Rapid and Efficient GeneticTransformation of Sorghum viaAgrobacterium-Mediated MethodPhat Tien Do,2,3 Hyeyoung Lee,1 Kimberly Nelson-Vasilchik,4 Albert Kausch,4

and Zhanyuan J. Zhang1,5

1Division of Plant Sciences, University of Missouri, Columbia, Missouri2Christopher S. Bond Life Sciences Center, Division of Plant Sciences, University ofMissouri, Columbia, Missouri

3Current address: Institute of Biotechnology, Vietnam Academy of Science andTechnology, Hanoi, Vietnam

4Plant Biotechnology Laboratory, University of Rhode Island, Kingston, Rhode Island5Corresponding author: ZhangZh@missouri.edu

Genetic transformation via Agrobacterium-mediated methodology has beenused in many sorghum studies. However, the transformation efficiency stillvaries significantly due to high dependence on sorghum genotypes and technicalexpertise. In this article, we describe a sorghum transformation procedurein sufficient detail using a public genotype, P898012. This system utilizes astandard binary transgenic vector carrying the bar gene as a selectable markerand immature embryos as starting explants. Glufosinate is employed as theselective agent during callus and shoot induction. This procedure is relativelyrapid, efficient, highly reproducible, and should be applicable for many othersorghum genotypes. C© 2018 by John Wiley & Sons, Inc.

Keywords: Agrobacterium-mediated � genetic transformation � P898012 �

sorghum � standard binary vector

How to cite this article:Do, P. T., Lee, H., Nelson-Vasilchik, K., Kausch, A., & Zhang, Z. J.(2018). Rapid and efficient genetic transformation of sorghum via

Agrobacterium-mediated method. Current Protocols in PlantBiology, 3, e20077. doi: 10.1002/cppb.20077

INTRODUCTION

Sorghum [Sorghum bicolor (L.) Moench] is known as the fifth most important cerealworldwide and the third most important cereal crop grown in the U.S. It is widelyused for food and feed production and in the biofuel industry. Since the first report onAgrobacterium-mediated transformation of sorghum by Zhao et al. (2000), even thoughsome studies have optimized media compositions and transformation parameters, therehas been limited progress in genetic transformation protocols for sorghum compared toother cereal crops.

Here, we describe a Basic Protocol for sorghum transformation via Agrobacterium-mediated method and also a Support Protocol for sorghum planting and managementunder greenhouse conditions.

Current Protocols in Plant Biology e20077, Volume 3Published in Wiley Online Library (wileyonlinelibrary.com).doi: 10.1002/cppb.20077C© 2018 John Wiley & Sons, Inc.

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BASICPROTOCOL

STABLE TRANSFORMATION OF SORGHUM

We present an improved sorghum transformation protocol employing Agrobacteriumtumefaciens harboring standard binary vectors for sorghum public genotype P898012.This protocol offers efficient and reproducible transformation results when implementedas described.

The protocol includes six main steps: (1) initiation of the Agrobacterium culture;(2) seed sterilization and immature embryo isolation; (3) heat shock, inoculation, andco-cultivation; (4) resting; (5) callus induction, selection, and shoot development; and(6) rooting and acclimation.

Materials

Agrobacterium tumefaciens (also called Rhizobium radiobacter) strains AGL1carrying a standard binary vector (e.g., pFGC5941, https://www.atcc.org/)

YEP agar plates containing appropriate antibiotics (see recipe)Immature seeds of sorghum public genotype P898012 (Support Protocol)50% (v/v) commercial bleachTween 20Inoculation medium (IM; see recipe)Co-cultivation medium (Co-M) and Co-M plates (see recipe)Resting medium (R) and R plates (see recipe)Callus induction medium (CIM) and CIM plates (see recipe)Shoot induction medium (SM) and SM plates (see recipe)Rooting medium (RM; see recipe)Soil mixture (e.g., Promix BX)

Dark culture incubators with temperature control and air circulation (PercivalScientific; one at 24°C for co-cultivation and one at 26°C for callus culture)15-ml conical centrifuge tubes (e.g., BD Falcon)Inoculating loopWater baths (Thermo Scientific Precision Circulating Water Bath CIR 89 - 89 liters)Spectrophotometer and cuvettesShakers2-ml microcentrifuge tubesForceps, sterile250-ml flasks, sterile100 × 15-mm and 100 × 20-mm Petri dishesMagenta GA–7 Vessel#15 razor blades (2976 #15 GF Health Sterile Surgical Blade)Dissecting microscope (e.g., Olympus stereo zoom microscope SZ40 MODEL)LMS-225R with 5-40× amplification and built-in light source (Leeds PrecisionInstruments)3M porous tapeSmall plastic planting potsHumidity domes (Hummert International, cat. no. 143850-1)Light incubator or culture room (settings 24°C 18 hr/light, 6 hr/dark)

NOTE: Autoclave and discard all contaminated Agrobacterium and sorghum cultureswhen observed.

Initiate Agrobacterium culture

1. Streak bacteria (AGL1) carrying a standard binary vector (e.g., pFGC5941) from−80°C glycerol stock on YEP agar plates with appropriate antibiotics (50 mg/liter

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Figure 1 Sorghum immature embryo isolation. (A) sorghum panicle at 12 days after pollination;(B) immature seed collection; (C) sorghum seed sterilization using bleach; (D) small cut in sorghumseed with scalpel blade #15; (E) an immature embryo; (F) immature embryos in inoculation medium.

kanamycin and 25 mg/liter rifamycin) and incubate plates in the dark 3 days at 28°Cto obtain single colonies.

2. Select a single colony and streak it on YEP agar plates containing appropriateantibiotics (for the vector and strain illustrated here, 50 mg/liter kanamycin and25 mg/liter rifamycin). Incubate plates in the dark 2 days at 28°C.

3. Add 5 ml filtered inoculation medium (IM) to a 15-ml conical centrifuge tube.

4. Transfer 2 to 3 full loops of bacteria from the YEP plates to tubes prepared in step3 and shake tubes to thoroughly suspend bacterial cells in the IM.

5. Add 1 ml bacterial suspension to spectrophotometer cuvettes and measure opticaldensity of the suspension at 550 nm (OD550). Adjust bacterial suspension to anOD550 of 0.4 (0.5 × 109 cfu/ml) by either adding more Agrobacterium cells ordiluting the culture with more IM.

6. Shake culture on a rotary shaker at 100 rpm, 4 to 5 hr at room temperature (e.g.,24°C).

Sterilize seed and isolate immature embryo

7. Collect immature seeds from sorghum panicles (Fig. 1A,B) 11 to 14 days afterpollination (with embryo size 1.0 to 1.5 mm; see Support Protocol).

The optimal size of immature sorghum embryos is between 1.0 to 1.5 mm. We found thatsmaller or bigger sized embryos decreased callus induction and regeneration frequency.The over-sized immature embryos also reduced the transformation efficiency. Becausesorghum flowers are pollinated from the top to the bottom of panicles, immature seedswill be collected from the top first then the bottom later to obtain optimally sized embryos.

8. Prepare 100 ml of 50% bleach and add 2 to 3 drops Tween20 (in sterilized 250-mlflask). Soak 200 to 300 immature sorghum seeds in the bleach solution, cover flask

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Figure 2 Sorghum transformation and regeneration. (A) sorghum immature embryos (top: Flatside face down, bottom: Flat side face up); (B) co-cultivation; (C) callus induction; (D) somaticembryos; (E) regenerated plants in selection medium; (F) rooted plants.

with sterilized aluminum foil, and incubate at room temperature 15 min with gentleagitation using vortexer (Fig. 1C).

9. Remove bleach solution completely and rinse seeds with sterile water four to fivetimes. Discard all floating and broken seeds. Briefly dry seeds using sterile filterpaper.

Use sterile technique for all of these steps and in tissue culture hoods to avoid contami-nation.

10. Make a small cut in the seed using sterile forceps and scalpel (blade #15), pressseed gently, isolate immature embryos (IEs), and transfer 50 to 70 IEs to a 2.0-mlmicrocentrifuge tube with 1 ml IM (Fig. 1D-F), then wash embryos with IM two tothree times.

A 1.5-mm cut at the stylar region (top) of sorghum seeds should ease immature embryoisolation and avoid damage. Place immature embryos immediately into IM to avoiddesiccation of the IEs. Keep only unbroken embryos (Fig. 2A). Approximately 200 to 300immature embryos should be isolated for each experiment.

Heat shock, inoculation, and co-cultivation

11. Prepare water baths at 43°C and 25°C for heat shock treatments.

12. Remove all IM from 2.0-microcentrifuge tube (from step 10) and merge IEs in freshIM; keep tubes with IEs at 43°C incubator for 3 min then immediately transfer to25°C incubator for at least 2 min.

Immerse the IEs with just enough IM to cover them; too much or too little IM will reduceheat shock efficiency.

13. Remove all IM from the tubes containing IEs after heat shock and immediatelyadd 1 ml Agrobacterium suspension (from step 6) to the tubes. Gently invert tubesDo et al.

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two to three times and keep them horizontal in the culture hood 10 min at roomtemperature.

Apply heat shock treatment only when the bacterial suspension is ready (see steps 1to 6).

14. Draw off Agrobacterium suspension using a pipet with a fine tip then spread embryos(30 to 50 IEs) evenly across the plate. Orient embryos with scutellum side up andembryo side face down on the co-cultivation medium (Co-M; Fig. 2B).

Importantly, immature embryos are co-cultivated with Agrobacterium on the Co-M,and the embryo must be embryo side down. Be sure to check the orientation of theembryos under a dissecting microscope. Embryos oriented with the scutellum side downsignificantly reduces sorghum callus induction.

15. Seal plates with Parafilm and incubate in the dark at 25°C, 3 days.

Rest

16. Transfer embryos with sterile forceps to resting medium (R) plates, and avoiddamaging the embryos.

17. Seal plate with Parafilm and incubate in the dark at 25°C, 10 days with a subcultureafter 5 days.

Typically, after 5 days on resting medium, phenolic compounds occur in the mediumsurrounding the embryos with black or brown colors. Phenolic compounds inhibit callusinduction and development. One subculture using new medium should reduce the negativeeffects on callus formation.

Callus induction, selection, and shoot development

18. Transfer embryos from resting medium with forceps to callus induction medium(CIM). Place 18 to 20 embryos per plate and seal plate with Parafilm. Incubateembryos in the dark 10 days at 26°C for callus induction and selection.

At the end of resting stage, a small callus can be observed forming from IEs; carefullytransfer IEs to the plates of CIM to avoid damaging the callus. After 20 days on CIM, thecallus becomes friable and somatic embryos will be observed as small white structures(Fig. 2C,D).

19. Use forceps and scalpel blade #15 to remove primary shoots and transfer 15 calli(with somatic embryos) to a shoot induction medium (SM) plate (Fig. 2D). Placeplates in a growth chamber under 80 µmol m−2sec−1 light conditions and 18 hr/light,6 hr/dark photoperiod at 24°C for shoot induction. Sub-culture embryogenic callievery 2 weeks using fresh SM for a total 6 to 10 weeks.

On CIM, primary shoots keep growing and they need to be removed before transferringcalli to shoot induction medium (SM). On SM, calli will be friable, somatic embryos andshoots will be formed. Non-transgenic calli will turn black and should be removed atevery subculture.

Rooting and acclimation

20. Transfer small shoots (with three to four healthy leaves) into Magenta boxes with30 ml rooting medium (RM) and incubate cultures under the same light conditionsas step 19 for 2 to 3 weeks.

Germinated shoots from somatic embryos may have roots. The roots should be cut beforetransferring the shoots to RM. Transfer three to four shoots per Magenta box.

21. Remove sorghum plants from Magenta boxes and carefully remove all medium fromthe roots by rinsing with room temperature tap water. Transfer small plantlets withhealthy roots to 4-inch (�10-cm) plastic pots with soil mixture, e.g., Promix BX Do et al.

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soil, in a light incubator or culture room at 24°C with an 18 hr/light, 6 hr/dark cyclefor acclimation.

For the first 5 days, plants should be covered by humidity domes to keep high humidconditions. Then, open the dome and water plants daily. Leaf paint assays to deter-mine glufosinate resistance can be performed as the second new leaf occurs to identifytransgenic events expressing the selectable marker bar gene.

SUPPORTPROTOCOL

GREENHOUSE SORGHUM PLANT MANAGEMENT

Greenhouse sorghum plant management is important to increase immature embryo andseed quality, eliminate false positive plants, and avoid loss of transgenic events.

Materials

Sorghum plants (seeds or small plantlets; see Basic Protocol)Soil mixture (e.g., Promix BX)Water source (avoid high concentrations of organic or inorganic compounds)Peters 20-20-20 fertilizer (Hummert International, cat. no. 07-5400-1)Iron sulfate (Hummert International, cat. no. 07-0851-1)Osmocote 18-6-12 or 19-6-12 fertilizer (Hummert International, cat. no. 07-6300-1

or 07-6330-1)

Greenhouse module (with maximal light exposure and intensity using combineddaylight and supplemental lighting from metal halide and high pressure sodiumlight bulbs; Hummert International)

3-gallon pots (Hummert International)Measuring spoon, �1 oz (�30 ml; Hummert International, cat. no. 060092-1)Tags for labeling pots (Hummert International)Jiffy pots (Hummert International)Tassel bags (Lawson Bags, cat. no. 402; https://www.lawsonbags.com)Stapling pliers (Hummert International, cat. no. 51-3800-1)

Greenhouse temperature and light settings

1. The temperature setting is: 20°C night time and 26°C daytime for vegetative sorghumgrowth (Fig. 3A). As the flag leaves emerge, move sorghum plants to the greenhouseat 20°C night time and 28°C daytime for reproduction stages.

The photoperiod is: 16-hour light and 8-hour darkness with a mixture of 50% metalhalide and 50% high-sodium-pressure light as supplemental lighting (24 lamps in threerows per room).

Figure 3 Sorghum growth in the soil. (A) sorghum plantlets in acclimation room; (B) sorghumplants under greenhouse conditions.Do et al.

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Light and temperature are critical for sorghum growth and development. For example,in Missouri, U.S., under greenhouse conditions, spring and summer are usually the besttime for sorghum growth and embryo production.

Plant

2. Use a paper towel to cover the holes at the bottom of a 3-gallon pot to prevent soilfrom leaking.

3. Fill 3-gallon pots with this soil mix (e.g., Promix BX).

4. Label each pot using a tag with date, sorghum cultivars, and treatment name usinga permanent marking pen.

5. Move pots to greenhouse, arrange them in neat rows, and water them three timesuntil soil within each pot is completely wet.

6. Insert seeds or small plantlets into the soil in the center of the pot and cover seedwith soil.

Transgenic plantlets from acclimation will be transferred from 4-inch plastic pots to 3-gallon pots. Prevent damage to small plantlet roots. Water pots after transplanting.

7. Water pot one more time after sowing and make sure the top of soil is flat.

Watering schedule

The watering schedule is important for sorghum growth, development, and pollination.Under watering during pollination reduces seed formation and the quality of immatureembryos and transgenic seeds. Avoid over-watering the pots during the first 3 weeks afterplanting to allow roots to develop well.

8. Check soil water status and plants daily. Water plants only as needed based onobservation of plants and soil.

Based on the soil and plant status, adjust watering suitably every day. If the soil and thepot are relatively light in weight, the top leaves will begin wilting, and more water shouldbe added to the pot daily. When the soil is still dark and wet, the pot is heavy, no morewater is needed for that day. Over watering for long periods of time will damage the rootsystem causing top leaves to wilt and turn yellow.

9. Make sure to provide plants enough water daily.

Under greenhouse conditions, plants will lose water dramatically if the pot is dry,especially during the summer. Three weeks after seed sowing, the osmocote 18-6-12or 19-6-12 (one third of a measuring spoon; ̴ 10 ml) will be added to each pot.

10. Once plants develop to the six-leaf stage, they require more water.

From this stage on, the plants require more water for panicle initiation, pollination, andseed formation (Fig. 3B). Plants should never experience drought conditions but also donot over water the plants. Water stress destroys root systems and plants may not recovercompletely. Poor water conditions will result in reduction in the number of seeds perplant and also reduce immature embryo quality.

Fertilize and apply iron sulfate

11. Apply fertilizer at the recommended amount of 1 teaspoon (15 ml) osmocote 18-6-12 or 19-6-12 and one-third teaspoon (5 ml) iron sulfate when the sorghum plant isin the six-leaf stage.

12. Water plants containing Peters 20-20-20 after application of iron sulfate, to allowsoil to absorb solutions evenly and continue the same watering regime each timewith Peters 20-20-20. Do et al.

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To use Peters 20-20-20, dissolve 1 cup (250 ml) of Peters 20-20-20 in 25 gallons of water.For use, mix this solution with water at a 1:3 ratio.

Cover panicle

13. When the panicle of a plant emerges and some florets bloom, use a tassel bag tocover each panicle.

Avoid damage to the flag leaves. Record the date when the panicle was covered todetermine when the immature seeds are ready for collection.

14. It takes �9 to 12 days for the embryo to develop to the desirable size (1.0 to1.5 mm) during the warm or hot seasons but it will take 11 to 15 days for an embryoto develop to a desirable size during the cool seasons.

To judge the right embryo size for harvest, randomly pick some immature seeds from thetop of panicles and isolate embryos to confirm the IE sizes.

Immature seed harvest

15. Harvest the top part of sorghum panicle first, then the middle and bottom parts.

Immature seed will be collected from the top of primary branches down to the bottom(Fig. 1B). This can reduce damage to the seeds.

16. Once immature seeds have been collected, use for tissue culture and transformationimmediately or within 1 day.

We found that storage of immature seeds at room temperature or in a refrigerator reducedcallus induction frequency.

17. After harvesting, discard wild-type sorghum plants and clean before being re-used.

Mature seed harvest

18. Once all seeds from sorghum panicles turn to anticipated colors depending on thecultivar (e.g., brown, red, lemon-yellow, or white to black), discontinue wateringand let plants dry.

For P898012 genotype, seed (grain) color is white.

19. When all seeds are dry, cut panicles, isolate seeds, place seeds in paper bags withlabels, and store in a cold room with low humidity for future work.

20. Discard wild-type plants with soil.

21. For transgenic experiments, autoclave all plant parts, soil, and unused seeds beforedisposal.

Package all transgenic plant parts and soil in appropriate plastic bags (biological safetybags) for autoclaving.

REAGENTS AND SOLUTIONS

Acetosyringone (AS), 1000× stock solution

Dissolve 0.196 g acetosyringone in 5 ml methanol. Add 5 ml double-distilled H2O(ddH2O) to make a final volume of 10 ml. Filter-sterilize and store at −20°C in 1 mlaliquots.

To dissolve AS, use methanol instead of dimethyl sulfoxide (DMSO) and store at −20oC. Useof methanol will avoid freeze-thaw steps encountered using DMSO. This will minimize thereduced activity of AS due to the freeze-thaw step.

B5 vitamin, 100× stock solution

10 g myo-inositol0.1 g nicotinic acidDo et al.

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0.1 g pyridoxine·HCl1 g thiamine·HClDissolve in 1000 ml distilled deionized H2OFilter-sterilizeStore at 4°C in the dark for up to 3 months

The vitamin stock is sensitive to light; store in brown glass bottles.

Recipe from Murashige and Skoog (1962).

6-Benzylaminopurine (BAP), 1 mg/ml stock solution

Dissolve 100 mg of 6-benzylaminopurine (BAP; Sigma-Aldrich) in 2 ml 1 N NaOHand adjust to a final volume of 100 ml with ddH2O. Store at −20°C for up to3 months.

Callus induction medium (CIM)

4.3 g/liter Murashige and Skoog (MS) salts30 g/liter sucrose1.0 g/liter L-proline0.5 g/liter 2-(N-morpholino)ethanesulfonic acid (MES)1.5 ml/liter 2,4-dichlorophenoxyacetic acid (2,4-D; see recipe)Adjust pH to 5.8 then add:8 g/liter agar (Sigma-Aldrich)10 g/liter poly(vinylpolypyrrolidone) (PVPP), then autoclaveAfter autoclaving, cool down to 50° to 55°CPrepare a solution containing:10 ml/liter vitamin B5 (see recipe)1 g/liter asparagine1 g/liter KH2PO4

300 mg/liter cefotaxime0.1 ml/liter CuSO4(see recipe)0.125 ml/liter glufosinate (see recipe)Filter-sterilize and add to the mediumPour medium into 15 × 100-mm Petri dishes (30 to 35 plates)Store plates up to 2 weeks at 4°C

Co-cultivation medium (Co-M)

4.3 g/liter Murashige and Skoog (MS) salts20 g/liter sucrose10 g/liter glucose0.7 g/liter L-proline0.5 g/liter 2-(N-morpholino)ethanesulfonic acid (MES)1.5 ml/liter 2,4-dichlorophenoxyacetic acid (2,4-D; see recipe)Adjust pH to 5.8 then add:8 g/liter agar (Sigma-Aldrich)10 mg/liter ascorbic acid10 g/liter poly (vinylpolypyrrolidone) (PVPP), then autoclaveAfter autoclaving, cool to 50° to 55°C then add:10 ml/liter B5 vitamin (see recipe)1 ml/liter 1000× AS (see recipe)Pour into 15 × 100-mm Petri dishesStore plates up to 3 days at 4°C

PVPP is not dissolved in the medium even after autoclaving. Therefore, mix the mediumwell before pouring into the plates.

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CuSO4, 16 mg/ml stock solution

Dissolve 160 mg CuSO4 in 10 ml ddH2O. Store at 4°C for up to 6 months.

2,4-Dichlorophenoxyacetic acid (2,4-D), 1 mg/ml stock solution

Dissolve 100 mg of 2,4-dichlorophenoxyacetic acid (2,4-D; Sigma-Aldrich) in 5 ml1 N NaOH and adjust to a final volume of 100 ml with ddH2O. Store stock solutionat −20°C for up to 3 months.

2,4-D is difficult to dissolve and may require shaking overnight to dissolve. Do notheat the solution while dissolving 2,4-D in NaOH.

Glufosinate, 5 mg/ml stock solution

Dissolve 200 mg glufosinate (Gold Biotechnology) in 40 ml ddH2O. Filter-sterilizeand store at −4°C for up to 3 months.

Indole-3-acetic acid (IAA), 1 mg/ml stock solution

Dissolve 100 mg indole-3-acetic acid (IAA; Sigma-Aldrich) in 2 ml 1 N NaOH andadjust to a final volume of 100 ml with ddH2O. Store at −20°C for up to 3 months.

Indole-3-butyric acid (IBA), 1 mg/ml stock solution

Dissolve 100 mg of indole-3-butyric acid (IBA; Sigma-Aldrich) in 2 ml 1 N NaOHand adjust to a final volume of 100 ml with ddH2O. Store at −20°C for up to 3months.

Inoculation medium (IM)

4.3 g/liter Murashige and Skoog (MS) salts (Sigma-Aldrich)68.5 g/liter sucrose36 g/liter glucose0.5 g/liter 2-(N-morpholino) ethanesulfonic acid (MES)1.5 ml/liter 2,4-dichlorophenoxyacetic acid (2,4-D; see recipe)10 ml/liter B5 vitamin (see recipe)Adjust pH to 5.2Filter-sterilize, make aliquots, and store at −20°C for up to 2 monthsRight before use, add 1000× acetosyringone stock (see recipe) to 1× final

concentration

Kanamycin sulfate, 50 mg/ml stock solution

Dissolve 500 mg kanamycin sulfate powder (Sigma-Aldrich) in 10 ml ddH2O. Filter-sterilize and store at −20°C for up to 2 months.

Resting medium (R)

4.3 g/liter Murashige and Skoog (MS) salts30 g/liter sucrose1.0 g/liter L-proline0.5 g/liter 2-(N-morpholino) ethanesulfonic acid (MES)1.5 ml/liter 2,4-dichlorophenoxyacetic acid (2,4-D; see recipe)Adjust pH to 5.8 then add:8 g/liter agar (Sigma-Aldrich)10 g/liter poly (vinylpolypyrrolidone) (PVPP), then autoclaveCool to 50° to 55°CPrepare a solution containing:10 ml/liter B5 vitamin (see recipe)1 g/liter asparagine1 g/liter KH2PO4

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0.1 ml/liter CuSO4 (see recipe)Filter-sterilize and add to the mediumPour medium into 15 × 100-mm Petri dishesStore plates up to 2 weeks at 4°C

Rifamycin, 25 mg/ml stock solution

Dissolve 250 mg rifamycin powder (Sigma-Aldrich) in 10 ml of 100% methanol andstore at −20°C for up to 2 months.

Rooting medium (RM)

4.3 g/liter Murashige and Skoog (MS) salts30 g/liter sucrose0.5 g/liter 2-(N-morpholino) ethanesulfonic acid (MES)Adjust pH to 5.8 then add:8 g/liter agar (Sigma-Aldrich)10 g/liter poly (vinylpolypyrrolidone) (PVPP), then autoclaveCool to 50° to 55°CPrepare a solution containing:10 ml/liter B5 vitamin (see recipe)300 mg/liter cefotaxime0.1 ml/liter CuSO4 (see recipe)1 ml/liter indole-3-butyric acid (IBA; see recipe)Filter-sterilize and add to the mediumPour medium into Magenta GA-7 vesselStore up to 2 weeks at 4°C

Shoot induction medium (SM)

4.3 g/liter Murashige and Skoog (MS) salts30 g/liter sucrose0.5 g/liter 2-(N-morpholino) ethanesulfonic acid (MES)Adjust pH to 5.8 then add:8 g/liter agar (Sigma-Aldrich)10 g/liter poly (vinylpolypyrrolidone) (PVPP), then autoclaveCool to 50° to 55°CPrepare a solution containing:10 ml/liter B5 vitamin (see recipe)1 ml/liter 6-benzylaminopurine (BAP; see recipe)1 ml/liter indole-3-acetic acid (IAA; see recipe)300 mg/liter cefotaxime0.1 ml/liter CuSO4 (see recipe)0.5 ml/liter glufosinate (see recipe)Filter-sterilize and add to the mediumPour medium into 20 × 100-mm Petri dishes (20 plates)Store plates up to 2 weeks at 4°C

YEP agar plates

10 g peptone10 g yeast extract5 g NaCl15 g Bacto agar (Thermo Fisher Scientific)Bring volume to 1 liter with waterAdjust pH to 7.0. Autoclave and cool to 50° to 55°C, then add:50 mg/liter kanamycin (see recipe)25 mg/liter rifampicin (see recipe) Do et al.

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Pour into 100 × 15-mm Petri dishesStore up to 1 month at 4°C

For other vectors and bacterial strains, different antibiotics may be used as appro-priate.

COMMENTARY

Background InformationSorghum [Sorghum bicolor (L.) Moench]

is known as an important cereal crop pro-viding a food staple for a half billion peo-ple in over 30 countries around the world.In addition, this crop has potential for bio-fuel production and other industrial applica-tions (Dahlberg, Berenji, Sikora, & Latkovic,2011). Various environmental factors such asbiotic and abiotic stresses have been shownto reduce sorghum yields. As a complement totraditional breeding methods, genetic transfor-mation has been shown as an important tool toaccelerate the breeding processes for sorghumvariety improvement.

Agrobacterium-mediated transformationhas been utilized in many plant speciesincluding sorghum. Although many improve-ments have been made in tissue culture andtransformation processes, sorghum transfor-mation efficiency is still limited comparedto other cereal crops such as rice or maize.The frequency of callus induction and plantregeneration varies widely depending onexplants and genotypes. Immature embryosare the most commonly used explant tissuesfor sorghum regeneration and transformation.The sorghum public genotype P898012 hasbeen widely utilized as a suitable variety forregeneration and transformation. This geno-type was first used for transformation throughparticle bombardment (Casas et al., 1993).Later, immature embryos of this genotypewere utilized for Agrobacterium-mediatedtransformation (Zhao et al., 2000). In theseearly reports, reporter genes were transferredinto sorghum at low frequency. Efforts havebeen made to optimize the transformationsystem for transferring genes of interest intosorghum but the transformation efficiencywas not much improved (Able, Rathus, &Godwin, 2001; Carvalho et al., 2004; Lu et al.,2009). By utilization of heat shock treatments,Gurel et al. (2009) reported a significantimprovement in sorghum transformationof 8.3% with public genotype P898012.Recently, the systematic improvements insorghum regeneration and transformationconditions enabled achievement of transfor-mation frequencies >14% with high qualityof transgenic events (Do, Lee, Mookkan,Folk, & Zhang, 2016).

The protocol presented here is based on ourpreviously published research in the improve-ment of sorghum transformation employingsorghum public genotype PI898012 and stan-dard binary vectors (Do et al., 2016). Theimprovement was achieved by utilizing opti-mized medium compositions, use of an appro-priate Agrobacterium strain, and binary con-structs in combination with the heat shocktreatments, leading to improved embryogeniccallus induction and shoot regeneration. Inaddition, a shortened tissue culture timeframe, higher transformation frequency, andgood quality of transgenic events have beenaccomplished.

Critical Parameters(1) The sorghum growing season is an

important factor that affects immature em-bryo quality. Under greenhouse conditions,immature embryos collected from sorghumplants grown during spring and summer time(from April to September in Missouri, U.S.)allow the highest transformation frequency.If sorghum panicles are small with less vi-able seeds, only the top part of panicles withlarge immature seeds should be collected forsorghum transformation.

(2) The quality of immature embryos iscritical for sorghum transformation. Strict at-tention should be paid to the examination ofthe quality of sorghum panicles and imma-ture seeds prior to all experiments. Under goodconditions, 10 to 12 days after pollination, im-mature embryos isolated from the top partsof panicles reach the optimal size of 1.0 to1.5 mm with fluid endosperm texture. Thesmaller size of immature embryos correlateswith higher senescence rates, likely caused bydamage during the heat shock and inocula-tion processes. By contrast, larger sized em-bryos promote the growth of primary shootsand reduce callus induction frequency. In somecases, it takes longer for immature embryos toreach a desirable size (1.0 to 1.5 mm). How-ever, seeds bearing these immature embryostypically have solid endosperm texture, an in-dicator of poor embryo quality, and thereforeshould not be used for transformation.

(3) It is critical to do heat shock treatmentaccurately. Heat shock treatments have been

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shown to increase the sorghum transformationefficiency. However, a prolonged heat shockat high temperature (43°C) damages imma-ture embryos whereas a shorter heat shocktime may not enable maximal enhancementof transformation. Agrobacterium inoculationimmediately after heat shock treatment willincrease sorghum transformation frequency.

(4) It is critical to conduct all required sub-cultures on a timely basis. Longer subcultureson callus induction medium (CIM) results inlower somatic embryo development and shootinduction. In addition, a delay in subculturewill result in high amounts of phenolic accu-mulation in the medium, especially during in-oculation and shoot induction stages. Accumu-lation of phenolics will inhibit callus, somaticembryo, and shoot developments. Therefore,it is desirable to subculture once within the5 days on resting medium to reduce explantexposure to high amounts of phenolics andincrease callus induction and transformationfrequency.

(5) Using PVPP and washed agar is criticalfor all sorghum transformation steps. Sorghumtissues produce high levels of phenolics duringthe transformation procedure. PVPP absorbsthe phenolics reducing the effects on sorghumtissue cultures. We found that washed agaris more suitable for sorghum tissue culturethan other gelling agents such as PhytagelTM

or gelrite. Use of PVPP and washed agar forthe entire sorghum transformation processeswill increase regeneration and transformationefficiency.

Troubleshooting(1) Accumulation of phenolics and less

swelled calli after resting: If immatureembryos turn to a brownish color and do notswell after resting time, it is likely that theembryo vigor is low. The smaller embryo size(<1.0 mm) is an important reason for thisresult. Therefore, larger immature embryosshould be used to overcome the situation.Moreover, longer time (>10 min) for inoc-ulation and higher bacterial density (OD550

>0.4) also cause the same problem. Theseparameters need to be adjusted to get theoptical values.

(2) Primary shoots become dominate andtherefore cause low callus induction fre-quency: When primary shoots develop domi-nantly along with small sizes of calli at the endof the callus induction stage, it is likely that oldimmature embryos were used for transforma-tion. To overcome this trouble, only immatureembryos with optical size at 10 to 14 days after

pollination should be collected. Under somestress conditions like drought, cold, and nu-trient deficiency, the immature embryos willbe <1.0 mm even at 14 days after pollination.These late collected embryos should not beused for transformation.

(3) Albino shoots occur on shoot inductionmedium: The delay in transferring embryo-genic calli to shoot induction medium mayincrease the occurrence of albino sorghumshoots. Therefore, do not keep calli on CIM for>4 weeks. In addition, subculture of sorghumexplant tissues in a timely fashion duringthe regeneration stage also reduces the albinoshoots.

(4) During the selection and regenera-tion stage, some shoots become glassy andswelling: Such a hyperhydricity (vitrification)inhibits shoot elongation, development, andalso root formation. To overcome this prob-lem, deep Petri plates (20 × 100 mm) andthe use of porous tape should be used for thecultures at the regeneration stage. In addition,reduction in the number of embryogenic callior shoots per plate will improve conditions.

(5) Most of calli turn black and senescesoon after is transferred to germinationmedium: This condition may be caused bypoor Agrobacterium infection or adverse im-pacts from the introduced transgenes. To pre-vent this problem all inoculation conditionsand steps need to be thoroughly implemented.Meanwhile, side-by-side experiments need tobe performed using an empty binary vec-tor (normally a backbone vector) to com-pare with the transgenic vector containinggenes of interest. If an expected transforma-tion rate is achieved from the empty controlvector, the low transformation frequency islikely due to the expression of the introducedtransgenes.

(6) Loss of individual transgenic events:It is possible to lose some transgenic eventsduring rooting, acclimation, and greenhousestages because of root damage or contami-nation. Therefore, it is essential to transferseveral plants (clones) from each event to thesoil and greenhouse. To do this, keep the extrashoots longer in the shoot induction mediumuntil multiple shoots are propagated, sepa-rated, and transferred to the rooting mediumto secure sufficient number of clones.

Anticipated ResultsFor good immature seeds, >90% of imma-

ture embryos will develop calli at the end ofthe resting stage. All calli should show so-matic embryos at the end of callus induction

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stage and are uniformly ready to be transferredto the shoot induction medium.

A thorough implementation of this proto-col should provide a stable transformation fre-quency as high as 14% using public genotypeP898012.

At least one plant (clone) of each event willproduce transgenic T1 seeds.

Time Considerations(1) The total time-frame from seed to seed

is about 9 to 11 months, depending on growthseasons and the effects of transgenes.

(2) In general, it should take 3 months toget immature seeds and 2.5 to 3 months forthe entire in vitro culture procedure. The extratime within the above time window normallycomes from regeneration and selection stagesof sorghum transformation.

(3) Under greenhouse conditions, it shouldtake >4 months to get dry mature sorghumseeds.

Acknowledgements

We want to thank Neng Wan (Zhanyuan J.Zhang’s lab) for technical assistance in pro-viding certain sorghum materials for this re-view work. This work has been supported, inpart, by the National Science Foundation PlantGenome Research Program # 1444478.

Literature CitedAble, J. A., Rathus, C., & Godwin, I. D.

(2001). The investigation of optimal bombard-ment parameters for transient and stable trans-gene expression in sorghum. In Vitro Cellular& Developmental Biology, 37, 341–348 doi:10.1007/s11627-001-0061-7.

Carvalho, C. H. S., Zehr, U. B., Gunaratna, N.,Erson, J., Kononowicz, H. H., Hodges, T.K., & Axtell, J. D. (2004). Agrobacterium-mediated transformation of sorghum: Factorsthat affect transformation efficiency. Genet-ics and Molecular Biology, 27, 259–269 doi:10.1590/S1415-47572004000200022.

Casas, A. M., Kononowicz, A. K., Zehr, U. B.,Tomes, D. T., Axtell, J. D., Butler, L. G., . . .Hasegawa, P. M. (1993). Transgenic sorghumplants via microprojectile bombardment. Pro-ceedings of the National Academy of Sciences ofthe United States of America, 90, 11212–11216doi: 10.1073/pnas.90.23.11212.

Dahlberg, J., Berenji, J., Sikora, V., & Latkovic,D. (2011). Assessing sorghum [Sorghum bi-color (L.) Moench] germplasm for new traits:Food, fuels and unique uses. Maydica, 56,85–92.

Do, P. T., Lee, H., Mookkan, M., Folk, W.R., & Zhang, Z. J. (2016). Rapid and effi-cient Agrobacterium-mediated transformationof sorghum (Sorghum bicolor) employing stan-dard binary vectors and bar gene as a selectablemarker. Plant Cell Reports, 35, 2065–2076 doi:10.1007/s00299-016-2019-6.

Gurel, S., Gurel, E., Kaur, R., Wong, J., Meng,L., Tan, H.–Q., & Lemaux, P. G. (2009). Ef-ficient, reproducible Agrobacterium-mediatedtransformation of sorghum using heat treat-ment of immature embryos. Plant Cell Re-ports, 28, 429–444 doi: 10.1007/s00299-008-0655-1.

Lu, L., Wu, X., Yin, X., Morrand, J., Chen, X., Folk,W. R., & Zhang, Z. J. (2009). Development ofmarker-free transgenic sorghum [Sorghum bi-color (L.) Moench] using standard binary vec-tors with bar as a selectable marker. PlantCell Tissue Organ Culture, 99, 97–108 doi:10.1007/s11240-009-9580-4.

Zhao, Z., Cai, T., Tagliani, L., Miller, M.,Wang, N., Pang, H., . . . Seltzer, J. (2000).Agrobacterium-mediated sorghum transforma-tion. Plant Molecular Biology, 44, 789–798. doi:10.1023/A:1026507517182.

Key ReferencesZhao et al. 2000. See above.A key milestone work establishing, for the first

time, that sorghum was stably transformed us-ing Agrobacterium tumefaciens aided by super-binary vector. The work also showed that thesame transformation system using super-binaryvector yielded a much higher sorghum transfor-mation efficiency using immature embryos com-pared with field grown sorghum plants.

Gurel et al. 2009. See above.A key milestone work demonstrating, for the

first time, that Agrobacterium-mediated stablesorghum transformation was quite efficient, asenhanced by heat shock treatment of immatureembryos, using a standard binary-vector.

Do et al. 2016. See above.A key milestone work illustrating that more rapid

and efficient Agrobacterium-mediated stablesorghum transformation than previous reportswas achieved by systematic optimization of re-generation and transformation conditions usinga standard binary vector.

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