Optimizing Wheat Tissue Culture Screening

Author: Shantel A. Martinez

There are multiple protocols published on how to transform wheat using an Agrobacterium construct. The general method relies on having the original immature embryo cells infected with agrobacterium that contains your gene of interest. Then those cells need to regenerate into a plants. The difficulty in wheat, is that regeneration rates are very low. For example, you may prep 600 embryos, and only 4 lead to complete regeneration into a new plant. On top of the low regeneration efficiency, there is an Agrobacterium infection rate which varies from construct to construct.

Most protocols transform either Bobwhite or Fielder wheat cultivars. However, some studies will need to transform a cultivar locally adapted to the region or a cultivar that expresses a specific gene of interest. This protocol is intended to identify the media combination that results in the optimal regeneration rate for your cultivar of interest. To achieve this, we will test three published protocols simultaneously:

Ishida Y, Tsunashima M, Hiei Y, Komari T (2015) Wheat (Triticum aestivum L.) Transformation Using Immature Embryos. In: Wang K (ed) Agrobacterium Protocols: Volume 1. Springer New York, New York, NY, pp 189–198

Wan Y, Layton J (2006) Wheat (Triticum aestivum L.). In: Agrobacterium Protocols, 2nd edn. Humana Press Inc., Totowa, NJ, pp 245–254

Bohorova N, Fenell S, McLean S, et al (1999) Plant Tissue Culture Protocol for Maize and Wheat. In: Laboratory Protocols: CIMMYT Applied Genetic Engineering Laboratory. CIMMYT, Mexico D.F., pp 3–36

Hayta S, Smedley MA, Demir SU, et al (2019) An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.). Plant Methods 15:121. https://doi.org/10.1186/s13007-019-0503-z

This following steps are written with the focus of taking steps in a consecutive order, in hopes to help users implement and execute the screening. It is also intended for people with little to no experience with tissue culture, resulting in extensive detail to the protocol. A short-hand protocol is provided in the supplementary material for quick reference.

1. Staggered Planting

Plant Material

Sowing Seeds

Sow six seeds for each cultivar of interest at weekly intervals. This will reduce time in having to wait for more plants to completely grow. For winter wheat, sow seeds initially in cell packs for 10-14 days to establish seedling emergence. Vernalize the seedlings for 6-8 weeks at 4C with a 12 hr photoperiod. Sufficient vernalization period varies from cultivar to cultivar, but vernalizing for eight weeks will ensure timely flowering for most cultivars. Transplant vernalized seedlings into pots in a greenhouse with a 16 hr photoperiod and a 20C day and 14C night temperatures. Ideally, the light intensity should be kept stronger than 1,000 μmol/m^2 /s. Many protocol state the plants were not sprayed with insecticides or fungicides at any growth stage.

It takes 4.5-5 mo from sowing to harvesting immature embryos of winter wheat. This is time that can be reduced by spending a short amount of time each week sowing new plant material. Throughout the protocol, there may be many steps that may need to be done multiple times to improve efficiency and precision. This is why staggered planting is necessary to keep a source of plant material available to redo any downstream steps or comparisons.

Embryos harvested from vigorous plants impact successful transformation. If transformation efficiency is low, start optimizing the growth conditions before checking media composition, vectors, or strains. Growth conditions should have sufficient light, temperature, and maintain disease and pest free growth periods.

2. DAA Range for your cultivar & Immature Embryo Harvest

Using the first sowed set of each cultivar, identify the number of days after anthesis (DAA) that the immature embryos reach a 1-2mm size, length-wise.

Table 1 -

Publication	DAA	Embryo Size (mm)	Cultivar
Ishida et al 2015	14	2.0 - 2.5	Fielder, Bobwhite
Wan and Layton 2006	14	-	Bobwhite
Bohorova et al 1999 (CIMMYT)	15–20	*	-
Ozias-Akins and Vasil 1982	10-14	1.0 ^	8 lines
Ozgen et al 1998	15	0.5–2.0	12 lines
Khanna and Daggard 2003	10-14	-	Veery5
Maddock et al 1983	“green seeds”	< 2.0 *	25 lines
Nizamani et al 2016	“immature”	-	4 lines
Sears and Deckard 1982	11-13	0.8 - 1.5	39 lines
Hayta et al 2020	14	1-1.5	Fielder

*endosperm is still relatively liquid; Translucent embryos
^tip of the coleoptile extended no more than half the length of the scutellum

Based on one greenhouse grow out and one field grow out, the dates for Cayuga and Caledonia’s time to a 1/5-2mm embryo size in listed below in the white lined box.

Figure 1 -

Table 2 - Soft White Varieties tested with the Small Grains group at Cornell University.

Variety	Type	I.E. Color Tested	I.E. Size Tested	I.E. Size (mm)	DAA
Fielder	Spring	Opaque	-	2.0-2.5	14
Glenn	Spring	Translucent, Opaque	0.8, 1.0, 1.5, 2.0 mm	2.0
Medina	Spring	Translucent, Opaque	0.8, 1.0, 1.5, 2.0 mm	2.0
Cayuga	Winter	Opaque	1.5, 2.0 mm	1.5-2.0	21
Caledonia	Winter	Opaque	1.5, 2.0 mm	1.5-2.0	17
Synthetic	Spring	Opaque	1.5, 2.0 mm	1.5-2.0	?
Opata	Spring	Opaque	1.5, 2.0 mm	1.5-2.0	?

First thing, is that you count on harvesting immature embryos 14 DAA. The date after anthesis is not the end goal, the embryo condition and size is. Therefore the first variation you need to test in for your cultivar is 1) Small Translucent 2) Opaque 1.5 mm 3) Opaque 2.0 mm immature embryos.

An initial experiment might be to get to know your cultivar by dissecting out embryos from 10-27 days after anthesis to know roughly how many days after anthesis your wheat cultivar reaches a 1-2mm embryo size.

In the greenhouse, I would have fine tweezers handing to dissect a seed to get an idea of what stage the embryos are at.

3. Callus Induction Media

All of the downstream tissue culture experiments will be done in the Transformation Facility in Weill Hall Rm B22.

The day before immature embryos are harvested, callus induction media should be prepared.

In general, media (without antibiotics? or agro?) can be stored for up to 7 days in 4C, but any longer increases the risk degradation of media components. There is a lot of light sensitive hormones, selection agents will break down. The soon you use the plates, you will get better/clearer results.

The three main wheat transformation protocols have four total callus induction media. CIMMYT uses either MSE3 or MSE5 “depending on the cultivar” (Bohorova et al 1999).

Media	Protocol	Sorrells Lab Tested?
MSE3	Bohorova et al 1999	No
MSE5	Bohorova et al 1999	No
WLS	Ishida et al 2015	Yes
CM4C	Wan and Layton 2006	Yes

Note: Write the published media names that are written in the protocol, not the author names as a replacement name. This keeps media names from the callus, regen, and rooting steps derived from the same publication separate and clear.

3a. Reserve hood space in the transformation facility.

The laminar flow hood will be needed to pour the plates. Since there is a limited number of hoods available, email Daniel a few days before to make sure there is room.

3b. Calculate the number of plates needed, plus a couple more. Determine how many mL of media will need to be made.

25mL per plate. Add roughly 30-50mL more to final volume so you don’t run out due to inaccurate pipetting.

Example: we need 750mL of CM4C

3c. Prep media bottles.

Sterile glassware: Need 250mL - 1L bottles, graduated cylinder for final volume, smaller graduated cylinder for adding in smaller amounts of water. magnetic stir bar

located in the windows shelves. Already high heat and sterilized in dish washer. Not autoclaved, but we will be autoclaving the media.

Label media: media name , initials, and date

It gets really difficult when you are making 3+ media at the same time, so just take this protocol really slow.

3d. Fill media bottle 1/2 - 2/3 full of ddH2O.

Turn on deionizer machine to “nonstop”, wait until it reaches 18.20 (microns?), then twist the black knob toward you to fill the large graduated cylinder. Pour in bottle.

3f. Add dry material first, then the liquid materials.

Adding the correct materials gets difficult when you have more than two media you are making in one sitting. Best advice is to take it really slow. I will also take the cap off of one bottle that I intend to pour the ingredient into, and immediately cap it once its poured in. I also make check marks on the recipe (see recipe below) to indicate which ingredient has been added. Mistakes happen, and adding incorrect ingredients to a media bottle happens. Just start over.

Picloram will sometimes crystalize. At the start, I check the picloram aliquots, and if it has come out of solution or crystalized, I will put the aliquot tubes on a 33C heating block until it is a solution again.

It is likely the dmso used to dissolve picloram. Using low dmso might result in the picloram to re-precipitate and not stay in solution.

Example diagram of media composition for 1L

The majority of dry reagents are in the upper cupboards, except sucrose and agar are in larger bins in the lower cupboards.

Use magnetic stir bar to stir on a stir plate as you are adding the ingredients.

Pull out spatulas from lower drawer and place on a clean kim wipe for easy access.

Most ingredients will use weigh paper, except when you have large volumes of sucrose or maltose. Then you can use small or large weigh boats.

Large are on a fridge, small are in a drawer

Tare scale between uses. For smaller precise weights, measure with the “air wall” on scale to avoid flow of air effecting measurements. Use new spatula between dry ingredients so stock material does not get contaminated.

For liquids, I tend to set the pipettes first before bringing the solution out of the fridge.

3g. Bring solution up to final volume with ddH2O then pH.

Pour liquid media into a graduated cylinder to assist with bringing the volume up to the total needed. Use a smaller secondary graduated cylinder to add ddH2O more accurately to large cylinder.

Use a new graduated cylinder for each solution.

You can use a second magnetic stir bar to hold the one in the bottle in place as you pour out the liquid. We still want to keep the stir bar because we still need to stir the media post autoclave.

Make sure to dissolve all of the ingredients into the liquid, even material stuck near the neck of the bottle. While pouring the liquid into the graduated cylinder, you can pour the liquid over the clumped powder to get it to dissolve and mix. You can also use a clean spatula to loosen up the powders on the side of the bottle.

The pH protocol is described in supplemental materials.

3h. Add gelling agent to the media.

3i. Autoclave. Cycles typically take 70 min.

Loosely tighten top, add autoclave tape. double check you’ve label your bottle.

Check water bath is on now, so it is warm when the autoclave is finished.

3j. Prepare the hood immediately before taking a break. This will shorten the pour time once the autoclave cycle is complete.

Wipe down the hood with ethanol, wipe down pipettes, and pipette boxes. Pull out sterile plates, label plates, and grab 25mL pipette tips.

3k. Once autoclave is finished, tighten the tops of the media and put the first media on a stir plate to begin cooling. Additional media should go into in a water bath until you are ready to cool and pour.

Cool media with time on the stir plate, or a quicker way is to run the bottle under cold water, stir a minute or two, then touch with hand to see if it is still hot but able to touch.

letting it get “warm” may result in gelling before you can pour all the plates. This happens more when the volume is much larger.

3l. Add any heat sensitive hormones or reagents in the hood, close cap, then stir on stir plate to mix.

Important to do everything from now on in the hood to keep media sterile.

3m. Use large pipette to pour 25mL into 9mm petri dishes. Let cool for a minimum of 15 minutes up to overnight.

To reduce excess condensation, I have been letting them sit in the hood overnight with the hood off.

If you do this, be sure to move the plates to the side, away from anyone else accidently touching the plates. We want to avoid any contamination.

Note that MSE3 does not use MS Salts, and has a lot of sucrose. Ozias-Akins and Vasil (1982) observed a higher concentration of sucrose, from 2g/L to 7g/L, was detrimental to callus formation.

Ozias-Akins and Vasil (1982) also observed casein hydrolysate suppressed precocious germination, but the scutellar callus produced was less compact, grew slowly, and showed a much reduced capacity for organogenesis.

Some protocols call for 4.4g of MS salts, and 4.3g of MS salts. The 4.4 g of MS salts, is MS salts + MS vitamins already mixed into the reagent. The protocol not only needs to be read carefully, but its important to report and read what type of MS salt was bought and used.

4. Immature Embryo Isolation and Plating

Ozias-Akins and Vasil 1982 Figure 2 : SC - Scutellum. The Embryonic axis in on the opposite side of the scutellum.

5. Callus Induction and Maintenance

Alikina et al (2016) Figure 1F - SE - somatic embryos, EC - embryogenic callus, AC - amorphous (non-regenerating) callus

Somatic embryo formation should be evaluated two weeks after immature embryo initiation using a stereo microscope. Tissue producing more than three somatic embryos is considered embryogenic.

Ozias-Akins and Vasil 1982

Produce friable and a compact, yellowish callus . compact callus was derived from the scutellum whereas embryo axis produced only friable callus. friable callus produced only roots when subcultured further.

6. Regeneration Media and Transfer

LSZ (Fielder; Ishida Yuji) MMS0.2C (Bobwhite; Wan and Layton) MSR (Bobwhite; CIMMYT)

Sears RG, Deckard EL (1982) Studies have shown that gradually reducing the 2,4-D concentration can improve tissue regeneration . It has also been shown on 39 different cultivars that moving calli with green totipotent regions to a 2,4-D free media with inadequate shoot formation, will result in root formations but inhibit the necessary shoot formation.

Gosch-Wackerle et al. (1979), the combination of IAA and zeatin at 1 mg/1 each was beneficial for shoot regeneration.

7. Regeneration and Maintenance

The percentage of plant regeneration is calculated based on the number of embryos regenerating out of the total number of embryos plated on the callus induction medium

8. Rooting Media and Transfer

LSF-P5 (Fielder; Yuji Ishida) MMS0C (Bobwhite; Wan and Layton) MSEB1C (Bobwhite; CIMMYT)

MS Alvina did 1/2 MS

Vector Screening

Author: Ella Taagen

Construct Assembly

Prepping Agrobacterium

Inoculation with Agrobacterium and co-cultivation

Supplemental Materials

pH

Turn on the pH reader

Twist off the safety bottle protecting the probe. Use water to rinse off excess liquid.

maneuver the probe to set inside the liquid. It may be helpful to take the cap off. I would advise not the take the bottle and the top off at the same time, since it could suction and break the probe.

Click measure for the probe to read, blinking means its measuring,

Starts at about 3.9 - 4’s. We want to bring it up to 5.8. So use

HCL brings it down

NaOH or KOH brings the pH up. The higher molarity, the bigger the jump.

Trick with solution. Water in cap with some 0.5M NaOH

Bring to pH.

If the probe reaches a steady reading, it will stop measuring. click measure again if the display is not blinking.

Once done with the probe, rinse well and clean with a kimwipe.

place buffered bottle back on probe

Power off.

version 2020.03.10