SEARCH

SEARCH BY CITATION

Keywords:

  • alkaloids;
  • codeine;
  • genetic engineering;
  • metabolic engineering;
  • morphine;
  • secondary metabolites

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Transgenic modification offers new opportunities to alter the content of important plant-based secondary metabolites, including pharmaceuticals, nutraceuticals and plant protection chemicals. Metabolic engineering seeks to modify the amounts or chemical structures of specific metabolites, for example through changes in the activities of biosynthetic enzymes or regulatory proteins responsible for the co-ordinate expression of pathway genes, or the introduction of novel enzyme activities. The opportunities and difficulties in the metabolic engineering of secondary metabolic pathways have been reviewed (Kutchan, 1997; Facchini et al., 2000; Sato et al., 2001; Hughes and Shanks, 2002; Verpoorte and Memelink, 2002; Trethewey, 2004). It has proven difficult to predict those steps in a pathway whose modification is most likely to influence product accumulation. The reasons for this are varied: biosynthetic intermediates may be transported across a number of subcellular compartments (De Luca and St Pierre, 2000); intermediates may be trafficked between cell types (Bird et al., 2003; Weid et al., 2004; Murata and De Luca, 2005); intermediates may be handled by interdependent multienzyme complexes (Burbulis and Winkel-Shirley, 1999); and there may be competition between alternative biosynthetic pathways (Liu et al., 2002).

Despite these complexities, there is now a growing list of examples of increased yields of various classes of secondary metabolite in transgenic plants. The provitamin A content of rice endosperm has been substantially increased with the over-expression of three transgenically introduced enzymes (Ye et al., 2000; Paine et al., 2005). The vitamin E content has been increased 6–15-fold in plants through the transgenic over-expression of single enzymes (Shintani and DellaPenna, 1998; Cahoon et al., 2002). When three different monoterpene synthases from lemon were introduced into tobacco, by the transformation of individual genes and subsequent crossing to pyramid the transgenes, the total level of monoterpenoids was increased 10–25-fold and the fragrance profile was changed drastically (Lucker et al., 2004). Over-expression of squalene synthase in Panax gingseng produced a marked increase in ginsenoside and phytosterols (Lee et al., 2004). Activating the flavonoid pathway with the transgenic over-expression of the maize regulators C1 and R, and suppression of a competing pathway through RNA interference (RNAi) silencing of flavonone 3′-hydroxylase, achieved a fourfold increase in isoflavones in soybean seed (Yu et al., 2003).

Alkaloid pathways have also been successfully modified by metabolic engineering. Increasing the activity of ornithine decarboxylase in tobacco can result in increased nicotine (Hamill et al., 1990). Over-expression of strictosidine synthase in cell cultures of Catharanthus roseus resulted in a greatly increased content of terpenoid indole alkaloids (Canel et al., 1998). When cDNAs for two Hyoscyamus nigrans tropane alkaloid enzymes, tropinone reductase and hyoscyamine-6β-hydroxylase, were expressed in tobacco, the transgenic plants produced the expected products of these enzymes, namely tropine and 6β-hydroxyhyoscyamine (Rocha et al., 2002); in addition, they accumulated other novel nicotine derivatives, such as bipyridine, nornicotine, myosmine and anabasine, which were not detectable in the controls. When the tobacco gene for putrescine:SAM N-methyltransferase (PMT), the first committed step in tropane alkaloid biosynthesis, was expressed behind a constitutive promoter in Datura and Hyoscyamus hairy root cultures, the production of hyoscyamine and/or scopolamine was increased (Moyano et al., 2003); however, in Atropa belladonna plants and roots, there was no effect (Rothe et al., 2003). Likewise, transgenic over-expression of hyoscyamine 6-hydroxylase (H6H) caused increased production of scopolamine: fivefold in Atropa hairy roots (Hashimoto et al., 1993); threefold in Duboisia hairy roots (Palazon et al., 2003); and greater than 100-fold in Hyoscyamus muticus hairy roots (Jouhikainen et al., 1999). Large fold increases in examples such as these are usually only achieved when the background levels of the product are very low.

Morphinan alkaloids are produced from the opium poppy, Papaver somniferum. These include morphine, codeine, oripavine and thebaine, which are either important in their own right or as feedstocks in the manufacture of semi-synthetic pharmaceutical analgesics or compounds used in the treatment of opiate poisoning and addiction. A higher alkaloid content in the harvested dried plant material allows more efficient extraction of alkaloids; a higher alkaloid content on a dry weight (DW) basis also achieves economies associated with land use, harvesting, transport, storage and waste disposal. The alkaloid content in harvested material is generally in the range 1.5%−2.7% DW.

We report here the first genetically modified poppies with increased morphinan alkaloid production. A commercial cultivar of opium poppy with high alkaloid yield was transformed with cDNA encoding P. somniferum codeinone reductase (PsCor1.1). Figure 1 summarizes the morphine biosynthetic pathway in poppy. The steps between thebaine and morphine involve two O-demethylations and a reduction; the alternative order of these O-demethylations results in a bifurcation of the pathway. Both pathways are active in the Tasmanian genotypes, as evidenced by the accumulation of both codeine and oripavine. Codeinone reductase is the penultimate step and the last step in the two alternative routes. This paper reports transgenic plants in which the level of Cor transcript is increased. The morphinan alkaloid content is shown to be increased in both glasshouse and field experiments and in a number of generations and backcross derivatives.

image

Figure 1. Summary of the morphine biosynthesis pathway showing key intermediates and the bifurcated pathway between thebaine and morphine in which codeinone reductase appears twice.

Download figure to PowerPoint

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Comparison of alkaloid content between glasshouse and the field

In order to determine whether the glasshouse performance of a genotype is a good predictor of field performance, 14 different non-transgenic poppy varieties, including CO58-34, were included in the glasshouse trials. Field data were sourced from the 4 years of variety trials conducted by Tasmanian Alkaloids Pty Ltd, Westbury, Tas., Australia. In general, only the morphine content of these varieties has been rigorously assessed in the field, and therefore only the morphine content was compared between glasshouse and field performance. Comparison of the 2003 glasshouse data with the field data over 4 years indicated that the ranking of performance was comparable across the years (data not shown). The morphine content in the glasshouse was in the range 1.23%−2.06% DW, and was generally only 87% of the content in the field, which ranged from 1.23% to 2.45% DW; however, the morphine content in the glasshouse was strongly correlated with the morphine content in the field with this set of genotypes (linear regression, r2 = 0.84; P < 0.0001). This result suggests that, when the glasshouse trials are conducted as described here, the glasshouse alkaloid contents are a useful predictor of field performance.

Cor transgenic events

Seven Cor transgenics were derived from four independently transformed hypocotyls. Southern hybridization patterns confirmed that even the plants derived from the same hypocotyl represented independent transformation events. Thus, all seven transgenics are independent events.

T1 segregation data and Southern analysis for the Cor transgenic lines are shown in Table 1. Although most of the transgenic lines had multiple copies of the NptII transgene by Southern analysis, the segregation was usually simple, suggesting that the multiple transgenes were inserted at the same locus and inherited as a single linkage block. The exception was #7, which had three copies and failed to segregate any susceptible individuals out of 20 T1 plants tested, suggesting that the transgenes were at two or three independent loci.

Table 1. Cor transgenic insertions
T0 plantSouthern copy numberScores of NptII T1 segregationHypothesis (transgene linkage blocks)Probability (chi-squared with Yates’ correction)
+
Parent  018
1a3–524 710.82
1b3411610.83
23451511.00
3317 510.86
4122 710.76
7320 020.91
8120 410.72

Alkaloid content of Cor transgenics

Two different seed sources were used for parental controls in the 2003 glasshouse and field trials: seed that was originally supplied for the transformations, and breeders’ seed from commercial variety trials. A variation of up to 5% for both morphine and total alkaloid content was observed between the seed sources, but this variation was not statistically significant; therefore, the control data were pooled.

Seven Cor transgenics in the background genotype CO58-34 were examined for alkaloid content in three trials (Table 2). The only transgenic line not represented in all three trials was #3, for which there was insufficient seed for the glasshouse 2002 trial. The total alkaloid contents for all seven transgenics were higher than the control background genotype in all trials. The increases were quite consistent across the three trials and sometimes highly significant (P < 0.01). The field trial demonstrated statistical significance for increased alkaloids in all but one of the transgenic lines. In the field trial, #2 showed a highly significant 28% increase in total alkaloid, 22% increase in morphine, 58% increase in codeine and 75% increase in thebaine. It should be noted that, in the greenhouse 2003 and field 2003 trials, six of the seven transgenic lines, including #2, are expected to have 15% of the population as non-transgenic segregants (see ‘Experimental procedures’).

Table 2.  Alkaloid content in glasshouse and field trials
TrialMorphine (% DW)Codeine (% DW)Thebaine (% DW)Total alkaloid (% DW)
MeansSignif.MeansSignif.MeansSignif.MeansSignif.
  1. Significance of the differences of the means from control CO58-34 in each of the three experiments using analysis of variance (anova) and Dunnett's post-test. *P < 0.05. P < 0.01.

  2. Glasshouse trial 2002. The control of CO58-34 consisted of 12 observations of 12 plants each (total of 144 plants). The transgenic data consisted of four observations of 12 plants each (total of 48 plants). Transgenic line #3 was not available for the glasshouse 2002 trial.

  3. Glasshouse trial 2003. The control of CO58-34 consisted of 12 observations of eight plants each (total of 96 plants). The transgenic data consisted of four observations of eight plants each (total of 32 plants).

  4. Field trial 2003. The control consisted of 18 replicate rows with about 20 plants per row. The capsules from each row were collected and processed as a single observation. The control means therefore represent 18 observations and about 360 plants. The transgenic lines were represented as six replicate rows, with each row processed as a single observation; the mean for a transgenic represents a total of about 120 plants.

Glasshouse 2002
CO58-341.69 0.27 0.36 2.24 
1a1.85 0.33 0.40 2.50 
1b1.95 0.39 0.53 2.75
21.97 0.38 0.622.89
3nd nd nd nd 
41.65 0.31 0.50 2.38 
71.88 0.36 0.36 2.56*
81.63 0.39 0.47 2.43 
Glasshouse 2003
CO58-341.62 0.26 0.28 2.18 
1a1.83 0.29 0.31 2.45 
1b1.88 0.33 0.55 2.76*
21.90 0.27 0.45 2.63 
31.88 0.35 0.49 2.74*
41.85 0.23 0.33 2.43 
71.87 0.30 0.32 2.49 
81.80 0.40 0.25 2.45 
Field 2003
CO58-342.83 0.19 0.21 3.25 
1a3.43*0.24 0.22 3.91*
1b3.32 0.31*0.32 3.97*
23.44*0.31*0.38 4.17
33.31 0.30*0.28 3.95*
43.38*0.27 0.26 3.92*
73.45*0.25 0.18 3.89*
83.16 0.22 0.19 3.60 

From two of the segregating T1 transgenic families, #1b and #2, dried capsule alkaloids were analysed on a single plant basis. The results were pooled on the basis of whether they were transgenic or not. Figure 2a shows that, for transgenic segregants of the #2 family, the increase in total alkaloids was evident and significant relative to that of the non-transgenic segregants.

image

Figure 2. T1 segregant and BC1 populations. (a) Alkaloid analysis of individual bulked T1 segregants in families #1b and #2. The columns represent dried capsule analysis of five non-transgenics (nulls), six transgenics in #1b and seven transgenics in #2. The major alkaloids morphine, codeine and thebaine are shown. The values are the means ± SEM. *P < 0.05 (anova and Dunnett's post-test). (b) Alkaloid analysis of populations derived from two cycles of crossing of three transgenic events into an elite high-morphine genotype, grown in a glasshouse trial in 2004. Results are shown for morphine, codeine and thebaine. Comparison is shown with the original recipient genotype CO58-34 and the elite recurrent parent. anova and Dunnett's comparison was made between the elite parent and the backcross derivative populations. Asterisks to the left of the individual alkaloids indicate their level of significance relative to the elite recurrent parent; asterisks over the top of the columns indicate the significance of differences in the total alkaloids relative to the elite recurrent parent. *P < 0.05. **P < 0.01.

Download figure to PowerPoint

Backcross derivatives

Two cycles of crossing of three transgenic events into an elite high-morphine genotype resulted in total alkaloid increases when assessed in a glasshouse trial in 2004. In the case of the highest alkaloid line, #2, the increases in morphine, codeine, thebaine (P < 0.05) and total alkaloids (P < 0.01) were all significant when compared with the recurrent elite parent (Figure 2b). All three BC1 populations significantly exceeded the CO58-34 parent in total alkaloid, morphine, codeine and thebaine.

Transcript levels in transgenics

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was undertaken with RNA prepared from leaves just prior to flower opening to confirm that the Cor transgenes were achieving an increase in Cor message (Figure 3). Two different experiments are shown involving segregating populations of #1b and/or #2. Equal amounts of RNA were used in Figures 3a and 3b, and the transgenic status of 18 individuals was determined with NptII primers. The corresponding intensity of the product using Cor primers demonstrated the increase in transcript in the transgenic individuals relative to the non-transgenic segregants and the CO58-34 controls. Figure 3c–e show another experiment with seven T1 individuals of #2, in which the comparable loading of RNA is demonstrated with ribosomal RNA (Figure 3c) and the comparable presence of derived amplifiable cDNA is demonstrated by multiplexing Cor and Actin primers (Figure 3e). The Cor primers used in these experiments were designed to amplify all known endogenous and transgenic Cor transcripts. It is evident that the Cor transcript is increased only in those individuals which are transgenic.

image

Figure 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) showing increased Cor transcript levels segregating with the transgene. Two experiments are shown. (a, b) Eight transgenic T1 individuals from each segregating T1 family of #1b and #2; also shown are two RNA preparations from two control CO58-34 plants, one a seed-grown plant and the other a non-transgenic tissue culture regenerant. (a) RT-PCR using NptII primers to show which individuals are non-transgenic segregants. (b) RT-PCR using general Cor primers designed to amplify all known endogenous and transgene Cor transcripts. (c–e) Seven individuals from the segregating T1 family #2. The comparable loading of RNA is demonstrated with ribosomal RNA and multiplexing with Actin primers. (c) Ribosomal RNA. (d) RT-PCR results with NptII primers; below this panel the transgenic status is denoted with + or – symbols. (e) Multiplexed RT-PCR with both Actin and Cor primers. Tracks marked with M are molecular marker tracks.

Download figure to PowerPoint

Real-time RT-PCR was used to quantify the increase in Cor transcript in the leaves of the transgenic plants. RNA was extracted from the leaves of six T1 individuals of each of the #1b and #2 families. Quantitative RT-PCR was performed using primers suitable for all known members of the Cor multigene family; independently, the same preparations were amplified with primers to Actin to normalize the results. Figure S1 (Supplementary material) summarizes the results, showing that the level of total Cor transcript in the leaves of both transgenic families is about 10-fold greater than that in the non-transgenic segregants.

Alkaloid contents of various plant tissues

Various plant tissues from the 2003 field trial were analysed for alkaloid content. The genotypes included in the analysis were control line CO58-34 and three Cor transgenic populations: #1b, #2 and #7. The purpose was to determine whether transgenic over-expression was causing changes in the distribution of alkaloids in various plant parts. The transgenic lines chosen all showed increases in capsule total alkaloids. Four replicate samples of the roots, lower stem (10–30 cm below the capsule), upper stem (0–10 cm below the capsule), capsule and pollen were collected, extracted and analysed by high-performance liquid chromatography (HPLC) for morphine, codeine, oripavine, thebaine, noscapine, papaverine and reticuline contents (Figure 4). Noscapine and papaverine were not detected in any tissue or genotype, and no unusual alkaloid peaks were observed in any tissue. The only significant differences observed were increases in codeine in the lower and upper stem. In general, the distribution of alkaloids across tissues was similar in the transgenics when compared with that in the CO58-34 non-transgenic controls.

image

Figure 4. Total alkaloids (a), morphine (b), codeine (c) and thebaine (d) contents (% dry weight) in various tissues (leaves, lower stem, upper stem, pollen, roots, seed) of three transgenic lines (#1b, #2 and #7) in comparison with control CO58-34. The plants are T2 generation grown in the field in 2003. The values are the means ± SEM (n = 4). Asterisks over the top of the columns indicate the significance of differences relative to the control CO58-34. *P < 0.05. **P < 0.01.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Several reports have demonstrated the transgenic perturbation of benzylisoquinoline alkaloid biosynthesis and accumulation. Transgenic cell cultures of Eschscholzia californica with anti-sense berberine bridge enzyme (BBE) showed a 7–10-fold decrease in total benzophenanthridines (Park et al., 2002). Transgenic cell cultures with anti-sense CYP80B1 showed a less dramatic decrease in benzophenanthridines (Park et al., 2002). Similarly, transgenic hairy root cultures of E. californica showed substantially reduced benzophenanthridines (Park et al., 2003). Conversely, over-expression of BBE resulted in a five- to sixfold increase in total benzophenanthridines, including a 33-fold increase in dihydrochelilutine, in E. californica hairy root cultures (Park et al., 2003). We have also recently shown that the transformation of opium poppy plants with anti-sense BBE can increase reticuline, dehydroreticuline, scoulerine, salutaridine, laudanine and laudanosine relative to morphine, codeine, thebaine and oripavine, in latex, with little effect on benzophenanthridine alkaloids in transgenic roots (Frick et al., 2004). The work reported here demonstrates statistically significant increases in morphinan alkaloids in transgenic whole plants in both glasshouse and field trials as a consequence of over-expression of codeinone reductase. The highest alkaloid transgenic event, when backcrossed into an elite high-morphine genotype, also resulted in significant increases in alkaloids relative to the recurrent elite parent. The validity and importance of these increases were emphasized by using growing conditions, extraction methods and alkaloid assays as employed in a very successful poppy breeding operation. The high-alkaloid transgenics and their backcross derivatives outperformed the highest alkaloid conventional germplasm in the high-morphine breeding programme.

The increase in total alkaloids observed was generally attributable to increases in morphine, codeine and thebaine. Increases in morphine and codeine can be expected from an increase in codeinone reductase; however, increases in thebaine were not expected given that this intermediate occurs prior to codeinone reductase in the pathway. Because there are still uncertainties about the cellular and subcellular localization of the various steps of the morphinan pathway, we can only speculate how this is happening. It may be that increases in morphine or codeine result in feedback inhibition of the demethylation steps or inhibition of thebaine transport to the vesicles in which codeine and morphine accumulate.

The success of secondary metabolic engineering is occurring despite the difficulties in predicting which enzymes will have the greatest effect on the metabolic flux. Metabolic control analysis (MCA) (Rees and Hill, 1994) is a modelling device for complex biochemical systems which assumes that control over flux is shared and dynamic amongst many enzymes, and aims to predict the most responsive steps to perturb in order to achieve desired outcomes. MCA assumes that the system is close to steady state, that the enzyme under investigation acts in only one reaction, and that the mutant or transgenic perturbation is not met by any form of compensatory changes in other enzymes. The complexities of whole plant systems and the possible inapplicability of the assumptions have so far precluded the success of MCA in guiding attempts at secondary metabolic engineering in plants (Hughes and Shanks, 2002). In comparison with microbial cultures, plant tissues and whole plants introduce extra complexities of developmental changes and the interplay of different cell types. Thus, for example, various branches of benzylisoquinoline alkaloid biosynthesis are localized to vesicles in distinct cell types, the laticifers (Bock et al., 2002); the early steps of the specific morphine branch appear to be occurring in different cell types to the later steps (Bird et al., 2003; Weid et al., 2004), suggesting the transport of proteins, metabolites and possibly even transcripts between different compartments and cells. Given such complex compartmentalization, it seems likely that there will be a multiplicity of constraints on flux, including enzymatic, structural and transport limitations. Furthermore, now that poppy transformation is relatively straightforward (Chitty et al., 2003), transgenic perturbation of morphine biosynthesis is proving to be an effective tool to investigate the control points and bottlenecks of synthesis and accumulation (see below). Mutants in the morphine pathway have been unavailable until recently (Millgate et al., 2004), and should now become a valuable resource for dissecting biosynthetic regulation.

Codeinone reductase is encoded by a multigene family, and both enzyme and transcripts are evident across tissues and developmental stages (Unterlinner et al., 1999; Huang and Kutchan, 2000; Facchini and Park, 2003). This may suggest that codeinone reductase is a poor target for metabolic engineering. However, this inference was challenged by our recent experiments in silencing Cor. The opium poppy was transformed with a chimeric cDNA hairpin RNA construct designed to silence all known members of the codeinone reductase multigene family through DNA-directed RNAi (Allen et al., 2004). We showed that the precursor (S)-reticuline accumulates in transgenic silenced plants at the expense of morphine, codeine, oripavine and thebaine. (S)-Reticuline is seven enzymatic steps upstream of the substrate codeinone. The loss of the codeinone reductase gene transcript was verified and 22-mer degradation products were detected in silenced lines. The accumulation of (S)-reticuline demonstrates that some form of negative feedback exists between the unique morphinan-specific pathway and the more general benzylisoquinoline pathway in which (S)-reticuline is the key branch point intermediate. Although the nature of this long-range feedback remains unresolved, it suggests the possibility of codeinone reductase being a control point whose over-expression may lead to increased morphinan alkaloid production. The results presented here confirm this speculation. Taken together with our silencing work (Allen et al., 2004), it seems that both increasing and decreasing codeinone reductase activity can have major effects on morphinan alkaloid synthesis.

The analyses of tissues other than capsules indicate that there are no major changes in alkaloid types or amounts across tissues that might be expected to have an ecological impact. Only codeine was significantly increased in the lower and upper stem. This is despite the fact that the introduced gene was driven by a constitutively expressing promoter. The normal tissue distribution of alkaloids is probably preserved by the fact that all other genes of the pathway are unchanged. This result is of practical significance for regulatory approval if these transgenic lines are to be utilized commercially in an agricultural setting. For example, a significant change in alkaloid type in the roots might affect the soil biology, whereas major changes in pollen alkaloids might conceivably affect bees or other pollinators. The results suggest that these transgenic poppies will have no adverse ecological impact if used agriculturally. We have recently reported other studies exploring the potential ecological significance of the transgenic poppies described here, such as their relative fitness, their modes of pollen dispersal and the characteristics of pollen flow under field conditions (Miller et al., 2005). These results, together with the current paper, suggest that Cor over-expression is an effective and ecologically benign way to achieve improved morphinan alkaloid production efficiencies.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Transformation

Our method of Agrobacterium-mediated transformation of poppy has been detailed elsewhere (Chitty et al., 2003). In brief, seedling hypocotyls were infected with Agrobacterium and cultured to callus, somatic embryos and plantlets in the presence of 25 µg/mL paromomycin as a selectable marker. The transgenics reported here are into CO58-34, a high-morphine variety (Tasmanian Alkaloids Pty Ltd).

The disarmed A. tumefaciens strain AGL1 was used for transformation experiments (Lazo et al., 1991). The binary construct is based on pPLEX X002 (Schunmann et al., 2003). The T-DNA consisted of two genes: S4S4 promoter, PsCor1.1 cDNA, and malic enzyme (Me1) 3′ terminator; and 35S promoter, NptII selectable marker gene interrupted by a catalase intron, and 35S terminator. The S4S4 promoter is derived from segment 4 of the subterranean clover stunt virus genome (Schunmann et al., 2003). The codeinone reductase 1.1 cDNA sequence was derived from P. somniferum (Unterlinner et al., 1999).

RT-PCR

RNA was extracted from plants or callus using a Qiagen (Hilden, Germany) RNeasy Plant Mini-Kit with DNase treatment. Plants or callus cultured with paromomycin selection were analysed for the expression of the NptII gene using a Qiagen One-Step RT-PCR Kit. The primers used were 5′-TATGACTGGGCACAACAGACAATCGGCTGCTC-3′ (NptII 64 F) and 5′-GGCGATACCGTAAAGCACGAGGAAGCGGTCAG-3′ (NptII 931 R).

The cycling conditions were 50 °C/30 min, 95 °C/15 min, followed by 30 cycles of 94 °C/45 s, 65 °C/45 s and 72 °C/1 min, with a final 72 °C/10 min extension step. The primers used for Actin RT-PCR as an internal standard were 5′-GGAGAAGATTTGGCATCACACTTTCTACAATGAG-3′ (Act 119F) and 5′-CTTCCTGATATCCACAATCACACTTCATGATGG-3′ (Act 1090R). The primers used for Cor analysis were 5′-GAGAGTAATGGTGTACCTATGATCACTCTCAGTTC-3′ (Cor1F) and 5′-GGGCTCATCTCCACTTGATTCACAACTG-3′ (Cor575R). For the multiplex analysis of transgene expression with actin, the cycling conditions were performed as above, except that 27 cycles of denaturation/annealing/extension were employed. Products were purified using the Promega Wizard PCR Preps Purification System and cloned into pGEM-T-easy (Promega, Madison, WI) for sequencing.

Real-time quantitative PCR

cDNA was prepared from 2 µg of RNA using an Invitrogen Thermoscript Reverse Transcriptase cDNA Synthesis Kit with oligo dT primers (Invitrogen, San Diego, CA). The levels of Cor and Actin transcript were determined by real-time PCR on a Rotor Gene 3000 real-time cycler (Corbett Research, Sydney, Australia). An aliquot of 0.5 µL of the 20-µL cDNA reaction was used as a template for quantitative RT-PCR in a total volume of 20 µL as follows: 10 µL of SYBR Green Jumpstart Taq Readymix (Sigma, St Louis, MO), 0.2 µL of forward or reverse primer at 20 pmol/µL and 9.1 µL of H2O. Samples were analysed in triplicate. Primers for the amplification of the Cor transcript were 5′-CGATAGAGGTCGGTTACAGACACTTCGATACAG-3′ (F) and 5′-GAAGAGCAGGGAGGACAAGATCAGCGTGAGC-3′ (R). Primers for the amplification of poppy Actin transcript were 5′-CTTTATGCCAGTGGTCGAACAACTGGTATTG-3′ (F) and 5′-CACGCTCAGCCGTGGTGGTGAATGAG-3′ (R). The cycling conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 15 s at 65 °C and 20 s at 72 °C. At the end of cycling, the samples were subjected to a melting curve programme of 72–95 °C in 1 °C increments with a 5 s hold at each temperature.

Southern blot analysis

Genomic DNA was extracted from 1–2 g of leaf tissue by the cetyltrimethylammonium bromide (CTAB) method (Lodhi et al., 1994). DNA (15 µg) was digested with HindIII, electrophoresed in 1% agarose and transferred to a Hybond N+ membrane. The membranes were pre-hybridized in sodium dodecyl sulphate (SDS)–phosphate buffer [0.5 m phosphate buffer, pH 7.2, 7% SDS, 10 mm ethylenediaminetetraacetic acid (EDTA)] and probed with an NptII PCR fragment of approximately 900 bp produced using the NptII primers described above and the plasmid vector as template. The fragment was labelled using a Ready-To-Go DNA Labelling Kit (Amersham Biosciences, Piscataway, NJ). Membranes were washed twice at 65 °C with 2 × saline sodium citrate (SSC), 0.1% SDS for 15 min, and then once with 0.1 × SSC, 0.1% SDS. Blots were exposed on Kodak Biomax MS film with an intensifying screen at −70 °C for 12 h, and developed with an Agfa CP 1000 developer.

Glasshouse trial 2002

The 2002 glasshouse trial was carried out in a 20 m × 8 m glasshouse covered in double inflated 200 µm film. The trial was designed with four randomized blocks. Each treatment consisted of two pots (200 mm in diameter) of six poppy plants each, and each block consisted of 75 treatments (five rows of 15 treatments each). A buffer area, two pots in width, surrounded the entire trial area and was sown to variety CO58-34. This was designed to eliminate edge effects on the growth of the trial plants.

The CO58-34 control consisted of 12 observations of 12 plants each (total of 144 plants). The transgenic data consisted of four observations of 12 plants each (total of 48 plants).

T1 selfed seed from the primary transgenic plant (T0) was sown in excess in pots. At the six- to eight-leaf stage, seedling leaves were painted with 1%−2% paromomycin (plus 0.1% Tween 80 as wetting agent) using a cotton bud to confirm whether individual plants were transgenic. Areas painted were marked with an indelible marker pen. Non-transgenic segregants were evident by senescence and browning of the painted area after 3–4 days, and these plants were removed from the pots. Enzyme-linked immunosorbent analysis (ELISA) of NptII protein was used to confirm the accuracy of paromomycin leaf painting.

Information, such as plant height, plant vigour, flowering date, number of capsules, straw weight and capsule weight, was collected, but not reported, because there were no significant differences observed. The trial was sown in early November 2001 (late spring) and harvested in early March 2002 (early autumn).

Glasshouse trial 2003

The design and management of the 2003 glasshouse trial was essentially the same as that for the 2002 glasshouse trial. The trial was sown in mid-October 2002 and harvested in late February 2003.

The CO58-34 controls consisted of 12 observations of eight plants each (total of 96 plants). The transgenic data consisted of four observations of eight plants each (total of 32 plants). T2 seed was sown, which was derived from the confirmed T1 transgenics of the 2002 glasshouse trial, the null segregants being eliminated. No paromomycin leaf painting was conducted before T2 plant thinning. As a consequence, where the transgenes were being inherited as a single linkage unit, the contributing T1 parents were expected to segregate in a hemizygous to homozygous transgenic ratio of 2 : 1, and the derived T2 progeny plants were expected in a non-transgenic to hemizygous transgenic to homozygous transgenic ratio of 1 : 2 : 3, i.e. only one plant in nine should be non-transgenic. If the line has more than one transgene insertion locus, non-transgenic segregants will be more infrequent.

Parental CO58-34 seed was obtained from two sources: seed collected from the 2002 glasshouse trial and seed from the Tasmanian Alkaloids Pty Ltd seed stocks that were used in the 2002 glasshouse trial. There were no significant differences between these CO58-34 seed controls, and so the data were pooled. In addition to CO58-34, another 13 morphine varieties, covering a range of morphine accumulation levels, were included in the trial. The seed for these lines was obtained from the same seed lots as used to sow the Tasmanian Alkaloids Pty Ltd variety trials, so that morphine production could be compared between glasshouse and the field.

Field trial 2003

The 2003 field trial used a three-block randomized design. Each block consisted of 21 plots of six rows each, and each row in a block was a different treatment. A 10-m buffer area surrounded the trial in every direction and was sown to a mixture of four commercial morphine varieties. The experimental area, including the 10-m buffer, was completely enclosed in bird-proof netting. The field trial was sown in early November 2002 and harvested in early March 2003. T2 seed collected from the 2002 glasshouse trial (or a parallel glasshouse generation of new lines) was sown in the field trial. As in the 2003 glasshouse trial (see above), the bulk T2 plant population was expected to segregate in a non-transgenic to hemizygous transgenic to homozygous transgenic ratio of 1 : 2 : 3 where the transgenes were being inherited as a single linkage unit. In instances in which the line contained more than one transgene insertion locus, non-transgenic segregants would be more infrequent.

In total, there were nine replicate rows of each control with about 20 plants per row. The transgenic lines were represented as three replicate rows (total of about 60 plants per line). The field trial also included four current commercial lines against which to benchmark the transgenic alkaloid productivity.

Each poppy line was planted in a randomized trial design using six-row plots. The water content of the soil was measured using a water tension meter (Watermark, Riverside, CA) approximately twice a week, and irrigation was applied if required. The trial was thinned where required at about the six- to eight-leaf stage and urea fertilizer was applied (40 kg N/ha). Weeds and downy mildew were controlled using standard industrial practice.

The capsules from each row were collected and processed as a single observation. Transgenic alkaloid content means were compared with control mean contents using analysis of variance (anova) and Bartlett's post-test (Graphpad Instat version 3; San Diego, CA).

All trials were conducted with the approval of, and according to the conditions set by, the Australian Office of the Gene Technology Regulator.

Alkaloid analysis

Both the glasshouse and field trials were harvested by collecting the capsules from each treatment by hand. Capsules were threshed and the seed and straw (remaining tissue) were separated using a thresher (Tamar Designs Pty Ltd, Exeter, Tasmania). Straw from each treatment was ground for alkaloid analysis using a sample mill (Retsch SM 2000, Hunslet, Leeds, UK). Ground samples were analysed for oripavine, morphine, codeine, thebaine, reticuline, noscapine and papaverine content. Two grams of air-dried ground straw was added to 50 mL of acid extractant (0.17% phosphoric acid, 10% methanol) in a 125-mL conical flask and shaken for 60 min at 175 r.p.m. Samples were filtered (Whatman no. 6 filter paper) directly into an autosampler vial for HPLC injection. Extracted alkaloid samples were run on an Alltech platinum C18 53 × 7-mm rocket column at a flow rate of 1.5 mL/min, and were analysed using a Waters 2487 dual-wavelength detector and a Millennium 32 version 3.05.01 data system with UV detection of peaks at 254 nm.

In addition, various tissues were sampled from the 2003 field trial of a subset of lines. Four replicate samples were taken of the root, leaf, lower stem (10–30 cm below the capsule), upper stem (0–10 cm below the capsule), pollen and seed. For each sample, extractant was used at 25 mL per gram of ground dried tissue and placed in 50-mL screw-top tubes on a rotating mixer/inverter. Pollen samples were extracted using buffer at 100 volumes per unit fresh weight.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We gratefully acknowledge the excellent technical contribution of Luke Henning and Patricia Hallam.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Allen, R.S., Millgate, A.M., Chitty, J.A., Thistleton, J., Miller, J.A.C., Fist, A.J., Gerlach, W.L. and Larkin, P.J. (2004) RNAi-mediated replacement of morphine with the non-narcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 15591566.
  • Bird, D.A., Franceschi, V.R. and Facchini, P.J. (2003) A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy. Plant Cell, 15, 26262635.
  • Bock, A., Wanner, G. and Zenk, M.H. (2002) Immunocytological localization of two enzymes involved in berberine biosynthesis. Planta, 216, 5763.
  • Burbulis, I.E. and Winkel-Shirley, B. (1999) Interactions among enzymes of the Arabidopsis flavonoid biosynthetic Pathway. Proc. Natl. Acad. Sci. USA, 96, 12 92912 934.
  • Cahoon, E.B., Ripp, K.G., Hall, S.E. and McGonigle, B. (2002) Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed. Plant Physiol. 128, 615624.
  • Canel, C., Lopes-Cardoso, M.I., Whitmer, S., Van Der Fits, L., Pasquali, G., Van Der Heijden, R., Hoge, J.H.C. and Verpoorte, R. (1998) Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta, 205, 414419.
  • Chitty, J.A., Allen, R.S., Fist, A.J. and Larkin, P.J. (2003) Genetic transformation in commercial Tasmanian cultivars of opium poppy, Papaver somniferum, and movement of transgenic pollen in the field. Func. Plant Biol. 30, 10451058.
  • De Luca, V. and St Pierre, B. (2000) The cell and developmental biology of alkaloid biosynthesis. Trends Plant Sci. 5, 168173.
  • Facchini, P.J. and Park, S.U. (2003) Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry, 64, 177186.
  • Facchini, P.J., Park, S.U., Bird, D.A. and Samanani, N. (2000) Toward the metabolic engineering of benzylisoquinoline alkaloid biosynthesis in opium poppy and related species. Recent Res. Dev. Phytochem. 4, 3147.
  • Frick, S., Chitty, J.A., Kramell, R., Schmidt, J., Larkin, P.J. and Kutchan, T.M. (2004) Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots. Transgenic Res. 13, 607613.
  • Hamill, J.D., Robins, R.J., Parr, A.J., Evans, D.M., Furze, J.M. and Rhodes, M.J.C. (1990) Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana-rustica can lead to enhanced nicotine accumulation. Plant Mol. Biol. 15, 2738.
  • Hashimoto, T., Yun, D.J. and Yamada, Y. (1993) Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry, 32, 713718.
  • Huang, F.C. and Kutchan, T.M. (2000) Distribution of morphinan and benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver somniferum. Phytochemistry, 53, 555564.
  • Hughes, E.H. and Shanks, J.V. (2002) Metabolic engineering of plants for alkaloid production. Metabolic Eng. 4, 4148.
  • Jouhikainen, K., Lindgren, L., Jokelainen, T., Hiltunen, R., Teeri, T.H. and Oksman-Caldentey, K.M. (1999) Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta, 208, 545551.
  • Kutchan, T.M. (1997) Natural Product Munitions – new prospects for plant protection. Trends Plant Sci. 2, 449450.
  • Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology, 9, 963967.
  • Lee, M.H., Jeong, J.H., Seo, J.W., Shin, C.G., Kim, Y.S., In, J.G., Yang, D.C., Yi, J.S and Choi, Y.E. (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol. 45, 976984.
  • Liu, C.J., Blount, J.W., Steele, C.L. and Dixon, R.A. (2002) Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. Proc. Natl. Acad. Sci. USA, 99, 14 57814 583.
  • Lodhi, M.A., Ye, G.N., Weeden, N.F. and Reisch, B.I. (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol. Biol. Rep. 12, 613.
  • Lucker, J., Schwab, W., Van Hautum, B., Blaas, J., Van Der Plas, L.H.W., Bouwmeester, H.J. and Verhoeven, H.A. (2004) Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon. Plant Physiol. 134, 510519.
  • Miller, J.A.C., Henning, L., Heazlewood, V.L., Larkin, P.J., Chitty, J., Allen, R., Brown, P.H., Gerlach, W.L. and Fist, A.J. (2005) Pollination biology of oilseed poppy, Papaver somniferum L. Aust. J. Agric. Res. 56, 483490.
  • Millgate, A.G., Pogson, B.J., Wilson, I.W., Kutchan, T.M., Zenk, M.H., Gerlach, W.L., Fist, A.J. and Larkin, P.J. (2004) Analgesia: morphine-pathway block in top1 poppies. Nature, 431, 413414.
  • Moyano, E., Jouhikainen, K., Tammela, P., Palazon, J., Cusido, R.M., Pinol, M.T., Teeri, T.H. and Oksman-Caldentey, K.M. (2003) Effect of Pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus. J. Exp. Bot. 54, 203211.
  • Murata, J. and De Luca, V. (2005) Localization of tabersonine 16-hydroxylase and 16-OH-tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus. Plant J. 44, 581594.
  • Paine, J.A., Shipton, C.A., Chaggar, S., Howells, R.M., Kennedy, M.J., Vernon, G., Wright, S.Y., Hinchliffe, E., Adams, J.L., Silverstone, A.L. and Drake, R. (2005) Improving the nutritional value of golden rice through increased pro-vitamin A content. Nat. Biotechnol. 23, 482487.
  • Palazon, J., Moyano, E., Cusido, R.M., Bonfill, M., Oksman-Caldentey, K.M. and Pinol, M.T. (2003) Alkaloid production in Duboisia hybrid hairy roots and plants overexpressing the H6H gene. Plant Sci. 165, 12891295.
  • Park, S.U., Yu, M. and Facchini, P.J. (2002) Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy. Plant Physiol. 128, 696706.
  • Park, S.U., Yu, M. and Facchini, P.J. (2003) Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol. Biol. 51, 153164.
  • Rees, T.A. and Hill, S.A. (1994) Metabolic control analysis of plant metabolism. Plant Cell Environ. 17, 587599.
  • Rocha, P., Stenzel, O., Parr, A., Walton, N., Christou, P., Drager, B. and Leech, M.J. (2002) Functional expression of tropinone reductase I (trI) and hyoscyamine-6 beta-hydroxylase (H6H) from Hyoscyamus niger in Nicotiana tabacum. Plant Sci. 162, 905913.
  • Rothe, G., Hachiya, A., Yamada, Y., Hashimoto, T. and Drager, B. (2003) Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase. J. Exp. Bot. 54, 20652070.
  • Sato, F., Hashimoto, T., Hachiya, A., Tamura, K., Choi, K.B., Morishige, T., Fujimoto, H. and Yamada, Y. (2001) Metabolic engineering of plant alkaloid biosynthesis. Proc. Natl. Acad. Sci. USA, 98, 367372.
  • Schunmann, P.H.D., Llewellyn, D.J., Surin, B., Boevink, P., Defeyter, R.C. and Waterhouse, P.M. (2003) A suite of novel promoters and terminators for plant biotechnology. Func. Plant Biol. 30, 443452.
  • Shintani, D. and DellaPenna, D. (1998) Elevating the vitamin E content of plants through metabolic engineering. Science, 282, 20982100.
  • Trethewey, R.N. (2004) Metabolite profiling as an aid to metabolic engineering in plants. Curr. Opin. Plant Biol. 7, 196201.
  • Unterlinner, B., Lenz, R. and Kutchan, T.M. (1999) Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J. 18, 465475.
  • Verpoorte, R. and Memelink, J. (2002) Engineering secondary metabolite production in plants. Curr. Opin. Biotechnol. 13, 181187.
  • Weid, M., Ziegler, J. and Kutchan, T.M. (2004) The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum. Proc. Natl. Acad. Sci. USA, 101, 13 95713 962.
  • Ye, X.D., Al-Babili, S., Kloti, A., Zhang, J., Lucca, P., Beyer, P. and Potrykus, I. (2000) Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 287, 303305.
  • Yu, O., Shi, J., Hession, A.O., Maxwell, C.A., McGonigle, B. and Odell, J.T. (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry, 63, 753763.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Quantitative real time PCR results of leaf RNA preparations of T1 transgenic individuals (Tg) of #1b and #2 compared to non-transgenic individuals (NTg). The amount of Cor transcript is shown relative to Actin message using arbitrary units. The RNA preparations were subjected twice to real-time PCR and the means used in the analysis. The results shown are means ± SEM of the same #1b transgenics and #1b transgenics as in Figure 2a. General Cor primers were used designed to amplify all known endogenous and transgene Cor transcripts.

FilenameFormatSizeDescription
pbi212_fS1.pdf41KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.