. 3 is pZmUbi-GUS (ZmUbis-HPH) plasmid transformation seedlings GUS staining conditions.As can be seen from Fig., through Buddha's warrior attendant
The resistance seedling that filters out nearly all is the transgenic seedling of homozygosis after sand auxiliary Agrobacterium-mediated Transformation green bristlegrass seed, and whole strain can be by
GUS is dyeed.
Fig. 4 is the optimization base program that green bristlegrass mature seed diamond dust auxiliary Agrobacterium directly converts.Seed activation
(active seeds) 5 days, contaminates and co-cultures (transformation) 5 days, low concentration screening (selection 1) 1 week,
High concentration secretly screens (selection 2) 2 weeks, high concentration light screening (selection 3) 2 weeks, seedling culture
(regeneration) 2 weeks.
Specific embodiment
With reference to embodiment, specific embodiment of the invention is described in further detail.Following examples are used for
The present invention is illustrated, but is not limited to the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally
According to normal condition, or according to the condition proposed by manufacturer.
Embodiment
1st, the treatment and activation of explant
Green bristlegrass strain A10 and the ME34 dry mature seed processed through dormancy is chosen, by each about 1 gram of kind after removal kind skin
Son is respectively charged into two 14ml centrifuge tubes, is processed with liquor natrii hypochloritis's sterilization of valid density 1%, sterile water wash
Afterwards, the bud induced fluid culture medium shaken cultivation of about half volume is added in centrifuge tube 5 days, condition of culture：Optical culture, culture
24 DEG C or so of temperature.Bud induced fluid culture medium is culture medium (micronutrient levels is doubled) based on modified MS medium,
PH is 5.8, and adds 2mg/L2,4-D+0.5mg/L KT.
2nd, the treatment of diamond dust wound and During Agrobacterium
According to seed volume ratio (1:1) diamond dust (120 mesh and each half weight of 320 mesh) is weighed, high-temperature sterilization is processed,
Fall the bud induced fluid culture medium in centrifuge tube, add diamond dust.Addition contains 0.6OD on a small quantity600nmThe dip-dye culture of Agrobacterium
Then base (liquid), high speed processing 2 minutes under turbula shaker add the dip-dye culture medium containing Agrobacterium, train dip-dye
The cumulative volume for supporting base is 10mL, and low-speed oscillation is contaminated 40 minutes, condition of culture：Optical culture, 24 DEG C or so of cultivation temperature.Contaminate training
Foster base is culture medium (a great number of elements content halves, and micronutrient levels is doubled) based on modified MS medium, and pH value is
5.2,2mg/L 2,4-D is added, acetosyringone (Acetosyringone) 40mg/L is added, add surfactant
Synperonic 0.1% (mass volume ratio).Agrobacterium is the bacterial strains of AGL 1, and conversion plasmid is pZmUbi-GUS (ZmUbis-
HPH Fig. 1 (GUS marks)) is seen, with band 50mg/L carbenicillins and the LB culture medium coated plates of 50mg/L kanamycins, at 20 DEG C
It is 0.6OD that light culture take bacterium block after 3~5 days to contaminate culture medium resuspended600nm, agrobacterium liquid jog activate 2~3 hours after be used for
Conversion.
3rd, co-culture
Seed after dip-dye is connected on the co-cultivation culture medium with filter paper, light culture 3~9 days, cultivation temperature is 20 DEG C.
It is the culture medium based on modified MS medium to co-culture culture medium, and a great number of elements content halves, and micronutrient levels is doubled, pH
It is 5.2 to be worth, and adds 2mg/L 2,4-D+0.5mg/L KT, adds acetosyringone (Acetosyringone) 40mg/L.Point
A10, ME34 seed are not taken in the different co-cultivation stages (3 days, 5 days, 7 days, 9 days) and do GUS dyeing transient expression detections, from Fig. 2
It can be seen that the seed GUS transient expressions of use diamond dust (emery) aid in treatment conversion are substantially stronger than not aid in treatment, therefore Buddha's warrior attendant
Sand aid in treatment can significantly improve the transformation efficiency of activated seed.
4th, antibacterial and screening and culturing
Seed after co-culturing 5 days is connected on antibacterial and screening and culturing medium, and antibacterial and screening and culturing medium is to improve MS
Culture medium based on culture medium, micronutrient levels is doubled, and pH value is 5.8, adds 2mg/L2,4-D+0.5mg/L KT, addition
Suppress antibiotic 150mg/L Te Meiding (Timentin) of Agrobacterium, addition selective agent 25mg/L hygromycin (Hygromycin).
In 1 week follow-up generation, was brought up on the antibacterial and screening and culturing medium of 45mg/L to hygromycin, was cultivated 4 weeks.Condition of culture：First light culture,
2 weeks optical cultures, 24 DEG C or so of cultivation temperature afterwards.Observation resistant embryogenic calli induction situation, counts resistance embryo callus subculture group
Number is knitted, inductivity is calculated, 1, resistant embryogenic calli inductivity (resistant embryogenic calli number/germinated seeds is the results are shown in Table
Number) A10 is up to 49%, ME34 up to 34%.
The resistant embryogenic calli of table 1 induces situation
| Green bristlegrass strain | Germinated seeds number | Resistant embryogenic calli number | Inductivity |
| A10 | 500 | 245 | 49% |
| ME34 | 500 | 170 | 34% |
5th, kanamycin-resistant callus tissue seedling culture
The resistant embryogenic calli that will be induced by screening stage, on subculture to seedling culture medium, condition of culture：Light is trained
Support, 24 DEG C or so of cultivation temperature.Seedling culture medium is the culture medium based on modified MS medium, organic and micronutrient levels
Double, sucrose 20g/L, pH value is 5.8, add 1.5mg/L KT, 20mg/ is added in addition 150mg/L Te Meiding (Timentin)
L hygromycin.Incubation time 2 weeks.Observation kanamycin-resistant callus tissue seedling culture situation, counts clump bud number, calculates inductivity, the results are shown in Table 2,
Substantially every piece kanamycin-resistant callus tissue can induce clump bud.
The plant regeneration situation of table 2
| Green bristlegrass strain | Resistant embryogenic calli number | Callus regenerates clump bud number | Inductivity |
| A10 | 150 | 147 | 98% |
| ME34 | 150 | 138 | 92% |
6th, culture of rootage
The bud for reaching about more than 1cm that will be regenerated on seedling culture medium individually takes out, and is connected on root media and takes root,
Condition of culture：Optical culture, 24 DEG C or so of cultivation temperature.Root media is the MS culture mediums of improvement, and a great number of elements content halves,
Cane sugar content halves, and pH value is 5.8, addition 150mg/L Te Meiding (Timentin), adds 20mg/L hygromycin.Incubation time 1
~2 weeks.Observation culture of rootage situation, counts number of taking root, and calculates rooting rate, the results are shown in Table 3.By by the transformation seedlings of screening regeneration
GUS dyeing detections are carried out, staining conditions are shown in Fig. 3.
The culture of rootage situation of table 3
| Green bristlegrass strain | Clump bud number | Clump bud is taken root number | Rooting rate |
| A10 | 120 | 113 | 94.1% |
| ME34 | 120 | 102 | 85% |
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, on the premise of the technology of the present invention principle is not departed from, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (1)
1. a kind of diamond dust aids in the Efficient Conversion method of Agrobacterium-mediated Transformation green bristlegrass seed, it is characterised in that its specific steps
It is as follows：
1), the treatment and activation of explant
The green bristlegrass dry mature seed processed through dormancy is chosen, the seed after kind of skin will be removed and loaded centrifuge tube, with effectively dense
The liquor natrii hypochloritis's sterilization spent for 1~3% is processed, after sterile water wash, by mass volume ratio：Seed：Bud induces liquid
Body culture medium=1:6~8 ratio adds bud induced fluid culture medium, shaken cultivation 1~7 day, condition of culture in centrifuge tube：
Optical culture, 24~26 DEG C of cultivation temperature；The bud induced fluid culture medium is the culture medium based on MS culture mediums, wherein micro
Constituent content is doubled, and pH value is 5.0~6.0, and adds hormone；The hormone is in 2,4-D, Dicamba, KT, 6-BA
Plant or various, its addition is respectively：Below 10mg/L；
2), the treatment of diamond dust wound and During Agrobacterium
The diamond dust of the medium mesh degree of equivalent is weighed according to seed volume, the bud induction in centrifuge tube is outwelled in high-temperature sterilization treatment
Fluid nutrient medium, adds diamond dust, is subsequently adding on a small quantity containing 0.6~1.0OD600nmThe dip-dye culture medium of Agrobacterium, is being vortexed
High speed processing 2~3 minutes under oscillator, add the dip-dye culture medium containing Agrobacterium, make the dip-dye culture medium containing Agrobacterium
Cumulative volume is 8~12 times of seed volume, and low-speed oscillation is contaminated 0.5~1 hour, condition of culture：Optical culture, cultivation temperature 24~
26℃.The dip-dye culture medium is the culture medium based on MS culture mediums, and wherein a great number of elements content halves, micronutrient levels
Double, pH value is 5.0~6.0, addition hormone, 20~60mg/L of acetosyringone, surfactant 0.1~1%；The hormone
It is 2, one or more in 4-D, Dicamba, KT, 6-BA, its addition is respectively：Below 10mg/L；
3), co-culture
Seed after dip-dye is connected on the co-cultivation culture medium with filter paper, light culture 3~9 days, cultivation temperature is at 20~28 DEG C
Between；The co-cultivation culture medium is the culture medium based on MS culture mediums, and wherein a great number of elements content halves, and trace element contains
Amount is doubled, and pH value is 5.0~6.0, addition hormone, 20~60mg/L of acetosyringone；The hormone be 2,4-D, Dicamba,
One or more in KT, 6-BA, its addition is respectively：Below 10mg/L；
4), antibacterial and screening and culturing
Seed after co-cultivation is connected on antibacterial and screening and culturing medium and is screened, screening is in two stages：First stage, first
The semilethal metering of selective agent is added in antibacterial and screening and culturing medium, is screened 1 week；Second stage, will sieve by the first stage
The seed subculture of choosing to addition the full lethal metering of selective agent antibacterial and screening and culturing medium in, screening 3~4 weeks；Condition of culture：First
Light culture, last 1~2 week optical culture, 24~26 DEG C of cultivation temperature；Described antibacterial and screening and culturing medium is with MS culture mediums as base
Basal culture medium, wherein micronutrient levels are doubled, pH value be 5.0~6.0, addition hormone, suppress Agrobacterium antibiotic 100~
500mg/L；The hormone is 2, one or more in 4-D, Dicamba, KT, 6-BA, and its addition is respectively：10mg/L with
Under；
5), kanamycin-resistant callus tissue seedling culture
The embryo callus subculture that will be induced by screening stage on seedling culture medium, condition of culture：Optical culture, culture temperature
24~26 DEG C of degree, incubation time 2~4 weeks；The seedling culture medium is the culture medium based on MS culture mediums, wherein organic element
Doubled with micronutrient levels, 15~20g/L of sucrose, pH value is 5.0~6.0, addition hormone, the antibiotic of suppression Agrobacterium
100~500mg/L, without or addition selective agent semilethal metering；The hormone is the one kind or many in 2,4-D, KT, 6-BA
Kind, its addition is respectively：Below 10mg/L；
6), culture of rootage
The bud for reaching more than 1cm that will be regenerated on seedling culture medium individually takes out, and is connected on root media and takes root, and cultivates bar
Part：Optical culture, 24~26 DEG C of cultivation temperature, incubation time 1~2 week；The root media is trained based on MS culture mediums
Base is supported, wherein a great number of elements content halves, and cane sugar content halves, pH value is 5.0~6.0, the semilethal metering of addition selective agent adds
Plus or without IBA, its addition is：Below 10mg/L.
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| CN201710016961.5A CN106755079A (en) | 2017-01-11 | 2017-01-11 | Diamond dust aids in the Efficient Conversion method of Agrobacterium-mediated Transformation green bristlegrass seed |
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Application publication date: 20170531 |