Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress.

Plant Cell Rep DOI 10.1007/s00299-014-1707-3

ORIGINAL PAPER

Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress Yinghui Dan • Song Zhang • Heng Zhong Hochul Yi • Manuel B. Sainz

•

Received: 26 August 2014 / Revised: 30 September 2014 / Accepted: 22 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Key message Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium-mediated plant transformation by mitigating oxidative stress. Abstract Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low

Communicated by Zeng-Yu Wang. Y. Dan (&) S. Zhang Institute for Advanced Learning and Research, 150 Slayton Avenue, Danville, VA 24540, USA e-mail: [email protected] Y. Dan Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA Y. Dan Department of Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA H. Zhong H. Yi M. B. Sainz Syngenta Biotechnology, Inc., 3054 East Cornwallis Road, Research Triangle Park, NC 27709, USA

stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacteriummediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P \ 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these compounds at different levels. Keywords Agrobacterium tumefaciens Anti-oxidative compounds Cell death Oxidative stress Plant transformation Tissue browning Abbreviations LA a-Lipoic acid MEL Melatonin ROS Reactive oxygen species STF Stable transformation frequency TB Tissue browning

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Introduction Plant transformation plays a crucial role in the development of transgenic crop products and in the assignment of gene function by functional genomics. However, genetic transformation is only marginally successful in many crops, and indeed in most elite cultivars of all crops. This limits the biotechnological improvement of many economically important plant species, lengthens product development timelines for crops where transformation systems are available, and impairs wider application of functional genomics. Conversely, improving crop transformation technology can reduce the time and resources required to produce transgenic events, boosting productivity as well as broadening the range of transformable crop species and cultivars. A commonly used transformation method for many crops requires that cells or tissues be inoculated with Agrobacterium tumefaciens and then maintained on culture media for several weeks to effect selection for the growth of the rare, stably transformed cells, followed by regeneration of transgenic plants from the undifferentiated tissue arising from such cells. One problem inherent in Agrobacterium-based transformation systems is that A. tumefaciens is a plant pathogen, resulting in a poor survival rate of target plant tissues following Agrobacterium-mediated transformation in many plant species (Barampuram and Zhang 2011). The plant defence response following attack by pathogenic bacteria, fungi and viruses is associated with the production of reactive oxygen species (ROS), mainly hydrogen peroxide (H2O2) (Doke 1983; Levine et al. 1994; Tenhaken et al. 1995). Subsequently, plant cells undergo an apoptotic-like programmed cell death (PCD) termed the hypersensitive response (HR) in intact plants, that acts to limit pathogen spread (Greenberg and Yao 2004; Mittler et al. 2004). ROS in the oxidative burst in response to pathogen attack is generated by at least two major enzyme complexes: NADPH oxidases similar to those of mammalian phagocytotic cells, and the pH-dependent generation of hydrogen peroxide by exocellular peroxidases (Jalali et al. 1999; Apel and Hirt 2004). The importance of this response is evidenced by inhibitors of NADPH oxidase and peroxidases that repress H2O2 production (Tenhaken et al. 1995; Otte and Barz 1996; Mitho¨fer et al. 1997) and defense gene induction (Tenhaken et al. 1995; Lin et al. 2005) in cultured suspension cells. The available evidence suggests that tissue browning and death of plant cells during Agrobacterium-mediated plant transformation is due to PCD caused by the plant– pathogen interaction. The interaction of Agrobacterium with plant cells causes an oxidative burst (Xu and Pan

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2000), and expression of anti-apoptosis genes can suppress PCD and increase plant transformation frequencies (Hansen 2000; Dickman et al. 2001; Lincoln et al. 2002; Chen and Dickman 2004; Li and Dickman 2004; Xu et al. 2004). Hydrogen peroxide (H2O2) plays an important role in PCD and oxidative stress. However, little is known about the biological and molecular mechanisms leading from H2O2 to the death of the tissues during plant transformation. Media conditioning to mitigate tissue browning in Agrobacterium-mediated transformation has focused on the use of antioxidants such as ascorbic acid, cysteine, dithiothreitol (DTT), a-lipoic acid, polyvinylpolypyrrolidone (PVPP), glutathione, selenite and a-tocopherol (Gupta 2011; Dan 2008). Perl et al. (1996) were the first to investigate antioxidant media additions in plant transformation. They tested a series of antioxidants in Agrobacterium-mediated transformation of grape, a notably recalcitrant species, and found that PVPP and DTT were effective in enhancing transformation frequency. A combination of cysteine, silver nitrate (AgNO3) and ascorbate was successful in enhancing Agrobacterium-mediated transformation of sugar cane (Enriquez-Obregoń et al. 1997, 1998) and the same compounds either singly or in combination were effective in rice (Enrı´quez-Obregoń et al. 1999). Subsequently, antioxidants have been shown to enhance Agrobacterium-mediated transformation in a considerable number of additional species (Olhoft and Somers 2001; Olhoft et al. 2001, 2003; Frame et al. 2002; Toldi et al. 2002; Dan et al. 2004, 2009; Zheng et al. 2005; Vega et al. 2008; Velcheva et al. 2010). Among the antioxidants used the efficacy of thiol compounds (Deneke 2000) is notable. Cysteine (Olhoft and Somers 2001; Olhoft et al. 2001, 2003; Frame et al. 2002; Toldi et al. 2002; Zeng et al. 2004) and a-lipoic acid (Dan et al. 2004, 2009; Dutt et al. 2011) are among the most efficacious in reducing tissue browning/death of transformed cells and in improving regeneration and lowering escape frequencies, thus enhancing transformation frequencies. Both LA and L-cysteine (like DTT and glutathione) are sulfur-containing compounds with free sulfhydryl groups or thiol bonds. We were interested in investigating effects of non-thiol compounds on mitigating tissue browning/ death in infected cells during Agrobacterium-mediated tomato transformation. The non-thiol compounds included antioxidant and non-antioxidant compounds, which inhibited ROS (mainly H2O2) generation mostly in mammalian cells through different mechanisms to suppress ROS and cell death from lipoic acid and cysteine (Table 1; Olhoft et al. 2001). Our hypotheses are that Agrobacterium causes tissue browning/death in infected cells; and the non-thiol compounds suppress the Agrobacterium-mediated cell browning/death and increased stable transformation.

A plant-derived phenolic compound

ROSA DFMO

DL-a-Difluoromethylornithine

TMZ

Thiamazole

Very strong antioxidant in mammalian cell cultures H2O2 detoxifying enzyme implicated in diverse oxidative stress responses; inhibitor of H2O2 accumulation in elicited plant cell cultures

BHA CAT DMT EPI RES MEL

N-(tert-butyl) hydroxyamine hydrochloride Catalase

1,3 Dimethylthiourea

Epicatechin

Resveratrol

Melatonin

Ubiquitous antioxidant and hormone with no pro-oxidant activity

Endogenous plant phytoalexin antioxidant; found in red wine

Endogenous plant flavan-3-ol antioxidant; found in green tea

H2O2 scavenger; inhibitor of signal transduction and H2O2 production in elicited plant cells

An antioxidant shown to reduce tissue browning in soybean

LA CYS

a-Lipoic acid

Herrera et al. (2007)

Jang et al. (1997)

Matsuzaki and Hara (1985)

Lin et al. (2005), Linas et al. (1985), Tenhaken et al. (1995)

Chen et al. (1993), Lin et al. (2005), Mitho¨fer et al. (1997), Otte and Barz (1996), Willekens et al. (1997)

Ratan et al. (1994)

Olhoft et al. (2001)

Dan et al. (2004, 2009), Suzuki et al. (1991)

Crescioli et al. (2007), Shen et al. (2010)

Antithyroid agent Ubiquitous disulfide antioxidant; demonstrated transformation enhancer

Wu et al. (1986)

Wu et al. (1986)

Lee et al. (2004)

Mitho¨fer et al. (1997), Otte and Barz (1996), Tenhaken et al. (1995) Mitho¨fer et al. (1997), Otte and Barz (1996), Rich et al. (1978), Schonbaum et al. (1971)

Mintz (1993)

Isom and Pegg (1979)

Makino et al. (2000)

Raneva et al. (2001)

Lin et al. (2005), Tenhaken et al. (1995), Umezawa et al. (1990)

Shen and Zhang (1991), Xia et al. (1998)

References

Dehydrogenase co-enzyme

Cysteine

Antioxidants or acting in antioxidant pathway

NADH

a-Nicotinamide adenine dinucleotide

Dehydrogenase co-enzyme

Soybean isoflavone

GEN NADPH

Inhibitor of peroxidase, mitochondrial alternative oxidase, and H2O2 production in elicited plant cells

SHA

Salicylyhydroxamic acid

Genistin

Inhibitor of plant NADPH oxidase, defense response signal transduction, and H2O2 production in elicited cells

DPI

a-Nicotinamide adenine dinucleotide phosphate

Serine protease inhibitor

AEBSF

Ornithine decarboxylase inhibitor

4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride Diphenyleneiodonium chloride

hydrochloride hydrate

Endogenous hydroxycinnamic acid and antioxidant; found in coffee

CAF

Caffeic acid (3,4-dihydroxy cinnamic acid) Rosmarinic acid

Tyrosine kinase inhibitor; inhibitor of signal transduction and H2O2 production in elicited plant cells

DHC

2,5-Dihydroxycinnamic acid methyl ester

Mammalian pterin nitric oxide synthase co-factor and antioxidant

Function

(6R)BH4

Code

(6R)-5,6,7,8Tetrahydrobiopterin dihydrochloride

Non-antioxidants

Compounds

Table 1 Description of candidate compounds including abbreviation (code) used in the text, putative function and relevant references

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In this study, we tested the hypothesis that Agrobacterium causes oxidative stress leading to browning and death of its transformed cells and anti-oxidative compounds reduce tissue browning and cell death of transformed cells and thus increase stable transformation frequency by mitigating oxidative stress. We selected a group of new candidate compounds predicted to have the potential to reduce tissue browning and to increase transient expression and stable transformation, based on their demonstrated ability to mitigate ROS accumulation in the plant defense response or antioxidant activity in human and animal studies. We investigated biological function of these compounds in comparison with LA using tomato cv. MicroTom transformation. We have proved the hypothesis that the Agrobacterium causes the observed browning/death of transformed cells, whose magnitude was triggered by anti-oxidative compounds. In addition, we have determined biological mechanistic relations among tissue browning, transient expression and stable transformation under antioxidant regulation. Our correlation study showed that melatonin (an antioxidant and a neurohormone in animals) had additional effect on promoting the regeneration of transformed cells into stable transgenic plants, besides its effect on reducing tissue browning in MicroTom transformation.

Materials and methods Description and preparation of candidate compounds New candidate compounds including 6 antioxidants and 12 non-antioxidant compounds were selected because they either reduced H2O2 production in mammalian cells or inhibited ROS signaling in plant (Table 1) and they could potentially reduce tissue browning and increase transformation frequency in plant transformation. The effects of the compounds on tissue browning, transient expression and stable transformation were investigated. The compiled list of compounds with their functions is shown in Table 1 and the structures of some of these compounds are illustrated in Fig. 1. Their functions, solution preparation and the three concentrations tested for each compound are described in Tables 1 and 2. The concentrations used in screening were chosen empirically based on analysis of the literature. Plant and Agrobacterium materials MicroTom seeds (Ball Seed Co., Chicago, IL, USA) were surface-sterilized and germinated using the method of Dan et al. (2006). Agrobacterium tumefaciens strain LBA4404 carrying plasmid V1 were used for all candidate compound screening in MicroTom. The V1 plasmid contains a gus gene driven by the CaMV 35S promoter and a neomycin

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(a) positive controls

α-lipoic acid cysteine (b) experimental compounds

dimethylthiourea melatonin

resveratrol epicatechin

N-tert butyl hydroxylamine Fig. 1 Structure of control and selected candidate compounds. Candidate compounds were tested for reduction of tissue browning and enhancement of transient expression in the MicroTom tomato system. a Positive control compounds a-lipoic acid and cysteine; b structures of some of the experimental compounds tested

phosphotransferase II (nptII) gene driven by a nos promoter. Agrobacterium strain LBA4404 containing plasmid pHB2892 (Molinier et al. 2000) was used to generate stable transgenic plants. Plasmid pHB2892 contains a green fluorescent protein (gfp) gene driven by a double CaMV 35S promoter, and a nptII gene driven by a nos promoter. The plasmid pHB2892 was used only for generating MicroTom transgenic plants for Southern blot analysis. Agrobacterium strain EHA101 harbors plasmid 17377 was used for soybean transformation, which contains a modified gene of soybean 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that confers glyphosate tolerance. The EPSPS gene is driven by Cestrum Yellow Leaf Curl Virus promoter. MicroTom transformation procedure MicroTom transformation experiments were performed according to Dan et al. (2006) for all experiments except soybean transformation. The transformation procedure is

Plant Cell Rep Table 2 Preparation of candidate compound solutions and tested concentrations. Working solutions at low, medium and high concentrations (based on the literature) were prepared from stock solutions at the concentrations and in the solvents indicated

a

All candidate compounds were purchased from Sigma

Compound abbreviationa

Concentrations tested

Solvent

Stock solution concentration

Low

Medium

High

MEL_1

10 lM

50 lM

100 lM

95 % ethanol

34 mM

MEL_2

100 lM

500 lM

1,000 lM

95 % ethanol

200 mM

BHA

10 lM

50 lM

100 lM

DMSO

1M

CAT

200 U

1,000 U

2,000 U

15 mM K2HPO4

88,500 U/mL

DMT

10 lM

50 lM

100 lM

95 % ethanol

100 mM

EPI

0.17 mM

0.34 mM

3.4 mM

DMSO

689 mM

CYS

50 lM

100 lM

3.3 mM

H2O

825 mM

RES

50 lM

100 lM

200 lM

95 % ethanol

100 mM

(6R)-BH4

10 lM

50 lM

100 lM

H2O

16 mM

DHC

10 lM

50 lM

100 lM

95 % ethanol

19 mM

CAF ROSA

2.78 mM 278 lM

5.56 mM 2.78 mM

11.12 mM 8.34 mM

95 % ethanol DMSO

555 mM 833 mM

DFMO

50 lM

100 lM

500 lM

DMSO

200 mM

AEBSF

100 lM

500 lM

1,000 lM

H2O

100 mM

DPI

5 lM

10 lM

50 lM

DMSO

16 mM

SHA

5 lM

10 lM

50 lM

DMSO

4.55 M

GEN

2 lM

10 lM

50 lM

DMSO

23 mM

NADPH

2 lM

10 lM

50 lM

0.01 N NaOH

16 mM

NADH

2.82 lM

14.1 lM

70.5 lM

0.01 N NaOH

35 mM

TMZ

10 lM

50 lM

100 lM

H2O

100 mM

composed of three stages including inoculation, co-culture and selection stages. Sterile seeds were cultured on MS medium for 6 days in the dark and 1 day under 44 lmol s-1 m-2 cool white florescent light for a 16 h photoperiod. V-shaped cotyledons were selected as explants and six pokes were made on each cotyledon with forceps. Cotyledons were inoculated with LBA4404 V1 or pHB2829 at a concentration of OD600 = 0.1 for 10 min. The inoculated explants were co-cultivated for 2 days and cultured on a selection medium SI-2 (Dan et al. 2006) supplemented with each of the candidate compounds (Table 1) at three different concentrations (Table 2) for 3 days, along with a negative control medium (SI-2 medium without the candidate compounds) and a positive control medium of SI-2 with 50 lM lipoic acid (LA). Selection medium SI-2 also contained 500 mg/L carbenicillin, 100 mg/L cefotaxime and 100 mg/ L kanamycin. LA was selected as a positive control for all compound screening because it was an effective antioxidant for reducing tissue browning and increasing stable transformation in MicroTom and other crops (Dan 2008; Dan et al. 2009). All cultures were placed in a growth chamber at 24 °C under the same light conditions described above. Soybean transformation procedure Soybean (Glycine max cv. Jack) stock plants were grown in a greenhouse. Pods are collected and sterilized by

immersing in 15 % CLOROXÒ bleach and rinsing with sterile tap water. The isolated seeds are further sterilized with 10 % CLOROXÒ bleach and followed by rinsing three times with sterile water. Sterilized seeds are used for preparing explants for Agrobacterium-mediated transformation according to Dan et al. (2013). Following infection, the explants are removed from the Agrobacterium suspension and placed on co-cultivation medium containing designated concentrations of melatonin (0, 1 10 and 100 lM). The co-cultivation plates are incubated for 3–5 days at 22 ± 1 °C in the dark. The experiments were conducted at seven different times with total of 395, 279, 418 and 422 explants for 0, 1, 10 and 100 lM, respectively. Candidate compound screening To test our hypothesis that anti-oxidative compounds reduce tissue browning and increase transformation frequency, we tested 18 candidate compounds including 12 non-antioxidant and 6 antioxidant compounds listed in Table 1 in MicroTom transformation system, rated transient GUS expression and tissue browning level, and evaluated their ability to significantly reduce the most extreme levels of tissue browning (MELB rating level 4). Three different concentrations plus a negative control (no added candidate compound) and a positive control using

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no browning on poke; B30 % of poke area having browning; 30–50 % of poke area having browning; 50–70 % of poke area having browning; and 70–100 % of poke area having browning.

Measurements of browning and transient expression were obtained for three concentrations of each compound (Table 2). Additionally, each experiment contained a negative control (no added candidate compound) and a positive control using 50 lM LA. The browning rating score was collapsed into two categories for purposes of statistical analysis. The first browning category encompasses ratings 0–3 and the second corresponds to a rating score of 4. The second category is the most extreme level of browning (MELB) observed, whereas the first category corresponds to a reduced amount of browning. Measurement of transient expression Transient GUS expression was measured on a five level scale from 0 to 4, similar to the browning level scale (Fig. 2). The rating scale was: 0 1 2 3 4

= = = = =

no GUS expression; B30 % of poke area having GUS expression; 30–50 % of poke area having GUS expression; 50–70 % poke area having GUS expression; and 70–100 % of poke area having GUS expression.

Levels 2–4 were considered to be high levels of GUS expression for transient expression analysis. Determination of causes of tissue browning To investigate the cause of tissue browning during Agrobacterium-mediated MicroTom transformation two independent experiments, each comprising five treatments with 60 cotyledonary explants per treatment, were designed. For each treatment, six pokes were made on each of the 7-dayold MicroTom seedling cotyledonary explants. The five treatments were:

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Level 3 50% - 70%

GUS expression

= = = = =

Browning

0 1 2 3 4

Level 2 30% -50%

Tissue browning within each poke of each cotyledonary explant from the five treatments was measured under a dissecting microscope using a five-level scale ranging from 0 to 4, at 3, 5 and 7 days after selection (Fig. 2). The rating scale was:

Level 1 ≤ 30 %

Measurement of tissue browning

Level 0 0%

50 lM LA were tested with a total five treatments for each compound (Table 2). Three independent experiments were performed with a total of 90 explants per treatment for each compound.

Level 4 70% - 100%

Plant Cell Rep

Fig. 2 Browning and GUS expression rating. 0 = no browning or GUS expression on poke; 1 = B30 % of poke area having browning or GUS expression; 2 = 30–50 % of poke area having browning or GUS expression; 3 = 50–70 % of poke area having browning or GUS expression; and 4 = 70–100 % of poke area having browning or GUS expression

Poke: pokes were made without further treatment (no treatment control); Mock: immersion in inoculation medium (mock inoculation control); Low 19: immersion in Agrobacterium suspension in inoculation medium at 1.24 9 108 CFU/mL (19, low dose treatment, OD600 = 0.1); Med 59: immersion in Agrobacterium suspension in inoculation medium at 6.21 9 108 CFU/mL (59, medium dose treatment, OD600 = 0.5); and

Plant Cell Rep

High 259: immersion in Agrobacterium suspension in inoculation medium at 3.11 9 109 CFU/mL (259, high dose treatment, OD600 = 2.5). The explants from the five treatments were co-cultivated for 2 days and transferred to selection medium with 100 mg/L cefotaxime and 500 mg/L carbenicillin to prevent overgrowth of the bacterium, but without kanamycin to prevent killing the explants in control treatments 1 and 2 for 3, 5 and 7 days. Tissue browning within each poke of each explant from the five treatments was measured as described above. The approach chosen for analyzing these data was to compute an average browning rating across the six pocks for each explant and then compare the average browning value across treatments using analysis of variance. Experiment and Agrobacterium dose were included as treatments under a factorial structure, and the data were stratified across day of collection. Since the inferential findings were consistent across each day on which data were collected, the data were pooled across all three collection days, and an analysis using the pooled data was conducted. Stable MicroTom transformation To examine effects of anti-oxidative compounds on stable transformation frequency (STF) promising compounds that either reduced tissue browning or increased transient expression were evaluated for STF. Three independent experiments were performed with a total of 90 explants per treatment. Explants were assayed 30 days after selection for histochemical GUS expression according to Jefferson (1987) and data were collected from each of the 540 pokes for the 90 explants of each treatment. A poke with GUS positive shoots or buds was counted as an independent transgenic event. For pokes that only produced calli a poke having more than 50 % of callus area that was GUS positive was counted as an independent transgenic event. Transformation frequency (TF) was measured as independent transgenic events divided by total cotyledons inoculated, then multiplied by 100 %. A poke without GUS positive shoots or buds and/or calli was counted as an escape event. Escape frequency (EF) was determined as escape events divided by total cotyledons inoculated, then multiplied by 100 %. Determination of correlation between tissue browning, transient expression and stable transformation To determine if there was a substantial inverse correlation between the occurrence of tissue browning and transient expression, two independent experiments were carried out comprising two treatments each with a total of 40 explants

per treatment. The two treatments included PokeMA1 (no LA) as described above and PokeMA1 ? LA (PokeMA1 plus 50 lM LA). The relationship was investigated at two different dosage of LA (0 and 50 lM). Each individual experiment consisted of samples of 20 explants, and 6 pocks were made from each explant. To determine the correlation between tissue browning and stable transformation, the similar experiments were conducted as the experiments described above, but an additional antioxidant, melatonin (MEL) was parallelly tested with LA. Following the treatments, both tissue browning and transient GUS expression from the same poke of each explants from the two treatments were measured under a dissecting microscope using a five level scale from 0 to 4 at 3 days after selection as described above. Both tissue browning and stable transformation from the same poke of each explant from the two treatments were measured using the methods described above. Browning ratings of 1 or 0 were indicative of a desirable result as were expression ratings of 2 or higher. The data were analyzed using the actual rating values. Explant, LA and MEL level were included as treatment variables. The browning rating was included as a linear and quadratic term in the prediction model. Frequencies for each of the combinations of browning and expression outcomes were computed, and the Phi coefficient, a measure of association for 2 9 2 tables, was analyzed. Determination of optimal transformation stage to apply MEL MicroTom transformation is composed of three stages: inoculation, co-culture and selection. Since MEL was only applied in selection stage in all experiments above to determine the optimal stage to apply MEL, the effects of different application treatments on tissue browning, transient expression and transformation frequency were examined. MEL at 100 lM was applied in six different treatments: (1) at inoculation alone; (2) at co-culture alone; (3) at selection alone; (4) at both inoculation and co-culture; (5) at both co-culture and selection; and (6) at inoculation, co-culture and selection. Ethanol was the solvent used to dissolve MEL; therefore, the amount of ethanol used to dissolve MEL was applied to all treatments regardless of whether MEL itself was applied. Three independent experiments were performed with a total of 90 explants per treatment. Tissue browning, transient GUS expression, TF and EF were measured as described above. Statistical data analysis For browning data analysis, the first step was to conduct a descriptive analysis of the data. Frequencies for each

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browning rating score were computed for each treatment, first stratifying by experiment and then pooling data across replicate experiments. Because of the ordered nature of the data, cumulative frequencies for the browning response also were obtained. Based on the statistical analysis of the original experimental data, a weighted least squares analysis of variance was performed where the weights were derived as the inverse of (the maximum binomial variance (0.50) divided by the number of pocks for that explant). Factors controlled in the analysis were experimental treatment and the identification number of the experiment. An interaction of these two factors was included to determine if the results of the experimental treatment was consistent across experimental replicates. The second analysis of the browning data was a comparison at the relative proportions of pocks within each explant exhibiting various browning levels. Three different boundary conditions (B1, 2 or 3) were used to group browning levels. The distribution of pocks classified as meeting the specified browning criterion and not meeting the criterion was determined for each of the boundary conditions. Using the criterion B3 yields a distribution suitable for analysis. Pocks which are classified at a level of 3 or less do not exhibit the most severe amount of browning. For transient expression data, the first step of analysis was the same as the browning data analysis described above. The next analysis was to compute the proportion of pocks from any explant yielding an ‘‘acceptable’’ rating (expression C2) and then use those proportions in an analysis of variance. The data were transformed using an arcsine transformation prior to any inferential analysis. Factors controlled in the analysis were experimental treatment and the identification number of the experiment. An interaction of these two factors was included to determine if the results of the experimental treatment were consistent across experimental replicates. For stable transformation data analysis, within these experiments, there were six treatments. The treatments consisted of inoculation only; co-culture only; selection only; inoculation and co-culture; inoculation and selection; and inoculation, co-culture and selection. Thirty explants were assigned to each experimental replicate. The entire experiment was replicated two additional times. Since the number of stable events appeared to follow a Poisson distribution, a data transformation was applied prior to any inferential analysis. The transformed value is defined as the square root of the quantity (observed value ? 0.375). The transformed data were then entered into an analysis of variance controlling for experiment, treatment, and the experiment by treatment interaction. Stable transformation frequencies were then statistically analyzed using Fisher’s LSD test (Hayter 1986).

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Southern blot analysis To determine if melatonin has any effect on T-DNA integration Southern blot analysis was conducted to examine copy number of transgene insertion. Plant genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit (catalog #69104, Qiagen, MD, USA). Southern blot analysis was performed by Lofstrand Labs Ltd. (Gaithersburg, MD 20879, USA). Approximately, 20 lg of genomic DNA from transgenic plants was digested with EcoR1 and probed with a probe for the nptII gene. The digested DNA samples were loaded onto a 350 mL 0.7 % Tris–borateEDTA agarose gel and the gel was electrophoresed at 50 V for 18 h. The gel was transferred to a Nytran Supercharge nylon membrane (Whatman/Schleicher and Schuell). Each membrane was UV linked and air-dried. The membrane was pre-hybridized using 69 SSC, 59 Denhardt’s solution, and 0.5 % SDS at 68 °C for 6 h. They were separately hybridized using random-primed, P32-labeled probes of nptII and gfp template DNA. Hybridizations were carried out at 68 °C for 27 h. The membranes was washed in 29 SSC ? 0.1 % SDS at 68 °C with three buffer changes over a period of 60 min; and 20 min at 68 °C with 0.19 SSC ? 0.1 % SDS. The membranes were autoradiographed for up to approximately, 41 h using an intensifier screen at -80 °C.

Results Determining the cause of tissue browning To clearly establish the cause for tissue browning, multiple experiments of co-cultivation of Agrobacterium at different concentrations with MicroTom cotyledonary explants were carried out, with browning ratings recorded during the course of the experiment. Statistical analysis of the pooled data showed a highly significant effect attributable to Agrobacterium concentration (P \ 0.0001; Fig. 3a). Overall, there was a highly significant increase in browning from day 3 to day 5 (P \ 0.0001), but the change in browning from day 5 to day 7 was not significant (P = 0.146; Fig. 3b). Substantial tissue browning was observed at all three Agrobacterium concentrations on all days tested. No tissue browning occurred for the no Agrobacterium control treatments on all days tested, with the exception that 2 out of 60 mock inoculated explants exhibited some browning on day 7 (Fig. 3a). These results provide strong evidence that tissue browning did not occur due to the methodology of making pokes or to incubation on growth media. Treatments with Agrobacterium caused tissue browning in vitro 3, 5 and 7 days after selection, while the treatments without Agrobacterium did not. The

Plant Cell Rep

(a)

(b) 200

4.0

3 dpi

3.8

Average browning rating

Total % poke area

100 0 200 100

5 dpi

0 200 100

7 dpi

3.5 3.3 3.0 days post inoculation

2.8

5

7

2.5 2.3 2.0 1×

0 Poke

3

Mock

Low

1× Treatment

Med

High

5×

25×

5×

25×

Relative Agrobacterium concentration

% Browning of total poke area = none ≤ 30% = 30% - 50% =50% - 70% =70% - 100%

Fig. 3 Effect of Agrobacterium concentration on cumulative tissue browning at different days after inoculation of MicroTom tomato cotyledons. a Cumulative percentage of tissue browning 3, 5 and 7 days after inoculation (dpi) of MicroTom cotyledons across two independent experiments. Treatments are Poke (uninoculated poke); Mock (poke inoculated with inoculation media); Low 13 (poke inoculated with 19 Agrobacterium suspension—1.24 9 108 CFU/ mL, OD600 = 0.1); Med 59 (poke inoculated with 59 Agrobacterium

suspension—6.21 9 108 CFU/mL, OD600 = 0.5); and High 259 (poke inoculated with 259 Agrobacterium suspension—3.11 9 109 CFU/mL, OD600 = 2.5). b The same data showing browning rating of inoculated MicroTom cotyledonary pokes 3, 5 and 7 days post inoculation of MicroTom cotyledons across two independent experiments of the three different Agrobacterium concentrations described above

tissue browning occurred consistently in the presence of Agrobacterium infection. In addition, we showed that tissue browning increased with increasing concentrations of Agrobacterium or co-culture time, indicating dosagedependent tissue browning by the bacteria in our MicroTom system (Fig. 3b). We hypothesized that necrosis (tissue browning) of cultured MicroTom cotyledonary explants inoculated with A. tumefaciens was due to activation of the plant defense response, potentially involving the generation of ROS and PCD.

linear relationship between these two variables from Phi coefficient analysis (data not shown). Since we found that browning occurs as a result of increasing levels of Agrobacterium infection, we hypothesized an infection threshold that cells needed to reach to detect gus gene expression. Therefore, the data were re-analyzed using the actual rating values. Explant and lipoic acid (LA) levels were included as treatment variables. The browning rating was included as a linear and quadratic term in the prediction model. Without LA addition, there was a trend that transient expression increased with increasing browning and 64 % of inoculated pokes had extreme tissue browning levels (level 4), with high transient expression (Fig. 4a). LA addition shifted the curve in that most tissue browning was intermediate (at level 1, 2 and 3) and associated with the highest levels of GUS transient expression (Fig. 4b). Transient expression was higher for moderate and extremely high levels of browning and lower for lower browning rating levels. Since the degree of browning is

Correlation among tissue browning, transient expression and stable transformation Our preliminary hypothesis was that there would be an inverse relationship between tissue browning and GUS transient expression, reasoning that browning cells would be dead or dying and thus unable to express the gus transgene. However, there was no evidence of an inverse

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Plant Cell Rep Fig. 4 Correlation between tissue browning and transient expression levels a without and b with addition of lipoic acid

(a)

18% 17%

18% 16%

15% 14%

0

12%

1 10%

10% 2

8% 6%

9%

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4%

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2%

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0

associated with Agrobacterium concentration and inoculation time, this indicated a direct correlation between presumptive Agrobacterium infection levels and transient GUS expression levels. Thus, we were interested in the effect of LA and melatonin (MEL) on the relationship between tissue browning and stable transformation. Pokes generated significantly (P \ 0.05) higher frequency of 68 % having the most extreme levels (rating level 4) of tissue browning (MELB) than the rest of tissue browning levels when LA was not present in culture medium (Fig. 5a). With the addition of LA or MEL to the media this relationship became curvilinear, in that 71.3 % of the poke browning levels were low (level 1: 10.9 %; level 2: 25.8 %; level 3: 34.6 %), and these levels were where both transient GUS expression and

123

3

3% 3%

0% 0%

2

3%

2%

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3%

0

8% 9% 5% 6%

1

2

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stable transformation were found to be highest (Fig. 5b, c). Stable transformation frequencies were significantly higher in tissues with browning levels 3 than the rest of browning levels for control and LA treatments. However, there was no significant difference among the stable transformation frequencies in tissues with browning levels 1, 2, 3 and 4 for MEL treatment although there was significant difference among the tissue browning frequencies (Fig. 5a–c). In addition, in the pokes with level 4 browning, the addition of MEL produced significantly (P \ 0.05) higher stable transformation frequency of 38 % than those (13 and 20 %, respectively) of the control and LA treatments (Fig. 5a–c). These results indicated that MEL had additional effect on promoting regeneration of transformed cells into stable transgenic plants while the transformed cells were having

Plant Cell Rep

severe tissue browning (level 4) beyond reducing tissue browning. Both LA and MEL also reduced browning from level 4 to levels 0–3, while increasing transient expression levels and stable transformation frequency (Fig. 5b, c). We speculate that this pattern may be due to LA and MEL rescuing cells that would otherwise undergo necrosis before exhibiting GUS expression.

Percentage of pokes having browning and stable events

(a) 80%

A

% of pokes having browning

70%

68%

% of pokes producing stable events

60% 50% 40%

B

30%

b CD10%

20% 10%

D 0%

c 0%

0%

0_NCK

3% 1_NCK

ab C

15%

Candidate compound screening

a 22% 20%

b 13%

7%

2_NCK

3_NCK

4_NCK

Tissue browning ratings across treatments


(b) 80% 70% 60%

% of pokes having browning % of pokes producing stable events

50%

b

40% 30%

C

20% 10% 0%

D

d

11%

c 16%

BC 29%

a

A

42%

44%

B

c

26%

20%

18%

1% 0% 0_LA

1_LA

2_LA

3_LA

4_LA

Tissue browning ratings across treatments


(c) 80%

% of pokes having browning

70%

% of pokes producing stable events 60% 50%

a

40%

33%

30% 20% 10% 0%

B C

b

3%

14%

a 40%

a A 39%

33%

B

a A 38% 30%

20%

0%

0_MEL

1_MEL

2_MEL

3_MEL

4_MEL

Tissue browning ratings across treatments Fig. 5 Association between tissue browning ratings and stable transformation frequencies for different treatments. Percentages of tissue browning ratings and stable transformation frequencies in MicroTom transformation under a standard conditions without antioxidants; b with the addition of a-lipoic acid to media; or c with the addition of melatonin to media. Treatments with different capital and small letters are significantly (P \ 0.05) different for tissue browning and stable transformation frequency, respectively, using Fisher’s LSD test

If plant defense responses are responsible for tissue browning and necrosis during Agrobacterium-mediated transformation, compounds predicted to reduce ROS generation and PCD should be able to reduce tissue browning and increase transformation frequencies. Among 12 non-antioxidant compounds tested 6 compounds, including thiamazole (TMZ), 2,5-dihydroxycinnamic acid methyl ester (DHC), DL-a-difluoromethylornithine hydrochloride hydrate (DFMO), diphenyleneiodonium chloride (DPI), genistin (GEN), a-nicotinamide adenine dinucleotide phosphate (NADPH) and a-nicotinamide adenine dinucleotide (NADH), highly significantly (P \ 0.01) reduced tissue browning by 1.2- to 2.0-fold (Fig. 6a) and three of them (SHA, DHC and DPI) highly significantly increased 1.7- to 1.8-fold of transient GUS expression (Fig. 6b). Regarding to six antioxidant compounds tested melatonin (MEL) highly significantly (P \ 0.01) reduced the most extreme levels (rating level 4) of tissue browning (MELB) at the concentrations of 10, 50, 100, 500 and 1,000 lM compared to negative control, ranging from 1.4fold at 1,000 lM to 3.3-fold at 100 lM (Fig. 7). The MELB reduction by MEL was even higher than that by LA (positive control, Fig. 7). Catalase (CAT) only at 1,000 U had a significant (P \ 0.05) 1.3-fold reduction in MELB compared to the negative control (Fig. 7). Resveratrol (RES) at 50, 100 and 200 lM and cysteine (CYS, positive control) at 50 and 100 lM and 3.3 mM highly significantly reduced MELB compared with the negative control, as did 1,3 dimethylthiourea (DMT) at 10, 50 and 100 lM, at a significant level (P \ 0.05). No significant reduction in MELB compared to the negative control was observed with the applications of hydroxylamine hydrochloride (BHA) and epicatechin (EPI, Fig. 7). In fact BHA at 100 lM and EPI at all concentrations tested had highly significant (P \ 0.01) higher percentage of MELB than the negative control, suggesting the possible toxicity of these compounds to the explants. However, in the transient expression assay, only hydroxylamine hydrochloride (BHA) at 10 lM and CYS at 50 lM significantly (P \ 0.05) increased transient GUS expression 1.3- and 1.6-fold, respectively, compared to the negative control. The rest of

123

Plant Cell Rep

(b)

H

M

80

40

20 10 0

Treatment

Effect of selected compounds on stable transformation Among 12 non-antioxidant compounds tested above four of them, which had significant reduction in tissue browning (TMZ, NADPH and NADH) and exhibited significant increase in transient expression (DHC), were further tested for their effect on stable transformation. All four compounds highly significantly (P \ 0.01) increased transformation frequency (TF) by 1.2- to 1.6-fold and reduced escape frequencies (EF) by 1.4- to 8-fold (Table 3).

123

dihydrochloride, DHC 2,5-dihydroxycinnamic acid methyl ester, DFMO DL-a-difluoromethylornithine hydrochloride hydrate, AEBSF 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, CAF caffeic acid, DPI diphenyleneiodonium chloride, ROSA rosmarinic acid, GEN genistin, NADPH a-nicotinamide adenine dinucleotide phosphate, NADH a-nicotinamide adenine dinucleotide. *Treatments were significantly different (p \ 0.05) compared to the negative control (N_CK) using Fisher’s LSD. **Treatments were highly significantly different (P \ 0.01) compared with negative control (N_CK) using Fisher’s LSD

N_CK

P_CK

M

L

H

100

**

80

20

* *

**

** **

40

** **

**

**

60

** **

Percentage of pokes having most extreme level of browning

the antioxidants had no significant effects on transient expression (data not shown). To evaluate possible additive or synergistic effect on tissue browning and transient expression between different compounds tested above, combinations of MEL with compounds that exhibited tissue browning reduction (DHC and DPI) or with antioxidants that enhanced transient expression (DHC, DPI and SHA) were investigated. However, no additive or synergistic effects on browning reduction or increased transient expression were observed while testing the five compounds in combination with MEL at optimal concentrations for each of the compounds (data not shown).

H

30

Treatment

Fig. 6 Effect of non-antioxidant compounds on frequency of extreme browning severity and high levels of transient expression (rating levels = 4). a Extreme browning severity; b transient expression levels were measured in 540 pokes per treatment from three independent experiments. Treatments were negative control (N_CK; no media addition); positive control (P_CK; 50 lM lipoic acid added) and low (L), medium (M) or high (H) concentrations of indicated candidate compounds added (for exact concentrations used see Table 2). TMZ thiamazole (also known as methimazole), SHA salicylhydroxamic acid, (6R)-BH4 (6R)-5,6,7,8-tetrahydrobiopterin

** **

0

50

M

** **

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**

**

20

60

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70 **

70

P_CK

80

** ** ****

L

**

P_CK

N-CK

90

**

N_CK

90

Percentage of pokes having high levels of transient expression

100

** ** **


100

** ** ** **

(a)

0 Mel_1 Mel_2

BHA

CAT

DMT

EPI

CYS

RES

Treatment

Fig. 7 Effect of antioxidants on frequency of extreme browning severity (rating level 4). Extreme browning severity was measured in 540 pokes per treatment from three independent experiments. Treatments were negative control (N_CK; no media addition); positive control (P_CK; 50 lM lipoic acid added) and low (L), medium (M) or high (H) concentrations of indicated candidate compounds added (for exact concentrations used see Table 2). Mel_1 melatonin at low concentration series, Mel_2 melatonin at high concentration series, BHA N-(tert-butyl) hydroxylamine hydrochloride, CAT catalase, DMT 1,3 dimethylthiourea, EPI epicatechin, CYS cysteine, RES resveratrol. *Treatments were significantly different (P \ 0.05) compared to the negative control (N_CK) using Fisher’s LSD. **Treatments were highly significantly different (P \ 0.01) compared with negative control (N_CK) using Fisher’s LSD

Plant Cell Rep Table 3 Effect of addition of candidate compounds at different concentrations on stable transformation and escape frequencies 4 weeks after selection in MicroTom transformation Treatment

Frequency of independent stable transgenic events (%)a

Frequency of escapes (%)

Positive control (50 lM LA)

175.6 % ± 27.4 A

6.7 ± 0.0

Negative control (no MEL)

88.1 % ± 25.6 B

11.7 ± 2.4

Table 3 continued Treatment

Frequency of independent stable transgenic events (%)a

Frequency of escapes (%)


308.9 ± 55.5 AB

6.7 ± 3.3

Negative control (no DHC)

234.4 ± 62.6 C

8.9 ± 6.9

DHC (10 lM)

344.4 ± 65.0 A

1.1 ± 1.9

DHC (50 lM)

320.0 ± 103.7 A

3.3 ± 5.8

DHC (100 lM)

263.3 ± 72.2 BC

1.1 ± 1.9


288.9 ± 52.1 AB

3.3 ± 3.3 4.4 ± 3.8

MEL (10 lM)

214.4 % ± 6.2 A

1.7 ± 0.0

MEL (50 lM)

221.3 % ± 47.7 A

0.0 ± 0.0

MEL (100 lM)

229.4 % ± 55.7 A

0.0 ± 0.0


183.3 % ± 0 A

Negative control (no NADPH)

243.3 ± 49.1 BC

Negative control (no MEL)

106.7 % ± 23.6 B

NADPH (2 lM)

297.8 ± 22.2 A

5.6 ± 5.1

MEL at 500 lM

121.7 % ± 1.7 B

NADPH (10 lM)

237.8 ± 22.2 BC

3.3 ± 3.3

MEL at 1000 lM

80.0 % ± 14.1 B

NADPH (50 lM)

214.4 ± 25.5 C

3.3 ± 3.3

288.9 ± 22.2 A

3.3 ± 3.3

208.9 ± 13.9 B

2.2 ± 3.8


196.7 ± 23.3 B

4.4 ± 5.1


Negative control (no BHA)

121.1 ± 13.5 C

11.1 ± 6.9

Negative control (no NADH)

BHA (10 lM)

245.6 ± 44.0 A

4.4 ± 5.1

BHA (50 lM)

222.2 ± 9.6 AB

6.7 ± 3.3

BHA (100 lM)

205.6 ± 10.2 AB

8.9 ± 1.9


363.3 ± 4.7 B

0.0 ± 0.0

Negative control (no EPI)

226.7 ± 4.7 C

6.7 ± 0.0

EPI (50 mg/L)

418.3 ± 25.9 B

1.7 ± 2.4

EPI (100 mg/L)

521.7 ± 16.5 A

0.0 ± 0.0

EPI (1000 mg/L)

NADH (2 mg/L)

295.6 ± 9.6 A

4.2 ± 3.8

NADH (10 mg/ L)

327.8 ± 29.1 A

1.1 ± 1.9

NADH (50 mg/ L)

201.1 ± 22.2 B

1.1 ± 1.9

a

51.7 ± 2.4 D

1.7 ± 2.4


306.7 ± 76.4 AB

3.3 ± 3.3

Negative control (no DMT)

231.1 ± 65.0 C

10.0 ± 6.7

DMT (10 lM)

354.4 ± 55.5 A

3.3 ± 3.3

DMT (50 lM)

312.2 ± 36.9 AB

3.3 ± 0.0

DMT (100 lM)

271.1 ± 33.6 BC

5.6 ± 1.9


325.6 ± 28.3 AB

2.2 ± 1.9

Negative control (no RES) RES (50 lM)

267.8 ± 10.2 BC

5.6 ± 3.8

297.8 ± 24.6 B

4.4 ± 7.7

RES (100 lM)

356.7 ± 10.0 A

1.1 ± 1.9

RES (200 lM)

227.8 ± 17.1 C

1.1 ± 1.9


212.2 ± 38.3 BC

8.9 ± 1.9

Negative control (no TMZ)

168.9 ± 28.3 C

TMZ (10 lM)

261.1 ± 12.6 A

TMZ (50 lM) TMZ (100 lM)

211.1 ± 13.5 BC 217.8 ± 17.1 AB

11.1 ± 6.9 7.8 ± 7.7 13.3 ± 3.3 11.1 ± 1.9

The treatments with different letters represent highly significant (P \ 0.01) difference using Fisher’s LSD test

For six antioxidant compounds tested above DMT, EPI, MEL and RES with significant tissue browning reduction and BHA with significant transient expression increase were further examined for their effect on stable transformation. The stable transformation results were summarized in Table 3. EPI at 50 and 100 mg/L highly significantly increased 1.9- and 2.3-fold of TF, respectively, compared with the negative control. At a tenfold higher concentration (1,000 mg/L) EPI reduced TF, indicating potentially toxicity at this concentration. MEL at 10, 50 and 100 lM highly significantly (P \ 0.01) increased 2.3-, 2.5- and 2.6fold of TF, respectively, compared with the negative control. MEL at 10, 50 and 100 lM dramatically reduced EF from 11.7 % in the negative control to 1.7 and 0 %, respectively. BHA at three different concentrations (10, 50 and 100 lM), highly significantly (P \ 0.01) increased TF by 1.6- to 2-fold compared to the negative control. BHA at 10 lM also reduced escape frequency (EF) 2.5-fold. Since MEL was optimal for reducing tissue browning and increasing TF it was further evaluated for its effect on soybean (cultivar Jack) transformation. MEL at 10 lM increased TF 2.2-fold in soybean (Fig. 8), although the observed effect was not statistically significant.

123

TMT_10 TMT_M_1 TMT_M_2 TMT_M_3 TMT_M_4 TMT_M_5 TMT_M_6

TMT_9

TMT_8

TMT_7

TMT_6

TMT_5

4.5

3.0

TMT_3

8

5.6

TMT_4

6.6

10

TMT_2

Non-transgenic

12

TMT_1

% of Transformation Frequency (TF)

Plant Cell Rep

6 4 2

9 kb

0 0 μM

1 μM

10 μM

100 μM

Melatonin Concentration

Fig. 8 Effect of melatonin on soybean stable transformation frequency

Southern blot analysis revealed that there was no negative effect of MEL on T-DNA integration and copy number of a transgene in MicroTom transformation (Fig. 9). Effect of MEL application at different transformation stages Since MEL and all candidate compounds were applied only in selection medium (selection stage) of Microtom transformation procedure for all experiments above we further evaluate what is the best stage of transformation to apply MEL for maximizing TF. First, to determine possible inhibition of MEL on Agrobacterium infection the optimal concentration of 100 lM MEL was added each to inoculation medium (inoculation stage), co-culture media (coculture stage) and both inoculation and co-culture media. Application of MEL at the stages highly significantly (P \ 0.01) reduced transient expression by twofold compared to no-application of MEL at these stages (Fig. 10a), suggesting that MEL had a significant inhibitory effect on Agrobacterium infection at these stages and MEL could not be applied in inoculation, co-culture or both stages. Second, to examine whether there is difference of tissue browning reduction among MEL application at different stages of transformation MEL was tested at six combinations of the different transformation stages, including inoculation (I), co-culture (C), selection (S), I ? C, C ? S and I ? C ? S. When applied MEL at all six combinations of the different transformation stages, no significant (P [ 0.05) difference on tissue browning reduction among the stages was observed (Fig. 10b). However, when evaluating TF among application of MEL at the same six stages above MEL application at the selection stage had a significant (P \ 0.05) increase in TF of 1.3- to 2.5-fold as compared to the rest stages (Fig. 10c). Media addition of

123

Fig. 9 Southern blot analysis for transgenic MicroTom plants generated with or without melatonin. NPTII probes were used. Transgenic MicroTom plants from TMT_1 to TMT_10 and TMT_M_1 to TMT_M_6 were generated without and with melatonin, respectively. Plant genomic DNA was extracted and approximately 20 lg of genomic DNA from transgenic plants was digested with EcoRI and probed with a probe for the nptII gene

just the solvent (ethanol) used to dissolve melatonin had no significant effect on tissue browning, transient expression or stable transformation frequency at all the six stages tested (data not shown). These results demonstrated that the optimal stage to apply MEL in MicroTom tomato transformation was at the selection stage.

Discussion The MicroTom browning/transient expression model system has been successfully used in the past to discover a novel transformation-enhancing compound, lipoic acid (LA; Dan et al. 2009). However, it had not previously been demonstrated that browning ratings in this model system are linearly correlated to Agrobacterium concentration and incubation time. Given the correlation between Agrobacterium concentration and tissue browning, we hypothesized that this response in the MicroTom model system is similar to HR that occurs during pathogen attack in intact plants and that is associated with ROS production. We demonstrated that browning occurred solely in response to the presence of the bacteria and that browning ratings increased with increasing concentration and incubation

Percentage of pokes having high levels of transient expression

Plant Cell Rep

A

(a) 90 80 70 60 50 40 30 20 10 0

73.9

B

B

B

36.7

38.9

37.6

INOC

CO-C

I+C

No MEL


Stage of application of melatonin

35 30 25 20

A

(b)

A A

A

28.9

A

23.9 21.7

21.9

A 19.4

16.1

15 10 5 0

Transformation frequency (TF)


50 45 40 35 30 25 20 15 10 5 0

A

(c)

40.4

B 23.5

C

C

16.5

16.9

INOC

CO-C

SEL

I+C

B 28.5

C+S

B 30.6

I+C+S


Fig. 10 Effect of melatonin application at different stages of MicroTom transformation on transient expression, percentage of most extreme level of browning and transformation frequency. a Transient expression as measured in 540 pokes per treatment from three independent experiments; b percentage of pokes having most extreme level of browning; c transformation frequencies as a percentage of inoculated pokes. Melatonin (MEL) at 100 lM was applied in six different treatments: (1) at inoculation alone (INOC); (2) at co-culture alone (CO-C); (3) at selection alone (SEL); (4) at both inoculation and co-culture (I ? C); (5) at both co-culture and selection (C ? S); and (6) at inoculation, co-culture and selection (ICS). The data represent the average of three independent experiments performed with a total of 90 explants per treatment per experiment. Treatments with different letters are significantly (P \ 0.05) or highly significantly (P \ 0.01) different using Fisher’s LSD test

time, indicating that tissue browning and death is caused by plant response to Agrobacterium in the MicroTom model system. Previous results in maize indicated that the death of Agrobacterium-inoculated cells during transformation was due to apoptosis, or programmed cell death (Hansen 2000). Much like intact plants under pathogen attack, cultured plant suspension cells can be elicited to produce H2O2 in response to virulent but not avirulent pathogens (Tenhaken et al. 1995). In studying the correlation between tissue browning and transient expression we showed that in the absence of the antioxidants LA or melatonin (MEL), most tissue browning was severe, and at these levels of browning GUS transient expression was highest and stable transformation frequency was lowest. This suggested that the plant tissue with high transient GUS expression level were severely damaged and exhibited extreme browning symptoms, and likely died following the severe Agrobacterium infection. When LA was added to the media most of the tissue browning observed was suppressed to intermediate levels, where transient GUS expression and stable transformation was found to be highest. We also found that stable transformation frequencies were significantly higher in tissues with browning levels 3 than the rest of browning levels for control and LA treatments. There was no significant difference among the stable transformation frequencies in tissues with all browning levels 1, 2, 3 and 4 for MEL treatment although there was significant difference among the tissue browning frequencies. Also, MEL significantly increased stable transformation frequency (STF) from cells with extreme tissue browning levels compared with of the control and LA treatments. These results indicated that MEL promoted regeneration of transformed cells into stable transgenic plants in addition to its effect on tissue browning reduction. Promotion of transformed cell regeneration by LA was reported in Agrobacterium-mediated Microtom transformation (Dan et al. 2009). A number of thiol and related sulfur-containing compounds have previously been successfully used as media additions to mitigate tissue browning in Agrobacteriummediated transformation, including cysteine; DTT; glutathione; sodium thiosulfate and LA (Perl et al. 1996; Enriquez-Obregoń et al. 1997, 1998, 1999; Das et al. 2002; Olhoft and Somers 2001; Olhoft et al. 2001, 2003; Dan et al. 2004, 2009). However, thiol compounds are important regulators of cellular redox status (Deneke 2000) in addition to being antioxidants and inhibitors of ROS-producing polyphenol oxidases and peroxidases (Olhoft et al. 2001; Dan et al. 2004, 2009; Dan 2008).

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Plant Cell Rep

Eleven (4 antioxidant and 7 non-antioxidant compounds) of our 19 compounds tested significantly reduced tissue browning and STF, and reduced escape frequency in MicroTom tomato. Three compounds, melatonin (MEL), N-(tert-butyl) hydroxylamine hydrochloride (BHA) and epicatechin (EPI), increased STF 2- to 2.6-fold. MEL was found to be approximately equivalent to LA (positive control) in reducing tissue browning and increasing STF in MicroTom tomato. Among MEL concentrations of 10, 50, 100, 500 and 1,000 lM tested 100 lM was optimal in term of transformation frequency (TF). However, it was shown that as MEL concentration increased, TF increased but has not reached the plateau at 100 lM because the next concentration tested was 500 lM, which might be too high to capture the potential higher TF at the concentrations between 100 and 500 lM. Similarly, 100 mg/L EPI has not reached the plateau at 100 mg/L because the next concentration tested was 1000 mg/L, which might be too high to obtain the potential higher TF at the concentrations between 100 and 1,000 mg/L. In contrast with BHA, although the lowest concentration of 10 lM tested was optimal for TF 10 lM might not be the lowest concentration for the potential higher TF. The optimal stage for applying cysteine has been shown to be at the co-cultivation stage in soybean (Olhoft et al. 2001) and maize (Frame et al. 2002). However, we found that the optimal stage to use MEL as a media addition to enhance MicroTom transformation was during selection, although application at other stages was found to have a positive effect on stable transformation frequency. In addition, the study of the effect of MEL at different stages of the process in combination supports the contention that selection is the best stage to apply the compound, and points to the importance of testing candidate transformation enhancing compounds at different stages of the process, as the optimal stage will likely vary depending on by crop, variety and transformation protocol. Given the diversity of compounds and postulated modes of action, we speculate that these compounds act in different ways to mitigate cellular oxidative stress in the transformation process. And they are likely involved in regulating ROS generation and detoxification, which causes the tissue browning and death during Agrobacteriummediated transformation in MicroTom. These compounds, which were able to reduce tissue browning and death, and increase STF in the MicroTom transformation, supported our hypothesis that oxidative stress caused by bacterial infection is an important limiting factor in Agrobacteriummediated transformation. The mechanism by which the antioxidant and nonantioxidant compounds evaluated in our study reduced tissue browning and death of Agrobacterium-infected cells is not currently known. Both LA and L-cysteine (like DTT

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and glutathione) are sulfur-containing compounds with free sulfhydryl groups or thiol bonds (Deneke 2000). Olhoft et al. (2001) showed that the frequency of transformed cells was increased not only by L-cysteine, but also by D-cysteine and other thiol-containing compounds, suggesting that the reduction in tissue necrosis is due to the activity of the thiol group. The thiol group inhibits the activities of the polyphenol oxidases and peroxidases, thereby reducing tissue browning by these compounds (Olhoft et al. 2001; Dan et al. 2004, 2009; Dan 2008). Apoptosis functions as an important defense strategy by host cells against viral invasion. Many viruses contain the antiapoptotic genes to block the defense-by-death response of host cells (Wang et al. 2004). To investigate our hypothesis that compounds capable of blocking plant defense responses can mitigate tissue browning and death in Agrobacterium-mediated transformation, we evaluated several compounds (DADPH, DADH, TMZ and DHC), which are not classically defined as antioxidants. We discovered that they were capable of reducing the incidence of severe tissue browning and enhancing transformation frequency while decreasing escapes. NADH and NADPH are enzyme co-factors that provide reducing power to multiple cellular reactions. DHC is a phenolic protein kinase inhibitor known to inhibit H2O2 production in elicited cultured rice cells (Lin et al. 2005); and TMZ, a thione compound with a free sulfhydryl group. The results with NADH, NADPH and thiamazole lend support to the hypothesis that compounds that affect cellular redox status, and in particular compounds with free sulfhydryl groups, can significantly mitigate Agrobacterium-mediated tissue browning, consistent with previously discovered compounds in this class such as cysteine and LA (Olhoft et al. 2001; Dan et al. 2004, 2009; Dan 2008). Regarding escape reduction by these compounds our previous study showed that LA significantly increased stable transformation frequency by significantly increasing transgenic shoot production and decreasing non-transgenic shoot production (Dan et al. 2009). Therefore, the growth of non-transformed cells or tissues was overwhelmed by the promoted growth of the transformed cells or tissues by LA under the selective competition, resulting in a significant decrease of shoot escapes and subsequent increase of stable transformation frequency in MicroTom transformation (Dan et al. 2009). Zheng et al. (2005) reported a similar result with the antioxidants, sodium selenite, DL-atocopherol, and glutathione, increasing the percentage of GUS-positive shoots without altering the total number of shoots regenerated in peanut Agrobacterium-mediated transformation. The results with these compounds further supported the previous evidence. MEL exists in plants, and is known as a neurohormone in animals. It plays specific roles during plant

Plant Cell Rep

development and exogenously applied MEL affects developmental processes during vegetative growth (Murch et al. 2001; Hernandez-Ruiz et al. 2004, 2005; Arnao and Hernandez-Ruiz 2007; Chen et al. 2009; Park 2011). However, the question still remains whether MEL acts directly or indirectly through effects on auxin biosynthesis. Our results showed that MEL reduced tissue browning and subsequently enhanced stable transformation, indicating that MEL plays a role in antioxidant defense against oxidative stress as observed in animals. Animal recognition of MEL is through multiple membrane receptors (Dobocovich and Markowska 2005). In plants no proteins showing similarity to animal MEL receptors have been identified to date (Park 2011). Identifying plant melatonin receptors or a receptor-independent mechanism will enable us to better understand how the MEL signal is perceived and transduced in plants. One hypothesis to explain the promotion of regeneration by antioxidants relates to their effect on the cellular redox environment, in that a cellular environment that is less oxidizing can promote cell division, proliferation and differentiation. There is evidence that, in the root apex, cells having relatively slower mitotic activity and reducing cell proliferation had a more oxidizing environment and were involved in root apical meristem establishment and maintenance (De Tullio et al. 2010). In contrast, cells with a mildly oxidizing or reducing environment correlated with an increase in mitoses and differentiation in the root apical cells. Indeed, redox gradients may underlie gradients in various activities along the root axis, including auxin regulation of root development. Perhaps, ROS balance in root meristems involves interactions with other plant growth regulators (De Tullio et al. 2010). This might explain that when LA and MEL are applied to the medium the majority of tissue browning distributed at the intermediate level where transient GUS expression and stable transformation was found to be highest. This result indicated that LA and MEL might not only reduce the oxidization caused by Agrobacterium but also maintain a reducing environment and interact with plant growth hormones that enabled transformed cells to divide, proliferate and regenerate. Both LA (Dan et al. 2009) and MEL significantly increased stable transformation in tomato and soybean. However, among all compounds tested MEL exists in plants, and is known as a neurohormone in animals. Our results showed that MEL application had an additional effect of promoting the regeneration of transformed cells into stable events, besides their effects on reducing tissue browning, suggesting that MEL may also act as a growth hormone in plant. However, the question still remains whether MEL acts directly or indirectly through effects on auxin biosynthesis or this effect is mitigated through known hormones such as auxin (Tan et al. 2012).

In conclusion our study addresses the tissue browning and subsequent death associated with Agrobacteriummediated plant transformation, a long-standing and high impact problem in plant biotechnology. Solving the problem of tissue browning and death in transformation and understanding its mechanisms associated with it is a proven route to improving Agrobacterium-mediated transformation. The efficient production of transgenic plants has great value in both our understanding of plant gene function and in delivering the genetically modified crop products of the future. Compounds that enhance the efficiency of plant transformation have an important role to improve existing transformation systems, and to broaden the range of transformable species and cultivars. The exact mechanisms underlying the function of these compounds, particularly LA and MEL, remain to be studied more precisely in the future. Author contribution statement As principal investigator YD co-directed this project, co-designed theexperiments, co-analyzed the data and co-wrote the manuscript. SZ performed the experimentsand collected the raw data. HZ co-designed the transformation experiments and performed thesoybean experiments. MS co-directed this project, co-designed the experiments, and co-wrote themanuscript. Hochul Yi performed the soybean transformation experiments and collected the raw data. Acknowledgments Funding for this project was provided by Syngenta Biotechnology, Inc. The authors would like to thank Drs. Liang Shi and Thomas Chenier from Syngenta Biotechnology, Inc., and Drs. Timothy Hawkes, Eric Clarke from Syngenta Jealott´s Hill International Research Centre for technical assistance and stimulating discussions. We gratefully acknowledge the contribution of Drs. Shujie Dong and John Ke from Syngenta Biotechnology, Inc. in critically reviewing this manuscript. Conflict of interest of interest.

The authors declare that they have no conflict

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