Nov 26, 2019
A protocol for Agrobacterium mediated transformation of Mimulus guttatus from leaf petiole explants
- 1Department of Plant & Microbial Biology, UC Berkeley;
- 2University of California, Berkeley
- Mimulus
Protocol Citation: Srinidhi Holalu, Benjamin Blackman 2019. A protocol for Agrobacterium mediated transformation of Mimulus guttatus from leaf petiole explants. protocols.io https://dx.doi.org/10.17504/protocols.io.8vghw3w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 30, 2019
Last Modified: November 26, 2019
Protocol Integer ID: 29320
Keywords: Agrobacterium, Mimulus guttatus, transformation
Abstract
This is a protocol for Agrobacterium mediated transformation of Mimulus guttatus from leaf petiole explants including the following procedures:
- Surface sterilization of seeds
- Agrobacterium culture preparation
- Agrobacterium infection and co-cultivation
- Callus induction and shoot induction
- Rooting of shoots
Attachments
Guidelines
- Check the contents on herbicide formula to verify the active ingredient (Basta or Phosphinothricin) before use so that you can modify the selection protocol as appropriate.
- A pilot kill-curve test may be necessary to choose the optimum concentrations of herbicide for each M. guttatus population.
- Antibiotics and hormones need to be filter sterilized and added to medium after autoclaving. For best results, add antibiotics and hormones when the contents cool to 45 °C — 50 °C or lower after autoclaving.
- Fresh medium should be used for best results. Leave the solid medium overnight at room temperature after pouring into petri-dish to cool and solidify. This will minimize condensation on the lids.
- This protocol was standardized for herbicide resistance selection.
Materials
MATERIALS
TimentinGold BiotechnologyCatalog #T-104-2
CefotaximeGold BiotechnologyCatalog #C-104-25
PhosphinothricinGold BiotechnologyCatalog #P-165-250
4-CPPUPhytotech LabsCatalog # C279
Meta-toplinPhytotech LabsCatalog # T841
Murashige & Skoog basal salts with Gamborg VitaminsPhytotech LabsCatalog #M404
AcetosyringoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #2478-38-8
Growth medium composition
| Murashige & Skoog basal salt medium (MS salts) | 4 g/L | |
| Sucrose | 20 g/L | |
| Calcium gluconate | 1.3 g/L | |
| MES 2-(NMorpholino) ethanesulfonic acid hydrate) | 0.25 g/L | |
| Adjust pH to 5.6 using KOH before adding gelrite | ||
| Gelrite | 0.25 % |
Agrobacterium virulence induction medium
| Murashige & Skoog basal salt medium (MS salts) | 2 g/L | |
| Sucrose | 10 g/L | |
| MES 2-(NMorpholino) ethanesulfonic acid hydrate) | 0.5 g/L | |
| Adjust pH to 5.5 using KOH and autoclave | ||
| Acetosyringone (Dissolved in DMSO and add before use) | 200 μM |
Co-cultivation medium
| Murashige & Skoog basal salt medium (MS salts) | 4 g/L | |
| Sucrose | 20 g/L | |
| Calcium gluconate | 1.3 g/L | |
| MES 2-(NMorpholino) ethanesulfonic acid hydrate) | 0.25 g/L | |
| Adjust pH to 5.6 using KOH before adding gelrite | ||
| Gelrite | 0.25 % | |
| CPPU* | 1 mg/L | |
| Acetosyringone* | 100 μM | |
| * Filter sterilize before adding to the medium |
Callus induction medium
| Murashige & Skoog basal salt medium (MS salts) | 4 g/L | |
| Sucrose | 20 g/L | |
| Calcium gluconate | 1.3 g/L | |
| MES 2-(N-Morpholino)ethanesulfonic acid hydrate | 0.25 g/L | |
| Adjust pH to 5.6 using KOH before adding gelrite | ||
| Gelrite | 0.25 % | |
| CPPU* | 1 mg/L | |
| Timentin (Ticarcillin-cluvanate)* | 200 mg/L | |
| Ceftaxime* | 50 mg/L | |
| Phosphinothrocin* | 6 mg/L | |
| * Filter sterilize before adding to the medium |
Shoot induction medium
| Murashige & Skoog basal salt medium (MS salts) | 4 g/L | |
| Sucrose | 20 g/L | |
| Calcium gluconate | 1.3 g/L | |
| MES 2-(NMorpholino) ethanesulfonic acid hydrate) | 0.25 g/L | |
| Adjust pH to 5.6 using KOH before adding gelrite | ||
| Gelrite | 0.25 % | |
| Meta-toplin* | 0.1 mg/L | |
| Timentin (Ticarcillin-cluvanate) | 200 mg/L | |
| Ceftaxime | 50 mg/L | |
| Phosphinothrocin | 6 mg/L | |
| * Filter sterilize before adding to the medium |
Root induction medium
| Murashige & Skoog basal salt medium | 2 g/L | |
| Sucrose | 10 g/L | |
| Calcium gluconate | 1.3 g/L | |
| MES 2-(N-Morpholino)ethanesulfonic acid hydrate | 0.25 g/L | |
| Adjust pH to 5.6 using KOH before adding gelrite | ||
| Gelrite | 0.25 % | |
| Napthelene AceticAcid (NAA)* | 0.1 mg/L | |
| Timentin (Ticarcillin-cluvanate)* | 200 mg/L | |
| Ceftaxime* | 50 mg/L | |
| Phosphinothrocin* | 6 mg/L | |
| * Filter sterilize before adding to the medium |
Safety warnings
For safety information and warnings, please refer to the SDS (Safety Data Sheet).
Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds
Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds
Collect mature seeds from plants grown in greenhouse or growth chamber to reduce contamination in tissue culture. Seeds collected from natural sites/fields may increase the risks of endogenous contaminations in cultures.
Place seeds in a 1.5 mL sterilized eppendorf tube(s) then fill the tube(s) with surface sterilization solution (5 % laundry bleach and a drop of hand-soap). Shake the tube(s) vigorously for 00:08:00 - 00:10:00 .
Move the tube(s) to laminar flow hood. Discard the sterilizing solution and add sterile water to rinse the seeds by shaking for at least 00:00:30 . Repeat rinse at least 5 — 6 times to completely wash-off bleach and soap from seeds. After the last rinse add at least 1 mL sterilized water to later plate the seeds.
Store the seeds at 4 °C for at least 2 — 3 weeks to cold stratify seeds.
Bring back tubes to laminar flow hood. Transfer the sterilized seeds onto growth medium. Spread the seeds uniformly. Grow around 5 — 6 seeds per jar (200 mL ) or polypropylene Deli jars (quart-size).
Incubate jars cultures at 20 °C — 21 °C under cool fluorescent lamps.
When the plants produce proliferating axillary shoots, harvest shoots with roots and subculture onto new jar. Adjust frequency of sub-culturing to avoid senescence.
Agrobacterium mediated transformation of explants: Agrobacterium culture preparation (4 days)
Agrobacterium mediated transformation of explants: Agrobacterium culture preparation (4 days)
Streak EHA105 Agrobacterium strain harboring binary plasmid on LB or YEP agar plate with rifampicin
(40 μg/mL ) and appropriate antibiotic resistance cassette on the binary plasmid. Incubate plates at 28 °C for two days.
Inoculate a single colony of Agrobacterium into 10 mL YEP liquid medium with rifampicin (40 μg/mL ) and an appropriate antibiotic (Kanamycin at 50 μg/mL or Spectinomycin at 100 μg/mL or Amplicillin at 100 μg/mL ). Shake the culture at 28 °C for 36:00:00 — 48:00:00 at 200 rpm .
Centrifuge the cultures to pellet Agrobacterium at 4000 rpm . Remove the medium and resuspend in 5 mL virulence induction medium for at least 03:30:00 — 04:00:00 with gentle shaking (50 rpm — 80 rpm ) in dark at Room temperature . Adjusting to5.5 is critical. Glucose can be substituted for sucrose, because glucose enhances virulence induction. However, glucose may enhance senescence of older leaves; if leaves are old, then use sucrose.
Adjust agrobacterium OD to 0.2 using liquid half-strength MS medium to achieve a very light color suspension in half-strength MS medium. Higher OD is not desirable.
Agrobacterium mediated transformation of explants: Agrobacterium infection and co-cultivation (3-4 days)
Agrobacterium mediated transformation of explants: Agrobacterium infection and co-cultivation (3-4 days)
Bring plant culture jar to laminar flow hood. Pull shoots onto a sterile petri dish. Using a sterile scalpel cut a leaf explant off the plant and dip the cut petiole in agrobacterium and plate on co-cultivation medium. Perform infections in small batches to avoid desiccation of petiole explants.
Incubate plates in dark or low intensity light at Room temperature for 2 — 3 days. Avoid overgrowth of agro on plates. Overgrowth of agrobacterium may kill explants, thus the use dilute agro culture to dip petioles.
On day 3, wash the explants in sterile timentin (100 mg/L ) + cefatoxime (50 mg/L ) in hood and transfer the explants to callus induction medium with phosphinothrocin. This is appropriate selection medium for plasmids with Blp resistance cassette. Incubate plates at 21 °C under cool fluorescent lamps.
Agrobacterium mediated transformation of explants: Callus induction and shoot induction (4-5 months)
Agrobacterium mediated transformation of explants: Callus induction and shoot induction (4-5 months)
Subculture explants after 21 — 24 days. Retain only small part of callus and culture on callus induction medium to allow callus growth. It is critical to grow long enough on CPPU with routine subculture (21 — 24-days interval) for sufficient growth and maturation of callus. Premature transfer to Meta-toplin may kill explants. Increasing sucrose to 2.5 % at later stages may help in getting larger callus. At least 3 — 4 subcultures are necessary.
When the compact callus turns friable with signatures of differentiation, pick the healthy callus and plate on shoot induction medium to regenerate shoots from callus.
Agrobacterium mediated transformation of explants: Rooting of shoots (3 weeks)
Agrobacterium mediated transformation of explants: Rooting of shoots (3 weeks)
When the shoots emerge and reach 0.5cm — 1cm tall, separate individual shoots and plate them on rooting medium.
For good rooting and hardening, subculture on half-strength rooting medium.
When roots proliferate, move the shoots to potting medium in greenhouse and leave the pots on a mist bench to harden for at least two weeks.