CN106854659A - A kind of method of sinocalamus latiflorus genetic transformation and plant regeneration - Google Patents
A kind of method of sinocalamus latiflorus genetic transformation and plant regeneration Download PDFInfo
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- CN106854659A CN106854659A CN201510887378.2A CN201510887378A CN106854659A CN 106854659 A CN106854659 A CN 106854659A CN 201510887378 A CN201510887378 A CN 201510887378A CN 106854659 A CN106854659 A CN 106854659A
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Abstract
The invention discloses a kind of sinocalamus latiflorus (Dendrocalamus latiflorus Munro) tissue cultures and genetic transforming method, by sinocalamus latiflorus flower pesticide induced embryonic callus, and carried out with this agriculture bacillus mediated, so as to obtain transfer-gen plant, it is related to Plant Tissue Breeding and genetic transforming method field.Key step includes：The collection and pretreatment of sinocalamus latiflorus flower pesticide, the acquisition of embryo callus, plasmid construction and Agrobacterium culture, Agrobacterium are infected, the screening of sinocalamus latiflorus embryo callus and differentiation, plant regeneration and acclimatization and transplantses.The genetic transformation regeneration system is genetic recipient material with the callus induced by flower pesticide, and simple with method, reliability is high, easy-operating feature, is that the genetic improvement of sinocalamus latiflorus opens new way.
Description
Technical field
The present invention relates to Plant Tissue Breeding and genetic transforming method, and in particular to one kind is induced by flower pesticide and is genetic recipient material with power of regeneration callus, carries out the genetic transformation of Agrobacterium, and then the method for producing transfer-gen plant.
Background technology
Sinocalamus latiflorus (Dendrocalamus latiflorus Munro) is grass family Bambusoideae Dendrocalamus sinocalamus latiflorus subgenus, originate in China, NATURAL DISTRIBUTION is saved in Fujian, Taiwan, Guangdong, Guangxi, Guizhou and Yunnan etc., is one of the important excellent, fast-growing in China south China and the southeastern coastal areas, the large-scale sympodial bamboo kind of double purposes.Sinocalamus latiflorus seldom blooms, and mainly based on nature choiceness seed selection, genetic improvement is restricted breed breeding.Because sinocalamus latiflorus tissue cultures are difficult to obtain regeneration plant, the application on sinocalamus latiflorus by genetic transformation is constrained.Recently, by continuous experimental exploring, sinocalamus latiflorus Regenerated Plantlets technology has breakthrough, the embryo callus obtained by flower pesticide induction, can directly break up as regeneration plant, is that the genetic transformation of sinocalamus latiflorus is laid a good foundation.It is genetic recipient material with the embryo callus with good regeneration plant ability, has method simple by Agrobacterium-mediated genetic transformation regenerating system, reliability is high, easy-operating feature, is that the genetic improvement of sinocalamus latiflorus opens new way.
The content of the invention
The invention provides a kind of sinocalamus latiflorus (Dendrocalamus latiflorus Munro) tissue cultures and genetic transforming method, by sinocalamus latiflorus flower pesticide induced embryonic callus, and carried out with this agriculture bacillus mediated, so as to obtain transfer-gen plant, it is related to Plant Tissue Breeding and genetic transforming method field.Key step includes：The collection and pretreatment of sinocalamus latiflorus flower pesticide, the acquisition of embryo callus, plasmid construction and Agrobacterium culture, Agrobacterium are infected, the screening of sinocalamus latiflorus embryo callus and differentiation, plant regeneration and acclimatization and transplantses.Basic process is as follows：
Step 1. gathers sinocalamus latiflorus prematurity flower pesticide, and evoked callus obtain genetic recipient material；
Step 2. build genes of interest carrier in and imported into Agrobacterium, obtain monoclonal simultaneously cultivated；
Step 3. infects by Agrobacterium；
Step 4. sinocalamus latiflorus embryo callus are screened and differentiation；
Step 5. sinocalamus latiflorus plant regeneration and acclimatization and transplantses.
Step 1 operating method is as follows：
It is the flower pesticide of mid-late uninucleate stage that sinocalamus latiflorus flower pesticide should select the developmental stage of sporidiole, and now from observing in appearance, small ear is in lavender, 1.2cm long or so, and top stylet head somewhat exposes, and flower pesticide is in faint yellow.Win (4-15 DEG C) pretreatment of low temperature that small ear first passes through 2-6 days, flowing water is rinsed 1 hour before inoculation, 70% ethanol surface sterilization, disinfecting time is 20-40 seconds, after aseptic water washing 3-5 times, 2% sodium hypochlorite carries out surface sterilization, and disinfecting time is controlled 10-15 minutes, aseptic water washing 3-5 times.Flower pesticide is peeled off from small ear, avoids flower pesticide injured as far as possible, be uniformly inoculated into antherderived callus inducing culture.Callus induces and is with proliferated culture medium composition：M8 basal mediums；Addition：2mg·L-1NAA, 0.5mgL-16-BA, 15mgL-1PAA, 7.5mgL-1STS, 500mgL-1CH, 100mgL-1Proline, 100mgL-1Glutamin, 5.4%Maltose, 0.8%Agar；pH5.0；Condition of culture is light culture, and temperature is 22-28 DEG C.Obtain embryo callus subculture after, callus is chosen be transferred to new culture medium carry out propagation it is standby.
Step 2 operating method is as follows：
By the Agrobacterium EHA105 monoclonals containing binary plant expression vector pBS1305RdcodA in containing 50mgL-1Kanamycins and 25mgL-1Rule on the YM culture mediums of rifampin, 28 DEG C are cultivated 3 days.Agrobacterium thalline is collected, is suspended in M8 fluid nutrient mediums, it is 0.3-0.6 constantly to rock after 30min to thalline OD600, you can for converting.
Step 3 operating method is as follows：
Each design parameter is as follows when infecting：Sinocalamus latiflorus callus is cut into the fritter of 2mm-3mm；Pre-incubation time 2-4 days；Time of infection 20-40min；2-4 days co-cultivation time；Addition acetosyringone concentration is 100-200mgL-1, hygromycin concentration is 25-30mgL-1。
Step 4 operating method is as follows：
Callus lines after co-cultivation are transferred into screening and culturing medium (the callus induction of addition hygromycin and cephalosporin and proliferated culture medium) to be screened, culture medium is changed once within 2 weeks, until kanamycin-resistant callus tissue grows stabilization, and then plumule is induced.
Step 5 operating method is as follows：
After callus induction goes out plumule, culture dish is transferred to illumination cultivation.Healthy and strong plumule is chosen, root media is moved into.Root media is constituted：B5+300mg·L-1Cef+30%Sugar+0.25%Gelrite, pH 5.8, illumination.Well developed root system is treated, seedling is transplanted when growing to more than 50mm-80mm, and cultivation matrix is peat soil: vermiculite=1: 1 (v/v), it is placed in the culture 1-2 months in illumination box, you can be transferred to greenhouse culture of sheltering from heat or light, can be cultivated into field forest land after the 3-5 months.
Brief description of the drawings
Fig. 1 is sinocalamus latiflorus callus conversion results, Figure 1A be it is transformed after kanamycin-resistant callus tissue, Figure 1B is that differentiation obtains resistant budses, and Fig. 1 C are that resistant budses are taken root, and Fig. 1 D are positive regeneration plants；
Fig. 2 is PCR augmentation detection Rd29A electrophoretograms；
Fig. 3 is PCR augmentation detection codA gene electrophoretograms；
Fig. 4 is Southern blots electrophoretograms, M：λ DNA/HindIII Marker, plasmid：PBS1305RdcodA, T1-T3：Transfer-gen plant, CK：Nontransgenic plants；
Fig. 5 is the RT-PCR analyses of codA gene expressions, CK：Non-transgenic sinocalamus latiflorus plant, T1, T2：Transgenic regenerated plant.
Specific embodiment
Embodiment
1. the sinocalamus latiflorus plant that blooms takes from Fujian Province's Yong'an City.1.2-1.4 centimetres long of small ear is chosen at the sinocalamus latiflorus florescence, is preserved 4 days at 4 DEG C, flowing water is rinsed 60 minutes before inoculation, 70% ethanol surface sterilization 30 seconds, aseptic water washing 3 times, 2% sodium hypochlorite 12 minutes, aseptic water washing 5 times.Flower pesticide is stripped out from small ear, antherderived callus inducing culture is uniformly inoculated into.Callus induces and is with proliferated culture medium composition：M8 basal mediums；Addition：2mg·L-1NAA, 0.5mgL-16-BA, 15mgL-1PAA, 7.5mgL-1STS, 500mgL-1CH, 100mgL-1Proline, 100mgL-1Glutamin, 5.4%Maltose, 0.8%Agar；pH5.0；Condition of culture is light culture, and temperature is 22-28 DEG C.When flower pesticide induce callus it is long to 2-3mm after, choosing callus and being transferred to new culture medium breed and squamous subculture repeatedly, obtains enough Callus materials.
2. plasmid is the double T-DNA carrier pBS1305RdcodA containing inducible promoter Rd29A guiding cold tolerance genes CodA；Agrobacterium strain is EHA105.Agrobacterium EHA105 monoclonals are in containing 50mgL-1Kanamycins and 25mgL-1Rule on the YM culture mediums of rifampin, 28 DEG C are cultivated 3 days.Agrobacterium thalline is collected, is suspended in M8 fluid nutrient mediums, it is 0.3 constantly to rock after 30min to thalline OD600, for converting.
3. sinocalamus latiflorus callus is cut into the fritter of 2-3mm, preculture 3 days.Choose callus lines to be placed in aseptic triangular flask, pour into 3 times of material volume containing 100mgL-1The Agrobacterium bacterium solution of AS infects 20 minutes, and period rocked 1 minute every 3 minutes, isolated callus lines, with Adsorption of Filter Paper it is unnecessary infect liquid, blown on superclean bench 20 minutes, be transferred to and contain 100mgL-1Light culture 3 days, is then transferred to contain 25mgL in the co-cultivation base of AS-1Hyg and 300mgL-1On the screening and culturing medium of Cef, every 2 weeks subcultures once, pick out kanamycin-resistant callus tissue, continue squamous subculture culture until differentiating adventitious bud.
4. whne adventitious bud it is long to 2-3cm when, move into root media.Root media is constituted：B5+300mg·L-1Cef+30%Sugar+0.25%Gelrite, pH 5.8, illumination.When regrowth is long to 50mm-80mm, when root system 80mm is long, seedling is extracted from the medium heart, culture medium is cleaned, is transplanted and is extremely transferred to hot-house culture through autoclaved matrix, cultivation matrix is through autoclaved vermiculite peat soil (ratio 1: 1), poured with Hoagland nutrient solutions, overlay film above opens aperture, sunshade greenhouse cultivation is transferred to after 1 week, you can enter conventional seedbed system program.Overall process is referring to Fig. 1.
5. the detection of transgenic regenerated plant：
(1) PCR detections
The STb gene for extracting resistance sinocalamus latiflorus regeneration plant using CTAB methods enters performing PCR and expands, plasmid pBS1305RdcodA is used as control identification Rd29A and codA genes, electrophoresis result shows as the positive, it was demonstrated that Rd29A genes (Fig. 2) and codA9 (Fig. 3) gene have been incorporated on sinocalamus latiflorus regeneration plant genome.
(2) Southern detections
To growth regeneration plant Southern hybridization analysis (Fig. 4) in order, the CodA genes with digoxigenin labeled can detect a genes of interest as probe in transfer-gen plant, and not have purpose fragment in non-transgenic.CodA gene integrations are illustrated in sinocalamus latiflorus genome, and is a single insertion event.
(3) RT-PCR detections
RT-PCR analyses (Fig. 5) are carried out to PCR positive plants, the expression of codA genes can be detected after 4 DEG C of low temperature stress 24h, and the expression of codA genes is not detected in plant before nontransgenic plants and stress.Illustrate that the codA genes of Rd29A guiding transcribe the corresponding mRNA of generation in sinocalamus latiflorus cell, i.e. codA genes have been incorporated into sinocalamus latiflorus genome.
Claims (6)
1. a kind of sinocalamus latiflorus (Dendrocalamus latiflorus Munro) tissue cultures and genetic transforming method, it is characterised in that the side
Method include using sinocalamus latiflorus flower pesticide induction obtain embryo callus as genetic recipient, and carried out with this it is agriculture bacillus mediated, so as to obtain
Obtain transfer-gen plant.Detailed process is as follows：
Step 1. gathers sinocalamus latiflorus prematurity flower pesticide, and evoked callus are chosen compact texture, carried out into granular embryo callus
Subculture expands numerous culture and obtains genetic recipient material.
Step 2. build genes of interest carrier in and imported into Agrobacterium, obtain monoclonal simultaneously cultivated；
Step 3. infects by Agrobacterium；
Step 4. sinocalamus latiflorus embryo callus are screened and differentiation；
Step 5. sinocalamus latiflorus plant regeneration and acclimatization and transplantses.
2. the sinocalamus latiflorus prematurity flower pesticide as described in step 1 in claim 1, refers to be sent out in sporidiole late period from most of pollen grain
Educate isolated flower pesticide in the sinocalamus latiflorus small ear of blooming in stage.
3. the Agrobacterium as described in step 2 in claim 1, refers to Agrobacterium EHA105, and is carried containing RD29A induction types
The plasmid pCAMBIA1305 of promoter guiding purpose functional gene expression.
4. the Agrobacterium as described in step 3 in claim 1 is infected, it is characterised in that sinocalamus latiflorus callus preculture 1-3 days is (dark
Culture, 25 DEG C of temperature), Agrobacterium concentration is OD600=0.3, and time of infection is 20-30 minutes, and the time that co-cultures is 2-3 days (dark
Culture, 25 DEG C of temperature).
5. the screening as described in step 4 in claim 1 and differentiation, it is characterised in that acetosyringone is added in screening and culturing medium
100mg.L-1-150mg.L-1, screening pressure hygromycin concentration is 25mg.L-1, breaking up screening and culturing medium is：M8+KT1mg·L-1+
NAA0.2mg·L-1+6-BA1mg·L-1+PAA15mg·L-1+STS7.5mg·L-1+CH500mg·L-1+pro100
mg·L-1+Gln100mg·L-1+ 5.4%Maltose+0.8%Agar pH5.8.Condition of culture is light culture, 25 DEG C of temperature,
Every 2 weeks subcultures once, until adventitious buds differentiation.
6. the plant regeneration and acclimatization and transplantses as described in step 5 in claim 1, it is characterised in that when adventitious bud it is long to 20mm when,
(root media is to be transferred into root media：B5+30%Sugar+0.25%Gelrite pH5.8)；Work as regrowth
Long when root system 80mm is long, to be transferred to hot-house culture to 50mm-80mm, cultivation matrix is through autoclaved vermiculite peat soil (ratio
Example 1: 1), is poured, overlay film above opens aperture with Hoagland nutrient solutions, sunshade greenhouse cultivation is transferred to after 1-2 weeks.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058344A (en) * | 2017-06-29 | 2017-08-18 | 福建农林大学 | Application of the corn LC genes in sinocalamus latiflorus transgenic breeding |
| CN107190019A (en) * | 2017-06-29 | 2017-09-22 | 福建农林大学 | A kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation |
| CN110951774A (en) * | 2019-12-31 | 2020-04-03 | 中国林业科学研究院亚热带林业研究所 | Method for genetic transformation and transgenic plant regeneration of red poplar |
| CN112813096A (en) * | 2021-01-08 | 2021-05-18 | 中国林业科学研究院亚热带林业研究所 | Method for in vitro regeneration and genetic transformation of young moso bamboo embryo |
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Patent Citations (2)
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| CN101006768A (en) * | 2007-01-24 | 2007-08-01 | 山东省林业科学研究院 | Agrobacterium-mediated Sophora japonica transgenic and tissue-culturing rapid propagation method |
| CN103155862A (en) * | 2011-12-12 | 2013-06-19 | 蒋晶 | Method of dendrocalamuslatiflorus Munro anther somatic embryo induction and for acquiring regenerated plant |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058344A (en) * | 2017-06-29 | 2017-08-18 | 福建农林大学 | Application of the corn LC genes in sinocalamus latiflorus transgenic breeding |
| CN107190019A (en) * | 2017-06-29 | 2017-09-22 | 福建农林大学 | A kind of agriculture bacillus mediated sinocalamus latiflorus method for transformation |
| CN107190019B (en) * | 2017-06-29 | 2019-09-03 | 福建农林大学 | A kind of sinocalamus latiflorus method for transformation of mediated by agriculture bacillus |
| CN110951774A (en) * | 2019-12-31 | 2020-04-03 | 中国林业科学研究院亚热带林业研究所 | Method for genetic transformation and transgenic plant regeneration of red poplar |
| CN112813096A (en) * | 2021-01-08 | 2021-05-18 | 中国林业科学研究院亚热带林业研究所 | Method for in vitro regeneration and genetic transformation of young moso bamboo embryo |
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Application publication date: 20170616 |