Maize (Zea mays) Hi-II Transformation via Agrobacterium ......Figure 3 Embryogenic calli develop on callus induction and selection medium. Left: embryo proliferation; right: close

Maize (Zea mays) Hi-II Transformationvia Agrobacterium-Mediated T-DNATransferHyeyoung Lee1 and Zhanyuan J Zhang1

1University of Missouri Plant Transformation Core Facility Division of Plant SciencesColumbia Missouri

Genetic transformation of maize via Agrobacterium tumefaciens is still more artthan science with different researchers achieving substantially different trans-formation results This article describes our advanced Agrobacterium-mediatedtransformation system in Hi-II maize The system utilizes simple binary vec-tors and immature embryos for the transformation employing the bar gene asa plant selectable marker in combination with bialaphos for subsequent cultureselection The transformation process is efficient and highly reproducible Cer-tain inbred maize lines can also be transformed with some modification of thesystem Ccopy 2016 by John Wiley amp Sons Inc

Keywords Agrobacterium tumefaciens binary vectors Hi-II maize

How to cite this articleLee H and Zhang ZJ 2016 Maize (Zea mays) Hi-II

transformation via Agrobacterium-mediated T-DNA transfer CurrProtoc Plant Biol 1121-137

doi 101002cppb20016

INTRODUCTION

Maize (Zea mays) is ranked as a top crop in the United States and in many parts of theworld Maize is also a model plant for genetic and molecular biological studies Sincethe initial report describing the successful transformation of maize (Gould et al 1991)efforts involving the genetic transformation of maize have increased substantially As aresult the demand for maize transformation procedures has swelled over the past decade

Here we present a Basic Protocol for stable maize transformation and a Support Protocolfor greenhouse maize management

BASICPROTOCOL

STABLE TRANSFORMATION OF MAIZE

In this protocol an improved method is described for Agrobacterium-mediated stabletransformation of maize Hi-II A times B using standard binary constructs This protocoloffers efficient and reproducible transformation results when implemented as described

This protocol includes five steps (a) initiation of the Agrobacterium culture (b) isolationof embryos inoculation and co-cultivation (c) resting (d) selection and (e) plantletregeneration

Materials

A tumefaciens (also called Rhizobium radiobacter) strain AGL1 carrying a simplebinary vector (eg pZY102 ATCC httpwwwatccorg)

ABC agar plates containing appropriate antibiotics (see recipe)YEP agar plates containing appropriate antibiotics (see recipe)

Current Protocols in Plant Biology 1121-137 May 2016Published online May 2016 in Wiley Online Library (wileyonlinelibrarycom)doi 101002cppb20016Copyright Ccopy 2016 John Wiley amp Sons Inc

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PHI-A (inoculation medium see recipe)Ears of maize Hi-II A times B (Support Protocol)30 (vv) commercial bleachTween 20PHI-B (co-cultivation medium plates see recipe)PHI-C (resting medium plates see recipe)PHI-D1 (selection medium I plates see recipe)PHI-D2 (selection medium II plates see recipe)PHI-E (maturation medium plates see recipe)PHI-F (regeneration medium see recipe)Soil mixture (eg Promix BX)

Dark culture incubators with temperature control and air circulation (PercivalScientific one at 20degC for co-cultivation and the other at 28degC for callus culture)

15-ml conical centrifuge tubes (eg BD Falcon)Inoculating loopSpectrophotometer and cuvettesShaker2-ml microcentrifuge tubesForceps sterile1-liter wide-mouth bottle sterile150 times 15ndashmm and 100 times 15ndashmm Petri dishes11 razor blades sterileMicrospatula sterileDissecting microscope such as Olympus stereo zoom microscope SZ40 MODEL

LMS-225R with 5-40times amplification and built-in light source (Leeds PrecisionInstruments)

3 M porous tape25 times 150ndashmm test tubesSmall plastic planting potsGrowth chamber settings 25degC 16 hrlight 8 hrdarkLight incubator or culture room settings 24degC 18 hrlight 6 hrdark

Additional reagents and equipment for maintenance and pollination of maize(Support Protocol)

NOTE Autoclave and discard all contaminated Agrobacterium and maize culturespromptly

Initiate Agrobacterium culture

1 Streak AGL1 carrying a simple binary vector (eg pZY102) from minus80degC stock onABC agar plates with appropriate antibiotics (for the vector and strain illustratedhere 100 mgliter spectinomycin and 30 mgliter rifampin) preparing a dilutionseries in order to obtain single colonies Incubate the plates in the dark for 3 days at28degC

The colonies on the ABC agar plate can be used for up to 1 month when stored at minus4degC

2 Select a single colony and streak it on YEP agar plates containing appropriateantibiotics (for the vector and strain illustrated here 100 mgliter spectinomycin and30 mgliter rifampin) Incubate the plates in the dark for 3 days at 20degC

3 Add 5 ml of sterile PHI-A (inoculation medium) to a 15-ml conical centrifuge tube

4 Transfer two full loops of AGL1 from the YEP plate to the tube prepared in step 3After 2 to 3 min shake the tube to thoroughly suspend bacterial cells

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Figure 1 Isolation of immature embryos (A) ear sterilization (B) removal of top halves of kernels(C) isolate immature embryos (D) an immature embryo on the spatula

5 Remove 1 ml of this suspension and place it in a spectrophotometer cuvette to checkthe optical density at 550 nm (OD550) Adjust the cell suspension to an OD550 of035 (05 times 109 cfuml) at room temperature (eg 24degC) by either adding moreAgrobacterium cells or diluting the culture with more PHI-A

6 Shake the culture in a shaker at 100 rpm for 4 to 5 hr at room temperature (eg24degC)

7 Aliquot 1 ml of the suspension into 2-ml sterile microcentrifuge tube

Embryo isolation inoculation and co-cultivation

8 Remove the husks and silk from ears which were harvested 10 to 13 days post-pollination (with embryo size of 15 mm see Support Protocol) Insert a pair offorceps into one end of the ear as shown in Figure 1A

It is crucial that the immature embryos be between 15 mm to 18 mm in length We havefound that as the size of the embryo increases the transformation frequency decreasesalthough the embryos are more tolerant of low-salt conditions in the medium (unpubobserv) Smaller embryos (less than 12 mm) require a full-strength salt medium to avoidhigh mortality Because it is much more difficult to isolate small embryos than large oneswe suggest using 15-mm embryos with a low-salt medium (see Critical Parameters) inorder to balance high transformation frequency with easier embryo isolation

9 Completely submerge the fresh Hi-II ears in a solution containing 05 liters of 30commercial bleach with a few drops of Tween 20 (in a sterile 1-liter wide-mouthbottle) for 20 min (Fig 1A)

If the maize plants are grown in the field to obtain immature embryos it is recommendedthat 50 commercial bleach be used for decontamination Embryos from the field aretypically transformed at a higher frequency than embryos grown in the greenhouse butmay have higher levels of contamination


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Figure 2 Size of immature embryos used for infection by Agrobacterium Left immature embryo15 mm in length right immature embryos in co-cultivation plate

10 Wash ears three times with sterile water (making sure ears are completely submergedin the water each time) and let the ear stand upright on a sterile 150 times 15ndashmm petridish

11 Remove top half of the kernels from each ear with a sterile 11 razor blade (Fig 1B)

Work on only two rows at a time to prevent the loss of moisture and maintain the vigor ofthe embryos in the remaining rows

12 Isolate 15-mm immature embryos (Fig 1CD) from the sterile ear with a sterilemicrospatula and transfer 50 to 100 embryos per 17- to 20-ml microcentrifuge tubeWash the embryos with 1 ml PHI-A solution three times to remove debris and starch

Typically 200 to 400 embryos can be isolated in each experiment

13 Immediately afterwards add 1 ml of the Agrobacterium suspension to the tubecontaining the immature embryos allow the tube to stand 5 min in the sterile hoodthen pour the entire contents including all of the embryos onto PHI-B (co-cultivationmedium) agar plate

14 Draw off Agrobacterium suspension using a pipet with a fine tip then spread theembryos evenly across the plate and place embryos with scutellum face up and flatside face down on the medium (Fig 2)

When the immature embryos are co-cultivated with Agrobacterium on the PHI-B mediumthe embryo should be flat side face down Be sure to check the orientation of the embryosunder a dissecting microscope

15 Seal the plate with parafilm and incubate in the dark at 20degC for 3 days

Resting

16 Transfer the embryos with a spatula to a plate of PHI-C (resting medium) Avoiddamaging the embryos


Selection

18 Transfer embryos with spatula or forceps to a plate of PHI-D1 (selectionmedium I) Place 25 embryos per plate and seal the plate Incubate the embryosin the dark at 28degC for the first 2-week selection (Fig 3)


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Figure 3 Embryogenic calli develop on callus induction and selection medium Left embryoproliferation right close up of Hi-II type II callus Note Hi-II type II callus is loose dry and fastgrowing which is in contrast to the type I callus which is compact and slow growing

Figure 4 Embryos before being transferred to maturation medium Left photograph showsopaque-color embryos while the arrow in the right photograph indicates a coleoptile structure

19 Transfer calli with forceps from the PHI-D1 plate to a plate of PHI-D2 (selectionmedium II) Subculture the calli every 2 weeks onto fresh PHI-D2 medium for atotal of 2 months using the incubation conditions in step 18

PHI-D1 medium and PHI-D2 medium select for resistance to the herbicide bialaphos

20 Bulk up the herbicide-resistant calli by growing them on fresh PHI-D2 medium foranother 2 weeks under the same conditions as in steps 18 and 19 until the diameterof the calli is about 10 cm

During this stage each large fast-growing Hi-II embryogenic callus can be divided(using forceps to cut up the callus mass) to select the best-quality part of the callus (quitedry friable and light in color and normally on the top half of the callus) Transfer thepart of the callus with the best characteristics to fresh medium In this way the fastestgrowing calli will be obtained selection stringency will be enhanced and it will bepossible to maintain callus cultures for a longer period of time

Maturation and regeneration

21 Using forceps transfer each entire callus mass containing opaque embryos (Fig 4)onto PHI-E (maturation medium) in 20 times 100 mm petri plates (wrapped with 3 M


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Figure 5 Mature (ivory) embryos on maturation medium (left) and subsequently regeneratedshoots (right)

Figure 6 Plantlet in rooting stage

porous tape) and place culture plates in the dark at 28degC for two 2 to 3 weeks toallow somatic embryos to mature

22 Transfer ivory-white calli (Fig 5 left) onto PHI-F (regeneration medium) andincubate at 25degC under 16-hr photoperiod until shoots and roots develop (Fig 5right)

23 Transfer each small plantlet to a 25 times 150ndashmm tube containing PHI-F (regenerationmedium) and grow at 25degC under 16-hr photoperiod for 2 to 3 weeks (Fig 6)

24 Transfer the plants to small plastic pots with soil mixture eg Promix BX soil in alight incubator or culture room at 24degC with an 18 hrlight 6 hrdark cycle (Fig 7)


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Figure 7 Plantlets in soil during acclimatization stage

SUPPORTPROTOCOL

GREENHOUSE MAIZE PLANT MANAGEMENT

Greenhouse maize plant management is critical to support efficient stable maize trans-formation increase plant fertility and avoid loss of transgenic events

Materials

Soil mixture (eg Promix BX)Osmocote 18-6-12 or 19-6-12 fertilizer (Hummert International cat no 07-6300-1

or 07-6330-1)Source of water (avoid high concentrations of organic or inorganic compounds)Maize Hi-II immature embryos derived from the self-pollinated ears (F2) of the F1

cross between Hi-II A and Hi-II B or between A188 and B73 are used as startingmaterial seeds of Hi-A (or A188) and Hi-II B (or B73) can be requested fromMaize Genetic Stock Center (httpmaizecoopcropsciuiucedu)

Iron sulfate (Hummert International cat no 07-0851-1)Peters 20-20-20 fertilizer (Hummert International cat no 07-5400-1)

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 (approximately 1 oz Hummert International cat no 060092-1)Tags for labeling pots (Hummert International)Jiffy Pots (Hummert International)Humidomes (Hummert International cat no 143850-1))


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Shoot bags (Lawson Bags cat no 217 httpwwwlawsonbagscom)Tassel bags (Lawson Bags cat no 402 httpwwwlawsonbagscom)Stapling pliars (Hummert International cat no 51-3800-1))

Greenhouse temperature and light settings

1 Set the greenhouse module to 20degC nighttime and 28degC daytime with a photoperiodof 16-hour light and 8 hr darkness with a mixture of daylight plus 50 metal halideand 50 high-sodium-pressure light as supplemental lighting (24 lamps in threerows per room)

There is a seasonal fluctuation of both temperature and lighting Spring is usually thebest time for maize plant growth and thus best for embryo production

Planting

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

3 Mix the soil (eg Promix BX) with fertilizer (Osmocote 18-6-12 or 19-6-12) at one-third measuring spoon (with a size equivalent to approximately 1 oz) per 3-gallonpot

4 Label each pot on a tag with date maize genotype treatment name and your initialsusing a permanent pen

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

6 Plant maize seed by inserting it into the soil to roughly a 20 cm deep in the centerof the pot and cover the seed with soil

Number of seeds to be planted per pot is dependent on seed germination rate normallyone seed per pot is enough when germination rate is good

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

Watering schedule

The watering schedule is critical for developing a good root system

8 Avoid watering the pots during the 3-week period after planting to allow roots todevelop well

9 Check the soil water status and plants daily Water the plants only as needed

Making a good judgment of whether or not the plant needs water is based on the soilcolor weight of the pot and plant morphology When the soil is light-colored and the potis light it means you need to water the plant If the soil is dark and the pot is heavy thatmeans that watering is not needed If soil is still wet and dark but plants are neverthelesswilted that means you either watered too much previously or if weather is not too hotthat plants have a root pathogen infection

10 When you water the plants make sure to give them enough water (saturating thesoil)

It is not a good practice to water the plants frequently but with insufficient water eachtime

11 Once plants achieve the 6-leaf stage provide sufficient water to saturate the soil

Make sure that you never allow plants to suffer from drought conditions but meanwhile donot over-water the plants because over-watering destroys good hairy root systems and theplants will become wilted despite supplying sufficient water Therefore the criteria is aslong as you never see plant leaves become wilted (even slightly) you are doing very well


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12 Harvest ears at the timepoint described in Basic Protocol 1 step 8

Once ears have been harvested they can be wrapped with a plastic bag and stored in arefrigerator up to 2 days

13 After harvesting autoclave the transgenic maize plants and dispose of them properly

Pots can be cleaned and reused

Transgenic plant greenhouse care

Select the best two to five plantlet clones from each event (see Basic Protocol) and placethem in separate culture tubes

14 Fill Jiffy Pots with Promix BX soil mix and label each pot with sufficient descriptiveinformation to distinguish each individual clone

15 Water the pot to compact the soil before transplanting

16 Remove the plantlet from each tube and wash off any adhering agar with tap waterAvoid using water that is cold to the touch

17 Make a hole in the soil so that the entire roots of a plantlet can be inserted into thesoil of the pot and then cover the hole with soil Plant one plantlet per Jiffy Pot

18 Water those pots when transfer is complete Cover the tray holding the pots with aHumidome or suitable plastic cover

19 Water as needed with water containing Peters 20-20-20 fertilizer that has beendiluted until a light blue color is just visible in the water

20 Gradually remove the plastic cover over a period of a few days by opening the lid14 of the way then 12 then 34 and then completely When plants have fullyacclimated transfer to 3-gallon pots in greenhouse

Removal of the plastic cover should take a few days and full acclimation usually takesaround 2 to 3 weeks

21 Mix the Promix soil with Osmocote 18-6-12 or 19-6-12 at one-half measuringspoon per pot Water the pot to compact the soil slightly Transfer plantlet with thesoil from the smaller Jiffy Pot to a 3-gallon pot and fill the hole with soil properly

22 Water the plants in the pot to saturation after transplanting

Fertilization and application of iron sulfate

23 When the maize plant is at the 6-leaf stage apply a full measuring spoon per pot ofOsmocote 18-6-12 or 19-6-12 fertilizer and one-third of a measuring spoon of ironsulfate

Osmocote is mixed with the soil before transplanting while iron sulfate is mixed into thesoil in the pot where plant is already growing

24 Dissolve one cup of Peters 20-20-20 in 5 gallons of water and further dilute this withwater at a 13 ratio (115 dilution overall) Water the plants with the diluted Peter20-20-20 right after iron sulfate application to allow soil to take it in and continuewatering as necessary with the diluted Peters 20-20-20

Pollination

Details for pollinating maize can be found in standard texts or on several Web sitesincluding httparchivemaizegdborgIMPWEBpollenhtm

25 When the tassel of a plant starts to emerge check each plant daily for any shoot thatis coming in


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26 Use a shoot bag to cover each shoot when it is long enough to allow you to do soand only use the top shoot for pollination

27 One day before pollination remove the top of the shoot with a sharp blade label theshoot bag with the date and cover the shoot Make sure that the Lawson side facesoutward (bag opening is facing down)

28 On the same day cover the tassel of the plant with a tassel bag to prevent pollenfrom drifting

29 On the day of pollination check the shoot to make sure that silks have grownuniformly and come out a little bit from the shoot (the silk has to be fleshy greenindicative of viable and fresh pollen) Collect good pollen with the tassel bag atabout 8 to 10 AM when the pollen is viable

30 Make a judgment of pollen quality

Light fresh yellow color indicates good pollen In contrast dark yellow pollen or clumpsof pollen indicate bad pollen

31 Dust collected pollen onto the silks uniformly to insure good pollination

32 Cover the ear with a tassel bag and label the bag with cross and date Staple the bagwith a stapling pliers to avoid detaching from the plant

It takes about 9 to 12 days for the embryo to develop to the desirable size (15 to 20 mm)during the warm or hot season but it will take 11 to 15 days for an embryo to develop toa desirable size during the cool season

To judge the right embryo size for the harvest use a spatula to remove the top half thekernel near the middle of an ear and take off an embryo to see its size

REAGENTS AND SOLUTIONS

Use deionized distilled water in all recipes and protocol steps

24-D 1 mgml stock

Dissolve 100 mg of 24-dichlorophenoxyacetic acid (24-D Sigma-Aldrich) in 5 mlof 1 N NaOH and adjust to the final volume of 100 ml with distilled deionized waterStore up to 3 months at 4degC

Do not heat the solution while dissolving 24-D in NaOH

AB buffer 20times

60 g K2HPO4

20 g NaH2PO4

Distilled deionized H2O to 1 literFilter-sterilizeStore up to 3 months at 4degC

AB salts 20times20 g NH4Cl6 g MgSO4

7H2O3 g KCl02 g CaCl2005 g FeSO4

7H2ODissolve in 1 liter distilled deionized H2OFilter-sterilizeStore up to 3 months at 4degC


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ABC agar plates

880 ml distilled deionized H2O39 g of 2-(N-morpholino)ethanesulfonic acid (MES)15 g Bacto agar (Fisher Scientific)Adjust pH to 56AutoclaveCool to 50deg to 55degC then add50 ml of 20times AB salts (see recipe)50 ml of 20times AB buffer (see recipe)20 ml Stock C (see recipe)100 mg spectinomycin (available from many vendors purity should be high)30 mgliter rifampin (available from many vendors purity should be high)Pour into 100 times 15ndashmm Petri dishesStore up to 1 month at 4degC

Recipe from Zhao et al (1999)For other vectors and strains than those used in this unit other antibiotics may be appropriate

Acetosyringone 1000times stock

Dissolve 0196 g acetosyringone (eg Sigma or Life Technologies) in 5 ml methanolAdd 5 ml distilled deionized water to make a final volume of 10 ml Filter-sterilizeand store up to 1 year at minus20degC in 1-ml aliquots

For dissolving acetosyringone it is recommended to use methanol instead of DMSOand to store at minus20degC Use of methanol will avoid freeze-thaw steps as will beencountered by the use of DMSO This will minimize reduction in potency ofacetosyringone due to the freeze-thaw

Bialaphos 5 mgml stock

Dissolve 200 mg bialaphos (Gold Biotechnology) in 40 ml distilled deionized waterFilter sterilize the stock solution and store at 4degC

Glycine 2 mgml stock

Dissolve 100 mg glycine in 50 ml distilled deionized water Filter-sterilize the stocksolution and store up to 1 month at 4degC

MS vitamins 1000times stock

5 g myo-inositol0025 g nicotinic acid0005 g thiamine HCl0025 g pyridoxine-HCl50 ml distilled deionized H2OFilter sterilizeStore at 4degC in the darkThe vitamin stock is sensitive to lightRecipe from Murashige and Skoog (1962)

N6 vitamins 1000times stock

001 g glycine005 g thiamine HCl0025 g pyridoxine HCl0025 g nicotinic acid

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50 ml distilled deionized H2OFilter sterilizeStore up to 1 month at 4degC

Recipe from Chu et al (1975)

PHI-A (inoculation medium)

2 gliter N6 salts (eg Sigma-Aldrich)685 gliter sucrose36 gliter glucose07 gliter L-proline05 gliter 2-(N-morpholino)ethanesulfonic acid (MES)15 mlliter of 1 mgml 24-D stock (see recipe)1 mlliter of 1000times N6 vitamins (see recipe)Adjust pH to 52Filter sterilizeStore with the above ingredients up to 2 months at minus20degCJust before use add 1000times acetosyringone stock (see recipe) to 1times final

concentration

PHI-B (co-cultivation medium) agar plates

2 gliter N6 salts (eg Sigma-Aldrich)30 gliter sucrose07 gliter L-proline05 gliter 2-(N-morpholino)ethanesulfonic acid (MES)15 mlliter liter of 1 mgml 24-D stock (see recipe)8 gliter agar (Sigma-Aldrich)Adjust pH to 58Autoclave cool to 50deg to 55degC then add1 mlliter of 1000times N6 vitamins (see recipe)01 mlliter of 85 mgliter silver nitrate (see recipe)1 mlliter of 1000times acetosyringone (see recipe)10 mlliter of a solution of 04 g L-cysteine and 0154 g DTT freshly dissolved in

10 ml of distilled deionized H2OPour into 15 times 100ndashmm Petri dishesStore plates up to 3 days at 4ordmC

PHI-C (resting medium) plates

4 gliter N6 salts (eg Sigma-Aldrich)30 gliter sucrose07 gliter L-proline05 gliter 2-(N-morpholino)ethanesulfonic acid (MES)15 mlliter of 1 mgml 24-D stock (see recipe)3 gliter Gelrite (Sigma-Aldrich)Adjust pH to 58Autoclave cool to 50deg to 55degC then add1 mlliter of 1000times N6 vitamins01 mlliter of 85 mgliter silver nitrate (see recipe)10 mlliter of a solution of 250 mgliter cefotaxime (Supplier cat no) freshly

dissolved in 10 ml of distilled deionized H2OPour into 15 times 100ndashmm Petri dishesStore plates up to 2 weeks at 4ordmC


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PHI-D1 (selection medium I) plates

4 gliter N6 salts (eg Sigma-Aldrich)30 gliter sucrose07 gliter L-proline05 gliter 2-(N-morpholino)ethanesulfonic acid (MES)15 mlliter of 1 mgml 24-D stock (see recipe)3 gliter Gelrite (Sigma-Aldrich)Adjust pH to 58Autoclave cool to 50deg to 55degC then add1 mlliter of 1000times N6 vitamins (see recipe)01 mlliter of 85 mgliter silver nitrate (see recipe)03 ml of 5 mgml bialaphos (see recipe)10 mlliter of a solution of 250 mgliter cefotaxime (Sigma-Aldrich) freshly


PHI-D2 (selection medium II) plates

4 gliter N6 salts (eg Sigma-Aldrich)30 gliter sucrose07 gliter L-proline05 gliter 2-(N-morpholino)ethanesulfonic acid (MES)15 mlliter of 1 mgml 24-D stock (see recipe)3 gliter Gelrite (Sigma-Aldrich)Adjust pH to 58Autoclave cool to 50deg to 55degC then add1 mlliter of 1000times N6 vitamins (see recipe)01 mlliter of 85 mgliter silver nitrate (see recipe)06 ml of 5 mgml bialaphos (see recipe)10 mlliter of a solution of 250 mgliter cefotaxime freshly dissolved in 10 ml of

distilled deionized H2OPour into 15 times 100ndashmm Petri dishesStore plates up to 2 weeks at 4ordmC

PHI-E (maturation medium)

43 gliter MS salts (eg Sigma-Aldrich)60 gliter sucrose3 gliter Gelrite (Sigma-Aldrich)Adjust pH to 56Autoclave cool to 50deg to 55degC then add1 mlliter of 1000times MS vitamins (see recipe)1 mlliter of 2 mgml glycine06 ml of 5 mgml bialaphos10 mlliter of a solution of 250 mgliter cefotaxime freshly dissolved in 10 ml of

distilled deionized H2OPour into 100 times 20ndashmm Petri platesStore plates up to 2 weeks at 4ordmC

PHI-F (regeneration medium)

29 gliter MS salts (eg Sigma-Aldrich)30 gliter sucrose

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3 gliter Gelrite (Sigma-Aldrich)Adjust pH to 56Autoclave cool to 50deg to 55degC then add1 mlliter of 1000times MS vitamins (see recipe)1 mlliter of 2 mgml glycine (see recipe)Pour into 100 times 20ndashmm Petri platesStore up to 2 weeks at 4ordmC

Silver nitrate 85 mgml stock

Dissolve 340 mg silver nitrate in 40 ml distilled deionized water Filter sterilizeand store up to 1 month at 4degC

Silver nitrate is sensitive to light therefore the chemical should be kept in the dark

Stock C

Dissolve 250 g glucose in 1 liter distilled deionized water Filter sterilize and storeup to 3 months at 4degC

YEP agar plates

10 g peptone75 g yeast extract5 g NaCl15 g Bacto agar (Fisher Scientific)Bring to 1 liter with waterAdjust pH to 70AutoclaveCool to 50deg to 55degC then add100 mg spectinomycin30 mgliter rifampinPour into 100 times 15ndashmm Petri dishesStore up to 1 month at 4degCFor other vectors and strains than those used in this unit other antibiotics may be appropriate

COMMENTARY

Background InformationUse of the Agrobacterium-mediated T-

DNA transfer method is a critical method fordelivering a transgene to the maize genomefor plant biology studies and genetic improve-ment Agrobacterium tumefaciens is a naturalgenetic engineering vehicle whose delivery of-ten leads to stable and good transgene expres-sion and inheritance

Maize transformation by Agrobacteriumtumefaciens has been extremely difficult be-cause maize was not thought to be the natu-ral host of Agrobacterium Historically maizegenetic transformation via Agrobacterium hasbenefited from experiments in rice transfor-mation by Hiei et al (1994) who employedembryogenic callus and a superbinary vectorcontaining an extra copy of virB virC andvirG Ishida et al (1996) was the first groupto make a breakthrough in the stable transfor-mation of maize inbred lines (A-188) at anefficiency of 55 using immature embryosand a superbinary vector In later studies Zhao

et al (1999 2002 2004) further improvedconditions for infecting maize Hi-II immatureembryos as starting explants These modifiedconditions included the use of the phenoliccompound acetosyringone low-pH mediumand a superbinary vector Later Frame et al(2002 2006) improved maize Hi-II and inbredtransformation by including the antioxidantL-cysteine during infection stage This im-proved protocol allows the use of a simple bi-nary vector without the need for a superbinaryvector We optimized inoculation conditionsby employing low-salt medium and optimizedco-cultivation conditions by employing the an-tioxidants L-cysteine and dithiothreitol (DTT)in combination with low-salt medium (Vegaet al 2008) These improvements made it pos-sible to transform maize Hi-II with over 12transformation efficiency using a simple bi-nary vector system

In order to enable transformation of maizeinbred or elite lines by Agrobacterium more


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efforts were made to employ different tar-get explant tissues such as mature embryos(Huang and Wei 2004) immature embryos(Huang and Wei 2005 Frame et al 2006)and the seedling nodal area from dry seeds(Sidorov et al 2006) Many laboratories haveadopted the maize Hi-II transformation usingimmature embryos and simple binary vectors(Frame et al 2006 Vega et al 2008) Theprotocol presented here is based on our previ-ously published advanced maize Hi-II trans-formation protocol (Vega et al 2008) Withminor modifications this protocol should bealso suitable for transformation of some maizeinbred lines (Lee et al 2007)

Critical Parameters1 Immature embryo vigor is critical to

achieve a high frequency of transformationImmature embryos from field-grown maizeplants or from greenhouse-grown plants dur-ing the spring season give the best transforma-tion rate In case ideal growth conditions arenot available for achieving a high frequency oftransformation F1 immature embryos can beused where the F1 crosses were made betweenHi-II A (or A188) and Hi-II B (or B73)

2 The size of immature embryos is criti-cal with 15 mm being optimal As the sizeof immature embryos increases the transfor-mation frequency declines By contrast thesmaller the embryos the higher the infectionrate achieved with Agrobacterium but this oc-curs at the expense of higher mortality of im-mature embryos and increasing difficulty inisolation of embryos

3 Use of one-half the manufacturerrsquos rec-ommended concentration (05times) of N6 salts inthe inoculation medium and use of L-cysteineand DTT combined with 05times concentrationof salts in the co-cultivation medium as wellas a low temperature (20C) are critical topromote high frequency of transformation Us-ing a higher temperature or a lower salt con-centration of the infection and co-cultivationmedia during the co-cultivation stage will in-crease the mortality of immature embryos andis therefore not recommended On the otherhand use of a higher salt concentration willdrastically decrease the transformation effi-ciency

4 It is also critical to recognize the cor-rect stages at which to transfer from embryo-genic callus induction and selection mediumto maturation medium as well as from mat-uration medium to regeneration medium Anopaque color of embryos suggests the righttime for culture transfer from embryogenic

callus induction and selection medium to mat-uration medium while an ivory color of theembryos indicates the proper time to transferembryos from maturation medium to regener-ation medium

Troubleshooting1 If you observe that a high percentage of

immature embryos do not swell after the rest-ing stage it is likely that your embryo vigoris weak Therefore efforts should be made toimprove your immature embryo vigor For ex-ample elevating greenhouse daytime temper-atures to 28C but maintaining them no lowerthan 22C in the nighttime during the wintershould be an effective way to enhance embryovigor

2 If a high percentage of calli becomebrown it is likely due to poor infection byAgrobacterium or impact from the gene of in-terest To overcome the poor infection prob-lem refer to the previous discussion on howto achieve high-frequency transformation Tofind out if your gene of interest negatively af-fects your transformation side-by-side trans-formation experiments in replicates will beneeded in which an empty control constructidentical to your gene of interest construct isused in parallel with your experimental geneconstruct If the empty vector controls yield theexpected transformation rate the poor trans-formation rate is likely due to expression ofyour transgene of interest

3 A high percentage of transgenic plantsoften do not produce tassels or shed pollengrains especially during an unfavorablegrowth season such as a cold winter or hotsummer Therefore it is essential to carry overmultiple clones from the same event to thegreenhouse to secure a high probability thatat least one clone will produce tassels andorshed pollen

4 Delay in maize ear harvest under high-moisture conditions could result in seed ger-mination which is undesirable Therefore assoon as the seeds are dry they should beharvested

Anticipated ResultsOver 90 of immature embryos should

swell with nodular structures on the scutel-lum by the end of resting stage suggest-ing that immature embryos are growingwell

By the end of 35 months after embryo in-fection all embryogenic calli should be readyto be moved on to maturation medium


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Good implementation of the protocol de-scribed here should provide for an approxi-mately 12 stable transformation efficiency

At least one clone out of five from eachevent should produce tassels andor shedpollen

Time Considerations1 The total time frame from seed to seed

is 10 to 14 months depending on the seasonand the impact of the transgenes

2 It normally takes about 25 months fromplanting Hi-II A times BF2 seeds to obtain F2immature embryos for the inoculation in themid-west region of the US The time framemay change if the maize plants are grownin different latitudes Embryogenic callus in-duction and selection requires about 2 to 3months Maturation regeneration and rootingtake about 3 weeks each

3 About 35 months are typically neededto obtain transgenic T1 dry seeds

AcknowledgementsWe thank Neng Wan for his greenhouse

work and Michelle Folta for proofreading ofthe manuscript This work was supported byUniversity of Missouri Research Board grant(to HL)

Literature CitedChu CC Wang CC Sun CS Hsu C Yin

KC Chu CY and Bi FY 1975 Establish-ment of an efficient medium for anther cultureof rice through comparative experiments on thenitrogen source Sci Sin 18659-668

Frame BR McMurray JM Fonger TM MainML Taylor KW Torney FJ Paz MMand Wang K 2006 Improved Agrobacterium-mediated transformation of three maize inbredlines using MS salts Plant Cell Rep 251024-1034 doi 101007s00299-006-0145-2

Frame BR Shou H Chikwamba RK ZhangZ Xiang C Fonger TM Pegg SEK LiB Nettleton DS Pei D and Wang K 2002Agrobacterium tumefaciens-mediated transfor-mation of maize embryos using a standard bi-nary vector system Plant Physiol 12913-22doi 101104pp000653

Gould J Devey M Hasegawa O Ulian ECPeterson G and Smith RH 1991 Trans-formation of Zea mays L using Agrobac-terium tumefaciens and the shoot apexPlant Physiol 95426-434 doi 0032-08899195042609$01000

Hiei Y Ohta S and Komari T 1994 Ef-ficient transformation of rice (Oryza sativaL) mediated by Agrobacterium and se-quence analysis of the boundaries of theT-DNA Plant J 6271-282 doi 101046j1365-313X19946020271x

Huang XQ and Wei ZM 2004 High-frequency plant regeneration through callus ini-tiation from mature embryos of maize (Zeamays L) Plant Cell Rep 22793-800 doi101007s00299-003-0748-9

Huang X and Wei Z 2005 SuccessfulAgrobacterium-mediated genetic transforma-tion of maize elite inbred lines PlantCell Tissue Organ Cult 83187-200 doi101007s11240-005-5772-8

Ishida Y Saito H Ohta S Hiel Y KomariT and Kumashiro T 1996 High efficiencytransformation of maize (Zea mays L) mediatedby Agrobacterium tumefaciens Nat Biotechnol14745-750 doi 101038nbt0696-745

Lee BK Kennon AR Chen X Jung TWAhn BO Lee ZY and Zhang Z 2007 Re-covery of transgenic events from two highlyrecalcitrant maize (Zea mays L) geno-types using Agrobacterium-mediated standardndashbinary-vector transformation Maydica 52457-469

Murashige T and Skoog F 1962 A revisedmedium for rapid growth and bioassays withtobacco tissue cultures Physiol Plant 15473-497 doi 101111j1399-30541962tb08052x

Sidorov V Gilbertson L Addae P andDuncan D 2006 Agrobacterium-mediatedtransformation of seedling-derived maize cal-lus Plant Cell Rep 25320-328 doi101007s00299-005-0058-5

Vega JM Yu W Kennon AR ChenX and Zhang Z 2008 Improvement ofAgrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binaryvectors Plant Cell Rep 27297-305 doi101007s00299-007-0463-z

Zhang W Subbarao S Addae P Shen AArmstrong C Peschke V and GilbertsonL 2003 Crelox mediated marker gene ex-cision in transgenic maize (Zea may L)plants Theor Appl Genet 1071157-1168 doi101007s00122-003-1368-z

Zhao ZY Gu W and Cai T 1999 Meth-ods for Agrobacterium-mediated transforma-tion United States Patent No 5981840

Zhao ZY Gu W Cai T and Pierce DA2004 Methods for Agrobacterium-mediatedtransformation United States Patent No963096 Pioneer Hi-bred International IncDes Moines Ia

Zhao ZY Gu W Cai T Tagliani L HondredD Bond D Schroeder S Rudert M andPierce D 2002 High throughput genetic trans-formation mediated by Agrobacterium tume-faciens in maize Mol Breed 8323-333 doi101023A1015243600325

Key ReferencesIshida et al 1996 See above

A ground-breaking work establishing that maizecould be transformed by Agrobacterium-mediated transformation


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Zhao et al 1999 See above

A milestone work establishing a basic protocol forsubsequent improvement of maize transforma-tion

Frame et al 2002 See above

A milestone work making it possible for the publicto transform maize Hi-II using standard binaryvectors

Vega et al 2008 See above

A milestone work making maize transformationmore efficient by combined use of low-saltmedium with antioxidants during the infectionstage using a standard binary vector system


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PHI-A (inoculation medium see recipe)Ears of maize Hi-II A times B (Support Protocol)30 (vv) commercial bleachTween 20PHI-B (co-cultivation medium plates see recipe)PHI-C (resting medium plates see recipe)PHI-D1 (selection medium I plates see recipe)PHI-D2 (selection medium II plates see recipe)PHI-E (maturation medium plates see recipe)PHI-F (regeneration medium see recipe)Soil mixture (eg Promix BX)

Dark culture incubators with temperature control and air circulation (PercivalScientific one at 20degC for co-cultivation and the other at 28degC for callus culture)

15-ml conical centrifuge tubes (eg BD Falcon)Inoculating loopSpectrophotometer and cuvettesShaker2-ml microcentrifuge tubesForceps sterile1-liter wide-mouth bottle sterile150 times 15ndashmm and 100 times 15ndashmm Petri dishes11 razor blades sterileMicrospatula sterileDissecting microscope such as Olympus stereo zoom microscope SZ40 MODEL

LMS-225R with 5-40times amplification and built-in light source (Leeds PrecisionInstruments)

3 M porous tape25 times 150ndashmm test tubesSmall plastic planting potsGrowth chamber settings 25degC 16 hrlight 8 hrdarkLight incubator or culture room settings 24degC 18 hrlight 6 hrdark

Additional reagents and equipment for maintenance and pollination of maize(Support Protocol)

NOTE Autoclave and discard all contaminated Agrobacterium and maize culturespromptly

Initiate Agrobacterium culture

1 Streak AGL1 carrying a simple binary vector (eg pZY102) from minus80degC stock onABC agar plates with appropriate antibiotics (for the vector and strain illustratedhere 100 mgliter spectinomycin and 30 mgliter rifampin) preparing a dilutionseries in order to obtain single colonies Incubate the plates in the dark for 3 days at28degC

The colonies on the ABC agar plate can be used for up to 1 month when stored at minus4degC

2 Select a single colony and streak it on YEP agar plates containing appropriateantibiotics (for the vector and strain illustrated here 100 mgliter spectinomycin and30 mgliter rifampin) Incubate the plates in the dark for 3 days at 20degC

3 Add 5 ml of sterile PHI-A (inoculation medium) to a 15-ml conical centrifuge tube

4 Transfer two full loops of AGL1 from the YEP plate to the tube prepared in step 3After 2 to 3 min shake the tube to thoroughly suspend bacterial cells


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Documents

Maize (Zea mays) Hi-II Transformation via Agrobacterium ......Figure 3 Embryogenic calli develop on callus induction and selection medium. Left: embryo proliferation; right: close