TITLE: A protocol for Agrobacterium mediated transformation of Mimulus guttatus from leaf petiole explants
AUTHORS: Srinidhi Holalu, Benjamin Blackman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This is a protocol for Agrobacterium mediated transformation of Mimulus guttatus from leaf petiole explants including the following procedures:</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Surface sterilization of seeds</li><li style = "counter-reset:ol0;">Agrobacterium culture preparation</li><li style = "counter-reset:ol0;">Agrobacterium infection and co-cultivation</li><li style = "counter-reset:ol0;">Callus induction and shoot induction</li><li style = "counter-reset:ol0;">Rooting of shoots</li></ol></div></div>

[STEPS]
?. [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.
?. [Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds]
Place seeds in a  sterilized eppendorf tube(s) then fill the tube(s) with surface sterilization solution ( and a drop of hand-soap). Shake the tube(s) vigorously for  - .
1.5 ml
[laundry bleach]
?. [Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds]
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 . Repeat rinse at least 5 — 6 times to completely wash-off bleach and soap from seeds. After the last rinse add at least  to later plate the seeds.
[sterilized water]
?. [Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds]
Store the seeds at  for at least 2 — 3 weeks to cold stratify seeds.
4 °C
?. [Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of 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 () or polypropylene Deli jars (quart-size).
200 ml
?. [Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds]
Incubate jars cultures at  —  under cool fluorescent lamps.
20 °C
21 °C
?. [Establishing invitro cultures for explant source (2.5-3 months): Surface sterilization of seeds]
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) ]
Streak EHA105 Agrobacterium strain harboring binary plasmid on LB or YEP agar plate with rifampicin () and appropriate antibiotic resistance cassette on the binary plasmid. Incubate plates at  for two days.
28 °C
?. [Agrobacterium mediated transformation of explants: Agrobacterium culture preparation (4 days) ]
Inoculate a single colony of Agrobacterium into  YEP liquid medium with rifampicin () and an appropriate antibiotic (Kanamycin at  or Spectinomycin at  or Amplicillin at ). Shake the culture at  for  —  at .
10 ml
28 °C
Centrifuge: 200 33
?. [Agrobacterium mediated transformation of explants: Agrobacterium culture preparation (4 days) ]
Centrifuge the cultures to pellet Agrobacterium at . Remove the medium and resuspend in  virulence induction medium for at least  —  with gentle shaking ( —  ) in dark at . Adjusting to 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.
Centrifuge: 4000 33
5 ml
Centrifuge: 50 33
Centrifuge: 80 33
28 Room temperature
?. [Agrobacterium mediated transformation of explants: Agrobacterium culture preparation (4 days) ]
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)]
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.
?. [Agrobacterium mediated transformation of explants: Agrobacterium infection and co-cultivation (3-4 days)]
Incubate plates in dark or low intensity light at  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.
0 Room temperature
?. [Agrobacterium mediated transformation of explants: Agrobacterium infection and co-cultivation (3-4 days)]
On day 3, wash the explants in sterile timentin () + cefatoxime () 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  under cool fluorescent lamps.
21 °C
?. [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  at later stages may help in getting larger callus. At least 3 — 4 subcultures are necessary.
?. [Agrobacterium mediated transformation of explants: Callus induction and shoot induction (4-5 months)]
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)]
When the shoots emerge and reach 0.5cm — 1cm tall, separate individual shoots and plate them on rooting medium.
?. [Agrobacterium mediated transformation of explants: Rooting of shoots (3 weeks)]
For good rooting and hardening, subculture on half-strength rooting medium.
?. [Agrobacterium mediated transformation of explants: Rooting of shoots (3 weeks)]
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.