chapter iii reducing caffeine content in c.canephora using...

Chapter III

Reducing caffeine content

in C.canephora

using post transcriptional

gene silencing (p.t.g.s)

Chapter 3 Reducing caffeine content in C. canephora using post transcriptional gene silencing

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Summary

Present chapter contains studies carried out to evaluate the effect and effectiveness of

different gene silencing constructs bearing a 360 bp fragment from the second exon of

the theobromine synthase gene. The region of the second exon from where the

fragment was cloned is conserved for all the N-methyltransferase genes involved in

caffeine biosynthesis. The constructs were used to transform somatic embryos using

Agrobacterium mediated gene silencing techniques. Molecular analysis confirmed the

stable integration of the transgene in the transformants. The effectiveness of the

silencing constructs was evaluated by transcript analysis of the targeted genes by RT-

PCR and northern blotting. The silencing efficiency was evaluated by estimating the

purine alkaloid content in the transgenic lines. The purine alkaloid content in the

transformants was analysed using high-performance liquid chromatography (HPLC).

The three constructs differed in their silencing efficiencies and specificity. Invert

repeat constructs were found to be most efficient in silencing the caffeine

biosynthesis. Though the constructs were not specific to a single N-methyltransferase,

transformants obtained were mainly affected in N-methyltransferase gene involved in

the third methylation. Results indicate that the use of homologous coding sequence in

the PTGS constructs resulted in a much higher efficiency in silencing of the caffeine

biosynthetic pathway. Though the silencing was efficient in the transformants, an

increased accumulation of caffeine metabolites was observed in regenerated plantlets

when compared to that in the somatic embryos. Invert repeat constructs were most

efficient is silencing caffeine biosynthesis however rate of survival of the transgenic

plants bearing invert repeat construct was extremely low. Based on these results it is

suggested that antisense should be the preferred method for decaffeination and

minimal caffeine lines preferred over completely decaffeinated plants.


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3.1 INTRODUCTION

Post-transcriptional gene silencing (PTGS), a sequence specific RNA degradation

mechanism, induced in plants by transforming them with antisense, co-suppression or

hairpin RNA constructs (Smith et al., 2000). Post transcriptional gene silencing is a

form of homology dependent gene silencing (HDGS) that requires homology between

the double stranded RNA and the protein coding regions (Vaucheret et al., 2001)

Antisense refers to short DNA or RNA sequences, termed oligonucleotides,

which are designed to be complementary to a specific gene sequence. The goal is to

alter specific gene expression resulting from the binding of the antisense

oligonucleotide to a unique gene sequence. Antisense is usually considered as a

mechanism for sequence- specific messenger RNA (mRNA) recognition that leads to

transcript degradation. Regulation of gene expression by antisense RNA was first

discovered as naturally occurring phenomena in bacteria. The antisense technology is

based on blocking the information flow from DNA via RNA to protein by the

introduction of an RNA strand complementary to (part of) the sequence of the target

mRNA. This so-called antisense RNA base pairs to its target mRNA thereby forming

double stranded RNA. Duplex formation may impair mRNA maturation and/or

translation or alternatively may lead to rapid mRNA degradation (Mol et al., 1990). In

eukaryotes, antisense RNAs can be produced by utilizing a DNA fragment that covers

the 5΄ end of the mRNA to block the capping reaction, or a DNA fragment which

covers an exon-intron (and intron-exon) junction to block the splicing reaction.

Alternatively, a DNA fragment which covers the poly (A) site of the mRNA may give

rise to antisense transcripts that block mRNA transfer from the nucleus to the

cytoplasm (Inouye, 1988). Antisense technology was first effectively used in plants to

alter the levels of various degradative enzymes or plant pigments.

In case of many endogenous plant genes, an over expression of the sense RNA

or mRNA leads to a drastic reduction in the level of expression of the genes

concerned; this phenomenon is called co-suppression. One way of achieving an over

expression of the mRNA is to introduce a homologous sense construct of the gene

concerned so that it also produces sense RNA or mRNA (in addition to the

endogenously present gene). The efficiency of co-suppression seems to vary among

plant genes. Co-suppression has never been observed for the Petunia chalcone


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isomerase gene, while tobacco glutamine synthetase nuclear gene is always co-

repressed; CHS gene (Petunia) represents the intermediate situation (Napoli et al.,

1990).

3.1.1 Post transcriptional gene silencing in plants

Post-transcriptional gene silencing (PTGS) was first reported in plants as a

coordinated and reciprocal inactivation of host genes and the transgene encoding the

same RNA (Napoli et al., 1990). A similar phenomenon observed in the filamentous

fungus Neurospora crassa was termed quelling (Romano & Macino, 1992). A major

breakthrough in the silencing history was the discovery of a gene silencing response

in the nematode Caenorhabditis elegans (Fire et al., 1998). This phenomenon, in

which experimentally introduced double stranded RNA (dsRNA) leads to loss of

expression of the corresponding cellular gene by sequence-specific RNA degradation,

was called RNA interference (RNAi) (Fire et al., 1998). The protective effect of RNA

silencing in reducing virus infectivity supports the view that PTGS has evolved as a

mechanism to defend plants against virus infection and also to moderate the possible

deleterious, genome-restructuring (insertional) activity of virus-like mobile genetic

elements e.g. retrotransposons. A growing body of biochemical and genetic data has

further demonstrated that plants, animals and yeasts share related mechanisms of

specific degradation of RNAs in which double-stranded forms of RNA are initiator

molecules (Dalmay et al., 2000; Fagard et al., 2000; Fagard & Vaucheret, 2000;

Brenstein et al., 2001).

The mechanism of co-suppression is not understood. According to a threshold

model, when RNA transcripts of a gene accumulate beyond a critical, threshold level,

they are selectively degraded by RNases. An accumulation of high levels of RNA

transcripts of a gene may lead to the production of aberrant sense RNA transcripts of

the transgene. An accumulation of aberrant RNA transcripts is proposed to activate

RNA-dependent RNA polymerase of plant origin, which transcribes the RNA

transcripts to produce antisense RNA. The antisense RNA transcripts would associate

with the accumulated normal and aberrant RNA transcripts of the transgene as well as

the endogenous gene. This will produce RNA duplexes, which present targets for

double-stranded RNA specific RNases like RNase H. Degradation of the RNA

transcripts of a gene is postulated to somehow lead to a hyper-methylation of the

RNAi sequences homologous to the degraded RNA sequences. This often leads to a


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drastic reduction in the level of expression of the transgene in question and also of

homologous endogenous gene(s).

RNA silencing is triggered by the presence of endogenous or exogenously

introduced double-stranded RNA (dsRNA), which is further cleaved into small RNAs

to become functional in a number of epigenetic gene-silencing processes (Zamore et

al., 2000; Eckardt, 2002 a, b). In plants, RNA silencing, as an efficient part of gene

silencing, but also plays important roles in the regulation of endogenous gene

expression (Voinnet, 2002). The signals of intracellular RNA silencing may be

transmitted systemically from cell to cell over a long distance through the phloem

(Uddin & Kim, 2011). Short interfering RNAs (siRNAs), aberrant RNAs, and

dsRNAs are the suggested candidates for such mobile silencing signals, although the

mechanism of their involvement in the process is not clear so far (Palauqui et al.,

1997; Voinnet et al., 1998).

The mechanisms of silencing have been discussed extensively by Yu &

Kumar (2003). The mechanism of RNA silencing induced by dsRNA occurs in two

major steps, viz., initiation and effector (Cerutti, 2003). The initiation step involves

the cleavage of the triggering dsRNA into siRNAs of 21–26 nucleotides with 2-

nucleotide 3` overhangs, which correspond to both sense and antisense strands of a

target gene (Hamilton & Baulcombe, 1999; Voinnet, 2002). In the effector step, the

siRNAs are recruited into a multi-protein complex referred to as the RNA-induced

silencing complex (RISC), in which the degradation of target mRNAs occurs with the

siRNA as a guide (Hammond et al., 2000; Zamore et al., 2000). Each RISC appears to

have a single siRNA, an RNase and an mRNA homology-recognition and binding

domain. The Dicer protein is involved in generating siRNA. The processing of a long

dsRNA into siRNA is mediated by an RNase-III-like dsRNA-specific ribonuclease,

designated Dicer, initially in Drosophila (Bernstein et al., 2001). The members of the

Dicer protein family may be functionally conserved in fungi, plants and animals

(Tijsterman et al., 2002).

Grafting experiments revealed that PTG silenced plants produce a sequence

specific systemic silencing signal that propagates long distance from cell to cell and

triggers PTGS in non silenced graft connected tissues of the plant (Palauqui et al.,

1997; Voinnet et al., 1998). The essence of RNAi technology is the delivery of

dsRNA as a potent activator of RNA silencing into an organism, or cell, with the


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purpose of triggering sequence-specific degradation of homologous target RNAs.

Double stranded RNA can be delivered by stably transforming plants with transgene

that expresses a self-complementary RNA separated by a spacer sequence (Hamilton

et al., 1998; Waterhouse et al., 1998). The resulting transcript hybridizes with itself to

form a hairpin structure that comprises a single-stranded loop region (usually

corresponding to an intron) and a base-paired stem, which mimics the dsRNA

structure that induces RNAi. It has been shown that transgene constructs encoding

intron-spliced RNA as spacer sequence with hairpin structure give stable silencing

with almost 100% efficiency when directed against viruses (Smith et al., 2000).

PTGS is a highly variable process dependent on the transgene, the host-plant

species, the developmental stage of the plants, and environmental factors (Meins,

2000). There appears to be strict species-specific requirements both in terms of the

length of sequence and the degree of homology shared, to initiate RNA silencing;

generally, more than 75% homology is needed to induce RNA silencing against a

specific gene (Sijen et al., 1996; Eamens et al., 2008). Silencing appears to be most

efficient when sequences of more than 300 base pairs are used to design hpRNA

constructs (Wang and Waterhouse, 2002). By choosing unique or conserved region, a

single or all members of gene families, respectively can be silenced (Wesley et al.,

2001). Silencing was found to be more efficient if cognate promoters for the target

genes were used to express the transgenes (Li et al., 2011). PTGS based on

untranslated regions is useful for inactivating individual alleles or gene family

members that have unique sequences in these regions. By contrast, PTGS based on

coding sequence homology is suited for silencing all members of gene families that

share high sequence homology in these regions (Kooter et al., 1999). Roughly, 60–

70% identity of sequence is required for PTGS of cDNAs representing members in

the same multigene family (Meins, 2000; Meins et al., 2005). One, some or all

members of a multigene family have been silenced by RNAi by targeting sequences

that are unique or conserved (Allen et al., 2004b; Fukusaki et al., 2004; Ifuku et al.,

2003; Huang et al., 2006).

The dsRNA-mediated silencing was first demonstrated in plants by the

simultaneous expression of antisense and sense gene fragments targeted against both

an RNA virus and a nuclear transgene (Waterhouse et al.,1998). The methodology of

the specific and heritable genetic interference by dsRNAs in Arabidopsis was further


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established in the investigation of several genes involved in floral development

(Chuang & Meyerowitz, 2000). In this respect, transformation vectors capable of

dsRNA formation were constructed by linking the gene-specific sequences in both

sense and antisense orientation under the control of a strong viral promoter. These

dsRNA expressing constructs, when delivered into Arabidopsis with Agrobacterium-

mediated transformation, created a heritable phenotypic series in the transformants,

which corresponded to mutant alleles of different strengths. Thus, the dsRNA

interference can generate transformants showing both reduction and loss of function,

with gradually reduced expression of a specific gene, which has proved to be an

effective tool in studying some dosage-dependent genes involved in plant

development and for those which are essential for plant viability (Hirai et al., 2010).

The application of dsRNA technology for large-scale investigation of gene functions

will be facilitated by making the critical experimental procedures fast and efficient

(Wesley et al., 2001; Hirai et al., 2007). The so-called “pHELLSGATE” system was

developed to convert a polymerase chain reaction product into a dsRNA structure that

includes a spacer intron in one simple step by using an in vitro recombinase system

(Wesley et al., 2001). This is based on the observation that inclusion of an intron as a

spacer between the sense and antisense arm of a dsRNA construct greatly increases

the silencing effect (Wesley et al., 2001; Hirai et al., 2007). Simple single-step

cloning method of dsRNA interference, termed ’silencing by heterologous 3′UTRs,

was shown to operate effectively in both Arabidopsis and Lycopersicon esculentum

systems (Brummell et al., 2003). In this method, an inverted repeat of the 3′UTR of a

heterologous gene was placed 3′ to a single-stranded targeting sequence, which

induced the effective degradation of endogenous target mRNAs homologous to the

target transgene, 5′ to a dsRNA region. (Sijen et al.,1996; Han & Grierson, 2002a, b).

3.1.2 Application of RNA silencing in crop plants

The practical use of RNA silencing to reduce gene expression in a sequence-specific

manner promises to be an essential and routine reverse genetics approach in plant

functional genomics. Technologically reliable and high-throughput methods of RNA

silencing have been developed by the recent progress on the understanding of the core

RNA silencing mechanism. Particularly, the discovery of dsRNA as an inducer of

RNA silencing has provided a scheme of dsRNA-mediated interference to direct

gene-specific silencing that is more efficient than antisense suppression or co


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suppression by over expression of target genes (Voinnet et al., 1999; Bass, 2000;

Hammond et al., 2000; Stam et al., 2000; Hammond et al., 2001; Vaucheret et al.,

2001; Matzke, 2002; Yu & Kumar, 2003; Herr & Baulcombe, 2004; Vinod et al.,

2004; Frizzi & Huang, 2010).

Gene silencing has been successfully done in wide range of plant species with

varied effects. Antisense techniques have been effectively used to down regulate

expression of specific genes like polygalacturonase in tomato (Sheehy et al., 1988),

Chalcone synthase (CHS) in Petunia (van der Krol et al., 1990). It has been used to

induce disease resistance in papaya (Tennant et al., 1994; Fan et al., 2009). Antisense

technique has been successfully applied to know function of genes (Yu & Kumar,

2003; Mahmood ur et al., 2008).

RNAi has also been successful in genetic modification of the fatty acid

composition of cotton seed oil (Liu et al., 2002). RNAi technology has also been used

to reduce alkaloid content in coffee (Ogita et al., 2003, 2004) and Opium poppy

(Allen et al., 2004b). RNAi has been successfully used to generate a dominant high-

lysine maize variant by knocking out the expression of the 22 kD maize zein storage

protein, a protein that is poor in lysine content (Segal et al., 2003). Reduction of

lysine catabolism specifically during seed development by an RNAi approach

improved seed germination (Zhu & Galili, 2004). Several others have also used RNAi

to demonstrate function of gene or to reduce accumulation of certain metabolites (Yu

& Kumar, 2003; Mahmood ur et al., 2008). Few significant examples have been listed

in Table 3.1.

Table 3.1: Examples for application of PTGS in crop plants

Trait Species Target gene Refrence

Altered starch

composition Potato

Granule bound starch synthase

(GBSS) Hofvander et al.,2004

Reduced toxicity Potato Solanidine glucosyl transferase McCue et al., 2003

Enhanced caroteinoid

and flavonoid content Tomato DET1 Davuluri et al. 2005

Increased essential

amino acids Corn

Lysine-ketoglutarate

reductase/saccharophine

dehydrogenase(LKR/SDH)

Houmard et al., 2007;

Reyes et al., 2009

Reduced lignin content Alfalfa 4-coumarate 3-hydroxilase (C3H) Reddy et al., 2005

Allergen reduction Soyabean P34 Herman et al., 2004

Rice 14-16 kDa protein Tada et al., 1996

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http://onlinelibrary.wiley.com/doi/10.1111/j.1467-7652.2010.00505.x/full#b65


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Rye

grass Lol p1 and Lol p2 Petrovska et al., 2004

Apple Mal d1 Gilissen et al., 2005

Peanut Ara h2 and Ara h6 Chu et al., 2008; Dodo et

al., 2008

Bean golden mosaic

virus Bean

Rep, TrAP, REn and movement

protein (MP) Bonfim et al., 2007

Rice tungro

bacilliform virus Rice Open reading frame IV Tyagi et al., 2008

3.1.3. Genetic engineering of Coffee plant

Genetic engineering of coffee to obtain new cultivar with desirable traits, such as

disease resistance and improved quality helps in overcome the lengthy time barrier

usually observed in the case of conventional breeding methods. Genetic

transformation of coffee opens a window of opportunity to introduce traits from

different species, adding characteristics which would be difficult or even impossible

to acquire using traditional breeding techniques, such as insect resistance (Leroy et

al., 2000), herbicide resistance (Ribas et al., 2006a) , cup quality and tolerance to

abiotic stress like drought or frost (Kasuga et al., 1999). Even though several research

groups around the world have produced genetically modified coffee plants (Ribas et

al., 2006b), there are only few examples for attempts made to improve the traits and

cup quality of coffee. CRY1-AC gene from Bacillus thuringiensis was introduced,

which is effective against the coffee leaf miner (Leroy et al., 2000). Use of specific 3′-

untranslated sequence for one of the coffee NMT genes (CaMXMT-1) in the design of

RNAi construct and reduction in theobromine and caffeine contents in C. canephora

and C. arabica was observed (Ogita et al., 2004).

Identification of genes encoding for the enzymes involved in caffeine

biosynthesis facilitated engineering of caffeine biosynthetic pathway to either

suppress or enhance production. The first approach was to construct transgenic coffee

plants with reduced caffeine content by RNAi method, in which the mRNA of the

target gene was selectively degraded by small double stranded RNA species (Ogita et

al., 2003; Ogita et al., 2004; Ogita et al., 2005). The 3′-untranslated region and the

coding region of CaMXMT cDNA were used to design the RNAi constructs. Two

different RNAi constructs, RNAi-S having a short insert with 150 bp, and RNAi-L

with a long insert of 360 bp, were inserted into the pBIH1-IG vector (Ohta et al.,

1990), which was introduced into the EHA101 strain of A. tumefaciens to transform


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C. arabica and C. canephora plants. The resulting transformed lines were assayed for

expression of N-methyltransferase genes by RT-PCR, and it was found that the

CaMXMT-RNAi construct suppressed transcripts for not only CaMXMT but also that

of the CaXMT and CaDXMT genes The reduced level of transcripts resulted in

decreased activity of the corresponding enzymes and this was confirmed by directly

measuring their products, theobromine and caffeine using HPLC. There is over 90%

homogeneity in the coding region between the three genes (Uefuji et al., 2003). This

suggests that the primary small double stranded CaMXMT-RNA progressively

produces many secondary small double-stranded RNAs spanning its coding region to

the adjacent sequence of the initiator region. This in turn destroys mRNAs of

CaMXMT and CaDXMT. Previously at C.F.T.R.I. homologous coding sequence has

been used to silence caffeine biosynthetic gene (Satyanarayana, 2006).

Present chapter deals with the evaluation of effect and efficacy of different

post-transcriptional gene silencing (PTGS) constructs. Sense, antisense and invert

repeat constructs were developed using a 360 bp fragment from the conserved region

of the coffee N-methyltransferase genes involved in caffeine biosynthesis. The

silencing constructs were mobilized into Agrobacterium tumefaciens (EHA101).

Sonication assisted Agrobacterium-mediated transformation of the somatic embryos

of C. canephora was performed, followed by regeneration of the transformants.

Molecular analysis and identification of flanking regions confirmed stable integration

of the transgenes in the transformants and their integration in non coding regions of

the genome, respectively. Effect and effectiveness of the individual silencing

constructs was evaluated by transcript analysis of the targeted genes by RT-PCR and

northern blotting. The silencing efficiency was further evaluated by estimation of the

purine alkaloid content in the transgenic lines using HPLC.


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3.2 MATERIALS AND METHODS

3.2.1 Plant material

Certified seedlings of C. canephora var. S-274 and C x R were purchased from

Central Coffee Research Institute (CCRI), India. Leaves were used as explants for

direct somatic embryogenesis.

3.2.2 Transformation of C. canephora somatic embryos

Somatic embryos were obtained from leaf explants (refer section 2.1). Sonication

assisted Agrobacterium-mediated transformation system was used (refer section 2.2,

2.3 and 2.4). The transformants were later regenerated (section 2.4).

3.2.3 Analysis of the transformants

Putative transformants were analyzed to check for the stable integration of the T-DNA

by GUS staining as (section 2.5). PCR based confirmation of the putative

transformants were carried out using hptII gene specific primers (section 2.6).

Genomic DNA was isolated from the hygromycin resistant PCR positive

transformants and untransformed plantlets using GeneElute Plant genomic DNA

isolation kit (Sigma-Aldrich, USA). DNA was digested using NdeI and SacII

restriction endonucleases for 4 hrs at 37°C. The digested DNA was size fractioned on

1% agarose (w/v) at 5V/cm2 for 1 hr. The digested DNA was transferred on to

HiBond positively charged nylon membrane (Amersham, USA) by electro-blotting

using Trans-Blot Semi-Dry electrophoretic transfer cell (Bio-Rad, IL, USA). PCR

amplicons of hpt gene biotinylated using Brightstar Psoralen-biotin non isotopic

labelling kit (Ambion, TX, USA) was used as probe. Hybridization was carried out at

58°C in a hybridization oven (Shell Lab model-1004-2E) at 50 rpm for 6 hrs.

Hybridization signal was developed by using Brightstar Bio-detect kit (Ambion, TX,

USA) and BCIP-NBT (Bangalore Genei, Bangalore) was used as substrate (Detailed

protocol in section 2.13).

3.2.4 Characterization of T-DNA integration site by genome walking

The site of integration of the T-DNA in the coffee genome was determined using PCR

based method used to determine the 5′ flanking sequences of the T-DNA (Figure 3.1)

(Cottage et al., 2001). The method is based on the genome walking method described

by Siebert et al. (1995).


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Figure 3.1: Analysis of T-DNA insertion sites by PCR-walking. Plant DNA was digested with

blunt-cutting enzymes. Adaptors were annealed to the blunt-ended fragments. A

walking primer (WP) was chosen 3′ to the left border sequence. The left border

sequence was not used for primer design as it is known that T-DNA insertion

frequently aborted before this sequence. The walking primer and adaptor primer

were used to amplify DNA sequence flanking the T-DNA insertion site and/or

inserted vector backbone sequences if present.

3.2.4.1 The principle of genome walking (Siebert et al., 1995)

The procedure for promoter isolation was the same as described by (Siebert et al.,

1995) which combines the ‘vectorette PCR’ (Lagerstrom et al., 1991) with

‘suppression PCR’ (Lukyanov et al., 1994). The ‘vectorette’ feature of the adaptor is

the presence of an amine group on the 3′- end of the lower strand. This blocks

polymerase-catalyzed extension of the lower adaptor strand, preventing the generation

of the primer-binding site unless a defined, distal, gene-specific primer extends a

DNA strand opposite the upper strand of the adaptor. In rare cases, the 3′end of the

adaptor gets extended, due to incomplete amine modification during oligonucleotides

synthesis or incomplete adaptor ligation. This creates a molecule that has the full-

length adaptor sequence on both ends and can serve as a template for end-to-end

amplification.

Without suppression effect, these rare events would lead to unacceptable

backgrounds due to the exponential nature of PCR amplification. However, in

‘suppression PCR’ the adaptor primer is much shorter in length than the adaptor itself

and is capable of hybridizing to the outer primer-binding site. During subsequent

thermal cycling, nearly all the DNA strands will form the ‘panhandle’ structure

(following every denaturation step due to the presence of inverted terminal repeats),


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which cannot be extended (Figure 3.2). At the appropriate annealing/extension

temperature, this intra-molecular annealing is strongly favoured over (and more stable

than) the intermolecular annealing of the much shorter adaptor primer to the

Figure 3.2: The suppression PCR effect

adaptor. The suppression PCR effect is lost or reduced if a primer with annealing

temperature lower than 60-65°C is used. However, when a distal gene specific primer

extends a DNA strand through the adaptor, the extension product will contain the

adaptor sequence only on one end and thus cannot form the ‘panhandle’ structure.

PCR amplification can then proceed normally.

3.2.4.2 Design of Oligonucleotide

Oligonucleotides were designed to isolate specific 5′ flanking regions of the T-DNA

by using PCR based genome walking method. Two adapter specific primers and non-

overlapping gene-specific primers were designed based on the adapter sequences and

pCAMBIA1301 T-DNA sequence using the software Oligo explorer (Figure 3.3).

The primers were used to amplify flanking regions of the T-DNA i.e. the 5′ regions

upstream to the T-DNA (Table 3.2).

Figure 3.3: Gene specific primer positions on the T-DNA used for gene walking


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Table 3.2 List of primers and adapters used in genome walking

Adapter Sequences

Adapter long arm-5′- CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT- 3′

Adapter short arm -5′- ACCTGCCC -NH2-3′

Adapter Specific Primers

ASP1 (5′- GGATCCTAATACGACTCACTATAGGGC-3΄)

ASP2 (5′- AATAGGGCTCGAGCGGC-3΄)

Gene Specific Primers:

WLB1-5′-ACACCACAATATATCCTGCCA WHG5-5′-AGCACTCGTCCGAGGGCAAA-3'

WLBA2- 5′-CCCCCGAATTAATTCGGCGT WHG6-5′-ACTCGCCGATAGTGGAAACC-3'

WPA3-5′-AGGGTTTCGCTCATGTGTTG-3′ WHG7-5′-TCCGCATTGGTCTTGACCAA-3'

WPA4-5′-GATAAGGGAATTAGGGTTCC-3′

3.2.4.3 Adapter annealing

The two adapters were dissolved in 25 µl of sterile deionised water and mixed

together. The mixture was kept in a boiling water bath for 5 min and then the mixture

was kept at room temperature to anneal slowly. The volume was made up to 100 µl to

obtain a 10X solution (50 µM). The double stranded adapter was used at 1X

concentration.

3.2.4.4 Construction of adapter ligated DNA libraries

DNA libraries were constructed by digesting the C. canephora plantlet genomic DNA

(3 μg) with 80 units of several blunt cutting restriction endonucleases for 3hrs. The

enzyme was heat inactivated at 65°C for 10mins. The DNA fragments were purified

by phenol: chloroform: isoamyl alcohol (25: 24: 1) extraction, followed by a

chloroform extraction and precipitated by addition of 1/10th

volume 3 M Sodium

acetate (pH 5.2) and two volumes of absolute ethanol. The mixture was vortexed and

centrifuged at 13,000 rpm for 15 mins and the pellets were washed with 70% ethanol

and aspirated and allowed to air dry. The DNA fragments were dissolved in 10 µl of

TE buffer (pH 8.0). DNA fragments (10 µl) were ligated to adapters at 4°C overnight

under the following conditions: 40 mM Tris-HCl, pH 7.8, 10 mM DTT, 0.5 mM ATP

and 10 U of T4 DNA Ligase (MBI Fermentas GmbH, Germany) in a total volume of

20 µl. The ligation reaction was terminated by incubating the tubes at 650C for 10

min. The ligation mix was diluted 1:10.


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3.2.4.5 PCR amplification of T-DNA flanking sequences

Primary PCR was carried out using DraI, EcoRV, PvuII and SspI adaptor ligated

DNA libraries as templates with specific primer ASP1 and gene specific primer

WHG5. Primary PCR reactions were carried out in 50 µl volumes containing 1µl of

ligated and diluted DNA, 10 mM TAPS (pH 8.8), 50 mM KCl, 1.75 mM MgCl2,

0.01% gelatin, 100 pM each of ASP1 and WHG5 primers, and 1U of Taq polymerase

(Sigma-Aldrich. USA). PCR was performed in a Primus 25 advanced thermocycler

(PEQLAB Biotechnologie, GmBH). The ‘touchdown’ PCR parameters used during

primary PCR reactions were as follows: 95°C, 15 s; 60°C, 3 min for seven cycles;

95°C, 15 s; 55°C, 3 min for 32 cycles; and a final hold at 72°C for 7 min. Touchdown

PCR involves using an annealing/extension temperature that is several degrees higher

than the Tm of the primers during the initial PCR cycles. Although primer annealing

(and amplification) is less efficient at this higher temperature, it is also much more

specific. The higher temperature also enhances the suppression PCR effect with

ASP1. This allows a critical amount of gene-specific product to accumulate. The

annealing/extension temperature is then reduced to slightly below the primer Tm for

the remaining PCR cycles, permitting efficient, exponential amplification of the gene-

specific template. An extremely short incubation at 95°C is necessary to preserve the

integrity of the larger genomic DNA templates required for long distance PCR in the

genomic walking protocol. The conditions for secondary nested PCR were as follows:

95°C, 15 s; 65°C, 3 min for seven cycles; 94°C, 2 s; 55°C, 3 min for 32 cycles; and a

final hold at 72°C for 7 min. Secondary PCR was conducted with 1 μl of a 50-fold

dilution of the primary PCR product using adapter specific primer ASP2 and the

nested gene specific primer WPA4.

Another round of PCR based walking was performed using a different gene

specific primer for primary PCR using DraI, EcoRV, PvuII and SspI adaptor ligated

DNA libraries as template with the adaptor specific primer ASP1 and gene specific

primer WPA4. Primary PCR reactions were carried out in 50 µl volumes containing

1µl of ligated and diluted DNA, 100 pm each of ASP1 and WPA primers, and 1 unit

of AccuTaq Long PCR DNA polymerase (Sigma-Aldrich, USA) having proof reading

activity. Secondary PCR was conducted with 1 μl of a 50-fold dilution of the primary

PCR product using adapter specific primer ASP2 and the nested gene specific primer


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WPA3. The PCR parameters used for primary and secondary PCR were same as

described earlier.

3.2.4.6 Cloning of walking products

Approximately, 10 µl of PCR products were size fractionated on 1% (w/v) agarose

gel. Largest PCR fragment from DraI adapter ligated DNA library was eluted from

the gel, purified using QIAQuick Gel Extraction Kit (QIAGEN, GmbH, Germany)

and T/A cloned using InsT/A clone PCR Product Kit (MBI Fermentas, GmbH,

Germany). The cloned PCR products were subsequently mobilized into E.coli strain

DH5α. The cloned PCR products were sequenced (Bio serve technologies, Genome

valley, HYD, India).

3.2.4.7 Insilco analysis of the 5′ flanking sequences

The sequence thus obtained by sequencing of the PCR fragment contained a portion

of the T-DNA and the flanking unknown gene fragment upstream to the T-DNA. The

flanking sequences were analysed using ExPASY to determine the presence of any

open reading frames. The sequences were then BLAST analysed against SGN Coffee

EST database (SGN, Cornell) and NCBI to identify a possible homology with

previously reported genes.

3.2.5 Transcript analysis of caffeine biosynthetic genes

3.2.5.1 RT- PCR analysis

Total RNA isolated from the hygromycin-resistant somatic embryos and plantlets

(section 2.14) was checked on 1.5% denaturing agarose gel and quantified using a

Nanodrop spectrophotometer (Model ND-1000, Nanodrop Technologies, Delware,

USA). DNA contamination of total RNA was avoided by treating with DNase (DNA

Free Kit, Ambion, TX, USA) before RT-PCR was performed. RT-PCR was

performed using specific primers for the amplification of the target gene CaDXMT1

and CaMXMT1.

Equal quantities of total RNA from each sample was used for first strand

cDNA synthesis. The first complementary DNA strand was synthesized from 1.5μg of

total RNA in 20-μl final volume, using AMV reverse transcriptase (Sigma- Aldrich,

USA) and oligo-dT (18mer) primer (Ambion, TX, USA). PCR amplifications were

performed using a PCR mixture (25 μl) that contained 1 μl of reverse transcriptase

reaction product as a template, 10× PCR buffer, 200 μM dNTPs (Fermentas, GmbH,


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Germany) and 1U of Taq DNA polymerase (Sigma- Aldrich, USA) at 60°C. The

PCR products obtained were separated on 1.2% agarose gel, stained with ethidium

bromide (0.001%) and documented (Chemidoc XRS Bio-Rad, Germany). The band

intensity of the PCR amplicons thus separated was measured using AlphaEaseFC

image analysis software (Alpha Innotech, USA). The amplicons were cloned and

sequenced. Ubiquitin gene (Accession No: SGN-U617292) was used as a control for

RT-PCR experiments.

3.2.5.2 Northern blot analysis

Total RNA isolated from transgenic plants was electrophoresed on 1.25% (w/v)

agarose gel and transferred on to HiBond H+ nylon membrane (Amersham, IL, USA)

using Northern Max kit (Ambion, TX, USA) following manufacturer’s instructions.

The cDNA amplicons were labelled using BrightStar psoralen-biotin labelling kit

(Ambion, TX, USA) and used as probes for hybridization. Bright Star Bio-Detect Kit

(Ambion, TX, USA) was used to detect hybridization signals. Detailed protocol is

provided in section 2.15.

3.2.6 HPLC analysis of transgenic somatic embryos and plantlets

3.2.6.1 Extraction of purine alkaloids

40-50 mg of fresh tissue (embryos/transformed plantlets) was used for alkaloid

extraction. The tissues were crushed in 80% v/v ethanol using a mortar and pestle and

the resultant slurry was homogenized using neutralized sand. The extract was

centrifuged for 10 min at 8000 rpm and the supernatant was collected. The pellet was

rewashed with 2-3 ml of ethanol and all the supernatant were pooled after

centrifugation. The extract was flash evaporated to dryness using a rotavapour

(Rotovapor R-200, Buchi Labortechnik AG, Switzerland) and the dried material was

taken in 1 ml of 80% ethanol prior to the estimation of caffeine and it’s metabolites by

HPLC.

3.2.6.2 HPLC separation of purine alkaloids

The purine alkaloids from hygromycin resistant embryos and plantlets were extracted

using 80% (v/v) ethanol as solvent. HPLC was performed on a Bondapak C18 column

(5μ X 250 mm) using isocratic solvent system (50 mM sodium acetate: Methanol:

Tetrahydrofuran in a ratio of 90: 9: 1) at a flow rate of 1 ml /min. A Shimadzu LC

20- A liquid chromatograph equipped with a dual pump and a UV spectrophotometric

detector (Model SPD –10 A) set at 270 nm was used. The recorder, a Shimadzu C–


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R7A chromatopac, was set at a chart speed of 2.5 cm/min. Injection volume was 10

µl, injected with Rheodyne 7125 injector. Peak identification was achieved by

comparing with the retention time of standards (Sigma-Aldrich, USA). HPLC analysis

was repeated for each treatment at least four times.


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3.3 RESULTS AND DISCUSSIONS

3.3.1 Transformation of Coffee

Somatic embryos were obtained from leaf explants of C. canephora by direct somatic

embryogenesis (Figure 3.4A & 3.4B). The somatic embryos were transformed with

the silencing constructs i.e. pSAT201, pSAT202, pSAT222 using Sonication Assisted

Agrobacterium mediated Transformation method (SAAT) (Vinod et al., 2006). The

transformed somatic embryos were selected for hygromycin resistance for a period of

4-6 months on a secondary embryogenesis selection media (Figure 3.4C). These

hygromycin resistant putative transformants were then transferred to plant growth

media for regeneration into plantlets (Figure 3.4D). The transformed plantlets were

hardened and transferred to green house (Figure 3.4F). Secondary embryos and

plantlets growing on 10 mgl-1

hygromycin selection medium were subjected to GUS

assay, PCR for hpt gene and Southern blot analysis to confirm transgene expression in

the T0 lines.

3.3.2 Analysis of transformants

3.3.2.1 GUS assay and PCR for hptII gene

Strong expression of intron uid A gene was observed in the tissue samples with

localized expression in leafs while certain portions did not show GUS staining (Figure

3.5A–D). GUS expression and staining may be lacking in certain tissues despite the

integration of T-DNA (Kumar et al., 2011; Wang et al., 2012). Hygromycin-resistant

plantlets showed positive PCR amplification for hptII gene with specific primers

(Figure 3.5E).

3.3.2.2 Southern blot anlysis

Southern blot analysis of the hygromycin-resistant regenerants confirmed the

integration of the T-DNA into the coffee genome and helped to eliminate false-

positives as well as to determine the number of transformation events that had

occurred. The position of SacII is located just outside the left border of T-DNA in

pCAMBIA 1301 and NdeI had only one restriction site within the T-DNA region.

Therefore presence of one band (expected to be above 1.1kb) in the Southern blot

analysis will be expected for each T-DNA integration event (Figure 3.5F & 3.5G).


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Figure 3.4: Different stages involved in somatic embryogenesis and regeneration of the

transformants. A: Direct somatic embryogenesis from leaf explants, B: Primary

somatic embryogenesis, C: Secondary somatic embryos in selection media, D: Elected

somatic embryos transferred to plant growth media, E: Regenerated transformed

plantlet, F: Hardened plant in Green house.

Figure 3.5: Analysis of transformants: (A) & (B) Gus staining in somatic embryos (C) Gus

staining in plantlet (D) Gus staining in plant leaf (E) PCR amplification of hpt II gene

from genomic DNA of transgenic plantlets. Cp:Control plamid pCAMBIA 1301 and

‘UT’: Genomic DNA from untransformed coffee plant used as control. (F) Southern

blot analysis of PCR positive transformants. Genomic DNA and control plasmid was

digested with SacII – NdeI and was transferred to positively charged nylon. Biotin-

labeled hpt gene was used as a probe for hybridization. C: Control plamid pCAMBIA

1301. T1: Control transformation using blank pCAMBIA 1301 vector. T4: Sense

transformant (pSAT 201). T6 &T7: Antisense transformant (pSAT202). T8&T9:

RNAi transformant (pSAT222), UT: Genomic DNA from an untransformed plantlet

(G) T-DNA region of pCAMBIA 1301 vector. T-LB: left border, T-RB: Right border,

P 35S: CAMV promoter.

1.1kb


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All the tested transgenic lines had 1-2 bands each of over 1.1kb when compared to the

control. Five of the six lines had a single band of different length and the remaining

one had 2 bands of different lengths (Figure 3.5G). No bands were observed in NdeI–

SacII digested genomic DNA from untransformed control. A similar method was used

to confirm the integration of transgene in the callus cells of Crataegus aronia using

HindIII (Al Abdallat et al., 2010).

3.3.2.3 Identification of flanking region by genome walking

Identification of the flanking sequences of the T-DNA was carried out in transformed

plantlets to determine site of integration of the T-DNA so as to ascertain that no

essential gene has been disrupted. This would dispel environmental concerns for field

trials of genetically modified crops and also fulfil environmental and legislative

requirements (Communities, 2001). Selecting plants having a stably expressed

transgene have greater chances of survival as a T-DNA insertion in an essential gene

may be lethal. Studies have shown that stably expressed transgenes contain relatively

simple T-DNA arrangements flanked at least on one side by plant DNA (Iglesias et

al., 1997).

The flanking sequences obtained by the modified genome walking method

analysed by using BLAST against NCBI nr database and coffee EST database at SGN

(Mueller et al., 2005) did not match with any known coding genes. This would

suggest that the integration must be into non-coding regions of the genome for all the

transformants. An ORF (Open Reading Frame) analysis using tool at ExPASy

proteomics tool (www.expasy.org/tool/B- CMsearchlauncher) revealed repeated stops

sites, which corroborated the BLAST results showing that the T-DNA insertion did

not disrupt any known gene or protein coding genes for all the regenerants except one.

T-DNA flanking region showed similarity to a non-annotated Populus trichocarpa

clone POP084-A05 (NCBI Blast) and SGN database showing similarity to unigene

SGN-E638105.

Further analysis of the flanking sequences revealed a pattern of integration in

which 3–45 bp were missing from the T-DNA left border and the flanking plant

nucleotides showed an integration pattern that is comparable to that observed in

transgenic plants of Maize and Barley (Stahl et al., 2002). The 3′ ends of the flanking

sequences were similar to that observed in transgenic plants of Maize and Barley

http://www.expasy.org/tool/B-%20CMsearchlauncher


104

(Figure 3.6). No vector backbone was observed in any of the transformants as its

presence would have raised serious environmental concerns during field trials.

Figure 3.6: Sequence alignment of left border flanking sequences of Barley (R&H series) with few

of that from coffee (C01-C05). The first three bases of the 3′ end of the flanking

sequence were found to be AT rich (marked in red)

3.3.2.4 Transcript analysis by RT-PCR

The effectiveness of silencing was evaluated by transcript analysis of the target genes

by both RT-PCR and Northern blot methods. Transcript accumulation of the target

genes was reduced in different silencing constructs (Figure 3.7). Maximum silencing

of the target genes was observed for RNAi constructs. Whereas antisense constructs

showed marginal decrease when compared to that noticed for RNAi constructs. Sense

constructs showed negligible decrease in transcript accumulation.

Figure 3.7: Expression of the NMT genes CaMXMT1 and CaDXMT in different transformants.

A: RT-PCR using gene specific primers with ubiquitin as control,

B: Northern blot using biotin-labeled cDNA amplicons as probes. rRNA was used as

the loading control.


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3.3.2.5 Quantification of purine alkaloids in caffeine metabolism

The effect of the silencing was evaluated by estimating the levels of different

metabolites involved in the caffeine biosynthetic pathway from hygromycin resistant

transformed somatic embryos and plantlets using HPLC. Purine alkaloids content

varied in the transformants among different post transcriptional gene silencing

constructs and a considerable increase in caffeine content was noted in older plants

(Table 3.3).

The sense construct resulted in 50% reduced theobromine content based on

average value when compared to that in control (pCAMBIA1301) somatic embryos.

Despite this reduction in its precursor content there was only a marginal decrease

(about 10%) in the caffeine levels of the individual transformants in comparison to

that in control, suggesting that theobromine synthase was mainly affected. In sense

knockdown plantlets there was marginal or no decrease in theobromine content which

may be attributed to the dual functional role of caffeine synthase in catalyzing both

theobromine and caffeine (Mizuno et al., 2003b; Uefuji et al., 2003). This enzyme

might be active and must have substituted for the main enzyme theobromine synthase

in caffeine biosynthesis. Similar observation was made for RNAi construct in Coffee

(Ogita et al., 2004) and in tea (Mohanpuria et al., 2011).

Antisense construct resulted in reduced contents of caffeine, theobromine in

the transformants. An 80% reduction in the theobromine content and 90% reduction

in caffeine content in antisense transformants when compared to that in control was

observed. The result seems to contradict with the observation of Tada et al., (2003)

who opined that antisense strategy is not suitable for completely suppressing the

expression of genes comprising a multigene family. In regenerated transformed

plantlets only 25-35% reductions in theobromine and caffeine was observed. The

presence of greater percentage of theobromine and caffeine in the leaf tissues of the

transformed plantlets when compared to that of the somatic embryos may be due to

the presence/ activation of by-pass pathways.


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Table 3.3 Purine alkaloid content in of transgenic C .canephora hygromycin resistant

somatic embryos1

1 Results are expressed as µg 100

-1 mg dry weight. Average values of at least four estimates are

provided

*Significant at 1% level over general mean ± SED (standard error deviation)

Table 3.4 Purine alkaloid content in transgenic C. canephora plantlets1

Construct Theobromine Caffeine Theophylline

pCAMBIA1301

(Control)

Line # 1 0.082 ± 0.0069 0.682 ± 0.0059 0.022 ± 0.0046

Line # 2 0.072 ± 0.0076 0.712 ± 0.0013 0.033 ± 0.0036

Average 0.0783 ± 0.0072 0.6856 ± 0.0341 0.0297 ± 0.0261

Sense (Co-

suppression)

pSAT201

Line # 1 0.082 ± 0.0033 0.067 ± 0.0037 0.025 ± 0.0056

Line # 2 0.071 ±0.0034 0.0655 ± 0.0041 0.025 ± 0.0057

Average 0.0733 ±0.0075 0.0664±0.0039 0.0266 ± 0.0041

Antisense

pSAT 202

Line # 1 0.062 ± 0.0012 0.041 ± 0.0023 0.0268 ± 0.0064

Line # 2 0.06 ± 0.0019 0.039 ± 0.0027 0.026 ± 0.0085

Average 0.0610 ±0.0016 0.0403 ± 0.0019 0.0264 ± 0.0005

RNAi

pSAT 222

Line # 1 0.048 ± 0.0058 0.032 ± 0.0045 0.019 ± 0.0097

Line # 2 0.071 ± 0.0067 0.038 ± 0.0054 0.0208 ± .0035*

Average 0.0540±0.0124 0.0372 ± 0.0197 0.0199 ± 0.0056

1 Results are expressed as µg 100

-1 mg dry weight. Average values of at least four estimates are

provided

Construct Theobromine Caffeine Theophylline

pCAMBIA1301

(Control)

Line # 1 0.228 ± 0.0082 * 0.770 ± 0.0081 0.023 ± 0.0034

Line # 2 0.098 ± 0.0024 0.784 ± 0.0042* 0.033 ± 0.0018 *

Average 0.146 ± 0.017 0.769 ± 0.0153 0.026 ± 0.0063

Sense

(Co-suppression)

Line # 1 0.084 ± 0.0060 * 0.675 ± 0.0196 0.028 ± 0.0030 *

Line # 2 0.074 ± 0.0035 0.705 ± 0.0055 * 0.023 ± 0.0030

Average 0.079 ± 0.0072 0.690 ± 0.0215 0.026 ± 0.0035

Antisense Line # 1 0.027 ± 0.0022 0.055 ± 0.0057 0.000 ± 0.0000

Line # 2 0.034 ± 0.0030 * 0.087 ± 0.0027* 0.000 ± 0.0000

Average 0.027 ± 0.0067 0.067 ± 0.0161 0.0

RNAi Line # 1 0.182 ± 0.0033 0.000 ± 0.0000 0.000 ± 0.0000

Line # 2 0.157 ± 0.0042 0.013 ± 0.0029 0.000 ± 0.0000

Average 0.178 ± 0.0197 0.004 0.0


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Increased accumulation of the precursors (theobromine) would have been

expected if the RNAi construct was specific to one of the enzymes but not in this case

wherein coding sequence used in the silencing constructs was conserved for all the

NMT genes involved in caffeine biosynthesis. Though there is accumulation of

precursors in invert repeat bearing transformed somatic embryos, no such

accumulation of precursors were observed in regenerated plantlets. This may be due

to the spreading of the RNAi signal to the secondary targets (Sijen et al., 2001;

García-Pérez et al., 2004).

In transformed somatic embryos more than 95% reduction in caffeine was

observed with RNAi when compared to 30% to 50% reduction reported by Ogita et

al., (2004). This could be primarily be due to the tighter loop of about 90 bp between

the two arms of the invert repeat when compared to the 517-bp GUS gene fragment as

spacer used by them. It is known that tightness of the hairpin loop can contribute to

enhanced RNAi silencing (Wesley et al., 2001). The use of homologous coding

sequence could have also been one of the reasons. But RNAi plantlets showed only

50-60% reduction in caffeine content may be due to the activation of bypass pathways

or gradual accumulation of caffeine over the period of growth.

Theophylline was observed only in control and sense transformed somatic

embryos, and not in antisense and RNAi constructs transformed plantlets. The fact

that trace amount of theophylline content was detected in transformed plantlet

suggests accumulation of caffeine in trace amounts during the development phase and

also ascertains that caffeine degradative pathway was active in transgenic plantlets of

C. canephora. It is known that theophylline is an intermediate in the catabolism of

caffeine rather than in de novo caffeine biosynthesis (Ito et al., 1997).

From the present study, it may be observed that even though the silencing

constructs were not specific, transformants obtained were mainly affected in only one

of the three NMTs. This is in contrast to the earlier report (Ogita et al., 2004),

wherein non-specific RNAi effect was observed with specific RNAi construct. They

observed a concomitant reduction of theobromine and caffeine contents to a range

between 30 and 50% to that of control. The non-specificity was assigned to the

possible effects of RNAi spreading from the initiator region into adjacent regions of

the target gene, and among genes whose sequences were closely related. Despite the

use of non-specific RNAi construct, transformants that were mainly affected in third


108

methylation step of the three NMTs. Such unexpected results are not unknown in

RNAi mediated gene silencing. Allen et al., (2004b) had observed the accumulation

of rare alkaloids several steps upstream to the target gene codeinone reductase (COR)

in opium alkaloid biosynthesis pathway, which was silenced with chimeric RNAi

construct; but surprisingly did not observe the accumulation of immediate precursors

as expected. Differences in the efficiencies and specificity of silencing observed in the

present study may be due to the different mechanisms of action involved in different

PTGS phenomena.

Although the basic concept of the application of transgene-based RNAi for

crop improvement has been developed, further studies are needed for its wider

application (Kusaba, 2004; Frizzi & Huang, 2010). The 100% elimination of caffeine

with accumulation of theobromine in the transformed somatic embryos bearing RNAi,

in spite of using highly homologous sequence for NMT gene family indicate that RNA

silencing may be subject to saturation. It is not yet known whether silencing in plants

is subject to saturation (Wang and Waterhouse, 2002; Frizzi & Huang, 2010) and this

possibility needs to be ascertained. The accumulation of theobromine, caffeine and

theophylline in transformed plantlets may due to the transcriptional activation of

related genes or bypass pathways during the course of development.

The possible transcriptional activation of other genes as observed in the

present study suggest that further studies are required before PTGS can be used as

tool for metabolic engineering of caffeine biosynthetic pathway. This will also help us

find possible intrinsic regulatory mechanism of the genes involved in the caffeine

metabolism in order to develop efficient silencing mechanisms to down regulate

caffeine biosynthesis.

chapter iii reducing caffeine content in c.canephora using...

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