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Chapter 2

Agrobacterium- and Biolistic-Mediated Transformationof Maize B104 Inbred

Jennifer A. Raji, Bronwyn Frame, Daniel Little, Tri Joko Santoso,and Kan Wang

Abstract

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due tovariations in cell competency to induce embryogenic callus, as well as the cell’s ability to receive andincorporate transgenes into the genome. This chapter describes two transformation protocols usingAgrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maizeinbred B104, excised from ears harvested 10–14 days post pollination, are used as starting explant material.Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-RadPDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agentbialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocolsdescribed here, average transformation frequencies (number of bialaphos resistant T0 callus events per 100explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16–21 weeks is needed using either protocolfrom the start of transformation experiments to obtaining putative transgenic plantlets with establishedroots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immatureears as transformation starting material and caring for transgenic plants for seed production are alsodescribed.

Key words Agrobacterium tumefaciens, Genetic transformation, Immature embryo, Inbred, Particlebombardment, Type I callus, Zea mays

1 Introduction

Maize is an important crop that is cultivated globally for a variety ofuses including human consumption, fodder, and industrial utiliza-tion [1]. The genetic history and divergence of maize from itsclosest wild relative teosinte is estimated between 6000 and9000 years ago [2]. Maize is one of the most studied model cropplants due in large part to its outcrossing nature and remarkablenatural genetic diversity that makes it suitable for basic and appliedresearch [3, 4]. Progress in genetic improvement of maize throughcombined application of biotechnology and conventional breeding

L. Mark Lagrimini (ed.), Maize: Methods and Protocols, Methods in Molecular Biology, vol. 1676,DOI 10.1007/978-1-4939-7315-6_2, © Springer Science+Business Media LLC 2018

15

approaches has provided powerful tools for creating superior vari-eties with better genetics for adaptation and tolerance to abioticand biotic stresses among other traits, thus improving productivityprospects to meet the growing demand of a rapidly expandingpopulation and global market [5–7]. For example, transgenicmaize expressing Bt (Bacillus thuringiensis) genes for insect resis-tance alone accounted for more than 48 million hectares cultivatedglobally in 2014, with the United States, Brazil, Argentina, SouthAfrica, and Canada emerging as five of the highest producingcountries worldwide [8].

Several genetic transformation methods exist that are based oneither physical or biological approaches. Among them, Agrobacter-ium tumefaciens and particle bombardment have remained themost commonly used methods. Agrobacterium tumefaciens is asoil-borne pathogen with a natural ability to transfer and integratea piece of its own DNA (T-DNA) into the plant’s genome. Thischaracteristic has been exploited for plant genetic transformationthrough T-DNA binary vector engineering techniques by substi-tuting the tumor-inducing genes with the gene of interest [9].Agrobacterium-mediated transformation is a popular transforma-tion method owing to its predictable gene integration and inheri-tance, as well as low copy number gene insertion among otherfeatures. Nevertheless, only a limited number of maize inbredlines have been transformed thus far viaAgrobacterium transforma-tion [10–13].

Particle bombardment methods generally rely on a high-velocity helium pulse for physical delivery of gold-bound DNAinto plant cells. The procedure for maize transformation using theBio-Rad PDS-1000/He device for micro-particle bombardmenthas been described extensively [14, 15]. Some of the positiveattributes of this method include ease of application, lack of needfor complex binary vector construction, flexibility of simultaneousdelivery of multiple genes, less genotype dependence, and highertransformation frequency [16, 17]. However, concerns about com-plex integration patterns including multiple transgene copy num-ber have also been documented [18, 19]. Although currentopinions regarding adverse effects of multiple copy delivery remaindivided [20, 21], several studies have proposed that effective vectordesign and delivery of lower DNA concentration into target cellscan mitigate multiple copy delivery [22, 23].

A limited number of maize genotypes have been transformedusing Agrobacterium-mediated transformation and micro-particlebombardment [24]. Transformation frequencies for these methodsare often determined by the regeneration competence of the geno-type, transformation procedure parameters, or a combination ofboth [11, 12, 25, 26]. Immature zygotic embryos (IZEs) are gener-ally used as target explants for maize transformation [11, 27, 28],but their embryogenic culture response and transformation

16 Jennifer A. Raji et al.

competence are highly genotype dependent. Typically, culture mediamodification and optimization for inducing high frequency somaticembryogenesis from IZE explant is an important first step whenattempting to transform an inbred line [11, 29]. Tissue cultureregeneration capabilities and cell types that differentiate from theseexplants vary extensively among maize inbred lines since formationof somatic embryos is believed to be under multiple and oftencomplex gene activity [30–32]. For instance, the maize hybrid Hi-II genotype produces primarily type II callus from the scutellum ofIZEs when cultured on N6 medium [33]. Inbred genotypes B104[11], A188 and A634 [28] produce type I embryogenic callus fromthe same explants when cultured on MS and LS media, respectively.Type I callus is often described as a dense and firm cluster of cells,while type II callus is more friable, loosely aggregated, and breaksapart quite easily. Both types of embryogenic culture can be trans-formed successfully once the appropriate culture media areidentified.

In this chapter, we describe genetic transformation of maizeinbred line B104 using both Agrobacterium-mediated transforma-tion and micro-particle bombardment methods. B104 is a publicinbred line that derives from BS13(S) C5, an Iowa Stiff StalkSynthetic (BSSS) population [34]. It also shares considerable(~60%) genetic similarity with B73 [35], an inbred line with apublicly available, complete genome sequence [36]. Agrobacter-ium-mediated transformation of B104 was initially reported in2006 [11] and 2011 [37]. Here an updated procedure is described.The particle bombardment protocol described in this chapter is thefirst report of the development of a biolistic-based procedure forinbred line B104. Following Agrobacterium infection/co-cultivation, or bombardment, explants are taken through callusinitiation and selection on the herbicide bialaphos. Transgenicplants are regenerated from glufosinate ammonium resistant calli,acclimated in a humidity controlled growth chamber, and estab-lished in the green house. An estimated period of 9 months isrequired to obtain transgenic seeds, which includes a duration of4–5.5 months for transformation and regeneration procedures inthe laboratory and 3.5 months plant maturation and seed produc-tion in the greenhouse.

2 Materials

2.1 A. tumefaciens

Strains, Binary

Vectors, and DNA

Constructs

1. EHA101 [38] (see Note 1): This is a disarmed, nopaline-typeAgrobacterium tumefaciens strain derived from A281, a hyper-virulent A. tumefaciens strain causing crown gall on plants.This strain has a kanamycin-resistant (npt II) gene on the Tiplasmid.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred 17

2. pTF101.1 [39] (seeNote 2): This is an 11.6 kb standard binaryvector that derives from the pPZP binary vector series [40]. Ithas a broad host range pVS1 origin of replication and aspectinomycin-resistant marker gene (aadA) for bacterial selec-tion. A herbicide (phosphinothricin and its derivatives such asLiberty) resistant gene, phosphinothricin acetyl transferasegene from Streptomyces hygroscopicus, is used as a selectablemarker gene for maize transformation [41]. The bar genecassette is regulated by a double 35S promoter from the cauli-flower mosaic virus (CaMV), a translational enhancer fromtobacco etch virus (TEV) and the soybean vegetative storageprotein terminator. A multiple cloning site containing sixunique restriction sites (BamH I, EcoR I, Hind III, Sac I,Sma I, and Xba I) between the right border region and theplant selectable marker gene allows for introducing gene ofinterest (GOI) into pTF101.1.

3. pAHC25 [42] (seeNote 3): This is a 9.6 kb plasmid DNA thatcontains a plant selectable marker bar gene driven by the maizeubiquitin promoter (Pubi) and a screenable marker gus genedriven by the same promoter (Pubi).

2.2 Plant Material

and Plant Growth

Supplies

1. Seed for maize inbred line B104 can be requested from theIowa State University Research Foundation (ISURF) at:http://www.cad.iastate.edu/. Greenhouse-grown B104embryo donor ears are harvested 11 (summer) to 14 (winter)days after cross pollination when immature zygotic embryos(IZEs) are 1.5–2 mm long. After harvest, maize ears (in theirhusks and inside their pollination bag) are stored in a labora-tory refrigerator crisper (4 �C) for 1–5 days prior to use.

2. Standard 804 inserts (T.O. Plastic, Clearwater, MN).

3. Plant flat with drain holes, STF-1020-OPEN (T.O. Plastic,Clearwater, MN).

4. Standard 7.6 L nursery pots (PT-2, Nursery Supplies Inc.,Chambersburg, PA).

5. Humi-dome (Hummert International, Earth City, MO).

6. Metro-Mix 900 potting mix (Sun-Gro Horticulture, Agawam,MA).

7. Pollination bag (Lawson Pollinating Bags, Northfield, IL).

8. Shoot bag (Lawson Pollinating Bags, Northfield, IL).

9. Epsom salts solution (2.4 g/L): Dissolve 2.4 g Epsom salts in1 L of distilled water.

10. Calcium Chloride solution (400 ppm): Dissolve 16 mL of Rot-Stop® (Bonide Products, Oriskany, NY) in 1 L of distilledwater.


http://www.cad.iastate.edu/

2.3 Equipment

and Supplies

1. Milli-Q® ultrapure water purification systems (EMD MilliporeCorporation, Billerica, MA, USA).

2. PDS 1000/He biolistic gun (Bio-Rad, Hercules, CA, USA).

3. Ultrasonic bath sonicator (Fisher Scientific, Rockford, IL,USA).

4. 0.6 μm gold particles (Bio-Rad).

5. Macro-carrier and macro-carrier holder (Bio-Rad).

6. Rupture disks 650 psi (Bio-Rad).

7. Stopping screen (Bio-Rad).

8. Ethanol (see Note 4): 100%, keep at �20 �C.

2.4 Stock Solutions 1. MS Vitamins stock for Agrobacterium-mediated procedure:1000� stock solution modified from Murashige and Skoog[43] by increasing thiamine and decreasing nicotinic acid con-centrations. In 1 L deionized water, dissolve 2.0 g glycine,0.5 g pyridoxine HCl, 0.5 g thiamine HCl, and 50mg nicotinicacid. Filter sterilized, and stored at �20 �C in 40 mL aliquotswhich are thawed and used over a period of weeks.

2. MS Vitamins stock for biolistic-mediated procedure [43]:1000� pre-sterilized liquid stock solution from the manufac-turer (PhytoTechnology Laboratories, Shawnee Mission, KS).

3. Dicamba (30 mM): 0.0663 g of Dicamba (3,6-dichloro-2-methoxybenzoic acid) is dissolved in 1 mL 1 N KOH on lowheat until completely dissolved. Deionized water is added up toa final volume of 10 mL. The stock solution is stored at 4 �C.

4. 2,4-D (2 mg/mL): 200 mg of 2,4-dichlorophenoxyacetic acid(2,4-D) powder is dissolved in 1 mL 1 N KOH on a magneticstirrer with low heat until completely dissolved. Deionizedwater is added up to a final volume of 100 mL. The solutionis stored at 4 �C.

5. Silver Nitrate (50 mM): 0.85 g of silver nitrate is dissolved in100 mL of deionized water. The stock solution is filter ster-ilized, aliquoted, and stored in the dark at 4 �C for up to 1 year.

6. Acetosyringone (AS, 100 mM): 0.392 g of AS is dissolved in10 mL of dimethyl sulfoxide (DMSO). This solution is diluted1:1 with deionized water and filter-sterilized. Aliquots(0.5 mL) of stock solution are stored at �20 �C for up to6 months (see Note 5).

7. Cysteine (100 mg/mL): This solution is made fresh each timeco-cultivation medium is prepared. The stock solution is filtersterilized and added to cooled, autoclaved co-cultivationmedium for a final concentration of 300 mg/L. Any unusedcysteine stock solution is discarded.


8. Bialaphos (1 mg/mL): 100 mg of Bialaphos (Gold Biotech-nology, Olivette, MO) is dissolved in 100 mL of deionizedwater. The stock solution is filter sterilized and stored at 4 �Cfor up to 6 months.

9. Glufosinate (1 mg/mL): 100 mg of glufosinate ammonia isdissolved in 100 mL of deionized water. The stock solution isfilter sterilized and stored at 4 �C for up to 6 months.

10. Carbenicillin (250 mg/mL): 2.5 g of carbenicillin is dissolvedin 10 mL of deionized water. The stock solution is filter ster-ilized and stored in aliquots at �20 �C for up to 1 year.

11. Cefotaxime (200 mg/mL): 1.0 g of cefotaxime is dissolved in5 mL deionized water. The stock solution is filter sterilized,aliquoted, and stored at �20 �C for up to 2 months.

12. Vancomycin (200 mg/mL): 1.0 g of vancomycin hydrochlo-ride is dissolved in 5 mL deionized water. The stock solution isfilter sterilized, aliquoted, and stored at �20 �C for up to2 months.

13. Kanamycin sulfate: (10 mg/mL): 200 mg of kanamycin isdissolved in 20 mL deionized water. The stock solution issterilized by filtration and stored in aliquots at �20 �C for upto 6 months.

14. Spectinomycin sulfate: (100 mg/mL): 1 g of spectinomycin isdissolved in 10 mL deionized water. The stock solution issterilized by filtration and stored in aliquots at �20 �C for upto 6 months.

15. CaCl2 (2.5 M): dissolve 11 g CaCl2 · 6H2O in 20 mL ofdeionized water, filter sterilize and aliquot in 1.5 mL Eppen-dorf tubes. Store at �20 �C.

16. Spermidine (0.1 M): dissolve 145.25 mg spermidine (Sigma-Aldrich, St. Louis, MO) in 10 mL deionized water, sterilizethrough 0.45 μm syringe filter, and aliquot in 1.5 mL Eppen-dorf tubes. Store at �20 �C.

17. Maize ear sterilization solution (60% bleach): Mix 600 mLcommercial bleach (6% hypochlorite) with 400 mL deionizedwater, add one drop of surfactant Tween-20.

2.5 Culture Media All media are autoclaved at 121 �C (30–40 min for 4 L). Antibioticsand other heat-sensitive components are added after the media arecooled to approximately 60 �C (see Note 6). All solid media use100 � 25 mm Petri plates and are stored at room temperature(22–25 �C) in the dark.

2.5.1 Media for

Agrobacterium-Mediated

Transformation

Media is modified from Carvalho et al. [44] and co-cultivationmedia is modified to include L-cysteine (300 mg/L).

1. YEP medium [45]: 5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl2, pH 6.8. For solid medium, add 15 g/L Bacto-agar.


Antibiotics are added to autoclaved and cooled medium. Anti-biotic concentrations used for the pTF101.1 vector in strainEHA101 are 50 mg/L kanamycin and 100 mg/Lspectinomycin.

2. Infection (liquid): 4.3 g/L MS Salts, 1 mL/L modified MSvitamin stock, 0.5 mL/L Dicamba, 0.7 g/L L-proline,100 mg/L casein-hydrolysate, 100 mg/L myo-inositol,68.4 g/L sucrose, and 36 g/L glucose, pH 5.2. This mediumis filter sterilized and stored at 4 �C. AS (100 μM final concen-tration) is added prior to use.

3. Co-cultivation: 4.3 g/LMS salts, 0.5 mL/LDicamba, 0.7 g/LL-proline, 100 mg/L casein-hydrolysate, 100 mg/L myo-inositol, 30 g/L sucrose, adjust pH to 5.8, add 2.3 g/L gelrite,and autoclave. The following stock solutions are added to thecooled medium to final concentrations: modified MS vitaminstock (1 mL/L), silver nitrate (88 μM), AS (100 μM), L-cyste-ine (300 mg/L), and 100 mg/L cefotaxime (see Note 7).

4. Agro Selection I: 4.3 g/L MS salts, 0.5 mL/L Dicamba,0.7 g/L L-proline, 0.5 g/L MES (2-(N-morpholino)ethane-sulfonic acid), 100 mg/L casein-hydrolysate, 100 mg/L myo-inositol, 30 g/L sucrose, adjust pH to 5.8, add 2.3 g/L gelrite,and autoclave. After the medium is cooled the following filtersterilized stocks are added to final concentrations: modifiedMSvitamin (1 mL/L), silver nitrate (88 μM), bialaphos (2 mg/L),cefotaxime (100 mg/L), and vancomycin (100 mg/L).

5. Agro Selection II: The same as Agro Selection I medium exceptthat the bialaphos concentration is increased to 6 mg/L.

2.5.2 Media

for Biolistic-Gun

Transformation

Media outlined below is adapted from Armstrong and Green [46],McCain et al. [47], and Frame et al. [27]. Antibiotics carbenicillin isadded to the following media as a precaution to prevent any bacte-rial contamination (see Note 8).

1. MSE (Embryo pre-culture medium): 4.3 g/L MS salts, 2 mg/L 2,4 D, 100 mg/L myo-inositol, 0.7 g/L L-proline, 30 g/Lsucrose, 100 mg/L casein hydrolysate, adjust pH to 5.8, add2.5 g/L gelrite and autoclave. After the medium is cooleddown to about 60 �C, the following filter sterilized stocks areadded to obtain the desired concentrations: carbenicillin(250 mg/L), MS vitamin (1 mL/L), and silver nitrate(50 μM).

2. MSosm (pre and post bombardment osmotic treatment): MSEmedium plus 37 g/L sorbitol and 37 g/L mannitol, adjust pHto 5.8, add 2.5 g/L gelrite and autoclave. After the medium iscooled down, the following filter sterilized stocks are added toobtain the desired concentrations: carbenicillin (250 mg/L),MS vitamin (1 mL/L), and silver nitrate (50 μM).


3. Resting: 4.3 g/L MS salts, 0.5 mg/L Dicamba, 100 mg/Lmyo-inositol, 0.7 g/L proline, 0.5 g/L MES, 30 g/L sucrose,100 mg/L casein hydrolysate, adjust pH to 5.8, add 2.5 g/Lgelrite and autoclave. After the medium is cooled down, thefollowing filter sterilized stocks are added to obtain the desiredconcentrations: carbenicillin (250 mg/L), MS vitamin (1 mL/L), and silver nitrate (50 μM).

4. Gun Selection 1: 4.3 g/L MS salts, 0.5 mg/L Dicamba,100 mg/L myo-inositol, 0.7 g/L proline, 0.5 g/L MES,30 g/L sucrose, 100 mg/L casein hydrolysate, adjust pH to5.8, add 2.5 g/L gelrite and autoclave. After the medium iscooled down, the following filter sterilized stocks are added toobtain the desired concentrations: carbenicillin (250 mg/L),MS vitamin (1 mL/L), silver nitrate (88 μM), and Bialaphos(3 mg/L).

5. Gun Selection 2: 4.3 g/L MS salts, 0.5 mg/L Dicamba,100 mg/L myo-inositol, 0.7 g/L proline, 0.5 g/L MES,30 g/L sucrose, 100 mg/L casein hydrolysate, adjust pH to5.8, add 2.5 g/L gelrite and autoclave. After the medium iscooled down, the following filter sterilized stocks are added toobtain the desired concentrations: carbenicillin (250 mg/L),MS vitamin (1 mL/L), silver nitrate (88 μM), and Bialaphos(6 mg/L).

2.6 Culture Media

for Regeneration

2.6.1 Regeneration

Media for

Agrobacterium-Mediated

Transformation

1. Agro Regeneration I [47]: 4.3 g/L MS Salts, 100 mg/L myo-inositol, 60 g/L sucrose, adjust pH to 5.8, add 3 g/L gelriteand autoclave. After the medium is cooled down, the followingfilter sterilized stocks are added to obtain final concentrations:modified MS vitamin stock (1 mL/L), cefotaxime (100 mg/L), and glufosinate ammonia (6 mg/L, see Note 9).

2. Agro Regeneration II [47]: The same as Agro Regeneration Iexcept that sucrose concentration is reduced to 30 g/L, 2 mg/L bialaphos is added after autoclaving (see Note 10).

2.6.2 Regeneration

Media for Biolistic-Gun

Transformation

All media outlined below are adapted from Armstrong and Green[46], McCain et al. [47], and Frame et al. [27].

1. Gun Regeneration 1: 4.3 g/L MS Salts, 100 mg/L myo-inositol, 60 g/L sucrose, adjust pH to 5.8, add 3 g/L gelriteand autoclave. After the medium is cooled down, the followingfilter sterilized stocks are added to obtain the desired concen-trations: MS vitamin stock (1 mL/L), carbenicillin (250 mg/L), and glufosinate ammonia (6 mg/L, see Notes 8 and 9).

2. Gun Regeneration 2: 4.3 g/L MS Salts, 100 mg/L myo-inositol, 30 g/L sucrose, 3 g/L gelrite, adjust pH to 5.8, add3 g/L gelrite and autoclave. After the medium is cooled down,the following filter sterilized stocks are added to obtain thedesired concentrations: MS vitamin stock (1 mL/L), carbeni-cillin (250 mg/L), and glufosinate ammonia (3 mg/L).


3 Methods

3.1 Growing Donor

Plants for Immature

Embryo Production

1. Donor plant production is conducted in the Iowa State Uni-versity Agronomy Department Plant Transformation Facility(PTF) greenhouse. The PTF greenhouse operates on a 14:10photoperiod. Daytime settings for heating and cooling are23.9 �C and 28.3 �C, respectively. Nighttime settings for heat-ing and cooling are 18.3 �C and 20 �C, respectively. Supple-mental light fixtures provide about 130 μmol/m2/s 1 m belowfixture. Light intensity was measured in February before sun-rise (see Note 11).

2. Fill standard 804 inserts with Metro-Mix 900 mix and placeinto 27 � 54 cm plant flat with drain holes. Water the pottingmix just enough that excessive water is coming out of thebottom of the inserts.

3. Place one B104 seed per cell of the 804 and bury it 2.5 cm deepin the potting mix. Cover the flat with a plastic humi-dome (seeNote 12).

4. Seeds should germinate in approximately 4 days. Check soilmoisture daily and water the plants only when the soil becomesdry.

5. Six days after planting, remove the plastic humi-dome andcontinue watering as needed.

6. To prevent calcium deficiency, spray Epsom salts solution onday 6 and calcium chloride solution on days 7, 10, and 13 (seeNote 13).

7. After approximately 2 weeks, transplant each seedling (~4–5leaf stage) into a large pot.

8. Standard 6.1 L nursery pots (PT-2, Nursery Supplies Inc.,Chambersburg, PA) are filled to the top with Metro-Mix 900potting mix.

9. The pots are watered with fertilizer solution (see step 1 inSubheading 3.6.3) until the potting mix is saturated.

10. To transplant, gently remove a plant with soil adhering to theroot ball from the small pot and lay it on the wetted soil surfacein the middle of the big pot. Place the plant in a hole about1 cm deeper than the soil surface of the pot and back-fill thehole.

11. Plants are placed on 90 cm tall benches for 2 weeks. After2 weeks, plants are moved to the floor in double rows with25 cm centers and 100 cm spacing between rows.

12. Plants are watered as needed (see Subheading 3.6.3).


13. Ears start to emerge approximately 60 days after germination.Embryo donor plants are sib-pollinated for producing imma-ture zygotic embryo donor ears for transformation.

14. Cover any emerging ears with a wax-treated paper shoot bag toprevent pollen-contamination of the silks before controlledpollination (see Note 14).

15. When a fair number of silks have emerged, lift the shoot bagandmake a cut using sterile scissors (seeNote 15) about 2.5 cmabove the cob and place the shoot bag back over the earimmediately. This will ensure an even distribution of silks forpollinating the following day.

16. The next morning, use a pollination bag to collect pollen froma sibling B104 plant. Use pollen from tassels for which antheremergence occurred within the last 5 days (see Note 16).Sprinkle the pollen on the tufts of the emerged silks.

17. Immediately cover the pollinated ear with the pollination bag.Label the pollination bag using a permanent marker with thecross ID (female plant ID �male plant ID) and the cross date.

18. Ears are harvested between 10 and 14 days when immatureembryos are between 1.5 and 2.0 mm (see Notes 17 and 18).

3.2 Agrobacterium-

Mediated

Transformation

3.2.1 Agrobacterium

Preparation

1. The vector system (GOI construct þ A. tumefaciens strain) isstored as a glycerol stock at �80 �C.

2. As needed, and at least every 4 weeks, a “mother” plate is re-initiated from this long-term glycerol stock by streaking thebacteria on a YEP plate (with antibiotics) and growing it for2 days at 28 �C (see Note 19).

3. The “mother” plate is then kept in the refrigerator (4 �C) andused as a source plate for plating Agrobacteria cells at 28 �C for1 day in preparation for infection experiments.

3.2.2 Embryo Dissection 1. After de-husking the ear, insert a pair of numbered forceps intothe tip of the cob. The forceps serve as a handle for cobmanipulation and as a way of labeling the cob throughout thedissection/infection experiment (see Note 20).

2. In a laminar flow bench, place up to 15 prepared ears in asterile, 4 L beaker. Avoid using ears showing signs of tip rotor extensive kernel browning (see Note 21).

3. Add ~2 L of bleach solution to completely submerge the earswhile leaving the forceps handles protruding.

4. At the start of the 25 min disinfection, gently tap the beaker onthe bench-top to dislodge air pockets to ensure thoroughsurface sterilization of the cobs. Pour off the bleach solution(seeNote 22) and rinse the ears three times using at least 2 L ofsterile deionized water at each rinse. After the final wash is


discarded, the beaker of ears is left (covered) in the bench untildissection begins.

5. Working in a laminar flow bench, a surface-sterilized ear is laidon the inside of a large (150� 15mm) sterile Petri plate (eitherbase or lid can be used) (see Note 23).

6. Using aseptic technique, and holding on to the forceps, standthe ear on its end, and carefully cut off the top 2 mm of thekernel crowns with a sharp scalpel blade (see Note 24). ASteriguard 350 bead sterilizer (Inotech Biosystems Interna-tional, Rockville, MD, USA) is used for repeated sterilizationof utensils throughout this protocol (see Note 25).

7. To excise an immature zygotic embryo (IZE), insert the end ofa sharpened spatula between the endosperm and pericarp at thebasipetal side of the kernel and pop the endosperm out of theseed coat to expose the embryo axis side of the untouchedembryo. Gently coax the IZE (nested in the endosperm) ontothe spatula tip and transfer it directly to liquid infectionmedium (see Notes 26 and 27).

3.2.3 Agrobacterium

Infection

1. Grow Agrobacterium cultures for 24 h at 28 �C on solid YEPmedium amended with antibiotics (see Note 28).

2. To begin an infection experiment, scrape one full loop (3 mm)of bacteria culture from the plate and suspend in a 50 mLFalcon tube containing 6 mL infection medium supplementedwith 100 μM AS.

3. Fix the tube horizontally or vertically to a Vortex Genie(Thermo Fisher Scientific Inc) platform head and shake onlowest setting for 2 h at room temperature (22–27 �C). Useliquid infection medium (with AS), to adjust OD550 tobetween 0.30 and 0.40 just prior to use (see Note 29).

4. Once theAgrobacterium preculture step is complete, dissect upto 75 I.E. directly into a 2 mL Eppendorf tube filled withAgrobacteria-free infection medium (with 100 μM AS) (seeNote 30). These wash tubes are prepared 2 h ahead of timeand stored at 4 �C until dissection begins.

5. Remove the first wash and wash the embryos a second timewith 1 mL of the same medium. Remove the final wash, andadd 1 mL of Agrobacterium suspension (OD550 ¼ 0.30–0.40).

6. Gently invert the tube 20 times before resting it on its side (inthe dark) for 5 min with embryos submerged in the Agrobac-terium suspension.

3.2.4 Co-cultivation 1. After the 5 min infection, empty the tube of embryos andAgrobacterium suspension onto the surface of the co-cultivation medium with a flick of the wrist (see Note 31).


2. Once all the embryos are plated, use a 1 mL narrow-boredpipet tip to carefully encircle each embryo to suck up all stand-ing bacterial suspension from around the margins of eachembryo.

3. Co-cultivation plates are left in the laminar flow bench (lidsajar) for 1–3 h to dry further. Then, with the aid of a stereomicroscope, use a sterile spatula to orient each embryo scutel-lum side up.

4. Wrap plates with vent tape (air permeable adhesive tape, VallenSafety Supply, Irving, TX, USA) and incubate at 20 �C (dark)for 3 days in a biological incubator (see Note 32).

3.3 Biolistic

Gun-Mediated

Transformation

3.3.1 Embryo Dissection

1. See Subheading 3.2.2 for embryo dissection. At step 5, Theembryo is plated with the rounded scutellum side facing up ona filter paper (Whatman No. 4, 5.5 cm, Fisher) placed atop theMSE medium.

2. Place up to 40 embryos in a square grid in the middle of theplate, wrap with Parafilm® or vent tape and incubate at 28 �C inthe dark for 3 days prior to bombardment.

3.3.2 Gold Particle

Preparation

1. Bio-Rad 0.6 μm gold particles are washed with 100% ethanol.Under aseptic conditions, add 500 μL 100% ice-cold ethanol(stored in �20 �C) to a 10� gold tube containing 15 mg goldparticles (see Note 33).

2. Sonicate the tube in an ultrasonic water bath for 15 s.

3. Tap the closed tube to ensure all droplets move to the tubebottom. Allow the tube to sit on bench for about 30 min untilall the gold particles settle to the bottom.

4. Centrifuge the tube in a tabletop microcentrifuge for 60 s at805 � g. Remove ethanol supernatant.

5. Rinse the gold particle with 1 mL sterile ice-cold sterile deio-nized water, finger vortex the tube to lightly disturb the parti-cles. Allow the gold to settle to the bottom of the tube beforecentrifugation as described in step 4. Remove the deionizedwater supernatant.

6. Repeat step 5 twice. Spin at 2236 � g for 15 s for the thirdwashing step.

7. Resuspend the gold in 500 μL sterile deionized water. Place thetube in an ultrasonic water bath for 15 s, then immediatelytransfer the tube onto a multi-head vortex (setting 3) to keepthe gold particles in suspension.

8. Aliquot the 10� gold solution while on the vortex shaker intomicrofuge tubes at 1� concentration. To evenly distribute thegold suspension, aliquot the first 25 μL to each of the tenempty tubes. Then, beginning with the last tube, start


backward, aliquot another 25 μL to each tube. The 1� goldtubes can be stored in �20 �C until use. Each 1� gold can beused for 8–10 bombardment shots.

3.3.3 Coat the Gold with

DNA

DNA-gold coating procedure is performed on the day of bombard-ment as follows:

1. Thaw 1� gold suspension tubes and ultra-sonicate for 20 s.

2. In the flow bench, add 4 μL DNA solution (250 ng/μL) to thegold tube, finger vortex or pipet up/down four to five times tomix thoroughly (see Note 34).

3. Add 50 μL 2.5 M CaCl2, gently pipet the suspension up/downonce, and place the tube on Vortex Genie (setting 3).

4. Add 20 μL 0.1 M spermidine to the tube on the vortex andallow it to shake for 30 s. Close the tube and mix thoroughly byfinger vortexing. Place the DNA-coated gold mixture back onthe Vortex Genie (setting 3–4) and let it shake for 10–15 min.

5. Remove the tube from the vortex and set the reaction on thebench for 10 min to allow the gold particles to settle.

6. Centrifuge the tube in a desktop micro centrifuge at 2236 � gfor 20 s, discard the supernatant by gently pipetting withoutdisturbing the gold pellet (see Note 35).

7. Add 250 μL ice-cold 100% ethanol (see Note 36) to wash theDNA-gold pellet. Finger vortex to dislodge the pellet, thenrock the tube back and forth until the gold achieves a very“silty” smooth consistency dispersed at the base of the tube.Let the tubes sit on the bench for 10 min to allow the gold tosettle.

8. Centrifuge for 15 s at 2236 � g. Remove the supernatant bygentle pipetting (see Note 35).

9. Resuspend the DNA-gold pellet in 120 μL 100% ice coldethanol. Finger vortex to ensure complete suspension of thegold pellet. Place the tube on a vortex genie set at a low shakingspeed (setting 2–3) to keep the gold in suspension and evenlydistributed throughout bombardment procedure.

3.3.4 Pre-bombardment

Embryo Treatment

1. After 3 days incubation at 28 �C, a raised ridge is visible at thebase of the swollen immature zygotic embryos indicating TypeI callus initiation is underway and embryo scutellar tissue isready for bombardment.

2. Using the open circle of the gene gun plate-holder as a guide,draw a circle on the bottom-center of each MSosm mediumplate with a permanent marker. This indicates the target area ofthe embryo grid for bombardment. This target area outline isabout 3.5 cm in diameter.


3. Four hours prior to bombardment, use two sterile forceps togently transfer the embryos andfilter paper ontoosmoticmedium(MSosm), centering the embryo grid, not the filter paper, on the3.5 cm diameter circle marked on the MSosm plate. Ensurecorrect orientation of the embryos with the rounded, scutellumside facing up at bombardment (seeNote 37).

3.3.5 Loading the

Macro-Carriers

1. Assemble the sterilized DNA macro-carrier and macro-carrierholder by fitting pre-sterilized macro-carriers (10 min soak in70% ethanol then air-dry overnight in a laminar flow bench)into the stainless steel holders in a sterile dish

2. Pipette 12 μL of the DNA-gold mixture from the tube on theshaking votex onto the center of each macro-carrier (see Note38).

3. Let the micro-carriers air dry for 5–10 min in the laminar flowbench before bombardment.

3.3.6 Micro-Projectile

Bombardment

1. Wearing safety glasses, operate the Bio-Rad PDS 1000/Hebiolistic gun according to the instruction manual.

2. Place a rupture disk (650 psi) in the retaining cap and handscrew the cap back in place. Tighten securely using the torquewrench.

3. Prepare a launch assembly by first laying in place a stoppingscreen followed by an inverted, pre-loaded macro-carrierholder (see Subheading 3.3.5). Screw on the launch assemblylid to hold these parts in place.

4. Place the launch assembly in the gun chamber by sliding it intoplace immediately below the helium nozzle, and set the gapdistance (6 mm).

5. Place an uncovered petri-dish containing pretreated maizeembryos onto the shelf at a preferred distance (6 cm) fromthe stopping screen.

6. Close the gun chamber door and activate the vacuum switchuntil it reaches 28 in. Hg. Press the fire switch until diskruptures with a pop sound in the chamber.

7. Press the vacuum release switch and remove the plate contain-ing the bombarded tissue.

8. Prepare the gun for the next bombardment by replacing thespent rupture disk, macro-carrier and stopping screen (biohaz-ard disposables).

9. Repeat steps 2–8 for the next shot (see Note 39).

3.3.7 Post-bombardment

Treatment

1. Gently wrap bombarded embryos (still on filter paper onMSosm) with parafilm or vent tape and incubate at 28 �C in adark incubator for recovery.


2. 20–24 h post-bombardment, transfer embryos individually offthe filter paper on MSosm directly onto resting medium tocontinue callus initiation. Position embryos scutellum side up(see Note 40).

3. Wrap plates with Parafilm® and incubate in a dark chamber at28 �C for 10 days. Resting can be extended for up to 2 weeks.

3.4 Selection

for Stable

Transformation Events

3.4.1 For Agrobacterium

Events

1. After 3 days on co-cultivation medium, use a sterile spatula totransfer all embryos to Agro Selection I medium (35 I.E. perplate) containing 2 mg/L bialaphos (see Note 41). Be sure allembryos are oriented scutellum side up. Wrap plates with venttape and incubate in a dark incubator at 28 �C for 2 weeks (seeNote 42).

2. Using sterile forceps, transfer each embryo to Agro Selection IImedium containing 6 mg/L bialaphos. Plates are wrapped withParafilm® and incubated in the dark (28 �C) for 3 weeks.

3. After the first 3-week period on Agro Selection II medium,quantitative and qualitative differences in the Type I callusproliferating from each still-intact IZE are evident. Using astereo scope and sterile scalpels, good quality (smooth, notgrainy-surfaced, and light, not dark-yellow), proliferating,embryogenic callus is transferred away from its IZE embryo-carcass and broken into smaller pieces on a fresh plate of AgroSelection II medium. Plates are incubated for another 3-weeksunder similar conditions (see Note 43).

4. The unique IZE lineage of each callus line is tracked by using apermanent marker pen to circle each single IZE-derived callus-line on the bottom of the petri-plate. Continued vigorousproliferation of embryogenic, Type I callus after this “picking”step indicates that the callus event is bialaphos resistant. Inde-pendent, putative callus events are subcultured every 2 weeksthereafter to fresh Agro SelectionMedium II until a full plate ofbialaphos resistant callus is achieved (see Note 44).

5. While some B104 putative Type I callus events can be identifiedby as early as 10 weeks after infection, events generally take4 months to bulk up to one full plate of callus in preparation forregeneration (see Note 45).

3.4.2 For Biolistic Gun

Events

1. After 10–14 days on resting medium, transfer bombardedembryos to Gun Selection 1 medium (3 mg/L bialaphos) tobegin the recovery of transformed cells. Wrap plates with Par-afilm® and incubate at 28 �C in the dark for 10–14 days.

2. Subculture the embryos to fresh Gun Selection 1 medium andincubate as described above.

3. After 3–4 weeks, fused callus pieces initiated on Gun Selection1 medium are transferred to Gun Selection 2 medium (6 mg/


bialaphos). Subculture three times on Gun Selection 2 mediumat 2-week intervals (see Note 46). Incubate in the dark at28 �C.

4. Similar to steps 3–5 in Subheading 3.4.1, resistant callus areoften distinguished from non-transformed ones by their vigor-ous growth and proliferation. Embryogenic callus can be iden-tified through a dissecting scope, carved up into small sizedpieces between 0.25 and 0.50 cm with a pair of sterile forceps,and transferred to fresh Gun Selection 2 medium. Continueselection for up to 4 additional weeks (2 subcultures) in thedark at 28 �C.

3.5 Regeneration

of Transgenic Plants

3.5.1 For Agrobacterium

Events

1. Using a stereo microscope, transfer 15–20 0.5 cm embryo-genic type I callus pieces (pried apart, not cut) from the surfaceof Agro Selection II medium to the surface of Agro Regenera-tion I medium (6 mg/L glufosinate ammonia). Multiplesomatic embryos may be fused together in one piece of callus.Wrap Petri plates with vent tape and incubate in the dark(25 �C).

2. After 3 weeks, most but not all of the callus pieces will produceone or more mature somatic embryo. B104 mature somaticembryos will appear opaque and white, but in many cases willbe fused together.

3. Using a stereo microscope, pry these mature somatic embryosapart from any unhardened (dark yellow, translucent) callusand from each other where possible without damaging embryointegrity.

4. Transfer these pieces (fused or not), 15 per plate, to AgroRegeneration II medium (2 mg/L bialaphos) for germinationin the light (25 �C, 80–100 μE/m2/s light intensity, 16:8photoperiod). Germinated B104 plantlets with roots andshoots are ready for transfer to soil 14 days later (seeNote 47).

3.5.2 For Biolistic Gun

Events

1. Similar to step 1 in Subheading 3.5.1, bialaphos resistantembryogenic callus pieces are transferred to Gun Regeneration1 medium (6 mg/L glufosinate ammonia). Plates are wrappedwith vent tape such as 3M surgical tape with micropores andincubated for 3 weeks at 25 �C in the dark.

2. Similar to steps 2 and 3 in Subheading 3.5.1, mature somaticembryos are transferred to Gun Regeneration 2 medium(3 mg/L glufosinate ammonia) and wrapped with vent tape.Plates are incubated in a 25 �C light chamber (80–100 μE/m2/s light intensity, 16:8 photoperiod).

3. Both shoot and root formation were visible as early as 1 weekafter light incubation (see Note 48). Regenerated rootedshoots are transferred to soil and acclimatized in a humiditycontrolled growth chamber.


3.6 Plant

Acclimatization

3.6.1 Moving Plants from

Petri-Dish to Soil

1. Transplanting is done on a per plant basis when plantlet leavesare ~5 cm long and a healthy set of roots have been developed.Smaller plantlets in the same petri-plate are returned to thelight until they are large enough for transplant.

2. See step 2 of Subheading 3.1 for pot/soil preparation.

3. In a laminar flow bench, remove a plantlet from the Petri dishby lifting it gently from the callus ball at the juncture betweenthe shoot and the root using a pair of sterile forceps (see Note49). Remove all media still attached to the root surface.

4. Create a hole in the potting mix with your finger. While hold-ing the plant by the callus ball, place all roots in the hole andgently cover the roots up to the bottom of the callus-ball.

3.6.2 Plant Hardening in

the Growth Chamber and

Greenhouse Acclimation

1. Cover the flat with a plastic humi-dome with ventilation holesopen and place the tray in a growth chamber (E-41L2, PercivalScientific, Perry, IA). The growth chamber is set to a 16:8photoperiod, 27 �C day and 22 �C night at a light intensity of130 μmols/m2/s on leaf surface.

2. The trays are checked twice daily and watered as needed withtap water stored inside the growth chamber. The plantletsshould neither be allowed to dry out completely nor shouldthey be overwatered (see Note 50).

3. The humi-dome is removed 4 days after transplanting. The flatis moved to the greenhouse 10 days after transplanting (seeNote 51).

4. In the greenhouse, the soil moisture is monitored daily andwatered as needed with a fertilizer solution (see step 1 inSubheading 3.6.3)

5. On the second day in the greenhouse, the plants are sprayedwith Epsom salts solution using a hand held sprayer.

6. On days 3 and 6, the plants are sprayed with Calcium Chloridesolution (see Note 52).

7. Plants are transplanted to larger pots 1 week after being movedto the greenhouse. See steps 7–12 in Subheading 3.1 fortransplant to big pots.

3.6.3 Watering/

Fertilization

1. Using tempered water, pots are watered on an as-needed basiswith a fertilizer solution of Jack’s Professional LX (J.R. Peters,Allentown, PA) at 75 ppm, N plus CalciumNitrate (Yara NorthAmerica, Stockton, CA) at 16 ppm, N plus Magnisal (HaifaChemicals, Haifa, Israel) at 9 ppm, N plus Sprint 138 (BeckerUnderwood, Ames, IA) red iron at 5 ppm Fe (see Note 53).

2. Watering is accomplished by slowly filling the pot until thewater overflows the top. Pots are not watered again until the


soil moisture is almost depleted but before plants becomewilted.

3. The frequency of watering will depend on the plant size and theseason. During the summer months, large plants may need tobe watered twice daily.

3.6.4 Pollination 1. Tassels from all transgenic plants are removed before anthershave a chance to emerge to ensure that no transgenic pollen ispresent in the greenhouse.

2. Upon ear emergence, the ear is carefully covered with a shootbag to ensure controlled pollination.

3. Follow steps 14–16 in Subheading 3.1 to perform transgenicplant pollination (see Note 54).

3.6.5 Dry Down and Seed

Harvest

1. Plants are watered as needed for 25 days after pollination. Donot overwater plants during this period as overwatering maycause seeds to sprout on the cob.

2. At 7–10 days post pollination, lift the pollination bag off the earto facilitate air drying of cob (see Note 55).

3. At day 25 post pollination, stop all watering and open the husksto allow the kernels to dry.

4. Check the seeds at day 40 post pollination for seed moisture.Seeds are normally ready for harvest (see Note 56).

5. Seeds are counted, placed in kraft-colored, labeled, coin envelopesand stored in a cold, dark, humidity controlled environment.

4 Notes

1. Other Agrobacterium tumefaciens strains such as EHA105,AGL-1, GV3101, and LBA4404 can be used for transforma-tion [48]. EHA101 has been used extensively and successfullyin our laboratory.

2. We have been successful with other standard vectors with thepPZP backbone [48]. However, it is interesting to note that wehave had limited and poor success with constructs derived frompCAMBIA-based vector backbone. We cannot explain the rea-son for this observation.

3. Plasmid DNA used for biolistic gun-mediated maize transfor-mation usually contains a selectable marker gene cassette and agene of interest (GOI) cassette. These two gene cassettes can beon the same plasmid or two separate plasmids. Bombardment ofa two-plasmid DNAmixture (mixed as equal molarity) is knownas co-bombardment. One of the most frequently used selectablemarker genes for maize transformation is the bar gene.


4. It is important to use pure ethanol (200 proof, 100%) for goldwashing, so no aqueous residue remains. Pure ethanol can bepurchased from Sigma Aldrich or other laboratory suppliesvendor.

5. AS stock solution retrieved from the freezer may contain pre-cipitate that can be redissolved by vortexing at room tempera-ture for 15 min.

6. Media containing MS salts and gelrite solidify readily. Makesure that plates are poured immediately after antibiotics areadded to the cooled media to avoid premature solidification.Media can be kept in 60 �C water bath for cooling. Media canalso be cooled for 30 min in a laminar flow bench (lids off)before antibiotics and other supplements are added.

7. This co-cultivation media is used the following day (1 day old).Left over co-cultivation medium is discarded.

8. Carbenicillin is added to forestall any bacterial cross contami-nation, especially in lab settings where shared equipment, uten-sils, media storage, and bench space are used for bothAgrobacterium and biolistic experiments. However, it doesnot have to be included in media throughout the entire processas the antibiotic is expensive and ecologically unfriendly.

9. We replace bialaphos (~$1200/g) with glufosinate ammonia (~$200/g) in this step to be cost effective.

10. We use 4 g/L gelrite in Regeneration II medium if the Petriplates are to be mailed to collaborators. The higher gelriteconcentration minimizes gelrite breakup in Petri plates duringshipment.

11. The greenhouse facility consists of two A-frame greenhousesdesigned by Ludy Greenhouse (Ludy Greenhouse Mfg. Corp.,New Madison, OH). Each greenhouse has approximately140 m2 of usable growing space. The rooftop is 16 mm twinwall acrylic (Acrylite Alltop, Evonik Cyro LLC, Parsippany,NJ). The room is equipped with the Argus Titan II (ArgusControl Systems Ltd., Surrey, British Columbia, Canada) envi-ronmental control system. Greenhouse location and exteriormaterial can influence management practices. Greenhouseplant-care protocols should be used as a reference guide andadjusted based on location, climate, or needs.

The PTF greenhouse is heated using hot water. Cooling isachieved using three different systems including ridge ventwindow openings, evaporative coolers, and vertical chilledwater air handlers.

Supplemental lighting is a 50/50 mixture of 1000 WMetalHalide and 1000 W High Pressure Sodium Fixtures mounted2.7 m above ground. Supplemental light fixtures are programedfor 14 h and provide about 130 μmol/m2/s at a height of 1 m


below fixture. An outdoor light sensor is mounted on the top ofthe greenhouse. The sensor will turn the lights off when naturallight reaches 350 W/m2 for over 10 min and lights will returnon when natural light drops below 300 W/m2 for longer than10 min.

12. To ensure a steady flow of donor plants, seeds are planted everyTuesday and Friday.

13. Inbred B104 is prone to calcium deficiency when grown undergreenhouse conditions, especially during the late fall season.Spraying plants with 400 ppm of CaCl2 can alleviate or reducethe symptoms. A third application of the CaCl2 solution can bemade 3 days later if needed. However, more than three applica-tions in a 2 week time period can cause yellowing. To ensureproper calcium absorption, an application of Epsom salts(MgSO4) is applied at a rate of 2.4 g/L 1 day prior to thefirst CaCl2 application.

14. Secure shoot bags over ear shoots to prevent them falling offdue to air movement. Any plant possessing an uncovered,silking ear is immediately discarded as it is assumed to be“contaminated” by unwanted pollen.

15. Scissors are cleaned with 70% ethanol prior to cutting silks toprevent cross-contaminating ears with fungal spores orbacteria.

16. The dates of tassel emergence, shoot emergence, and antheremergence (pollen) are recorded on a shoot bag that is stapledaround the stalk of the plant.

17. Rate of embryo growth can vary based on temperature andexterior weather conditions. Warm, sunny days will produceembryos more quickly than long periods of cold and cloudyconditions.

18. Harvested greenhouse ears are stored and used within 5 days.For example, maize ears harvested Thursday through Mondayare used in Tuesday experiments. Ears harvested Mondaythrough Thursday are used in Friday experiments.

19. Mother plates can also be started from stab cultures or streakedplates.

20. The ear # assigned at infection is cross-linked with greenhousecrossing information for that donor plant so we can traceplanting date and harvest date of ears, as well as assign embryosfrom the same ear to different treatments if testing infectionparameters.

21. If only a few kernels show browning, remove the whole kerneland spray exposed area with 70% ethanol prior to surfacesterilization with bleach.


22. Do not pour the bleach solution down the sink as it is a strongcorrosive reagent. The waste solution should be properly col-lected, labeled, and disposed per institutional safety proce-dures. The 60% bleach solution can be reused three timeswithout losing its efficacy for maize ear disinfection.

23. Use a fresh Petri plate for each new ear as spent kernel caps andextracted endosperm waste interferes with dissection.

24. A new scalpel blade is used every third ear as B104 kernel seedcoats are thick and tend to dull scalpel blades quite easily.

25. Work with at least three pairs of scalpels so each pair is suffi-ciently cooled before use after each re-sterilization.

26. Avoid digging too deeply with spatula or you will dislodge thewhole kernel, not just the endosperm. Optimal immatureembryo size ranges from 1.5 to 1.8 mm. Do not use if IZE is<1.2 mm or >2.0 mm.

27. The embryo dissection step requires a bit of practice to avoidbreaking or causing injury to the embryo as this may affectsubsequent callus formation.

28. Pre-streaking bacteria at 28 �C for 1 day offers more schedul-ing flexibility for experiments than does 19 �C/3 days. Cur-rently, we make co-cultivation medium and streakAgrobacterium plates on Monday and Thursday. We infect onTuesday and Friday, and take off embryos to selection mediumon Mondays and Fridays (after 3 days co-cultivation).

29. When diluting Agrobacterium culture, consider the number oftubes of embryos you plan to dissect so that you have enoughdiluted Agrobacterium suspension to work with.

30. Fill the wash tubes to the brim such that simply gliding thespatula holding the extracted IZE across the surface of theliquid allows surface tension to transfer the IZE into thewash. Submerging the spatula into the wash each time anIZE is extracted is not only time consuming but also leads toalready extracted embryos sticking to the spatula and beingcarried back out of the wash.

31. Reserve some of theAgrobacterium solution in a 1mL pipet tipto use for rinsing out any embryos adhering to the inside of thetube. Alternatively, extra media for tube-rinsing can bepipetted off the surface of the co-cultivation medium if needed.

32. Transgenic events have also been successfully recovered fromco-cultivation plates incubated at 25 �C.

33. It is a good idea to prepare an even number of tubes of gold atone time so that they balance each other in the centrifuge steps.

34. Typically, 100–150 ng DNA (single plasmid) is used for asingle shot. If co-bombardment (bombarding a DNA mixture


of two plasmid DNAs) is performed, a 1:3 ratio of DNAs(300 ng of selectable marker DNA þ threefold more GOI)will be mixed.

35. To avoid disturbing the tear-shaped gold pellet, tilt the micro-fuge tube slightly and pipette away from the pellet.

36. It is very important not to remove the 100% ethanol from�20 �C freezer too soon because it must be freezing cold.For convenience, aliquots in microfuge tubes may also bestored in a prechilled freezer block rack to help maintain theethanol at freezing cold after removal from �20 �C freezer andprior to use.

37. For embryo arrangement and orienting, use a rounded microspoon (spatula) instead of gripping the fragile embryo withsharp nosed forceps. Use a dissecting microscope to confirmcorrect orientation of the embryos. Ascertaining that the scu-tellum side is facing up during bombardment is crucial becausecallus formation and subsequent transformed cells start at thissurface.

38. For this step, pipette the gold mixture onto the center of themacrocarrier disk and spread gently in a circular motion withthe pipette tip to ensure an even distribution of the suspensionover the inner, target circle. It is important that this step iscarried out as quickly as possible to avoid evaporation of theremaining suspension.

39. When the bombardment is complete, close the valve on thehelium tank, release all pressure in the line, turn off the vacuumpump, and switch off the gene gun. Clean bombardmentchamber and gun components with 70% ethanol. Autoclavemacro-carrier holders for reuse. Dispose of all plasmid waste inbiohazard bags and autoclave before discarding

40. Arrange up to 30 evenly spaced embryos per plate, transfer allembryos regardless of size and appearance.

41. To expedite the recovery of putative events, we no longer use aresting period as described previously [37]. Instead, embryosare transferred directly to Agro Selection I after co-cultivation.In three separate experiments, %TF observed for IZE rested1 week prior to selection was the same as that for IZE that weretransferred directly to Agro Selection I after co-cultivation.Note that optimal embryo size and a 3-day rather than 2-dayco-cultivation contribute to the success of this revisedprotocol.

42. Embryogenic callus induction frequency (% ECIF) should be100% after 2 weeks on Agro Selection I Medium.

43. Callus piece size, typically 0.5 cm or less, often depends on howmuch good quality callus is harvested from any given IZE


44. Callus piece size is no bigger than 0.5 cm during bulking toensure rigorous, ongoing selection for bialaphos resistant callussectors, i.e., to “clean up” the callus event. A 2-week subcul-ture cycle is preferable to 3 weeks to ensure proliferation ofgood quality, embryogenic callus.

45. AverageAgrobacterium-mediated transformation frequency (%TF) for B104 using this protocol is 4%, or four independent,bialaphos-resistant type I callus events per 100 infected (andselected) IZEs.

46. B104 callus culture should be transferred to fresh medium14 days or earlier, to avoid formation of soft mushy callusthat are often non-embryogenic.

47. Regeneration II media plates will dry out after 2 weeks in thelight if rooted/shooted plantlets have not yet formed by then.To avoid desiccation of plant tissues, subculture all callus piecescontaining germinating embryos to a fresh Regeneration IImedia plate until plantlets are fully elongated.

48. Biolistic gun-mediated transformation frequencies by experi-ment (12 experiments of 250 bombarded embryos each) usingthis protocol ranged from 6 to 13% (or, depending on theexperiment, between 6 and 13 independent, bialaphos-resistant type I callus events per 100 bombarded and selectedIZEs).

49. Perform this step under an aseptic condition to ensure remain-ing cultures in the plate sterile while returned for continuousincubation. If the plates are no longer needed after transplant,this step can be done in non-aseptic environment.

50. May have to reposition the plantlets after the first watering.Typically only one or two waterings are needed.

51. Humi-domes may be left on longer if the plantlets are havingproblems establishing. But do not leave it on for longer than17 days.

52. Nutrient deficiency will start to show after about 14 days.Plants should be about 16 cm from soil surface to leaf tip.

53. Actual fertilizer rate is based on weekly soil Electrical Conduc-tivity (EC) and pH readings. Using the pour-thru procedure,check EC and pH for container plants [49]. EC should bebetween 1.8 and 2 and pH should be between 5.5 and 6.5.Sulfuric Acid can be added to the fertilizer if pH is above 6.5and lime can be added to the potting mix if pH is below 5.5.

54. We always use wild-type non-transgenic pollen to pollinatetransgenic plant silks. We do not perform transgenic self-pollination for two reasons: (1) male and female flowers donot always emerge at the same time from tissue culture-derived


maize plants; (2) male flowers are removed to prevent trans-genic pollen contamination in greenhouse.

55. Pollination bags are only lifted when there is little risk of crosspollination. Silks can remain viable for up to 17 days, so bagsshould remain down if pollen exposure is a possibility.

56. Seeds may take more time to dry under high humidity condi-tions. If needed the cob can be removed, placed on a table, andallowed to dry.

Acknowledgments

The authors wish to thank Marcy Main, Haleigh Summers, SarahSalmon, Stephanie Widener, Aaron Brand, and Katey Warnberg fortheir contributions to this work. This project was partially sup-ported by the USDA National Institute of Food and Agriculture,Hatch project number # IOW05162, and by State of Iowa funds.

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