Abstract Sonication-assisted Agrobacterium-mediatedtransformation (SAAT) tremendously improves the effi-ciency of Agrobacterium infection by introducing largenumbers of microwounds into the target plant tissue. Us-ing immature cotyledons of soybean as explants, we eval-uated the effects of the following parameters on transientβ-glucuronidase (GUS) activity: cultivars, binary vectors,optical density of Agrobacterium during infection, dura-tion of sonication treatment, co-culture conditions, lengthof explant preculture and addition of acetosyringone dur-ing co-culture. The extent of tissue disruption caused bysonication was also determined. The highest GUS expres-sion was obtained when immature cotyledons were soni-cated for 2 s in the presence of Agrobacterium (0.11OD600nm) followed by co-cultivation with the abaxial sideof the explant in contact with the culture medium for 3 daysat 27°C. The addition of acetosyringone to the co-culturemedium enhanced transient expression. No differenceswere observed when different cultivars or binary vectorswere used. Cotyledons sonicated for 2 s had moderate tissue disruption, while the longer treatments resulted inmore extensive damage.

Key words Agrobacterium tumefaciens · Glycine max ·Transient expression · SAAT · Transformation

Introduction

Agrobacterium provides one of the main vehicles for in-troducing foreign DNA into plants. A number of plant spe-cies (Wordragen and Dons, 1992; Fisk and Dandekar,1993) and even yeast (Bundock et al. 1995) have beentransformed by Agrobacterium. However, Agrobacterium-mediated transformation of many plants, including soy-bean, remains inefficient. Agrobacterium-mediated trans-formation of soybean has been reported using cotyledo-nary nodes (Hinchee et al. 1988) and immature cotyledons(Parrott et al. 1989), but the transformation frequencieswere very low and the plants recovered were often chi-meric (Parrott et al. 1989).

In order to enhance transformation rates, improvementshave been made in the delivery of the bacterium (Bidneyet al. 1992), and vectors have been modified to provideconstitutive expression of vir genes (Hansen et al. 1994;Ishida et al. 1996). Although transformation rates havebeen significantly improved using these modifications, in-creases in efficiency are still needed.

Recently, sonication has been used to enhance Agrobac-terium-mediated transformation of many different plantspecies (Trick and Finer 1997). Sonication-assisted Agro-bacterium-mediated transformation (SAAT) consists ofsubjecting the target tissue to ultrasound while immersedin an Agrobacterium suspension. The enhanced transfor-mation rates using SAAT probably result from micro-wounding, where the energy released by cavitation (Friz-zel 1988) causes small wounds both on the surface of anddeep within the target tissue. Sonication enhances the de-livery of naked DNA into tobacco protoplasts (Joersbo andBrunstedt 1990) and seedlings (Zhang et al. 1991) but hasonly recently been shown to enhance Agrobacterium-me-diated transformation of plant tissue (Trick and Finer1997).

Plant Cell Reports (1998) 17: 752–759 © Springer-Verlag 1998

Received: 1 October 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998

E. R. Santarém · H. N. Trick · J. S. Essig · J. J. Finer

Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression

Communicated by J. M. Widholm

E. R. Santarém1 · H. N. Trick2 · J. S. Essig2 · J. J. Finer (½)Department of Horticulture and Crop Science, Plant Molecular Biology and Biotechnology Program, OARDC,The Ohio State University, Wooster, OH, 44691, USAFax: 330–263–3887e-mail: finer.1@osu.edu

Present addresses:1 Universidade de Cruz Alta, Laboratório de Cultura de Tecidos Vegetais in vitro, Campus Universitario, Caixa Postal 858, Cruz Alta – RS 98025-810, Brazil2 Department of Plant Pathology, 3729 Throckmorton Plant Sciences Center, Kansas State Univer-sity, Manhattan, KS, 66506-5502, USA

Mention of a trademark or proprietary products does not constitutea guarantee or warranty of the product by OSU/OARDC, and alsodoes not imply approval to the exclusion of other products that maybe suitable.OARDC Journal Article No. 116-97

In this report, we describe the optimization of Agrobac-terium-mediated transient transformation of immature cot-yledons of soybean using SAAT. The effects of SAAT onthe structural integrity of the explants were also evaluated.

Materials and methods

Plant material

Immature pods of soybean [Glycine max (L.) Merrill], cvs ‘Jack’, ‘Chapman’ and ‘Kunitz’, were collected from plants grown in thegreenhouse under a 14-h light photoperiod at 28°C. Pods were disin-fested in a 20% commercial bleach solution containing 0.04% Tween-20 for 20 min and rinsed four times in sterile water. Immature seeds(4–5 mm in length) were aseptically removed from the pods and theend containing the embryonic axis was cut off and discarded. The twocotyledons were then removed from the seed coat, separated andplaced on D40 medium (Santarém et al. 1997) consisting of MS salts(Murashige and Skoog 1962), B5 vitamins (Gamborg et al. 1968), 6%sucrose and 40 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), solidi-fied with 0.2% Gelrite™ (Merck & Co, Rahway, N. J.), pH 7. Ex-plants were maintained on D40 medium with or without 0.4 M man-nitol for approximately 3 h until SAAT treatments were performed.

Bacterial strains, plasmids and culture conditions

Agrobacterium tumefaciens, strain EHA105 (Hood et al. 1993), wasused as host for all the plasmids. Plasmid pIG121Hm (Hiei et al.1994) or Vec035 were used for optimization work (Fig. 1). PlasmidVec035 was constructed by replacing the β-glucuronidase (GUS)gene in the plasmid pBIG-Hyg (Becker 1990) with an intron-con-taining GUS gene from p35SGUSINT (Vancanneyt et al. 1990).

Agrobacterium was grown overnight in a modified Luria-Berta-ni medium (Sambrook et al. 1989) containing half the normal levelof NaCl (5 g/l) and supplemented with 5 g/l sucrose and 50 mg/l ka-namycin. Bacteria were centrifuged at 1500 g for 10 min, washedtwice with equal volumes of liquid D40 medium and re-centrifugedas above. The bacterial pellet was finally resuspended in liquid D40medium, and the OD600nm was determined.

SAAT treatment

Ten cotyledons were placed in 1.5 ml microcentrifuge tubes contain-ing 0.5 ml of the Agrobacterium suspension. Cotyledons were gent-ly resuspended and placed in a float at the center of a bath sonicator(PC5 model, 55 kHz, L&R Manufacturing Company, Kearny, N. J.).The sonicator was controlled by an electronic timer. After SAATtreatment, cotyledons were removed from the tubes, placed on ster-

ile filter paper to blot off excess bacteria and then transferred to co-culture medium.

Pre- and post-SAAT culture conditions

To evaluate the effects of sonication duration, cotyledons were sonicated for 0.1, 0.5, 1, 2, 5, 7 or 10 s using Agrobacterium at 0.1OD600nm, and transient expression levels were recorded. In a subsequent experiment, a combination of OD600nm (0.05, 0.1 and 0.2) with different co-culture periods (2, 3 and 4 days) and temper-atures of 23°C and 27° C was evaluated using a 2-s sonication treat-ment.

To determine the effects of acetosyringone (AS) during the co-culture period, we evaluated transient GUS expression using cotyle-dons co-cultured on D40 medium with or without 100 µM AS, aftersonication treatments of 2 s and 5 s.

The effect of explant orientation was evaluated by placing thecotyledons adaxial side facing up or down during co-culture after a2-s sonication treatment. Transient expression was scored on bothsides of the cotyledons.

A preculture treatment period was evaluated using cotyledonsthat were excised and either sonicated immediately or maintained onD40 medium for 1, 2, 3 or 4 days before a sonication treatment of2 s at 0.2 OD600nm.

After co-culture in all cases, cotyledons were rinsed in sterile wa-ter, blotted dry on sterile filter paper and placed on D40 medium sup-plemented with 500 mg/l cefotaxime to prevent Agrobacteriumgrowth.

Wounding assessment

The contribution of sonication and Agrobacterium to overall tissueresponse was determined by sonicating the cotyledons with or with-out Agrobacterium. Control treatments consisted of either no treat-ment or Agrobacterium with no sonication. Sonication was per-formed for 2 s and 0.2 OD600nm. In an attempt to control tissue dis-ruption from the sonication, D40 medium containing 0.4 M manni-tol was used for maintaining cotyledons prior to SAAT. Mannitol(0.4 M) was also added to the D40 medium used to resuspend thebacteria. The OD400nm of the plant/bacterial exudate after sonicationwas measured as a means of damage assessment.

Histochemical GUS assays

Histochemical GUS assays were performed 2 days after transfer ofthe cotyledons to the medium containing 500 mg/l cefotaxime. Cot-yledons were placed in a GUS assay mix (Jefferson 1987) and incu-bated overnight at 37°C with shaking at 80 rpm. The GUS assay mixwas removed, and the tissue was rinsed twice with 70% ethanol. GUSactivity was then determined by placing the cotyledons on a grid andestimating the percentage of the cotyledon surface that showed bluesectors under a dissecting microscope.

753

HindIIIXbaI

Sal ISacI Sal I EcoRI HindIII

PIG121HM

1 kb

Vec035

BamHI Pst I Bgl I

HindIII

NcoI SnaBIKpnI HindIIISacI EcoRI

BglIBgl I

HPTIIGUS INT 35S TNos

LBTNos35STNosNPTIIPNos

RB

RBTNos HPTII Pnos 35S GUS INT TNos

LB

Fig. 1 T-DNA region ofpIG121Hm and Vec035. Ar-rows indicate the direction oftranscription. RB Right border,LB Left border, HPTII hygro-mycin resistance gene, NPTIIneomycin phosphotransferasegene, GUS INT intron-contain-ing GUS gene, PNos promoternopaline synthase, 35S promot-er 35S of CaMV, TNos termina-tor nopaline synthase

Electron microscopy

For scanning electron microscopy, cotyledons were fixed in 0.2 M

potassium phosphate buffer (pH 7.0) containing 3% glutaraldehyde,2% paraformaldehyde and 1.5% acrolein for 2 h at room tempera-ture. Samples were then dehydrated in an ethanol series (50–100%ethanol at 10 min each), critical point-dried, sputter-coated with plat-inum and viewed on a ISI-40 scanning electron microscope as de-scribed earlier (Trick and Finer 1997).

Data analysis

Experiments were performed with three replicates per treatment.Means of percentage of the cotyledon surface covered with blue sec-tors were calculated. Transformed data (sq.rt+0.5) was analyzed byANOVA. Treatments were separated using Fisher’s Least SignificantDifference test (α =0.05).

Results and discussion

SAAT treatment

The duration of sonication had a dramatic effect on thetransient expression of GUS in immature soybean cotyle-dons (Fig. 2A, Fig. 3A–D). When the cotyledons weretreated with Agrobacterium EHA105 (Vec035) without

sonication, an average of less than 1% of the surface of thecotyledons expressed GUS (Fig. 2A; Fig. 3B). A tremen-dous enhancement of GUS expression was observed whensonication was applied along with Agrobacterium(Fig. 3C–D). Treatments ranging from 0.5 s to 2 s gave thehighest transient expression (Fig. 2A), although somebrowning of the tissue resulted from the sonication treat-ment.

Scanning electron microscopy revealed large amountsof microwounding of the cotyledons (Fig. 4A–H). Cotyle-dons treated with Agrobacterium without sonicationshowed no surface microwounding (Fig. 4A,B), while witha 2-s sonication treatment, limited microwounding was ob-served on the cotyledons (Fig. 4C,D). With the longer son-ication treatments of 5–10 s, the entire surface of the cot-yledon became covered with microwounds (Fig. 4E–H).When cotyledons were SAAT-treated for more than 10 sbut less than 30 s, 30% of the cotyledons turned white afew days following culture and did not survive, while son-ication treatments of more than 30 s resulted in severe tis-sue disruption and death of all cotyledons 5 days after son-ication (data not shown).

Although high-intensity ultrasound results in immedi-ate cell lysis (Joersbo and Brunstedt, 1992), sublethal dosesresult in temporary suppression of RNA and protein syn-thesis as well as moderate rupture of the cell walls (Joersbo

754

0 0.1 0.5 1 2 5 7 100

5

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25

Treatment duration (s)

d

c

Kunitz Jack Chapman0

5

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Cultivars

Vec 035 pIG121HmA B

abcbcbc

a a a a

aaa

a a

0 2 50

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SAAT treatment (s)

- AS + AS

cc c

a

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ABAXIAL ADAXIAL0

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10

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Cotyledon surface facing the medium

GUS onAbaxial

GUS onAdaxial

aa

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Tran

sien

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Fig. 2A–D Frequency of tran-sient GUS expression of SAAT-treated immature soybean coty-ledons using Agrobacterium at0.1 OD600nm. A Effect of dura-tion of sonication, B effect oftwo different binary vectors andthree cultivars on GUS expres-sion using a 2-s SAAT treat-ment, C effect of the presenceor absence of acetosyringoneduring the co-cultivation peri-od. D effect of explant orienta-tion in relation to the co-culturemedium on transient GUS ex-pression. Data represents thepercentage of the cotyledonsurface area showing blue sec-tors as an average of three rep-lications with ten cotyledonseach. Different letters show sig-nificant difference among treat-ments according to Fisher’sLeast Significant DifferenceTest (α =0.05)

and Brunstedt, 1992). The cell-wall disruption caused bythe lower energy ultrasonic frequency utilized in thepresent study is apparently very useful for Agrobacterium-mediated transformation. The wounding may aid in theproduction of signal phenolics (Stachel et al. 1985) and en-hance the accessibility of putative cell-wall binding fac-tors (Lippencott and Lippencott 1969) to the bacterium.

Although the average GUS expression obtained with 2- and10-s sonication treatments did not differ statistically, thelarge extent of microwounding observed with the longertreatment (Fig. 4C–H) indicated that the 2-s treatment wasmore suitable for further experiments.

The enhancement of transient GUS expression due toSAAT treatment was not genotype specific. No differencewas observed among the three cultivars (‘Jack’, ‘Chap-man’ and ‘Kunitz’) tested (Fig. 2B). Furthermore, therewas no difference in transient GUS expression when eitherof the two different binary plasmids were used (Fig. 2B).The effect of plant genotypes has been reported for severalspecies. Wordragen and Dons (1992) suggested that thedifference in response among cultivars could be caused bydifferential response to the wounding stress. However, our

755

Fig. 3A–F Transient expression of GUS in the adaxial surface ofSAAT-treated immature cotyledons of soybean using Agrobacteri-um (Vec035) at 0.1 OD600nm with a 3-day co-culture. A Control (nosonication; no Agrobacterium). B Agrobacterium-treated cotyledons(no sonication), C, D SAAT-treated cotyledons on D40 medium (C 2 s, D 10 s), E, F SAAT-treated cotyledons on D40-mannitol me-dium (E 2 s, F 10 s). Bars: 1 mm

756

Fig. 4A–H Scanning electron microscopy of adaxial surface of son-icated immature cotyledons of soybean. A, B Agrobacterium-treat-ed cotyledons; no sonication. C–H SAAT-treated cotyledons (C, D

2 s, E, F 5 s, G, H 10 s). Bars on A, C, E, and G represent 100 µmand on B, D, F, and H represent 10 µm

results indicated that the use of sonication to mediate theAgrobacterium infection could minimize or alleviate thesedifferences. The cultivar ‘Jack’ and EHA105 containingthe binary vector Vec035 were used for the remaining ex-periments.

Pre- and post-SAAT culturing conditions

Transient GUS expression in SAAT-treated cotyledons wasaffected by both temperature and length of co-culture pe-riod but was not significantly influenced by the density ofthe bacterial suspension (Table 1). The highest GUS ex-pression was observed after 3- or 4-days of co-culture at27°C, regardless of the OD600nm used. At 23°C, the high-est GUS expression was also observed after a 3- or 4-dayco-culture period; however, transient expression was lowerthan at 27°C. Although a 2-day co-culture is commonlyused (Holford et al 1992; Muthukumar et al 1996), a longerco-culture period of 3 or 4 days can improve transforma-tion efficiency using Agrobacterium (Ducrocq et al. 1994).De Bondt et al. (1994) reported that a 4-day co-culture ofMalus explants with Agrobacterium could be used but thatbacterial overgrowth became problematic with this longerco-culture period. Temperature also affects the efficiencyof T-DNA transfer (Fullner and Nester 1996). In stem seg-ments of soybean infected with Agrobacterium, co-cultureat 25°C enhanced transient expression while higher tem-peratures suppressed the transfer of T-DNA (Kudirka et al.1986). Temperature can also affect pilus formation in Ag-robacterium, which may be involved in T-DNA transfer(Fullner et al. 1996). Pilus formation was observed at 19°Cbut was only rarely observed at 28°C. The results obtainedwith soybean immature cotyledons indicated that the bestconditions for co-culture after SAAT treatment were 3 daysat 27°C.

Co-culture of soybean cotyledons with acetosyringonegreatly increased transient expression following SAATtreatment (Fig. 2C). Although some blue foci were ob-served in control cotyledons (no AS), with or without son-

ication, a significant increase in transient expression re-sulted from SAAT treatments followed by co-culture withAS. Acetosyringone has been shown to enhance the tran-sient expression of GUS in different species (Atkinson andGardner 1991; James et al. 1993) due to activation of thevir genes (Stachel et al. 1985). Although the pH used inthe plant medium in the present study has been reported toinhibit vir gene induction (Stachel et al. 1986), a microen-vironment favorable for vir gene induction could be estab-lished on the surface of the soybean cotyledons which wasnot in contact with the pH 7 medium. Our results conclu-sively show that the addition of acetosyringone afterwounding enhances transient expression regardless of thelength of the sonication treatment.

The location of GUS activity was strongly influencedby the orientation of the explants during the co-cultivationperiod. The highest transient expression was always ob-served on the side of the cotyledon that was not in contactwith the culture medium (Fig. 2D). It is unclear if transientexpression was reduced on the side of the cotyledon in con-tact with the culture medium or enhanced on the other sideof the cotyledon as a result of the rapid divisions in thistissue (Santarém et al., 1997). As high pH is inhibitory tovir gene induction (Stachel et al. 1986), it is possible thatthe low GUS expression on the side of the cotyledon incontact with the medium resulted from a localized high pHinhibition of vir gene induction. Restricted culture aera-tion, on the side of the tissue in contact with the mediumcould also have resulted in reduced vir gene induction (Stachel et al. 1986). Sonicated explants were thereforecultured abaxial side facing the co-cultivation medium sothat the highest expression would be in the adaxial surfacetissue, which typically gives rise to large numbers of so-matic embryos (Santarém et al. 1997).

Since DNA synthesis appears to be required in hostcells for the incorporation of T-DNA (Villemont et al.1997), the effects of induction of cell division in the tar-get tissue were evaluated prior to the SAAT treatment byperforming a preculture timecourse using D40 medium.The highest transient expression was observed using ex-plants that were sonicated immediately after explant ex-cision (29.5%). Cotyledons that were precultured for 1, 2,3 or 4 days and then SAAT-treated had 12.8%, 11.2%,4.2% and 4.3% respectively of their surface showing GUSexpression. In Datura innoxia and Vigna ungiculata thehighest transformation efficiency was obtained after a 1-day preculture and decreased 50% after 8 day (Ducrocqet al. 1994; Muthukumar et al. 1996). In Arabidopsis thal-iana, the highest transient GUS expression was observedafter 4 days of preculture (Sangwan et al. 1991). Kudirkaet al. (1986) did not observe an enhancement of transfor-mation efficiency of the stem segments after the precul-ture period. Our results indicated no beneficial effect ontransient transformation efficiency from the preculture ofsoybean cotyledons on induction medium. The effects ofpreculture may need to be re-evaluated for stable trans-formation work as the mitotic state of the target tissue mayhave different effects on transient and stable transforma-tion.

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Table 1 Effect of OD600nm, temperature and duration of the co-cul-ture period on transient expression of immature cotyledons of soy-bean sonicated for 2 s

Temperature Co-culture Transient expression (%) a

period OD600nm of Agrobacterium(days)

0.05 0.1 0.2

23 °C 2 0.3d b 0.4 d 5.5 cd3 18.8 b 19.8 b 19.5 b4 20.0 b 22.6 b 21.3 b

27 °C 2 4.5 cd 8.2 c 12.8 c3 28.8 a 36.0 a 32.0 a4 29.3 a 32.3 a 36.0 a

a Percentage of surface area showing blue sectors as average of threereplications with ten cotyledons eachb Means followed by the same letters are not significantly differentat the 0.05 level according to Fisher’s Least Significant Differencetest

Damage assessment

Although SAAT of soybean cotyledons resulted in highlevels of transient expression, browning and callus forma-tion was apparent in many cases. To determine what wasinvolved in the browning, we sonicated the explants in thepresence or absence of Agrobacterium. Based on observa-tions of embryo induction and browning, it appeared thatAgrobacterium alone was not detrimental to tissue growthcompared to the minus-Agrobacterium control. On theother hand, sonication of the cotyledons either with or with-out Agrobacterium resulted in callus formation with somebrowning of that tissue. When sonication treatments longerthan 5 s were used, the primary response of the immaturecotyledons to form somatic embryos was diminished oreliminated. In order to obtain stable transformation of thistarget tissue, the intensity of the sonication treatmentshould be carefully monitored to control microwoundingand cell disruption.

To estimate the extent of wounding caused by sonica-tion, we measured the OD400nm of the bacterial suspensionafter various sonication treatments (Table 2). Light absor-bance of a plant/bacterial exudate at 400 nm gives an ap-proximation of tissue disruption. Longer sonication treat-ments resulted in higher plant exudate concentrations andconsequently higher optical density readings (Table 2).When cotyledons were excised and SAAT-treated in D40medium containing 0.4 M mannitol, a decrease in OD400nmwas observed in this exudate solution indicating less tis-sue wounding (Table 2). Scanning electron microscopy ofcotyledons excised and SAAT-treated in either D40 me-dium or D40 medium with 0.4 M mannitol revealed pro-tection of the mannitol-treated tissue from wounding (datanot shown). However, the transient expression of the cot-yledons treated with mannitol was also reduced (Table 2,Fig. 3E, F). Mannitol could act to protect the target tissueby causing cell plasmolysis, reducing turgidity and dimin-ishing sonication-induced microwounding. Although os-motic treatment enhanced particle bombardment-mediatedtransformation of soybean and maize suspension culturematerial (Vain et al. 1993a, b), it was clearly not effective

in enhancing transient transformation of soybean cotyle-dons using SAAT. The effectiveness of the SAAT treat-ment was apparently influenced by the water concentra-tion or turgidity of the target tissue.

We report here a new technique which enhances the ef-ficiency of Agrobacterium infection of plant cells. Imma-ture cotyledons of soybean were very responsive for tran-sient expression studies and were used for optimization ofSAAT. While stable transformation of embryogenic sus-pension cultures of soybean has already been obtained us-ing SAAT (Trick and Finer 1997, 1998), the stable trans-formation of soybean cotyledons as well as other plantsand target tissues are still being evaluated.

Acknowledgments We thank Beth Hood (ProdiGene Inc, CollegeStation, Tex.) for the Agrobacterium tumefaciens, strain EHA105,Tim Hall (Texas A&M University) for the construction Vec035, Bar-bara Norris and Lloyd Ringley Jr for their technical assistance andMasood Z. Hadi for his valuable comments. Salaries and researchsupport were provided by the United Soybean Board, Conselho Na-cional de Desenvolvimento Científico e Tecnológico (CNPq), Bra-zil, and by State and Federal funds appropriated to The Ohio StateUniversity/Ohio Agricultural Research and Development Center.

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Table 2 Comparison between immature cotyledons excised andsonicated in D40 medium or D40 0.4 M mannitol. Sonication treat-ment was 2 s with the Agrobacterium diluted to 0.2 OD600nm

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10 0.995 0.433 25.2 a 1.4 b

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