Modifying poppy growth and alkaloid yield with plant growth regulators by Geoffrey J. Dean B. Agr. Sc. (Hons) Submitted in fulfilment of the requirements for the degree of Master of Agricultural Science University of Tasmania Hobart June 2011 I, Geoffrey Johnson Dean, declare that this thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief, no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Signed: Date: This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Signed: Date: ii ABSTRACT Poppies (Papaver somniferum L.) are a major crop in Tasmanian broad-acre cropping rotations. Morphine has been the major alkaloid produced but now approximately half of the State‟s production is derived from thebaine-producing poppies. Yield potential in poppies is enhanced with earlier sowing, however this commonly results in excess vegetative growth and crop lodging. The effects of a range of plant growth regulators on plant growth and alkaloid yield of thebaine poppies were evaluated in three preliminary field trials conducted in northern Tasmania from 2002 - 04. Application of Slow Grow (maleic hydrazide) reduced seed weight in most trials and this result is potentially beneficial to industry as thebaine poppy seed is of limited commercial value due to thebaine residues. Application of Moddus (trinexapac) and Sunny (uniconazole) showed the greatest potential by altering alkaloid profile and reducing plant height respectively. In a subsequent field trial with morphine poppies, single and split applications of Sunny across three growth stages were compared and while plant height was reduced, effects on plant lodging were inconsistent. Whereas lodging was decreased with the split application, the single application increased lodging, perhaps a result of the large height difference between main stem and lateral capsules. The split application of Sunny also resulted in a greater capsule yield compared with single and nil treatments through increased capsules/m2. Despite a reduction in individual seed weight, seed yield from Sunny treatments was also greater due to a large increase in the number of seeds per capsule. It is proposed that the reduction in plant height and lodging with application of Sunny leads to reallocation of assimilates to yield components. Alkaloid content, in particular morphine, was also increased with application of Sunny and this is likely to be a result of extended alkaloid biosynthesis through delayed maturity. The alkaloid, thebaine, is of higher value than oripavine and application of Moddus in three additional rate and timing trials consistently increased thebaine content. Associated with this, oripavine content decreased and this effect was rate responsive. In contrast with Sunny, Moddus had little effect on plant height or maturity. Instead, seed yield was reduced and straw yield tended to increase and combined with alkaloid effects, this consistently increased thebaine yield around 25%. Multiple and split iii applications of Moddus tended to further increase thebaine content. As total alkaloid did not vary with single applications, it was postulated that Moddus inhibits the biosynthesis of oripavine from thebaine. Results from this study and further trials conducted by industry have led to the registration of Moddus on poppy crops and close to 100% adoption rates by thebaine poppy growers. The potential of Sunny as a plant growth regulator (now marketed as Sumagic) has also been verified and in addition the benefit of split applications of both Sunny and Moddus demonstrated. iv ACKNOWLEDGEMENTS There are a number of people who I wish to sincerely thank for helping complete this thesis. In particular I thank my more recent supervisors, Tina Acuna and Meixue Zhou for their persistence and good humour over the last year and also Neville Mendham for travelling the long haul where others couldn‟t see the light. I also thank Sergey Shabala for his assistance in dealing with administrative issues. Tony Fist has provided me with invaluable support and comment on poppy matters as have other staff at Tas Alkaloids. Fellow higher degree students have been helpful in bouncing ideas and providing suggestions, advice on thesis writing and with formatting, in particular Angela Merry. I also thank Simon Munford and Brett Davey for technical assistance with field trials and Anne Lucadou-Wells and Margaret Quill for their library support. Thank you to Horticulture Australia Limited and Tasmanian Alkaloids for the funding and thus enabling me to work in an interesting area. My gratitude also to the growers on whose properties these trials were conducted. In many ways this was an example of how not to do a higher degree: start later in life; attempt to do it while actively working and completely change topics after 5 years! However after a number of years this stage of my life is completed and no one will be more happy than my wonderful wife Jo (who I thank for her understanding and my reduced household chores) and my two lovely boys Jack and Laurie who will see more of their father and who may find they can now be noisier before bedtime. v CONTENTS ABSTRACT ............................................................................................................................... III ACKNOWLEDGEMENTS ......................................................................................................... V CHAPTER 1. INTRODUCTION ............................................................................................. 1 CHAPTER 2. LITERATURE REVIEW ................................................................................. 4 2.1 Poppy production and alkaloid biosynthesis ................................................................................... 4 2.1.1 Introduction .................................................................................................................................... 4 2.1.2 Cultural requirements .................................................................................................................... 5 2.1.3 Poppy alkaloids and biosynthesis ................................................................................................... 6 2.2 Crop lodging ................................................................................................................................... 10 2.2.1 Introduction .................................................................................................................................. 10 2.2.2 Crop losses from lodging .............................................................................................................. 11 2.2.3 Plant characteristics associated with reduced lodging ................................................................. 12 2.2.3.1 Stem characteristics .............................................................................................................. 12 2.2.3.2 Root characteristics .............................................................................................................. 13 2.2.4 Lodging and crop management .................................................................................................... 13 2.3 Use and effects of plant growth regulators .................................................................................... 15 2.3.1 Introduction .................................................................................................................................. 15 2.3.2 Effects of plant growth regulators on plant hormones and secondary metabolites ................... 16 2.3.2.1 Gibberellic acid metabolism ................................................................................................. 17 2.3.2.2 Abscisic acid metabolism ...................................................................................................... 20 2.3.2.3 Ethylene metabolism ............................................................................................................ 21 2.3.2.4 Cytokinin metabolism ........................................................................................................... 22 2.3.2.5 Flavonoid metabolism .......................................................................................................... 22 2.3.3 Effects of plant growth regulators on plant morphology and development ................................ 23 2.3.3.1 Stem characteristics .............................................................................................................. 23 2.3.3.2 Root characteristics .............................................................................................................. 24 2.3.3.3 Maturity ................................................................................................................................ 26 2.3.4 Variability in effects of plant growth regulators........................................................................... 27 2.3.4.1 Plant species and variety ...................................................................................................... 27 2.3.4.2 Crop stage of development/timing of application................................................................ 28 2.3.4.3 Chemical uptake, activation and duration of activity ........................................................... 29 vi 2.3.4.4 Rate of chemical applied ...................................................................................................... 29 2.3.4.5 Weather and growing conditions ......................................................................................... 30 2.3.4.6 Crop management ................................................................................................................ 30 2.3.5 The role of canopy size in PGR function ....................................................................................... 31 2.3.6 Summary and scope of thesis ....................................................................................................... 34 CHAPTER 3. SCREENING OF PLANT GROWTH REGULATORS TO REDUCE LODGING IN POPPIES ......................................................................................................... 35 3.1 Introduction ................................................................................................................................... 35 3.2 Materials and methods .................................................................................................................. 37 3.2.1 Field trial design and treatments.................................................................................................. 37 3.2.2 Cultural details.............................................................................................................................. 38 3.2.3 Trial measurements and harvest procedure ................................................................................ 39 3.2.4 Sample analysis ............................................................................................................................ 39 3.2.5 Statistical analysis ......................................................................................................................... 40 3.3 Results............................................................................................................................................ 40 3.3.1 Preliminary PGR screening trial: Cressy 2002-03 ......................................................................... 41 3.3.1.1 The effect of PGR application on plant growth and yield components ................................ 41 3.3.1.2 The effect of PGR application on alkaloid ............................................................................. 41 3.3.2 PGR screening trial: Symmons, 2003-04 ....................................................................................... 43 3.3.3 PGR screening trial: Hagley, 2004-05 ........................................................................................... 43 3.3.3.1 The effect of PGR application on plant growth and yield components ................................ 43 3.3.3.2 The effect of PGR application on alkaloid ............................................................................. 43 3.4 Discussion ...................................................................................................................................... 46 3.4.1 The effect of PGR application on plant growth ............................................................................ 46 3.4.2 The effect of PGR application on yield components .................................................................... 48 3.4.3 The effect of PGR application on alkaloid .................................................................................... 50 CHAPTER 4. THE EFFECT OF APPLICATION RATE AND TIMING OF THE PLANT GROWTH REGULATOR MODDUS ON POPPIES .......................................................... 53 4.1 Introduction ................................................................................................................................... 53 4.2 Materials and Methods .................................................................................................................. 54 4.2.1 Trial design and treatments.......................................................................................................... 54 4.2.2 Cultural details, measurements and processing .......................................................................... 55 vii 4.2.3 Statistical analysis ......................................................................................................................... 55 4.3 Results............................................................................................................................................ 56 4.3.1 Trial at Symmons, 2003-04 ........................................................................................................... 57 4.3.1.1 The effect of Moddus application on growth and yield components .................................. 57 4.3.1.2 The effect of Moddus application on alkaloid ...................................................................... 57 4.3.2 Trial at Hagley, 2004-05 ................................................................................................................ 57 4.3.2.1 The effect of Moddus application on growth and yield components .................................. 57 4.3.2.2 The effect of Moddus application on alkaloid ...................................................................... 58 4.3.3 Trial at Symmons, 2004-05 ........................................................................................................... 58 4.4 Discussion ...................................................................................................................................... 62 4.4.1 The effect of Moddus application on growth and yield components .......................................... 62 4.4.2 The effect of Moddus application on alkaloid .............................................................................. 64 CHAPTER 5. THE EFFECT OF SOWING RATE AND SPLIT APPLICATION OF SUNNY ON MORPHINE POPPIES ..................................................................................... 67 5.1 Introduction ................................................................................................................................... 67 5.2 Materials and methods .................................................................................................................. 68 5.2.1 Field trial design and treatments.................................................................................................. 68 5.2.2 Cultural details, measurements and processing .......................................................................... 69 5.2.3 Statistical analysis ......................................................................................................................... 70 5.3 Results............................................................................................................................................ 70 5.3.1 The effect of sowing rate and Sunny application on plant growth .............................................. 71 5.3.2 The effect of sowing rate and Sunny application on yield components ...................................... 75 5.3.3 The effect of sowing rate and Sunny application on alkaloid content ......................................... 79 5.4 Discussion ...................................................................................................................................... 80 5.4.1 The effect of sowing rate on growth, yield and alkaloid .............................................................. 80 5.4.2 The effect of Sunny application on growth, yield and alkaloid .................................................... 83 CHAPTER 6. GENERAL CONCLUSIONS AND RECOMMENDATIONS ..................... 89 REFERENCES .......................................................................................................................... 92 APPENDIX 1 ......................................................................................................................... 109 viii CHAPTER 1. INTRODUCTION Poppies (Papaver somniferum L.) are historically recognised for production of opioid drugs and were probably one of the earliest domesticated plants grown for medicinal value. The analgesic and narcotic properties of these plants have been known since the beginning of civilization (Duke, 1973; Hagel and Facchini, 2010). Poppy seed is also a valuable end product and is used for culinary purposes in bakery and confectionery industries (Duke, 1973; Bernath and Nemeth, 2009). The history of poppy production in Australia and, in particular, Tasmania is relatively recent and has been discussed previously by Laughlin et al. (1998), Fist (2001) and more recently, Frappell (2010). Early production in Australia in the 19th century was restricted to medicinal use by doctors and recreational opium smoking by, in particular, Chinese immigrants (Laughlin et al., 1998; Frappell, 2010). It was not until morphine shortages associated with World War II that more detailed investigations on morphine poppy production were conducted. However, plans for developing an industry were not seriously addressed until the early 1960s when a pharmaceutical subsidiary of the multinational Glaxo was keen to develop a more reliable source of opiate raw material than production from Western Asia and Eastern Europe (Laughlin et al., 1998; Frappell, 2010). Traditionally, alkaloids are extracted by manual lancing of the capsule wall and collecting the latex as raw opium. In 1928, the development of a break-through method to extract alkaloid from mechanically harvested dry poppy straw significantly reduced labour costs enabling broad-acre morphine poppy production (Hagel et al., 2007). Out of season production and a relatively dry harvest period over summer compared with England were key criteria in selecting Australia as a potential supplier of poppy opiates (Laughlin et al., 1998). With cooperation from the local Department of Agriculture, field trials in Tasmania were commenced in 1960 with Glaxo (now GlaxoSmithKline) beginning commercial production in the late 1960s (Frappell, 2010). Poppy production in the State further expanded in 1975 with the establishment of Tasmanian Alkaloids, this company being subsequently purchased by Johnson and Johnson (Fist, 2001; 1 Frappell, 2010). A third local company, TPI Enterprises, commenced operations in 2004 (TPI, 2010). Poppies are now an important crop in Tasmanian broad-acre cropping rotations and one of the State's major agricultural commodities with a farm gate value of between $60 and $80 million (Frappell, 2010). Through focused research and development (as well as effective extension practices), Tasmania is now considered the world's most efficient international producer of poppies with the highest yield per hectare of opiate-producing countries (PACB, 2010). Tasmania has a number of cultural advantages in maintaining this leading edge: the temperate climate is generally not conducive to serious poppy diseases or pests; long daylight hours increase floral initiation and crop yield (Frappell, 2010); the harvest period over summer is relatively dry ensuring high alkaloid content; and the industry has assembled considerable skills and intellectual capacity. In addition, one strategic advantage with poppy production in Tasmania is the island isolation and capacity to control illegal activities. Licit poppy production is monitored and controlled by the International Narcotic Control Board (INCB) with powers to reduce and expand supply depending on existing global production (Hagel et al., 2007; Laughlin et al., 1998). Australia, as a signatory to the 1961 Single Convention on Narcotic Drugs, abides by these supply requirements and hence production is tightly regulated. Licences to grow poppies in Tasmania require security clearances from the State controlled Poppy Advisory and Control Board (PACB). Poppy crops are regularly monitored during the growing season and post-harvest operations are required to ensure suitable treatment of harvest residue and poppy re-growth (Laughlin et al., 1998; PACB, 2010). The estimated area of poppy production in 2010 in Tasmania is over 24 000 ha (INCB, 2009) but increased demand for land has resulted in production moving to less well drained soils that are more prone to restricted root growth from water-logging (Chilvers, 1996). Farming systems using raised beds were introduced into Tasmania in the late 1990‟s to help alleviate water-logging. While this has enabled establishment of poppies in winter and early spring (Fist, 2001), earlier sowing commonly results in excess vegetative growth and subsequent lodging towards the end of the season. Furthermore, refinements in general poppy crop nutrition, disease control and irrigation have led to increased yield potential but also additional crop growth and greater potential for crop lodging. 2