Agrobacterium-mediated high-frequency ... - Springer Link

2 downloads 0 Views 1MB Size Report
Jul 26, 2014 - Myeong-Je Cho • Emily Wu • Jackie Kwan • Maryanne Yu • Jenny Banh •. Wutt Linn • Ajith Anand • Zhi Li • Susan TeRonde • James C. Register ...
Plant Cell Rep (2014) 33:1767–1777 DOI 10.1007/s00299-014-1656-x

ORIGINAL PAPER

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line Myeong-Je Cho • Emily Wu • Jackie Kwan • Maryanne Yu • Jenny Banh • Wutt Linn • Ajith Anand • Zhi Li • Susan TeRonde • James C. Register III Todd J. Jones • Zuo-Yu Zhao



Received: 21 June 2014 / Accepted: 4 July 2014 / Published online: 26 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Key message An improved Agrobacterium-mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. Abstract This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction

rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

Communicated by Prakash Lakshmanan. M.-J. Cho (&)  J. Kwan  M. Yu  J. Banh  W. Linn DuPont Agricultural Biotechnology, DuPont-Pioneer, 4010 Point Eden Way, Hayward, CA 94545, USA e-mail: [email protected] J. Kwan e-mail: [email protected] M. Yu e-mail: [email protected] J. Banh e-mail: [email protected] W. Linn e-mail: [email protected] E. Wu  A. Anand  Z. Li  S. TeRonde  J. C. Register III  T. J. Jones  Z.-Y. Zhao DuPont Agricultural Biotechnology, DuPont-Pioneer, 8305 NW 62nd Avenue, P. O. Box 7060, Johnston, IA 50131, USA e-mail: [email protected]

A. Anand e-mail: [email protected] Z. Li e-mail: [email protected] S. TeRonde e-mail: [email protected] J. C. Register III e-mail: [email protected] T. J. Jones e-mail: [email protected] Z.-Y. Zhao e-mail: [email protected] Present Address: W. Linn MetroHealth Medical Center, 2500 Metrohealth Dr, Cleveland, OH 44109, USA

123

1768

Keywords Maize inbred  Transformation  Agrobacterium  Green regenerative tissue medium  Transformation frequency  Transgenic plants

Introduction Maize (Zea mays L.) is one of the largest and most important cereal crops produced in the world. In 2012, the total production of maize was 872 million metric tons worldwide (Food and Agriculture Organization of the United Nations, Statistics Division 2012). It is the leading source of livestock feed and bio-energy crop for ethanol production. Maize is also very important for food production. It is used for production of starch, oil and sweeteners and is a staple food in many regions of the world. Therefore, a successful, highly efficient maize transformation protocol is a prerequisite to develop a commercial, genetically modified (GM) maize product conferring herbicide and/or insect resistance, drought tolerance or high carbohydrate content to increase the yield of maize for food and bio-fuel production. Maize transformation has been reported via electroporation (D’Halluin et al. 1992), PEG (Golovkin et al. 1993), silicon carbide whisker (Frame et al. 1994; Petolino et al. 2000), micro-projectile bombardment (Fromm et al. 1990; Gordon-Kamm et al. 1990; Wan et al. 1995) and Agrobacterium (Ishida et al. 1996, 2007; Zhao et al. 1998; 2001; Negrotto et al. 2000; Frame et al. 2002, 2006; Huang and Wei 2005; Ombori et al. 2013). The first successful Agrobacterium-mediated transformation was reported using A188, an amenable maize inbred (Ishida et al. 1996). However, A188 is an inbred with poor agronomical value. There were also previous reports of successful maize transformation in Hi-II, a hybrid (A188 9 B73) line (Zhao et al. 1998, 2001; Frame et al. 2002; Vega et al. 2008). In addition, Huang and Wei (2005) developed an Agrobacterium-mediated transformation system for maize inbred lines using EHA105 with optimal culturing and infection conditions. They reported transformation frequencies ranging from 2.4 to 5.3 % in four inbred lines, 9046, Mo17, 414 and Qi319. Another transformation system was established for three inbred lines, B104, B114, and Ky21 with 2.8–8 % transformation frequencies using MS (Murashige and Skoog 1962) salts as the basal tissue culture medium (Frame et al. 2006). The amenability of Agrobacterium-mediated transformation in tropical maize inbreds and hybrid lines was also evaluated (Ombori et al. 2013); immature zygotic embryos and embryogenic calli were infected to achieve a 1.4 % transformation frequency. An efficient in vitro tissue culture system was previously established to proliferate highly regenerative, green tissues in several monocotyledonous crops such as barley (Cho

123

Plant Cell Rep (2014) 33:1767–1777

et al. 1998; Lemaux et al. 1999), wheat (Cho et al. 1999a; Kim et al. 1999; Li et al. 2009), sorghum (Cho and Lemaux 2001), oat (Cho et al. 1999b), tall and red fescues (Cho et al. 2000), Kentucky bluegrass (Ha et al. 2001), orchardgrass (Cho et al. 2001), creeping bentgrass (Cho et al. 2003) and rice (Cho et al. 2004). These green regenerative tissues could be initiated and maintained to regenerate multiple shoots on MS-based media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high concentrations of copper. The establishment of an efficient Agrobacterium-mediated transformation method in commercial maize elite inbreds can facilitate faster and more effective transgene evaluation and GM product development. In addition, transformation into elite commercial maize inbreds could enable quick screening of transgene efficacy, faster introgression of the transgenes into hybrids and earlier initiation of regulatory studies. In this study we report a high-frequency Agrobacterium-mediated transformation protocol for a recalcitrant, commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media based on regenerative green tissues. PHR03 is a non-stiff stalk, yellow, dent corn inbred and is best adapted to the Eastern part of the United States (Oestreich 1995). PHR03 was selected from multiple inbreds based on a preliminary in vitro culturing response and T-DNA delivery efficiency screening. Different ratios and combinations of key tissue medium components such as copper, BAP, and glucose were tested to significantly increase transformation frequencies of PHR03. Four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 were also tested to evaluate the effect of Agrobacterium strains on transformation frequency and event quality.

Materials and methods Plant material A commercial maize elite-inbred line, PHR03, was used for Agrobacterium-mediated transformation optimization. Seeds of PHR03 were sown in pots containing 50:50 Metro-Mix 838 (Sun Gro Horticulture Canada CM Ltd., Agawam, MA, USA) and Turface Athletics MVP (Profile Products LLC, Buffalo Grove, IL, USA) and grown in a greenhouse at 16/8-h photoperiod intervals (300 lmol m-2 s-1), 21–27 °C and 16–21 °C, respectively. Seedlings were moved into two gallon pots of Metro-Mix 838 after 2 weeks and placed in a greenhouse (14/10-h photoperiod at 29 °C day and 21 °C night temperature regimes). At the reproductive stage, shoots were self- or sib-pollinated, and immature ears were harvested 10–11 days after pollination (DAP) as immature embryo (IE) donors. The IEs were

Plant Cell Rep (2014) 33:1767–1777

surface-sterilized for 15–20 min in 20 % (v/v) bleach (5.25 % sodium hypochlorite) plus one drop of Tween 20 followed by three washes in sterile water. Agrobacterium strains and vectors Two different binary vectors, PHP24600 and PHP32269 (Wu et al. 2014), were used in PHR03 transformation experiments; PHP24600 contains DsRed driven by the maize ubiquitin 1 promoter and its first intron, and phosphinothricin acetyltransferase (PAT) driven by the CaMV35S promoter (Fig. 1a) while PHP32269 contains two gene cassettes, maize codonoptimized PAT (moPAT) fused to yellow fluorescent protein (YFP), moPAT::YFP (a translational fusion), and phosphomannose isomerase (PMI), both driven by the maize ubiquitin 1 promoter and its intron (Wu et al. 2014, Fig. 1b). Experiments comparing Agrobacterium strains LBA4404, AGL1, EHA105 and GV3101 were completed using construct PHP24600 or PHP32269. Agrobacterium strains were streaked from glycerol stocks stored at -80 °C. Using standard microbiological technique, bacteria were streaked to produce single colonies on LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Bacto-Agar) with 100 mg/L spectinomycin in a 100 9 15 Petri dish, incubated at 28 °C with the plate in the dark for 2–3 days. From this master plate, 1–3 colonies were picked to streak on a fresh plate of YP medium (5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl, 15 g/L Bacto-agar) with 100 mg/L spectinomycin, incubated at the same conditions for 1 day overnight. Preparation of Agrobacterium suspension for immature embryo infection Agrobacterium was collected from the YP plate and placed in a solution of PHI-I infection medium (Zhao et al. 2000)

1769

with 100 lM acetosyringone. The solution was vigorously shaken and/or vortexed to produce a uniform Agrobacterium suspension. The Agrobacterium suspension was adjusted to a final absorbance (A550nm) of 0.35 and used in transformation of IEs. Medium compositions and modifications PIA medium (605J; Lowe et al. 2007) solidified with agar (Sigma-Aldrich Corp., St Louis, MO, USA) was tested against green tissue (GT) DBC3 medium (Cho et al. 1998, Table 1) in PHR03 IEs, isolated at 1.3–2.0 mm, and evaluated for tissue culture/callus induction optimization (Tables 2, 3). PIA medium was the standard, previously published culture medium containing 20 g/L sucrose and 0.6 g/L glucose with AgNO3. PI medium was equivalent to PIA medium but agar was substituted with Phytagel (Sigma-Aldrich Corp., St. Louis, MO, USA) as a gelling agent. PIBC2 medium was the equivalent of PI medium with the addition of 0.1 mg/L BAP and additional copper (509) amounts (5.0 lM final concentration). The PI scheme with additional modifications tested were (1) PIBC2-Ag, the same as PIBC-2 with AgNO3 removed; (2) PIBC2(SG)-Ag, the same as (1) but increased glucose from 0.6 to 10 g/L (Table 1). Modified DBC3 media were tested with various sugar combinations: (1) DBC3(30M10G), the same as DBC3 with 10 g/L glucose; (2) DBC3(20M10G), a 20 g/L maltose and 10 g/L glucose combination; (3) DBC3(S), the same as DBC3 but sucrose replacing maltose at 30 g/L; (4) DBC3(30S10G), the same as DBC3(S) with 10 g/L glucose; (5) DBC3(20S10G), a 20 g/L sucrose and 10 g/L glucose combination. To minimize the ear-to-ear variation, IEs derived from the same ear were evenly distributed per treatment and incubated under the same conditions to test the effect of medium modifications.

Fig. 1 Schematic diagram of PHP24600 (a) and PHP32269 (b) used for PHR03 transformation. RB right border, CAMV35S TERM CaMV35S terminator, PAT phosphinothricin acetyltransferase, CAMV35S Pro CaMV35S promoter, UBI1ZM Pro maize ubiquitin 1 promoter, UBI1ZM 5UTR maize ubiquitin 1 50 UTR, UBI1ZM INTRON1 maize ubiquitin 1 intron, LB left border, PMI phosphomannose isomerase, PINII TERM potato proteinase inhibitor II terminator, moPAT maize codon-optimized PAT

123

1770

Plant Cell Rep (2014) 33:1767–1777

Table 1 Compositions of media used for transformation Name

Ingredients

PIBC2(SG)-Ag

PIA-AgNO3 ? 0.1 mg/L BAP ? 10 g/L glucose ? 1.22 mg/L CuSO45H2O

DBC3

4.3 g/L MS salts, 30 g/L maltose, 1.0 mg/L thiamine-HCl, 0.25 g/L myo-inositol, 1.0 g/L casein hydrolysate, 0.69 g/L L-Proline, 1.0 mg/L 2,4-D, 0.5 mg/L BAP, 1.22 mg/L CuSO45H2O, 3.5 g/L Phytagel, pH 5.8

DBC3 PMI

DBC3 medium, reducing maltose to 5 g/L and supplemented with 12.5 g/L of D-mannose

MSB rooting medium

4.43 g/L MS salts with vitamins (Phyto Technology Laboratories, Shawnee Mission, KS, USA, catalog # M519), 1 g/L myo-inositol, 20 g/L sucrose, 0.5 mg/L indole-3-butyric acid (IBA), pH 5.8, 2.5 g/L Phytagel

PHR03 transformation PHR03 ears were collected from donor material described above. IEs from PHR03, at an optimal size of 1.5–1.8 mm, were aseptically isolated intact from kernels into 1.5 mL microfuge tubes of PHI-I liquid medium (Zhao et al. 2000) containing 100 lM acetosyringone suspension and allowed to sit for 20 min. The solution was drawn off and the IEs were then infected with 1.5 mL of Agrobacterium suspension containing a binary vector (PHP24600 or PHP32269), followed by a brief vortex and 5 min suspension in the bacterial solution prepared previously. The suspension of Agrobacterium and IEs was then poured onto PHI-T co-cultivation medium (Zhao et al. 2000) and the Agrobacterium liquid drawn off. IEs were arranged scutellum-side up on the medium; IEs derived from the same ear were evenly distributed per treatment for all transformation experiments to minimize seasonal and earto-ear variation of the donor material. The plate was sealed with Parafilm and incubated in the dark at 21 °C for 3 days of co-cultivation. IEs were transferred scutellumside up onto DBC3 or PIBC2(SG)-Ag resting medium (Table 1) without a selective agent and incubated at 26 ± 2 °C under dim light (10–30 mE m-2 s-1, 16/8h day and night photoperiod). The medium contained 100 mg/L carbenicillin (ICN, Costa Mesa, CA, USA) for LBA4404 or 100 mg/L cefotaxime (PhytoTechnology Lab., Shawnee Mission, KS, USA) and 150 mg/L timentin (PhytoTechnology Lab., catalog number T869) for AGL1, EHA105, and GV3101. After 4 days, the coleoptiles were removed from the IEs and the tissues were transferred to DBC3 medium containing 3 mg/L bialaphos (Meiji Seika Kaisha, Yokohama, Japan) for bar/PAT/moPAT selection and appropriate antibiotic(s). Two weeks after 1st round

123

selection, tissues were transferred to fresh DBC3 medium supplemented with 5 mg/L bialaphos ? antibiotic(s) and sub-cultured at 2–3 week intervals. At the 3rd round selection, tissues were broken into small pieces to place on the same medium containing bialaphos for further proliferation until sufficient amount of tissues was obtained. Regenerative green tissues were then transferred to regeneration medium PHI-X (Zhao et al. 2000) removing carbenicillin and adding 1.22 mg/L CuSO45H2O containing 5 mg/L bialaphos ? antibiotic(s) and incubated at 26 ± 2 °C under dim light for 2–3 weeks. The shoots were transferred to MSB rooting medium (Table 1) containing 3 mg/L bialaphos ? antibiotic(s), and exposed to higher light intensity (50–150 mE m-2 s-1, 16/8-h day and night photoperiod) for rooting. The plantlets were then transferred to soil. In experiments using PMI selection, after 3 days on PHI-T co-cultivation medium, the resting stage is extended to a total of 10–11 days on the appropriate resting medium before transferring to 1st round selection, DBC3 PMI medium (Table 1) containing mannose (SigmaAldrich Corp, St Louis, MO, USA) with antibiotic(s). Tissues were cultured for 2–3 weeks before transferring to fresh DBC3 PMI medium for 2nd round selection and repeated until sufficient amount of tissues was obtained. Regenerative green tissues were transferred to PHI-XM medium (Wu et al. 2014) under the same conditions described above. Shoots were then transferred to MSB rooting medium with antibiotic(s) as described above without a selection agent. Transformation frequency (%) at the T0 callus tissue level was measured 6 weeks after Agrobacterium infection and calculated as 100 % 9 (# of IEs with transgenic sectors/# of IEs infected). Transgenic sectors were identified as actively growing tissue exhibiting DsRED or YFP expression. Multiple sectors formed on a single embryo were counted as one event. The transformation frequencies at the T0 plant level was measured after regenerating T0 plantlets and was calculated as 100 % 9 (# of IEs regenerating plantlets/# of IEs infected). Multiple plantlets formed by a single embryo were counted as one event. Quality event (QE) is defined as single copy for transgenes without Agrobacterium T-DNA backbone. Usable quality event (UQE) is defined as transformation frequency multiplied by QE ratio. Copy number analysis of transgenic plants qPCR for copy number analysis of transgenes and binary vector backbone presence was performed following Wu et al. (2014). Genomic DNA from leaf tissue was extracted

Plant Cell Rep (2014) 33:1767–1777

1771

from T0 plants for analysis, as well as wild-type negative controls from PHR03.

Results and discussion Initial transformation improvement using GT medium in resting and selection steps

Fig. 2 Transformation frequencies of PHR03 at the T0 plant level using green regenerative tissue medium. Scheme 1 used PI medium in the resting step for 3 days and for selection while scheme 2 used DBC3 medium for 3 days in the resting step and for selection. Thirty to 35 immature embryos per treatment were used for transformation. Each histogram represents the mean level (±standard error) from nine replicates

Fig. 3 Transgenic green tissue, shoots, roots, leaf, pollen and seeds derived from PHR03. a Transgenic green tissue expressing DsRed Bar 2 mm, b regenerating transgenic plantlets, c transgenic root and d leaf expressing DsRed, e pollen grains from a T0 transgenic plant

Initial transformation frequency of PHR03 with PHP24600 was 2.6 % (8/310) at the T0 plant level using PI medium through resting and selection stages (Fig. 2). When the medium scheme was combined with GT medium using DBC3 for resting and selection, transformation frequency significantly increased up to 25.8 % (80/310) at the T0 plant level. Transgenic events were confirmed by checking for DsRed expression using fluorescence microscopy followed by qPCR/PCR analysis as previously mentioned (Wu et al. 2014). The T0 plants had a normal phenotype

with and without expression of DsRed Bar 0.1 mm, f normal phenotype and g tassels of T0 plants, and h T1 ear showing good seed set

123

1772

Plant Cell Rep (2014) 33:1767–1777

exhibiting normal height, good tassel quality, and excellent seed set (Fig. 3) comparable to wild-type PHR03. Tissue culture medium test and modification/ optimization for PHR03 To test, improve and optimize the tissue culture medium compositions, two basic corn tissue culture media were compared: PIA [605J medium based on MS (Murashige and Skoog, 1962) ? N6 salts (Chu et al. 1975)] and DBC3 (GT medium based on MS salts; Cho et al. 1998). The key components of BAP and copper from DBC3 medium were tested in PI medium. The gelling agent used for the media was also tested, PIA vs PI; PIA uses Sigma agar while PI uses Phytagel as a replacement of agar. Slightly better tissue quality was observed on PI than on PIA (Table 2). Subsequent PI-based media tested [PIBC2, PIBC2, PIBC2Ag and PIBC2(SG)-Ag] used Phytagel (Tables 2, 3). Between the two basic tissue culture media, PI-based

media was generally better in tissue induction using smaller-sized immature embryos, 1.3–1.5 mm, compared to DBC3 medium (Table 2). DBC3 medium was better in induction of good quality tissue, but the initial tissue induction surface area/embryo was lower than that of PI media. When BAP and additional copper were added to PI medium (PIBC2), PHR03 tissue quality was improved through formation of green regenerative tissues (Table 2). These green regenerative tissues contained multiple lightgreen structures similar to shoot meristems. Similar results by a combination of cytokinin and copper were observed in other monocot crops (Cho et al. 1998, 1999a, 1999b, 2000, 2001, 2003, 2004; Kim et al. 1999; Lemaux et al. 1999; Li et al. 2009; Cho and Lemaux 2001; Ha et al. 2001; Wu et al. 2014). More specifically, in barley, these tissues had physiological and developmental similarities with shoot meristematic tissues cultured from the excised shoot apices on medium containing 2,4-D and BAP (Lemaux et al.

Table 2 Green regenerative tissue induction on different culturing media and effect of immature embryo size on tissue induction and quality in PHR03 IE size

Medium

PIA

1.5–2.0 mm

GT induction frequencya

42 %

58 %

100 %

83 %

GT induction surface areab

30–40 %

40–50 %

50–70 %

20–30 %

GT quality GT induction frequency

??(?) 25 %

??? 35 %

????(?) 50 %

????? 45 %

GT induction surface area

30–40 %

20–40 %

20–40 %

10–20 %

GT quality

??(?)

???

???(?)

?????

1.3–1.5 mm

PI

PIBC2

DBC3

IEs derived from the same ear were placed onto each medium and incubated under the same conditions to test the effect of medium modifications a

GT induction frequency: # IEs inducing GT from scutellum tissue/# IEs plated

b

GT induction surface area: % range of induction surface area observed from IEs with GT induction

Table 3 Effect of component modifications on percentage of immature embryos with tissue induction from scutellum in PHR03 Media

Formulations

Sucrose PI-based

DBC3-based

Tissue induction rate (%)a

Key media components Maltose

Glucose

AgNO3

PIA

20



0.6

?

22.5 ± 2.8

PIBC2

20



0.6

?

43.8 ± 4.6

PIBC2-Ag PIBC2(SG)-Ag

20 20

– –

0.6 10

– –

42.5 ± 2.3 88.8 ± 6.1

DBC3



30





33.8 ± 6.7

DBC3(30M10G)



30

10



80.0 ± 2.3

DBC3(20M10G)



20

10



73.8 ± 1.2 46.3 ± 5.1

DBC3(S)

30







DBC3(30S10G)

30



10



91.7 ± 2.1

DBC3(20S10G)

20



10



88.8 ± 2.3

Sixteen IEs, 1.3–1.8 mm, derived from the same ear were placed on each medium incubated under the same conditions to test the effect of medium modifications a

Values represent mean ± standard error from 5 replicates except DBC3(30S10G) with 3 replicates

123

Plant Cell Rep (2014) 33:1767–1777

1773

Fig. 4 Improvement of green regenerative tissue induction from immature embryos of PHR03. Bar 2 mm. Photos were taken 10, 11 and 24 days after immature embryo isolation on two different tissue induction media, DBC3 and PIBC2(SG)-AG

Transformation Frequency (%)

60

50

40

30

20

10

0

Scheme 1

Scheme 2

Fig. 5 A side-by-side comparison to improve PHR03 transformation frequency at the T0 tissue level using LBA4404/PHP32269 and two different resting medium schemes. Scheme 1 used DBC3 medium for 10–11 days in the resting step while scheme 2 used PIBC2(SG)-Ag for 4 days and DBC3 for 6–7 days in the resting step. Schemes 1 and 2 used 583 and 603 IEs in total with three replicates for transformation, respectively. Each histogram represents the mean level (±standard error) from three replicates

1999). The results of these analyses were consistent with the visual observations that these tissues were organogenic rather than embryogenic (Lemaux et al. 1999). Highly regenerative green tissues with a high percentage of totipotent cells have been shown to be capable of sustained

cell division and competence for regeneration over long periods. This was also observed in PHR03 in which highly regenerative tissues could be maintained for more than 6 months with minimal loss in regenerability. The use of relatively large IEs (1.5–2.0 mm) on the above mentioned medium considerably increased the tissue induction frequency from scutellum after IE isolation (Table 2) mostly due to higher IE survivability. When IEs larger than 2.0 mm were used, however, there was lower quality tissue induction. Overall, PIBC2 was best in terms of green tissue induction frequency and surface area while DBC3 was best for tissue quality and proliferation (Table 2). Further improvements and optimizations were made by testing two other key components: AgNO3 and glucose. AgNO3 has been used extensively for plant tissue culture to reduce ethylene effects (Beyer 1976). In wheat, the addition of AgNO3 improved embryogenic callus frequency and callus growth but did not affect callus induction frequencies in genotypes Chuannong16, Chuanyu16, Y1496 (Wu et al. 2006). In maize, AgNO3 was first tested in the later stages of maize tissue culture and results showed that it increased plant regeneration frequency (Songstad et al. 1988). However, for maize elite inbred PHR03, AgNO3 did not affect GT induction (Table 3) but it had a slightly negative effect on tissue proliferation. When glucose was added as a component of the medium, tissue induction

123

1774

Plant Cell Rep (2014) 33:1767–1777

Table 4 A side-by-side comparison to improve PHR03 transformation frequency at the T0 plant level using LBA4404/PHP32269 and two different resting medium schemes Culturing schemea

Resting medium

# embryos

# events

T0 tissue level transformation frequency (%)b

# T0 plants

T0 plant level transformation frequency (%)b

1

DBC3 (10–11 days)

146

35

24.0 ± 10.9

9

6.2 ± 4.6

2

PIBC2(SG)-Ag (4 days) ? DBC3 (6–7 days)

147

47

32.0 ± 9.7

27

18.4 ± 3.9

About 35 IEs, 1.5–1.8 mm, derived from the same ear were used for each treatment incubated under the same conditions to test the effect of culturing scheme modifications a

Scheme 1 used DBC3 medium for 10–11 days in the resting step while scheme 2 used PIBC2(SG)-Ag for 4 days and DBC3 for 6–7 days in the resting step Values represent mean ± standard error from four replicates

frequency and tissue proliferation of IEs were greatly affected. All media containing 10 g/L glucose, PIBC2(SG)-Ag, DBC3(30M10G), DBC3(20M10G), DBC3(30S10G) and DBC3(20S10G), dramatically increased tissue induction frequency even when smallsized IEs were used (Table 3). Tissue induction frequency of embryos on PIBC2-Ag was 43 %; however, when 10 g/ L of glucose was added to medium PIBC2(SG)-Ag, tissue induction frequency increased to 89 %. The same trend was seen for DBC3 media containing maltose or sucrose. Tissue induction frequency was 34–46 % in DBC3 media containing only 30 g/L of maltose or sucrose [DBC3 or DBC3(S)] and when 10 g/L of glucose was added [DBC3(30M10S) or DBC3(30S10S)], frequency increased to 80–92 %. When 20 g/L of maltose or sucrose were used in conjunction with 10 g/L of glucose [DBC3(20M10G) and DBC3(20S10G)] there was very small or no significant difference in tissue induction frequency compared to 30 g/ L of maltose or sucrose. It was observed that PHR03 was sensitive to glucose at the step of IE isolation because a high percentage of IEs did not survive on medium containing 0–0.6 g/L of glucose. IE survival, especially for small-sized IEs, was better when 10 g/L of glucose in the medium was used. The increased survival rate on high glucose medium for smaller-sized IEs could be due to the fact that small-sized IEs more easily utilize glucose, a monosaccharide, as a carbon source rather than sucrose or maltose, a disaccharide. The above data suggest glucose is critical for IE survival at early stages following isolation. In general, medium containing 1–10 g/L glucose was found to be most effective for 1.5–2.0 mm IEs in terms of survival rate for short-term culturing. Overall, PIBC2(SG)Ag was identified as optimum medium for tissue survival, initial tissue induction and tissue proliferation from IEs (Table 3; Fig. 4). DBC3 was found optimal for tissue proliferation since prolonged culturing on PIBC2(SG)-Ag resulted in poor quality tissue with excess root formation and long-term culturing on high glucose containing

123

Transformation Frequency (%)

b

30 25 20 15 10 5 0 LBA4404

AGL1

EHA105

GV3101

Fig. 6 PHR03 transformation frequencies at the T0 plant level using four Agrobacterium strains containing PHP32269. About forty IEs, 1.5–1.8 mm, derived from the same ear were used for each Agrobacterium strain incubated under the same conditions to test the effect of Agrobacterium strains. Each histogram represents the mean level (±standard error) from four replicates

medium had an adverse effect on tissue culture and transformation of PHR03. Improvement of PHR03 transformation frequency using a modified resting medium PIBC2(SG)-Ag Based on the tissue culture medium test results, two different resting media in Agrobacterium-mediated transformation experiments were tested to compare the modified resting medium PIBC2(SG)-Ag with DBC3 resting medium using PMI selection and LBA4404. The modified resting step had a 4-day incubation period on PIBC2(SG)Ag and an additional 3- to 7-day incubation period on DBC3 after co-cultivation. The results show that the transformation frequencies at the T0 tissue level were 46.3 and 24.2 % for modified and DBC3 resting media, respectively; the use of PIBC2(SG)-Ag resting medium improved transformation frequency 1.9-fold (Fig. 5). A separate experiment showed that PIBC2(SG)-Ag resting medium significantly increased transformation frequencies

Plant Cell Rep (2014) 33:1767–1777

1775

Fig. 7 Improvement of PHR03 transformation using AGL1 vs LBA4404/PHP24600 with bialaphos selection. About 50 IEs, 1.5–1.9 mm, derived from the same ear were used for each Agrobacterium strain incubated under the same conditions to test the effect of Agrobacterium strains. Each histogram represents the mean level (±standard error) from three replicates

at the T0 plant level in PHR03 nearly three times compared to DBC3 resting medium (Table 4). Effect of Agrobacterium strains on PHR03 transformation frequencies Experiments were conducted in PHR03 comparing four different Agrobacterium strains. LBA4404, AGL1, EHA105 and GV3101 were tested using PHP32269 containing PMI and moPAT::YFP using bialaphos selection. In all experiments AGL1 had the highest transformation frequency at an average of 23.3 % compared to three other Agrobacterium strains (Fig. 6). LBA4404 had the 2nd highest transformation (5.4 %), but there was no significant difference among LBA4404, EHA105 and GV3101. Experiments comparing Agrobacterium strains AGL1 and LBA4404 were further conducted using construct PHP24600 containing DsRed and PAT and the bialaphos selection protocol as described above. As shown in Fig. 7, average transformation frequency of AGL1 at the T0 plant

level was 57.1 % (88/154) and LBA4404 was at 21.7 % (33/152) over a 2.6-fold increase using a standard construct PHP24600 with bialaphos selection. A similar trend was observed in sugarcane (Cho et al. 2013) and sorghum (Wu et al. 2014). The regeneration rate was also higher in T0 events generated by AGL1 than LBA4404 (Fig. 7). IEs transformed with AGL1 showed more multiple events per IE which could increase the potential for successful regeneration when compared to LBA4404. Additional experiments comparing Agrobacterium strains AGL1 and LBA4404 were conducted using construct PHP32269 containing PMI and moPAT::YFP using PMI selection for molecular data of T0 plants. It was consistent that transformation frequency at the T0 plant level using AGL1 was 1.9-fold higher than that obtained with LBA4404; 49.3 % (1,347/2,734) and 26.3 % (705/ 2,680) with AGL1 and LBA4404, respectively (Table 5). Leaf punch samples were submitted for QE analysis using qPCR. Out of 1,347 and 705 T0 events, 554 and 326 events were tested for qPCR for AGL1 and LBA4404, respectively. Less than 1 % of events were escapes. QE ratios of AGL1 and LBA4404 were 25.6 % and 30.1 %, respectively, with AGL1 slightly lower (Table 5). Backbonenegative events were 55.4 % for AGL1 and 72.7 % for LBA4404, with LBA4404 having less backbone presence. Similar results were observed in sorghum (Wu et al. 2014). Overall, the UQE ratio of AGL1 was 12.6 %, about 1.6fold higher than LBA4404, 7.9 % mainly due to high T0 transformation frequency (Table 5). In conclusion, the present study demonstrates significant improvements in transformation of a previously recalcitrant commercial maize elite inbred, PHR03, using optimized media components, tissue culture conditions and Agrobacterium strain. The use of DBC3 GT medium containing high copper and cytokinin in resting and selection steps resulted in dramatic improvement in transformation of PHR03. Further improvements were made by extending modifications to the resting medium by adding high glucose [PIBC2(SG)-Ag] together with DBC3 GT selection medium and the Agrobacterium strain, AGL1. This improved protocol can be applied to other recalcitrant commercial maize inbreds.

Table 5 Molecular analysis data from transgenic plants generated using AGL1 vs LBA4404 containing PHP32269 using PMI selection Agrobacterium strains

T0 plant level transformation frequency (%)

QE event (BB-, SC events) frequency (%)

Backbone minus event frequency (BB-, %)

Usable quality event frequency (UQE, %)

LBA4404

26.3

30.1

72.7*

7.9

AGL1

49.3*

25.6

55.4

12.6*

BB- Agrobacterium backbone-negative, SC single copy event, QE quality event, UQE transformation frequency 9 QE ratio * p B 0.01 ([2,500 embryos were infected for each Agrobacterium strain, involving two independent transformations and multiple infections)

123

1776 Conflict of interest of interest.

Plant Cell Rep (2014) 33:1767–1777 The authors declare that they have no conflict

Ethical standards The experiments comply with the current laws of the country in which they were performed.

References Beyer EM (1976) A potent inhibitor of ethylene action in plants. Plant Physiol 58:268–271 Cho M-J, Lemaux PG (2001) An efficient system for transformation and plant regeneration of sorghum using highly regenerative, green tissues. In Vitro Cell Dev Biol 37:38A Cho M-J, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138:229–244 Cho M-J, Buchanan BB, Lemaux PG (1999a) Development of transformation systems for monocotyledonous crop species and production of foreign proteins in transgenic barley and wheat seeds. In: Application of transformation technology in plant breeding. 30th Anniversary Korean Breeding Society, Suwon, Korea, November 19, pp 39–53 Cho M-J, Jiang W, Lemaux PG (1999b) High-frequency transformation of oat via microprojectile bombardment of seed-derived highly regenerative cultures. Plant Sci 148:9–17 Cho M-J, Ha CD, Lemaux PG (2000) Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues. Plant Cell Rep 19:1084–1089 Cho M-J, Choi HW, Lemaux PG (2001) Transformed T0 orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds. Plant Cell Rep 20:318–324 Cho M-J, Le VK, Okamoto D, Kim YB, Choi HW, Lemaux PG (2003) Generation of transgenic plants of creeping bentgrass (Agrostis palustris Huds.) plants from mature seed-derived highly regenerative tissues. In: Mujib A, Cho M-J, Predieri S, Banerjee S (eds) In vitro applications in crop improvement recent progress. Science Publishers, Enfield, pp 67–77 Cho M-J, Yano H, Okamoto D, Kim H-K, Jung H-R, Newcomb K, Le VK, Yoo HS, Langham R, Buchanan BB, Lemaux PG (2004) Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed. Plant Cell Rep 22:483–489 Cho M-J, Klein TM, Zhao Z-Y (2013) Methods for tissue culture and transformation of sugarcane. US patent application 2013/0055472 A1 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY (1975) Establishment of an efficient medium for anther culture in rice through comparative experiments on the nitrogen sources. Sci Sinica 18:659–668 D’Halluin K, Bonne E, Bossut M, De Beuckeleer M, Leemans J (1992) Transgenic maize plants by tissue electroporation. Plant Cell 4:1495–1505 Food and Agriculture Organization of the United Nations, Statistics Division 2012, http://faostat.fao.org/site/567/DesktopDefault. aspx?PageID=567#ancor Frame BR, Drayton PR, BagNall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA, Wang K (1994) Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation. Plant J 6:941–948 Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize

123

embryos using a standard binary vector system. Plant Physiol 129:13–22 Frame BR, McMurray JM, Fonger TM, Main ML, Taylor KW, Torney FJ, Paz MM, Wang K (2006) Improved Agrobacteriummediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:1024–1034 Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Biotechnology 8:833–839 Golovkin MV, Abraham M, Morocz S, Bottka S, Feher A, Dudits D (1993) Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts. Plant Sci 90:41–52 Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O’Brien JV, Chambers SA, Adams WR Jr, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603–618 Ha CD, Lemaux PG, Cho M-J (2001) Stable transformation of a recalcitrant Kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues. In Vitro Cell Dev Biol Plant 37:6–11 Huang X, Wei Z (2005) Successful Agrobacterium-mediated genetic transformation of maize elite inbred lines. Plant Cell Tissue Organ Cult 83:187–200 Ishida Y, Satto H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750 Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2(7):1614–1621 Kim H-K, Lemaux PG, Buchanan BB, Cho M-J (1999) Reduction of genotype limitation in wheat (Triticum aestivum L.) transformation. In Vitro Cell Dev Biol 35:43 Lemaux PG, Cho M-J, Zhang S, Bregitzer P (1999) Transgenic Cereals: Hordeum vulgare (barley). In: Vasil IK (ed) Molecular improvement of cereal crops. Kluwer, Dordrecht, The Netherlands, pp 255–316 Li Y-C, Ren J, Cho M-J, Zhou S, Kim Y-B, Guo H, Wong JH, Niu H, Kim H-K, Morigasaki S, Lemaux PG, Frick OL, Yin J, Buchanan BB (2009) The level of expression of thioredoxin is linked to fundamental properties and applications of wheat seeds. Mol Plant 2:430–441 Lowe KS, Cahoon RE, Scelonge CJ, Yumin T, Gordon-Kamm WJ, Bruce WB, Newman LJ (2007) Wuschel (wus) gene homologs. US patent 7256322 B2 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497 Negrotto D, Jolley M, Beer S, Wench AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803 Oestreich DC (1995) Inbred corn line PHR03. US patent 5436390 Ombori O, Muoma JMO, Machuka J (2013) Agrobacterium-mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines. Plant Cell Tissue Organ Cult 113:11–23 Petolino JF, Hopkins NL, Kosegi BD, Skokut M (2000) Whiskermediated transformation of embryogenic callus of maize. Plant Cell Rep 19:781–786 Songstad DD, Duncan DR, Widholm JM (1988) Effect of l-aminocyclopropane-l-carboxylic acid, silver nitrate, and norbornadiene on plant regeneration from maize callus cultures. Plant Cell Rep 7:262–265 Vega JM, Yu W, Kennon AR, Chen X, Zhang ZJ (2008) Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors. Plant Cell Rep 27:297–305

Plant Cell Rep (2014) 33:1767–1777 Wan Y, Widholm JM, Lemaux PG (1995) Type-I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.). Planta 196:7–14 Wu LM, Wei YM, Zheng YL (2006) Effect of silver nitrate on the tissue culture of immature wheat embryos. Plant Physiol 53:530–534 Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu Y, Cho M-J, Zhao Z-Y (2014) Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants. In Vitro Cell Dev Biol Plant 50:9–18 Zhao Z-Y, Gu W, Cai T, Tagliani LA, Hondred DA, Bond D, Krell S, Rudert ML, Bruce WB, Pierce DA (1998) Molecular analysis of

1777 T0 plants transformed by Agrobacterium and comparison of Agrobacterium-mediated transformation with bombardment transformation in maize. Maize Genet Coop Newslett 72:34–37 Zhao Z-Y, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798 Zhao Z-Y, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breed 8:323–333

123