Improving fruit quality in tomato (Lycopersicum esculentumMill) under ...

Plant Cell Tiss Organ Cult (2015) 121:153–166 DOI 10.1007/s11240-014-0691-1

ORIGINAL PAPER

Improving fruit quality in tomato (Lycopersicum esculentum Mill) under heat stress by silencing the vis 1 gene using small interfering RNA technology Ehab M. R. Metwali • Hemaid I. A. Soliman Michael P. Fuller • Omar A. Almaghrabi

•

Received: 14 June 2014 / Accepted: 10 December 2014 / Published online: 17 December 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Constantly elevated temperatures cause an array of physio-biochemical changes in tomato (Lycopersicum esculentum Mill.) which make the fruit ripen quickly and up to 50 % yield loss. The development of tomato cultivars, using genetic engineering approaches which delay ripening, offers a new way to keep tomatoes healthy under heat stress. Over-expression of small heat shock protein gene, viscosity 1 (vis 1) plays a role in increasing juice viscosity, early ripening and tissue soften which emphasizes the importance of this gene in premature ripening. The aim of this work was therefore to develop a useful system for silencing the vis1 gene using small interfering RNA strategy. Agrobacterium strain GV3101 harbouring the binary vector pICBV19 containing the gus and bar genes was used to adapt the transformation process in this study. The primers were designed to amplified the first exon of the vis 1 gene and the amplified

E. M. R. Metwali (&) O. A. Almaghrabi Biological Science Department, Faculty of Science, NorthJeddah, King Abdulaziz University, Jeddah 21589, Saudi Arabia e-mail: [email protected]; [email protected] E. M. R. Metwali Botany Department, Faculty of Agriculture, Suez Canal University, Ismailia 41522, Egypt H. I. A. Soliman Plant Genetic Resources Department, Desert Research Center, El-Matariya, Cairo 11753, Egypt M. P. Fuller School of Biological Sciences, Faculty of Science and Environment, Plymouth University, Plymouth PL4 8AA, UK

fragment was used for cloning into the pFGC5941 at XhoI/ NcoI site at sense orientation then additional fragment was subsequently cloned at BamHI/XbaI to form sense/antisense cloned fragment interrupted by the CHSA-intron Agrobacterium strain LBA4404 with the binary vector pFGC5941 which harbors vis1 gene under the control of the 35S promoter containing bar gene under the control of a mannopine synthase 20 (Mas20 ) as selectable marker, was used to reduce the expression of vis1 gene in fruit. Polymerase chain reaction (PCR), RT–PCR and northern blotting analysis were applied to detect putative transgenic plants. Significantly, silencing of vis 1 gene was potently occur and new transgenic tomato cultivars were produced with enhanced ripening qualities for recommendation for growing under heat stress. Keywords Agrobacterium tumefaciens Gene expression Gene viscosity 1 Heat shock protein In vitro regeneration Small interfering RNAs Tomato Transgenic Abbreviation BAP 6 Benzylaminopurine dsRNA Double strand RNA IAA Indole-3-acetic acid IR Inverted repeat LB Luria broth medium MCS Multiple cloning site NAA 1 Naphthaleneacetic acid nt Nucleotide RISC RNA induced silencing complex RNAi RNA interference shRNA Short hairpin RNA siRNA Short interfering RNA ZEA Zeatin

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Introduction Tomato (Lycopersicon esculentum Mill.) is an important crop, grown all over the world and is a key part of the salad vegetable industry in most countries. It is also considered a model species for the introduction of agronomically important genes into dicotyledonous crop plants (Wing et al. 1994). In fleshly fruits such as tomato ripening is a dynamic transitional period that encompasses a myriad of biochemical and physiological changes that predisposition the mature fruit to be ready for human consumption, it is a process which includes three stages beginning with a mature green stage followed by a breaker stage then a turning stage (Wusirika et al. 2003). For tomato fruit, when the temperature rises higher than 30 °C the production and accumulation of toxic ROS (Reactive Oxygen Species) is stimulated and abnormal ripening quickly occurs including lack of lycopene accumulation and an increase in cell wall depolymerization enzymes which result in fruit membrane injury causing fruit tissues to soften prematurely and leading to up to 50 % yield loss (Miller et al. 2001; Yueju et al. 2006) and a reduction in fruit shelf life (Maria 2009). The most prominent changes that occur in cell walls during fruit ripening are depolymerization of polyuronides and hemicelluloses (Brummell et al. 1999; Propper et al. 2011). Ripening-related depolymerization and solubilization of tomato fruit cell walls are intimately associated with juice viscosity (Takada and Nelson 1983). Taken together, these observations indicate that viscosity juice would provide a reasonable estimate of fruit cell wall solubilization. This is controlled by several expressed sequence tags (ESTs) that are differentially expressed during fruit ripening in tomatoes and characterization of one of these genes has been designated as viscosity 1 (vis1). Expression of this gene in immature green tomato fruit is rapidly induced and/or enhanced under high temperature. The molecular function of vis1 has been studied in transgenic tomato lines expressing sense and antisense transcripts of vis1 under the control of CaMV 35S promoter. Fruit with ectopic expression of vis1 significantly enhanced juice viscosity and early ripening, while impaired expression of vis1 resulted in significantly lowered juice viscosity in several independent transgenic lines which emphasizes the importance of this gene in premature ripening (Wusirika et al. 2003). Given these results, it is evident that the development of tomato cultivars with delayed ripening could lead to plants remaining healthy for long time under heat stress (Wusirika et al. 2003). Genetic engineering approaches such as small interfering RNA (siRNA) strategies offer new ways to silence a majority of genes controlling ripening such as MADS-RIN and vis1 (Graham et al. 2013). The previously studies of gene silencing via double-stranded RNA (dsRNA) in plants reported efficient

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Plant Cell Tiss Organ Cult (2015) 121:153–166

gene silencing for the improvement fruit and seed quality traits (Grierson et al. 1991; Dipak et al. 2014), agronomic traits (Fu et al. 2007; Ying and Paula 2014), biochemical traits (Ogita et al. 2003; Nan et al. 2013), resistance to biotic stress (Tomson and Manjula 2011; Simona et al. 2013) in crops. The mechanism of siRNA biogenesis and function are highly sequence specific and highly conserved in plants where siRNAs are produced from dsRNA by Dicer-like (DCL) proteins and then incorporated into a RNA induced silencing complex (RISC) to degrade homologous mRNA (Wang and Carmichael 2004). Synthetic siRNAs of 21–22 nucleotide (nt) RNA duplexes have been reported to play a crucial role for strong and specific RNA silencing (Elbashir et al. 2001a, b). Some experimental evidence implies a functional role of 21nt-secondary siRNAs, rather than the longer siRNAs, as a mobile silencing signal in transitive RNAi (Himber et al. 2003). In basic and practical studies of siRNA in tomato, a successful transformation protocol is essential. Several studies have reported improvement in the technique of transformation in tomato (Sun et al. 2005) but standardization of the various transformation procedures still need to be improved (Wie and Gregory 2001). Advances in recent tissue culture techniques have led to the development of improved transformation protocol for plants with certain genes (Ahmed et al. 2008). The main goal of the present research was to develop tomato cultivars with improved fruit quality and longer shelf life under heat stress by reducing the expression of vis1 gene. This demanded an optimized protocol for the in vitro regeneration of hypocotly and cotyledon explants, transfer of the vis 1 gene by Agrobacterium, and use of the technology of small interfering RNAs (siRNAs) in posttranscriptional RNA silencing of vis1. The system was tested, to confirm the success in silencing the vis1 gene using polymerase chain reaction (PCR), quantitative RT– PCR and northern blotting analysis.

Materials and methods Plant material Seeds of tomato (Lycopersicon esculentum Mill.) cultivars Pusa Ruby and Super-strain B were obtained from Ball Seeds Company, West Chicago, Illinois, USA and Vegetable Department, Agriculture Research Center, Giza, Egypt, repectively. This present study was conducted during the year 2013/2014. Agrobacterium strains and plasmid vector Agrobacterium strain GV3101 harbouring the binary vector pICBV19 containing the gus and bar genes (Fig. 1) was


LB

Apa1

Apa1

TOCS

BAR

155

Sac1 Cla1

PNOS

P35S

BamHl

GUS

Xbal

Pstl Hind3

TNOS

RB

Fig. 1 Schematic presentation of pICBV19 binary vector containing the bar and gus genes

used to adapt the transformation process in tomato. The Binary vector, pFGC5941 containing the first exon of the visI gene, containing 23 nt both in sense and antisense interrupted by CHSA-intron under the control of the 35S promoter containing bar gene under the control of a mannopine synthase 20 (Mas20 ) as selectable marker (Fig. 2), and transformed in Agrobacterium tumefaciens strain LBA4404 using an electroporation method according to (Nagel et al. 1990) was also used. Design of RNAi-inducing transgene for targeted gene By using known complementary DNA (cDNA) sequences or predicted gene sequences corresponding to the target gene for insertion, primers were designed to amplify a portion of the cDNA by using reverse transcription-PCR (RT-PCR). Cloned cDNA fragments corresponded to

targeted mRNAs. Using RT-PCR a 225 bp portion was typically amplified of a given cDNA and then this fragment was cloned twice, in inverted orientation, within the plasmid pFGC5941. The construction of the inverted repeat transgene was involved in a two-step ligation procedure facilitated by pairs of restriction endonuclease sites included in the primer sequences constituted at the ends of the PCR fragment. In the first cloning step, the PCR product was cleaved at the innermost restriction sites (XhoI and NcoI) at each end of the PCR product and ligated into the BamHI/XbaI digested pFGC5941 plasmid. The resulting plasmid then served as the template for a second PCR amplification using the same primers. The resulting PCR product was cleaved with XhoI and NcoI site at sense orientation then additional fragment was subsequently cloned at BamHI/XbaI to form sense/antisense cloned fragment interrupted by the CHSA-intron (Fig. 3).

Fig. 2 Physical map of the binary vector pFGC5941

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Fig. 3 Production of the vis1 RNAi cassette. The fragment was directionally cloned into the RNAi vector pFGC5941. Shown are vis1 sense (vis1s), vis1 antisense (vis1as) amplification fragment, the Caulifower mosaic virus 35S promoter (P35S), the Agrobacterium

tumefaciens promoter (PMAS), gene encoding BASTA (BAR), polyadenylation signal PA, ChsA intron (from the petunia chalcone synthase A gene), T-DNA left border (LB) and T-DNA right border (RB)

Cloning gene constructs

AAA CAT T-30 were designed for cloning the full length vis 1 gene from tomato plants, and the PCR primers used were as follows: forward primer: 50 CCGTTTGT GTGAACAACG-30 , reverse primer: 50 -GCACAGCA CATCA AAGAG-30 were designed for the gus gene. The primers used to detect the sequences of the bar gene were: 50 -CTCGAGTCAAATCTCGGTGACGGG-30 and 50 -CG AGTCTACCATGAG CCCAGAACG-30 . The primers, Ex-F 50 -GGCTCATTGCTTATCAAGATTTCCA-30 and Ex- R 50 -ACCAACAGGGGCCATTTGGGGTAG-30 were designed from the first exon of the vis 1 gene, containing both in sense and antisense interrupted by CHSA-intron and to be cloned the binary vector pFGC5941 under the control of CaMV35S promoter (P-35S) and an octopine synthase 30 end.

Suppress or reduce the expression of the vis 1 gene in tomato fruit, a post transcription gene silencing method (PTGS) was used. The primers were designed to amplified the first exon 225 nt of vis 1 gene, and amplified fragment was used for cloning into the RNAi vector pFGC5941 under the control of the Cauliflower mosaic virus CaMV35S promoter. Two pairs of primers were used to PCR-amplify a portion of the short segments of vis1 gene, yielding fragments with BamHI-XbaI and NcoI-XhoI ends. Two copies of each amplified fragment in sense and antisense direction interrupted by the CHSA intron between them to form small RNA in a shape of stem loop for Dicer to work on and produce functional siRNA (Fig. 4). Recombinant plasmids were screened using PCR and restriction digestion with the enzymes used for cloning the double stranded RNA is expected to enhance the Dicer to digest it into siRNA and forming fragments of *21–26 nt. The siRNA was associated with the protein complex RNAinduced silencing complex (RISC) where it serve as guides to select the trigger RNAs and affect their degradation. Primers used for screening putative transgenic plants The primers vis 1-F 50 -ATG GCT CAT TGC TTA TCA AGA TT-30 ; Vis1-R50 -TCA TTG AAC ATT AAT GTC

LB

BAR P-35S

In vitro regeneration protocol Explant preparation Tomato (Lycopersicon esculentum Mill.) cv. Pusa Ruby and Super strain B seeds were used as explant sources. Seeds were surface sterilized for 25 min, in commercial bleach solution (2.5 % NaClO) and rinsed three times in distilled water according to (Metwali 2006). The seeds were germinated on hormone free MS (Murashige and Skoog 1962)

RB

Transgene integrated into plant genome

siRNA species

Degradation of the targeted RNA transcript by Dicer and RISC

Targeted gene

Fig. 4 Strategy for transgene-induced RNAi of targeted genes. After random integration of the transgene into the plant genome, the strong 35S promoter drives expression of transcripts that are self-complementary through the inverted orientation of target gene sequences

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(open boxes), generating a double-strand RNA stem, which is processed by Dicer complex into 22-to26-nt short interfering RNA (siRNA) species. The siRNAs, in turn, direct degradation of highly homologous RNA transcripts by the RISC complex


157

Root formation and acclimatization

medium solidified with 0.25 % (w/v) phytagel and incubated at 27 °C in the dark for 3 days then they were maintained under photoperiod of 16 h illumination for another 5 days. The hypocotyls and cotyledon explants of in vitro grown seedlings were excised from the 10-day-old seedling and the cotyledons were cut into two parts followed by removing the bud from the proximal part by a sharp scalpel were incubated on different regeneration media to identify the best formulation for inducing shoot organogenesis. The compositions of these different regeneration media as described in Table 1 in order to optimize the best regeneration media. The MS media were adjusted to pH 5.8 prior to autoclaving and 25 mL were dispensed into Petri dishes and cultures maintained in growth chamber at 28 ± 2 °C, 16 h photoperiod with an irradiance of 13.5 lmol m-2 s-1 provided by white fluorescent tube lights. A total of 15 explants per plate were cultured, ten plates per treatment were used in a completely randomized block design, and the whole experiment was repeated three times. Frequency of regeneration (No. of regenerating explants/No. of plated explants 9 100) and the number of shoots and shoot primordial per explant were assessed after 6 weeks.

The elongated shoots were cultured on MS medium supplemented with 0.5, 1.0, 1.5 and 2.0 mg L-1 IBA alone or combined with 0.5 mg L-1 NAA. Hormone free MS medium was used as a control. For each treatment twenty shoots were used and incubated in growth-chamber conditions for 3–4 weeks. Rooted plantlets were transferred into pots containing a soil mixture of peat: sand (1:1), and covered with plastic bags in a controlled greenhouse. The temperature was adjusted to 28 °C and the plants were hardened by removing the plastic bags gradually over a 7–10 day-period. Agrobacterium mediated transformation of tomato plants Hypocotyl and cotyledon explants were prepared and soaked in a suspension of overnight grown A. tumefaciens strain GV3101 with pICBV19 for 15 min, blotted dry on sterile filter paper, then co-cultivated for 2–3 days at 28 ± 2 °C in the dark on the shoot regeneration medium. The explants were then transferred to the selection medium supplemented with 3.0 mg L-1 bialaphos (PPT) to select putative transgenic shoots and 250 mg L-1 cefatoxime to prevent Agrobacterium growth. After 4 weeks shoots that developed were sub-cultured on the optimized elongation medium and grown on to reach a suitable shoot length. Shoots were then transferred to rooting medium for 4 weeks and then acclimatized in the greenhouse. Control hypocotyl and cotyledons explants were passed through all

Shoot elongation Shoots were transferred to MS medium supplemented with different concentrations(0.5 and 1.0 mg L-1) of Gibberellic acid (GA3) alone or in combination with 0.5 mg L-1 of 2-isopentenyl adenine (2iP) and cultured for 3–4 weeks until they reached a suitable length of (1.5–2.0 cm).

Table 1 Effect of MS medium supplemented with different growth regulators on shoot formation from cotyledon and hypocotyl explants of tomato cvs. Pusa Ruby and Super strain B Mean followed by the same letter(s) in each column are not differ significantly (p B 0.05) Bold value indicates the highest percentage obtained for this treatment compared to other treatments ZEA zeatin, BAP 6-benzylaminopurine, IAA indole-3-acetic acid, NAA 1-naphthaleneacetic acid. ZEA zeatin–SN Silver nitrate, Cot. Cotyledon, Hyp. Hypocotyls

Code no. MS medium with different combination of growth regulators (mg L-1) ZEA

NAA

IAA

BAP

SN

Percentage of forming shoots primordial/healthy explants (%)

Mean number of shoots primordial/healthy explants

Pusa Ruby

Super strain B

Pusa Ruby

Cot.

Cot.

Cot.

Hyp.

Hyp.

Super strain B

Hyp. g

g

Cot.

0.00g

MS1

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.00

0.00

MS2

1

0.1

0.0

0.0

0.0

85

84

90

92

1.25e

1.05f

0.56f

0.98f

86

d

1.38

e

1.24

d

1.68b

1.52

d

1.75

a

1.98a

1.58

d

0.96

e

1.22d

a

1.23

d

1.45c

MS3 MS4 MS5

2 1 1

0.1 0.1 0.0

0.0 0.0 0.1

0.0 0.0 0.0

0.0 5 0.0

80 90 91

78 89 90

83 96 90

98 92

1.35

d

1.48

c

1.66

a

0.00

Hyp. g

MS6

1

0.0

0.1

0.0

5

94

93

87

91

2.28

2.12

MS7

0.0

0.1

0.0

1

0.0

58

62

69

72

1.02f

1.00f

0.45f

0.85f

78

e

1.36

e

0.88

e

1.02e

1.78

c

1.58

c

1.67b

1.96

b

1.62

b

1.72b

MS8 MS9 MS10

0.0 0.0 0.0

0.1 0.0 0.0

0.0 0.1 0.1

1 1 1

5 0.0 5

76 79 83

73 68 81

71 77 88

89 90

1.28

b

1.89

b

1.92

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158

stages starting from seed germination to the transfer to the selection plates without co-cultivation with Agrobacterium, and were cultured on regeneration medium without selection agents (antibiotics and PPT).


clone and published sequence in the GenBank. Purified plasmids were used as a template in the sequencing reaction using Big Dye Terminator Ready Sequencing kit. Confirmation of transgenic plants

Transformation with RNA silencing construct of viscosity 1 gene Cloning the vis 1 gene from tomato fruit DNA extracted from tomato fruits cultivar Pusa Ruby was used to amplify the visI gene using its specific primers (vis 1-F and vis 1-R). Amplified fragments with the expected molecular weight (2.25 kb) were cloned into the PCR cloning vector pGEMÒ-T Easy Vector System (Promega, USA) according to the manufacturer manual. The ligated reaction (cloning vectors plus inserts) was used to transform E. coli bacterial cells (XL-1blue). Transformation of the competent cells Fifty-lL of the competent cells was thawed on ice. Ligation mixture was added to the cells, mixed briefly and incubated on ice for 30 min, then heat pulsed in 42 °C water bath for 30 s followed by incubation on ice for 2 min. After that, 500 lL of LB medium was added and incubated at 37 °C for 1 h with shaking at 250 rpm. 100 lL of transformation mixture was plated using a sterile spreader onto LB-plates containing 100 lg mL-1 ampicillin, 40 lg mL-1 X-gal, and 0.5 mM IPTG (Isopropyl-beta-D-1-thiogalactopyranoside) and plates were incubated overnight at 37 °C. Screening of transformed cells

PCR screening To detect the presence or absence of a target DNA by PCR analysis, total genomic DNA was isolated from leaf material of the regenerated plants using a cetyltriethylammonium bromide (CTAB) extraction method (Stacey and Isaac 1994). The DNA extracted was subjected to the polymerase chain reaction (PCR) as described by Lassner et al. (1989). Total RNA of the T1 generation were extracted from fruits using the Trizol reagent from Invitrogen. Polymerase chain reaction (PCR) was performed using 1 lL (50–100 ng lL-1) of template DNA in a reaction mixture containing 10 mM TRIS–HCl pH 8.8, 50 mM KCl, 1.5 mM MgCl2, 0.1 % (v/v) Triton X-100, 200 lM of each dNTP, 0.3 lM of forward and reverse primers, 20–500 ng of DNA, and 0.66 U of Taq DNA polymerase. Reverse transcription-PCR (RT-PCR) was performed with the access RT-PCR system from Promega. RT-PCR analysis of the first exon (Forward primer 50 -GGCTCA TTGCTTATCAAGATTTCCA-temperature 57 °C, was carried out and the products were detected in a 0.8 % (w/v) agarose gel. The amplified fragment lengths were 225 bp. Thermocycling conditions of RT-PCR were as follows: 35 cycles, denaturation at 94 °C for 30 s, annealing for 30 and (Reverse primer 50 -ACCAA CAGGGGCCATTTGGGGTAG-30 ), annealing 30 s, and extension at 72 °C for 2 min.

Detection of transformed competent cells with pGEMÒ-T Easy was carried out by blue/white colony screening as the vector has a ligated fragment at its multiple cloning site (MCS). The MCS of Blue script lies within the LacZ gene, which when expressed, in response to the presence of synthetic inducer IPTG, produces the enzyme b–galactosidase. This releases an indigo dye from the chromomgeric substrate, X-gal, resulting in the formation of a blue colony. Insertion of a DNA fragment within the MCS would result in the failure of LacZ expression, and the consequent formation of white colony, thus, cells transformed with pGEMÒ-T Easy derivatives were plated on LB-plates, to select and identify recombinants.

GUS histochemical assay

Sequencing of viscosity 1 gene

Total RNAs were extracted from tomato fruits of Pusa Ruby at the turning stage according to Biggs et al. (1986). Total RNA extracted was quantified by spectrophotometric measurements and diluted to 200 ng lL-1. The cDNA insert of vis1 was labeled using an oligo-labeling kit

The recombinant plasmids were subjected to nucleotide sequencing to ensure that the cloned fragment belongs to vis1 gene and to identify the correlation between the isolated

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The expression of the gus gene in tomato explants cocultivated with Agrobacterium carrying plasmid pICBV19 was detected by incubation with GUS buffer [50 mM NaPO4 (pH 7.0), 1 mM EDTA, 0.1 % SDS, 100 lM ferrocyanid PO4] containing X-gluc (1 lg mL-1) as a substrate at 37 °C O/N (Jefferson et al. 1987). After that the explants were bleached in 50 % Clorox for 5 min and the explants that turned blue were identified as a positive result and counted. RNA extraction and probe labelling


(Pharmacia) and radiolabeled probe purified on a Sephadex G-50 column and used in hybridization with northern blots. Hybridized membranes were washed two times for 15 min each in 49 SSPE and 0.1 % (w/v) SDS at room temperature followed by a 10 min wash in 49 SSPE and 0.1 % (w/v) SDS at 55 °C. Then, three 10-min washes in 0.19 SSPE and 0.1 % (w/v) SDS at 65 °C were performed. Northern blotting and hybridization Total RNAs (50 lg) was separated and size fractionated on 1.2 % (w/v) agarose denaturing formaldehyde gels and blotted to Hybond-N nylon membrane (Roche Molecular Biochemicals) using 0.05 M NaOH according to Sambrook et al. (1989). Blots were hybridized to a9-32P-labeled vis1 probe at the same conditions described above. After hybridization, membranes were washed twice for 15 min each in 29 SSC and 0.1 % (w/v) SDS at room temperature and then twice for 10 min each in 0.29 SSC and 0.1 % (w/v) SDS at 62 °C. Statistical analysis The experiments were repeated two times and data were analyzed at 5 % significance level using analysis of variance (ANOVA). The differences among means for all treatments were tested for significance at 5 % level (Duncan 1955) and multiple range test according to Snedecor and Cocharn (1967). Means followed by the same letter are not significantly different at p B 0.05.

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cotyledon sections after co-cultivation with Agrobacterium. In the present study, differences between Pusa Ruby and Super Strain B were not statistically significant, and this may be due to varietal difference (Jaskani et al. 2005) and/or explants type (Ajenifujah-Soleba et al. 2013). In spite of our results recorded, non significant differences between hypocotyls and cotyledon in response to produce shoot was recorded in previous studies (Mohamad et al. 2011), these results are inconsistent with Ajenifujah-Soleba et al. (2013) who reported different regeneration frequency of various explants of tomato in order to leaf \ cotyledon \ hypocotyls. In comparing our results with previous studies (Habib et al. 2009; Mahmoud et al. 2013), we find that the best results for shoot regeneration are from either hypocotyls or cotyledon explants. Shoot elongation The data indicated that the best results for shoot elongation for both cultivars were observed with MS media containing 1.0 mg L-1 GA3 and 0.5 mg L-1 2iP compared to the other treatments (Figs. 7, 8). Amber et al. (2009) reported that GA3 enhanced the shoot elongation in tomato by stem internode extension and this effect had an invigorating effect and significant variation on shoot elongation in the presence of GA3 in combination with the cytokinins BA in the culture medium for tomato (Lycopersicon esculentum) cvs genotypes Avinash, Pusa Ruby and Pant Bahr. Rooting and acclimatization

Results and discussion In vitro regeneration of tomato The frequency of adventitious shoot regeneration differed depending on the type of explant and the concentration of growth regulators added to the regeneration medium. MS6 medium was the most effective in induction of adventitious shoots (94 and 93 %) and shoot primordial numbers per explants (2.28 and 2.12) from cotyledons and hypocotyl explants in the cultivar Pusa Ruby (Table 1; Figs. 5, 6). However, in the cultivar Super Strain B, MS4 medium was the most effective in induction of adventitious shoots (96 and 98 %) and mean number of shoots primordial per explants (1.75 and 1.98 from cotyledons and hypocotyls explants respectively). Our results are in agreement with Praveen and Rama (2009) who reported that 1 mg L-1 zeatin individually and also in combination with 0.1 mg L-1 IAA increased the shoot formation for tomato explants and they used this medium as a selective regeneration medium to obtain transformed tomato from the

Isolated single shoots were treated to induce rooting as specified in Table 2. The highest value of shoots forming root (97 %) and number of roots per explants (4.98) of Pusa Ruby was obtained with MS medium with 1.5 mg L-1 IBA while, the percentage of shoots forming roots reached the highest value (98 %) and mean number of roots per explants (5.82) of Super Strain B, were obtained on MS medium with 1.0 mg L-1 IBA ? 0.5 mg L-1 NAA compared to the other treatments (Fig. 9). Other researchers have also reported root induction at different concentrations of IBA and NAA (Sakthivel and Manigandan 2011; Indrani et al. 2013). In present study, the percentage of surviving plantlets after transfer to greenhouse conditions was 83 and 85.5 % for Pusa Ruby and Super Strain B, respectively. Some current studies have reported good response of tomato plants to acclimation in the range between 60 and 63 % survived plantlets (Devi et al. 2008; Sakthivel and Manigandan 2011). Only, Mahmoud et al. (2013) indicated that the percentage of survived tomato plantlets was up to 100 %.

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160


Fig. 5 Establishment of shoot formation from hypocotyl/cotyledon sections of tomato on MS medium supplemented with 1 mg L-1 zeatin, 0.1 mg L-1 IAA and 5 mg L-1 silver nitrate. a Pusa Ruby and b Super strain B cultivar

Fig. 6 Establishment of shoot formation from cotyledon sections of tomato on MS medium supplemented with 1 mg L-1 zeatin, 0.1 mg L-1 NAA and 5 mg L-1 silver nitrate. a Pusa Ruby and b Super strain B cultivar 5

mean length of shoots

Fig. 7 Effect of different concentrations of growth regulator on shoot elongation of two cultivars of tomato, Pusa Ruby and Super Strain B

Pusa Ruby

Super strain B

4 3 2 1 0

0.5mg/LGA3

1.0mg/LGA3

0.5mg/LGA3+0.5mg/L2iP 1.0mg/LGA3+0.5mg/L2iP

Different concentrations of GA3 (mg/L)

Transformation of tomato plantlets with bar and gus genes In order to identify transformed plants that have been growing on selective medium, it was necessary to have an easily assayable reporter gene (E. Coli b glucuronidasegus) (Parasharami et al. 2006). In this study a number of putative transformant explants which were bialaphos-

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resistant (data not presented) were randomly selected to examine the sequences and expression of the gus gene by PCR and histochemical assay, respectively. The results indicated that the gus gene encoding sequences were detected in a number of putative transformant regenerated at 1000 bp after PCR amplification (Fig. 10). Also, incubating the plant segments of bialaphos resistant plant materials with b-glucorondiase substrate X-Gluc at 37 °C


161

Fig. 8 Shoot elongation on MS medium containing 1 mg L-1 GA3 and 0.5 mg L-1 2iP (b) and control (a)

Table 2 The efficiency of shoots forming roots for tomato cvs. Pusa Ruby and Super strain B after growing on MS nutrient medium supplemented with different auxin concentrations Auxin conc. (mg L-1)

% of shoot forming roots

Mean number of roots/ explant

IBA

NAA

Pusa Ruby

Pusa Ruby

Super strain B

0.0

0.0

3

0.00f

0.00f

1.0

0.0

56

2.38d

2.28d

a

e

0.0 91

Super strain B

1.5

0.0

97

69

4.98

1.75

2.0

0.0

95

87

3.36c

3.58c

b

a

1.0

0.5

89

98

4.28

5.82

1.5

0.5

82

90

1.68e

4.25b

2.0

0.5

87

92

1.74e

4.36b

Mean followed by the same letter(s) in each column are not significantly different (p B 0.05) Bold value indicates the highest percentage obtained for this treatment compared to other treatments

for 24 h subsequently developed blue color and all tested explants that were obtained from the Agrobacterium treatment were 100 % GUS-positive, while 90 % of explants were bialaphos positive (Fig. 11) confirming a correlation between GUS enzymatic activity and bialaphos resistance. Metwali et al. (2012) indicated that the transient GUS assay approach either by histochemical or PCR is easy reliable methods of establishing conditions of transformation. Isolation and cloning the viscosity 1 gene from tomato fruits White colonies were picked up, plated on a master plate, and cultured in liquid media for DNA minipreps preparations. Recombinant plasmids were screened using PCR with the same primers and recombinant plasmids gave

positive results (Fig. 12). Vis 1 gene was performedusing vis1 gene specific primer (vis 1-F 50 -ATG GCT CAT TGC TTA TCA AGA TT-30 and Vis1-R50 -TTG AAC ATT AAT GTC AAA CAT T-30 ) and formation of sharp bands at (2.2 kb) confirm the presence of transgene (Vis1). Wusirika et al. (2003) reported that isolated vis 1-hta small heat shock protein (vis1) gene, complete cds from Lycopersicon esculentum (GenBank: AY128101.1). Sequencing of viscosity 1 gene The sequence results revealed 2,224 nucleotides and the sequence was compared to those in public database (Altschul et al. 1990; BLAST comparison with GenBank) in order to attribute biological function and it was found that the 50 end of the first exons for the vis1 gene were identical. Alignment showed 100 % match with vis1-hta isolate and 97 % with vis1-lta gene with three exons interrupted with two introns (Figs. 13, 14). The nucleotide sequences for the two genes showed 100 % similarities in the first, therefore these sequences were used to design siRNA sequences to silence both genes (Wusirika et al. 2003). Transgene-mediated RNA silencing of viscosity 1 gene Total DNA was extracted from tomato fruits and was used to amplify the first exons of the vis 1 gene. Fragments of 225 bp were successfully amplified as shown in (Fig. 15) and the amplified fragment was cloned and sequenced. The constructed plasmids were introduced into Agrobacterium tumefaciens strain LBA4404 and were used to transform the plants with different explants. The plasmid pFGC5941 was used as positive control in all transformation experiments. The construct was used to transform tomato plants to study its influence in reducing the expression of the vis 1 gene. Recombinant plasmids were identified by double

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Fig. 9 Rooting and acclimatization of tomato plants: elongated shoots showing roots after 4 weeks from culturing on rooting medium a Pusa Ruby, b Super strain B and c acclimatization of tomato plants in greenhouse

digestion with NcoI/EcoRI (Fig. 16). The constructed plasmid was used for tomato plant transformation and transgenic plants expressing the siRNA were evaluated for fruit quality. Real time PCR (qRT-PCR) has been regarded as the most powerful tool for the detection and quantification of GMO’s despite its high expense (Tripathi 2005). Confirmation of transgenic plants Fig. 10 Agarose gel electrophoresis of PCR amplification of a 1 kbp fragment of the gus gene by specific primers from transformed plant genomic DNA. Lane M: 1 kb DNA Ladder (promega); lane P positive sample; lanes 1–6 putative transgenic tomato plants

During micropropagation all explants were selected on herbicide glufosinate ammonium and leaves or shoots were taken for DNA isolation and analysed for stable integration

Fig. 11 Histochemical GUS assay for Agrobacterium-mediated transformed tomato plants for a leaf and b root

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Fig. 12 PCR results for plasmids isolated from white colonies for screening of recombinant plasmids with vis1 gene using vis1-F and vis1-R. Lane M 1 kb DNA ladder (fermentas); Lanes 1–7 vis1 gene

and expression of bar transgenes. A partial coding region of bar gene was amplified by basic PCR methods using specific primers of bar gene (50 -CTCGAGTCAAATCTC GGTGACGGG-30 and 50 -CGAGTCTAC CATGAGC Fig. 13 Nucleotide sequence of the cloned vis1 gene with nucleotide sequence of the three exons of the vis1 gene first exon (red color), second exon (blue color), third exon (green color) and two intron (black color). (Color figure online)

Fig. 14 Nucleotide sequence alignment for the three exon of the vis1 gene; first exon of the vis1 gene (red color), second exon (black color) and third exon (blue color). (Color figure online)

atggctcatt actcctctct tgaaagacat atggcccctg atcgtataat ttatatatat tttctaaagt tttgatgatc catataaaca tatatatctt tttgtggcag gagtcataga taaattggaa atttactgca agccacaagt atatataatt tcattgattt tgaattatct ttatgattaa agaatctttg tttaaaatga gaagatgtca tatttagatg tctaaacaaa ttttccttcg tactcgtaca tattgatgat tatcgggtac gacatgccag aaaaaatggt aattgctttg actataccta

gcttatcaag tccaatttct aatcttgatc ttggtaagaa ttgattttag tatatatgat taaaggtcct aattttcgaa ggtttattat taacttgtct ggttatggga ggatcctctt gaaaatgatt tccccccacc ccccattata acttcattta tttaatattt tgtatattta aataattaaa taattaaatt aaggactact tacgtaaaaa tttgatctgc taaaaataaa ttccaataag tcaaaattat cgatttaact agaaggggaa gaatgaccaa gaagaataat cctgaaaata aggctagtag

aaatattcca ttgaaggtca actcctcaac ccaacagcaa cttgctttta tgggagatca aaagaagatg gtgaagaata agaattgctt gtattgtata caatga

CCAGAACG-30 ). The data showed that putative transgenic plants were positive for presence bar gene which appear of the fragments on the expected size 520 bp while non transgenic plants have proved absence of bar gene sequence as described previously (Fig. 4). This is still no final proof that tested plants were genetically transformed because the positive signal can be result of residual Agrobacterium (Nevena et al. 2005). Robertson et al. (2004) detected that bar gene is conferring tolerance to glufosinate-ammonium could sometimes be silenced in a limited proportion of population (Fig. 17). The putative transgenic plants were grown in green house under heat stress (37 °C) were used to detect the expression of vis 1 gene at mRNA level (Fig. 18). The expression of vis 1 genes either in transgenic plant or nontransgenic plants was confirmed by Northern blot analysis. Transcript of one size fragments was presented in high

atttccaatt agtagaaaat acttacaaag tcgatacatc ctgatattta ataatactaa aggtgaagtt gagggggtgg tatttttttc ctagttttgg taggtttcca gcttttaatg tttttttaaa tactgatttg ttctggctta acattttttg taaattatca agacataaat ttaatttcaa gagtaattaa ttttacgtag ttttcaatca tagatgatat ataaataaga agtacatcaa ttattcggac cattgatcgt ggacaccatg agaagatgtt gaaaaagagg ttgactttga caatcccaaa

actcctctct tggttgttga ctagaaaaag ggacagtcca atggagggcc aagaaaatga ttaaagtttg atgaaaaaga tgcctgaaaa ttactatacc

tctcaaccta atcaaggtaa acaaaacaag ctcttttcca tggatcatac gttagacata catgttattt gccaatatgc ttaggttatt gcaattttct acagcaagga gaggttgggt aaaaatgatt ccccgaccct tacttctctt gattttgaaa attagtgtga tagatataat tactaactaa gttaatttga ttaataaata aatttaaatt ataaatggta aacttagatt aattatttat tttgtattta acgctacatt ggagatcaaa aaagtttggt aagagtggtc aaagattaag gtgtttgaca

tccaatttct tgaaagacat aacaccccaa acaaatgatg atcgaccgat aggagaatac gttagaagag ggaagagtgg tattgacttt taaggctagt

ttttattttc ttataagaaa actcctcaac cttattattt attaatcaaa aaattgtcaa ttgttcatca acaaaccatt atttaatgaa tttagtaatt cagtccaaca tgtaggtata tttctaatag ggttccacct tagataatgt tctaatatat cctttataac gatttatttc ttaaagataa aaaataaaaa tacatatata atttgactcg ggtgattttt tgttgttcta tccaacttaa tgtatatata cacttcgatt gaaaatgaag tagaagagaa agctaagagc gctgaggtta ttaatgttca

agtagaaaat aatcttgatc atggcccctg gacacaatgg gatatcgggt aaaatgaggt aaaatgttgg tcagctaaga gaaaagatta agcaatccca

aaacaatcca ttgaaggtca ctagaaaaag ttaatttttt atagttaaat ataaaataaa taacacatga tttatagttg aattataaaa ttgatctttg aatgatggac tatagtaaat gagcagaata ctttgaccat tttatctttt ttatttttta tctttttgct cttactaatt aattagaaga atttctcaaa aattttattt tgaaaggtgt agttaattat tattttagg a tatttatgta gctatacttc acagggccat gagaatacaa aatgttggtt tatggaaaat aagatggtgt atga

atcaaggtaa acttacaaag ttgggttatg acagagtcat acagaagggg ttgacatgcc ttgtaaaagg gctatggaaa aggctgaggt aagtgtttga

aaatattcca tggttgttga aacaccccaa ga tataaggt ttatataaaa tatttatgca tgtcaaagtc aagtcctata taaaaaaaat ctatgttcat acaatggaca ttttaatttt acaaatctaa ctcaatccta ttgctaggta ttataaattt ccaaattaag gatgttaaat ttattttaaa atttaattaa taattttttt cacaagtaaa gtaatgtact aatgttttct tatatagcta gtatcgaaaa cgaccgatga aatgaggttt gtaaaaggtg ataatacaag attgtatatt

ttataagaaa acaaaacaag ggataggttt agaggatcct aaggacacca aggaatgacc tgaaaaaatg atataataca taaagatggt cattaatgtt

120 180 240 300 360 420 480 540 600 660 666

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Fig. 15 A single-stranded cDNA was generated by reverse transcription from tomato total RNA (2 mg) using an oligonucleotide complementary to exon 1 as a primer. The cDNA was used as template in the PCR using a pair of primers corresponding to 20 nucleotides of exon 1. Lanes 2, 4, 6, 9, 11, 13 and 14 present the first exon while in the other lanes there is no first exon. M: 100 bp DNA marker (Promega) Fig. 18 Transgenic tomato plants with tomato fruits under heat stress (37 °C) in greenhouse

Fig. 16 Restriction digestion of the cloned short segment of vis1 gene in the binary vector pFGC5941 under the control of Caulifower mosaic virus 35S promoter with NcoI and EcoRI. Lane M: 1 kb plus DNA ladder; Lane 1, 2, 4 and 7 represents cutting the vector only while the other lanes represent recombinant clones Fig. 19 Total RNAs from transgenic turning stage of tissues of transgenic and non-transgenic tomato cv. Pusa Ruby under heat stress (37 °C) in greenhouse were size fractionated on agarose gel and blotted to Hybond-Nylon membranes (Amersham) and hybridized with a9-32P labeled vis1 cDNA. Lines C1 and C2 tomato nontransgenic and lines from T1 to T4 transgenic tomato plants

Fig. 17 PCR analysis to prove the presence of the transgenes with primer specific for the bar gene with expected size 520 bp. Lane T1, T3 and T5 from putative transgenic tomato plants cv. Pusa Ruby, Lane T2, lane T4 and T6 non-transformed tomato plant, Lane M DNA marker: 1 kb plus DNA ladder

level in non transgenic plant C1 and C2, while transgenic tomato plants T1, T2, T3 and T4 had no detectable transgene encoded protein as revealed by Northern blot analysis, presumably resulting from silencing of transcription of vis

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1 gene (Fig. 19). We found that the dsRNA interfere the vis 1 gene function in the transgenic tomato plants and about 136 out of 168 tomato plants transformed with RNAi construct containing vis 1 sequences. The detection of the transgene of interest and the study of mRNA transcribed from the introduced DNA provides more information than a screening method (Marouane et al. 2014).Wusirika et al. (2003) show that the absence of vis1 expression in high juice viscosity tomato genotypes is not attributable to presence of an early stop codon in the vis1-coding region, 90–100 % of independent transgenic plants showing silencing and the gene silencing mechanisms are becoming powerful tools for reducing gene expression. Recent work has demonstrated the potential for constructs encoding self complementary ‘hairpin’ RNA (hpRNA) to efficiently


silence genes (Wesley et al. 2001) and the gene silencing offers an alternative and complementary approach for large scale functional analysis of individual genes by knocking down the expression of endogenous genes (Burch-Smith et al. 2004).

Conclusion Plants are exposed to many types of abiotic stress but the most serious environmental stresses specially in Africa and Arab Gulf countries are heat stress which affects plants during their life cycle from the start of their growth in the field until time of the harvest and during transport to the market. Under this stress the plants are exposed to many changes in their metabolism and gene expression which leads to a decrease in growth and increase in damage to the fruits to become under heat stress the fruits ripen quickly and the tissues to soften prematurely resulting in up to 50 % yield loss. Given this observation the hypothesis of this research was to choose tomato for the work because of its economic importance for all tropical and sub-tropical regions of the world and to improve tomato fruit survival under heat stress using new genetic engineering strategies to silence the heat shock protein gene (vis 1) controlling fruit ripening and fruit quality. To achieve this goal, we succeeded first to establish a useful protocol for the production of sufficient number of transgenic tomato plants; Pusa Ruby and Super- strain B using Agrobacterium. After that significant finding from this study was development of tomato cultivars with improved fruit quality and longer shelf life by silencing the expression of vis1 gene. The results is first record to assess the effectiveness of a siRNAs strategy as an advanced technique to switch off vir1 gene expression and influence fruit ripening in tomato. Acknowledgments This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Saudi Arabia under Grant Number (965-003-D1434). The authors, therefore, acknowledge with thanks DSR technical and financial support.

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