Reducing Plant Stress Using Australian Melaleuca A report for the Rural Industries Research and Development Corporation by Dr. Bodapati, P. Naidu and Dr. Donald F. Cameron CSIRO Tropical Agriculture December 1999 RIRDC Publication No 99/148 RIRDC Project No. CSC-55A

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© 1999 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 57937 7 ISSN 1440-6845 Reducing Plant Stress Using Australian Melaleuca Publication no 99/148 Project no.CSC-55A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Dr. Bodapati P. Naidu CSIRO Tropical Agriculture Cunningham Laboratory 306 Carmody Rd, ST LUCIA QLD 4069

Phone: (07) 3214 2285 Fax: (07) 3214 2288 Email: naidu.bodapati@tag.csiro.au Website: http://www.tag.csiro.au

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4539 Fax: 02 6272 5877 Email: rirdc@rirdc.gov.au Website: http://www.rirdc.gov.au

Published in October 1999 Printed on environmentally friendly paper by Canprint

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Foreword Australia is the driest continent with about third of its lands affected by salinity and/or sodicity. A practical way to remedy the effects of stress in agricultural plants is of vital importance to Australia.

Recent research shows that some plant species have a greater capacity to tolerate stress and this capacity is mainly due to the accumulation of organic compounds called Osmoprotectants. Research at CSIRO Tropical Agriculture reveals that the levels of osmoprotectants and thus stress tolerance could be increased in agricultural crops simply by external application.

Glycinebetaine is currently imported from Finland for external application and world-wide shortage is predicted for this solute. Australian Melaleucas are a good source of osmoprotectants. This project was funded for three years to test the agronomic possibility of growing Melaleucas for the production of osmoprotectants. It forms part of the Corporation's Essential Oils and Plant Extracts program which aims to support the growth of a profitable and sustainable essential oils and natural plant extracts industry in Australia. Most of our diverse range of over 400 research publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html Peter Core Managing Director Rural Industries Research and Development Corporation

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Acknowledgments We sincerely thank Mr & Mrs. Lewis of Tyagarah (NSW), Mr. W. DalSanto and Mrs. A. Villanuva (Dimbulah, Qld) for their excellent co-operation, and for their time and material in conducting field trials on their properties. We also thank Dr. John Doran of CSIRO for the help in collecting M. bracteata provenances.

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Contents

Foreword ...................................................................................................................iii

Acknowledgments....................................................................................................iv

Executive Summary..................................................................................................vi

1. Introduction............................................................................................................1

2. Objectives...............................................................................................................3

3. Methodology ..........................................................................................................4

4. Results....................................................................................................................7

5. Discussion .............................................................................................................9

6. Implications..........................................................................................................11

7. Recommendations ..............................................................................................12

8. Tables ...................................................................................................................13

9. References ...........................................................................................................22

10. Appendix ............................................................................................................24

List of Tables Table 1: Seed sources for eight species of Melaleuca .............................................4 Table 2 : Plant height (cm) at Tyagarah ..................................................................13 Table 3: Plant height (cm) on Mr. DalSanto’s property near Dimbulah,

north Qld ........................................................................................13 Table 4: Dry matter production (kg/ha) and its partition at Tyagarah......................14 Table 5: Dry matter production (kg/ha) and its partition on Mr. DalSanto’s

property near Dimbulah, north Qld............................................................14 Table 6: Concentration of osmoprotectants (% of dry matter) in Melaleuca

species at Tyagarah, NSW. .....................................................................15 Table 7: Concentration of osmoprotectants (% of dry matter) in Melaleuca

species on Mr. DalSanto’s property near Dimbulah, north Qld. ................15 Table 8: Potential yield (kg/ha) of osmoprotectants at Tyagarah............................16 Table 9: Potential yield of osmoprotectants (kg/ha) on Mr. DalSanto’s property

near Dimbulah, north Queensland. ...........................................................16 Table 10: Potential yield of MHP from M. bracteata on Mrs. Villanuva’s property

near Dimbulah, northern Qld ....................................................................16 Table 11: Effect of water stress on the enhancement of betaine analogues in

Melaleuca species. ...................................................................................17 Table 12: MHP extraction efficiency from biomass:..................................................18 Table 13: M. bracteata provenances, their collection locations and leaf MHP

content. ........................................................................................19 Table 14: Average gross returns from M. bracteata plantation.................................20

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Executive Summary Osmoprotectants such as glycinebetaine are externally applied to minimise stress effects in crops. Glycinebetaine is currently imported from Finland and world-wide shortage is predicted for this solute. Australian Melaleucas are good alternate source of osmoprotectants. We field tested 8 Melaleuca species at Tyagarah (northern NSW), at two sites near Dimbulah (northern Qld), and at Samford near Brisbane. We found that M. bracteata was well adapted and produces an osmoprotectant N-methyl-4-hydroxy-proline (MHP). M. bracteata produced highest amount of MHP compared to any other species. At rain-fed Tyagarah, we recorded MHP yield of 673 kg/ha while at Dimbulah the maximum yield was only 313 kg under irrigated conditions. MHP level in M. bracteata increases with age, and stress level of plants. Highest concentration of MHP recorded in M. bracteata was up to 6.1% at Tyagarah under rain-fed conditions. MHP is a highly water soluble and non-volatile compound. Crushing the spent biomass in water following distillation to remove essential oil, appears highly efficient with over 95% of MHP extracted. The yield of osmoprotectants from other species was significantly lower. On the other hand, Tea Tree Oil yielding M. alternifolia did not have the capacity to produce any osmoprotectants. M. bracteata has the capacity to produce both osmoprotectant and valuable essential oil. A test of 46 families from northern NSW and QLD revealed a 10 fold variation for MHP content, and significant variation in oil concentration and composition. This suggets a possibility of selecting a superior genotype which can produce high levels of both MHP and essential oil. Gross return from the cultivation of dual purpose M. bracteata was estimated to be $13,458/ha while it is only about $9, 000 from the currently cultivated M. alternifolia .

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1. Introduction Australian agriculture often suffers from long periods of water stress, salinity, and unfavourable temperature. Any practical way to increase the tolerance of plants to these stresses would stabilise and increase the national income from agriculture. Plants resistant to cold, salinity or drought accumulate a large quantity of nitrogenous organic compatible solutes or osmoprotectants. Considerable varietal differences for solute accumulation and associated stress tolerance have been demonstrated in a number of plant species. Amino acids such as proline (Aspinall and Paleg, 1981) and its analogues (Naidu et al. 1987), and fully N-methyl amino acids generically known as betaines (Wyn Jones and Storey, 1981; Hanson et al. 1986; Naidu et al. 1992a) comprise the important osmoprotectants. These solutes protect plant processes under laboratory conditions against freezing, heat, drought or salinity (Stewart and Lee, 1974; Wyn Jones and Storey, 1981; Paleg et al. 1985; Jolivet et al. 1982; Zao et al. 1993). Breeding is the suggested way to improve stress tolerance in crops with the natural ability to accumulate osmoprotectants (Aspinall and Paleg, 1981). In crops with poor or no solute accumulating ability, genetic engineering is a way to increase stress tolerance. Alternatively, Naidu et al. (1992) investigated the possibility of glycinebetaine foliar application and found increase in economic yield of grapevines (+48%), potatoes (+10%), buckwheat (+14), and pasture (+30% biomass) in Tasmania under cold and intermittent moisture stress. This work is being patented internationally by the company Finnsugar Bioproducts, Helsinki. Betaine was foliar applied on sugarcane to reduce cold stress under field conditions (Campbell et al. 1996), and fed to the roots of sugarcane to increase early growth rate (Campbell et al. 1999). In both the cases the growth and yield advantage was about 30-40%. In a recently funded RIRDC project (project no. CST-2A) glycinebetaine foliar application has doubled rice grain set under cold stress conditions compared to untreated and cold stressed control (Naidu unpublished data). Problems associated with seed germination, seedling vigour and seedling survival of crops under a range of environmental stresses can variously affect crop establishment and subsequent yield. Germination and vigour of salt affected commercial crops such as wheat and cotton, and pasture legumes were significantly increased by treating the seed with betaine (Naidu, 1995). Cotton field trials with betaine seed treatment increased yield by 18-22% in Queensland, Australia (Naidu et al. 1996; 1998). Finnsugar Bioproducts (a Cultor Company) based in Helsinki, Finland produces betaine as a by-product from sugar-beet industry. Finnsugar produces an estimated 90% of the world production and for this reason it is difficult to obtain accurate figures on the usage and production. Estimates range from a low of 10, 000 up to 35, 000 tonnes per annum. The current price in Australia is about $25 up from $4 in 1991. The major use of betaine is currently limited to animal nutrition as a feed additive. The demand in this area exceeds the supply with regional distribution subject to quotas. Finnsugar is well advanced in a program to utilise betaine (Agboma et al. 1997 a, b, c; Makela et al. 1998) for foliar application on various crops based on the work of Naidu et al. (1992) in Tasmania. Commercial sale of betaine commenced in Europe on tomatoes with reported yield increases of

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40%. An increase in demand for betaine of at least 100 fold is predicted if the foliar application technologies become successful on other crops also. On this basis, current solute production from sugar-beet would not be sufficient to also provide for seed treatment of a significant proportion of Australian field and horticultural crops. This suggests a need to look for alternative sources for these solutes. Melaleucas as a source of betaine: From the Waite Agricultural Research Institute, SA, Naidu et al. (1987; 1999) surveyed about 120 Melaleuca species for their content of betaine/methylated proline in relation to their stress tolerance. N-methyl proline (MP), trans-4-hydroxy-N-methyl proline (MHP), and trans-4-hydroxy-N-dimethyl proline (DHP) were the 3 analogues of proline isolated from these species (Naidu et al. 1987). These compounds comprised up to 4-6% of the dry wt of the tissue. However, terpinen-4-ol yielding spp. such as M. alternifolia, and M. linariifolia accumulated none of these analogues and M. dissitiflora showed only a trace amount of these analogues.

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2. Objectives

• To establish the agronomic and commercial feasibility of growing Melaleuca species containing betaine/betaine (proline) analogues by conducting field trials.

• To develop lab-scale methodologies which could be scaled up for industrial prototype for

the extraction of betaines from Melaleuca spp.

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3. Methodology Seed collection and seedling growth Based on the previous work of Naidu et al. (1987) we identified several Melaleuca species with the capacity to accumulate betaine or betaine analogues. We found that obtaining seed of these species was difficult as these species are not commercially cultivated. However, we sourced the following 8 species from CSIRO Forestry and Forest Products; Queensland Tree Seeds P/L, Moura, Qld; and Tyagarah Tea Tree Plantation, NSW.

Table 1: Seed sources for eight species of Melaleuca Species Seed Source

1 M. alternifolia Tyagarah Tea Tree Plantation

2 M. bracteata Queensland Tree Seeds

3 M. cuticularis Queensland Tree Seeds

4 M. glomerata Queensland Tree Seeds

5 M. lanceolata Queensland Tree Seeds

6 M. lateriflora CSIRO Forestry and Forest Products

7 M. pauperiflora CSIRO Forestry and Forest products

8 M. uncinata Queensland Tree Seeds

Seedlings were grown in a glasshouse at Samford Research Station near Brisbane from October 1995 to February 1996, and a second batch from March to July 1996. Plastic trays containing cells (12 x 12 rows) were filled with 1:1 mixture of peat and sand. Each cell also received 5 slow release nutrient granules (Osmocote). About 10 seed were placed on the surface of soil in each cell by using a salt-shaker. Soil surface (with seeds) was firmly pressed and kept moist by sprinkling with water 4 times daily for 10 days until germination was visible. Later, watering was done twice a day only. Four weeks after germination, seedlings were thinned to one per cell. Seedlings grew to 10 to 20 cm in 5 months depending on the growth rate of each species. Seedlings from Samford were transported to Tyagarah and Dimbulah for field planting.

Establishment of Field trials

First field trial was planted in March 1996 at Tyagarah Tea Tree Plantation near Byron Bay, NSW. Second field trial was planted in December 1996 near Dimbulah in northern Queensland on the property of Mr. Walter DelSanto. Third and fourth field trials were planted on the property of Mrs. Alda Villanuva near Dimbulah and at Samford Research Station in February 1997.

Field trials were conducted in a randomised block design with 4 replications. Each plot consisted of 4 rows of seedlings planted 1m apart and with an intra row spacing of 30 cm. The field trials

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on Mrs Villanuva’s property and at Samford were not replicated. These, particularly the Samford site, were intended to supply plant material for the development of the analytical and extraction techniques. No measurements were made from Samford site. Field trial at Tyagarah was rain-fed while the other sites were irrigated once a week with a sprinkler system.

Plant height was measured from ground level to the tip of the tallest growing point once in 3 months. Youngest fully expanded leaves were collected for solute analysis. From the second sampling time, i.e, 6 month after transplanting, ten plants were also harvested for biomass estimation. Plants were separated into thick stem (>2 mm diameter), fine stem (<2 mm diameter) and leaves. Biomass was oven dried for a minimum of 24 h at 60o C prior to weighing.

Water stress and changes in betaine content:

Four month old plants of 8 species that have been included in the field trials were used for a glasshouse experiment. Plants were grown in plastic pots with 4.5kg of soil and pruned to normalise biomass/transpiring leaf area between the species. Once the plants were established, pots were watered to the field capacity and further water was withheld. Soil surface was covered with 3 cm thick layer of vermiculite to prevent evaporation. Pots were regularly weighed to determine water use. Leaves were sampled when all the available soil moisture was utilised by the plants. Methylated proline analogues (betaines) were analysed using the following method developed in our laboratory.

Development and standardisation of analytical methodology:

Solute accumulation in Melaleuca was studied using the NMR method of Jones et al. (1986). However, for rapid estimation of solutes, a simple method using high performance liquid chromatography (HPLC) was developed and the results were recently published in Australian Journal of Plant Physiology (reprints attached).

Development of solute extraction methods from plant biomass:

One year old M. bracteata plants grown at Samford were used to develop a solute extraction technique from the biomass. The following steps were followed to determine the extraction efficiency.

1) Plants were chopped into 10 cm pieces and two lots (1 kg each) of this biomass were distilled for 1 hour in two separate experiments. The reflux water at the bottom of the distillation vat was collected and volume measured. MHP content of the reflex water and biomass were estimated.

2) Biomass originating from one experiment was soaked in water (70o C) equal to the volume of

biomass at room temperature for an hour. Later, water was drained and volume noted. MHP content was analysed in both water and plant material. After soaking, the biomass was crushed in water (equal to the biomass volume) in a mortar at room temperature and MHP was measured in both water and biomass.

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3) Biomass from the second experiment was crushed directly in water without a pre-soaking in warm water. The MHP content in both biomass and water was estimated.

After the above extraction process, MHP was isolated/purified using cation exchange column to the purity of 98%. Dowex (a strong cation exchange resin) was loaded into 1 L glass column, thoroughly washed and activated with 4 M HCl. Filtered plant extract was passed through resin at a flow rate of 5 L/h. The column was thoroughly washed with distilled water and then eluted with 2 L of 2 M ammonia solution. The eluent was reduced in volume using rotary evaporator.

Assessment of M. bracteata for variation in MHP content:

A plant collection team from CSIRO Forestry and Forest Products collected 51 leaf and 46 seed samples of M. bracteata provenances from their natural locations in NSW and Queensland (Map 1; Table 2). The 16 collection locations provide a reasonable representation of the wide climatic and edaphic conditions for which this species is adapted. Seed was collected from up to five individual trees at each location to provide information on variation within the populations.

We analysed the leaf samples for the content of MHP and seed was germinated in trays similar to that described earlier to plant at Tyagarah and Dimbulah.

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4. Results

Plant growth under field conditions Of the 8 species planted, M. lateriflora and M. pauperiflora did not survive at Tyagarah. At Dimbulah M. lateriflora and M. glomerata did not survive. M. bracteata plants were the tallest of all the species under the rain-fed conditions at Tyagarah (Table 2). Under fully irrigated conditions at Dimbulah M. alternifolia was the tallest, although not significantly different to M. bracteata and M. cuticularis (Table 3). All the other species were significantly shorter than these 3 species at both the sites. Significant differences were recorded in dry matter production between the species at each of the sampling times both at Tyagarah (Table 4) and Dimbulah (Table 5). Over the two years of experimentation at Tyagarah, M. bracteata was consistently the highest yielding species. At the final harvest it produced 13790 kg dry matter/ha, more than twice the amount of dry matter produced by M. alternifolia (Table 4). M. bracteata plants were of a bushy habit with higher numbers of branches (data not presented) than any other species. However, at Dimbulah M. alternifolia gave the highest dry matter production. It was not significantly different to M. bracteata at the first two harvests but the final yield of 21, 700 kg/ha in November 1997 was significantly different from the 18, 333 kg/ha accumulated by M. bracteata (Table 5). M. uncinata produced lowest amount of dry matter at both the field sites (Tables 4 & 5). M. bracteata contained highest proportion of leaf in its dry matter compared to any other species at both the sites (Tables 4 & 5). Thick stem constituted from 40 to 60 % of the dry matter of the species. Fine stem content varied from 20 to 35%. Osmoprotectant content Four osmoprotectants, proline, MP, MHP and DHP were identified in the field grown Melaleucas (Tables 6 & 7). Proline levels were very low (<0.01%) in all the species (data not presented). MP was accumulated only in M. cuticularis. The levels were very low in young plants (about 0.1-0.2%). MP concentration increased up to 1.5% as the plants matured. MHP was found in M. bracteata, M. glomerata, M. lanceolata, M. pauperiflora, and M. uncinata (Tables 6 & 7). Highest concentration of MHP was recorded in M. lanceolata up to 9.8% on leaf dry weight basis. Next best species were M. bracteata (up to 6.1% at Tyagarah) and M. pauperiflora (up to 5.8% near Dimbulah).

DHP was found in M. glomerata and M. uncinata (Tables 6 & 7). M. uncinata (up to 5.3%) contained a higher concentration than M. glomerata (2.2%).

In most cases the concentration of the osmoprotectants increases from thick stem <fine stem ≤ leaf. Concentration of the osmoprotectants increased with plant age at both sites. It appears that the concentration peaked one year after transplanting. Osmoprotectant yield

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MHP yield was higher than MP or DHP (Tables 8,9, &10). M. bracteata produced highest amount of MHP compared to any other species. At Tyagarah we recorded MHP yield of 673 kg/ka while at Dimbulah the maximum yield was 313 kg. This difference between the sites reflected the much higher MHP concentration in the plant at Tyagarah. M. lanceolata was the next best species for MHP, yields ranging from 231 kg/ha at Tyagarah to 287 kg/ha at Dimbulah. Leaves had the highest concentration of MHP in both species. MP and DHP yields were much lower than MHP at either site. The yield of MP ranged from 28 to 52.8 kg/ha in M. cuticularis, while DHP yield ranged from 43.5 (Tyagarah) to 125 (Dimbulah) kg/ha in M. uncinata.

It is note worthy that M. alternifolia contained none of the methylated osmoprotectants.

Water stress and levels of osmoprotectants

MHP content in M. bracteata and M. lanceolata increased by 20, and 5 fold, respectively in response to water deficit under glass house conditions (Table 11). DHP content in M. glomerata and M. uncinata increased by 4.5 and 7 fold, respectively. MP content was not altered by water deficit.

Extraction of osmoprotectants from the biomass

All these osmoprotectants are highly water soluble and non-volatile compounds. Crushing the spent biomass in water following distillation (commercial extraction of osmoprotectant would likely follow a distillation to remove essential oil) appears highly efficient with over 95% of MHP extracted (Table 12). In two separate experiments, MHP measured in reflux water of the distillation vessel was 3.8-6.8%. Soaking the spent biomass with 70 oC water (equal to the biomass volume) for an hour at room temperature extracted 46.7% of MHP. After soaking, crushing this biomass in water (equal to the biomass volume) in a mortar at room temperature extracted most of the remaining MHP (42.4% of total ). Crushing biomass directly in water without a 70 oC pre-soaking released 91.6% of MHP so pre-soaking may not confer any advantage. Following crushing, only trace amounts of MHP remained in the leaves and fine stems and the thick stems retained a small amount of MHP (4.2-4.6%).

Purification of osmoprotectants

After the above extraction process, MHP was isolated using a cation exchange column. This procedure gave MHP with a purity of 98%, established using both thin layer chromatography and HPLC .

MHP content of M. bracteata provenances

MHP content of M. bracteata collected from natural populations ranged from 0.1 to 2.15 % on a dry weight basis (Table 13). Most of the samples (45 of 51) contained low levels of MHP, ranging from 0.1 to 0.75%. The remaining six samples contained higher MHP levels, ranging from 0.75 to 2.25%.

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5. Discussion

Osmoprotectants (N-methyl amino acids or betaines) play a crucial role in improving the tolerance of plants to environmental stresses (Rhodes and Hanson, 1993). Osmoprotectants are used in two ways in agronomy (foliar or seed treatment) and plant improvement (conventional breeding or genetic engineering) to increase crop productivity (Naidu et al. 1998). With the success of glycinebetaine external application/agronomic use on various crops, we projected a world wide shortage of the supply of betaine. We have conducted field trials to assess the possibility of producing osmoprotectants from Australian native Melaleuca species which are known to contain high levels of N-methylated proline analogues/osmoprotectants (Naidu et al. 1987). We selected 8 species for field testing. M. alternifolia (commercial Tea Tree Oil producing species) was included as a control to compare growth. This species did not accumulate any of the methylated proline analogues, except very low levels of proline. M. bracteata was the most vigorous of all the species tested under rainfed conditions of Tyagarah. However, under irrigated conditions in north Qld, M. alternifolia growth tended to be a little better than M. bracteata. Of the 3 osmoprotectants (MP, MHP, and DHP) in Melaleuca, MHP yield was the highest. MHP yield of 673 kg/ha was achieved from rainfed Tyagarah plantation while under irrigated conditions of north QLD, the yield was only up to 313 kg/ha. This difference is attributable to the difference in the concentration of the solute. At Tyagarah, the concentration in leaf and stems was about 2 fold greater than at the Qld sites. In glasshouse experimentation the concentration of these compounds increases under moisture stress (Table 11). Rainfed cultivation of the species at Tyagarah, with attendant periods of moisture stress, may have promoted greater accumulation of MHP. Once accumulated, the N-methylated analogues stay in the biomass as semi-permanent end products (Naidu et al 1992; Wyn Jones and Storey, 1981). Because of this stress responsive nature of the osmoprotectants, imposition of water stress could be a very important tool in agronomic management. In irrigated culture withholding water prior to harvesting could substantially increase the yield of the osmoprotectants. The other potential species for the production of MHP is M. lanceolata. This species is well adapted to winter rainfall southern regions of the continent and may have potential in the south. Production of MP and DHP would not be economical in north Qld as the yield of these solutes was low. However, production of these compounds in the winter rainfall regions could be worthy of investigation. Apart from its high MHP yielding potential, M. bracteata also produces essential oil. Some provenances from the field collection contained relatively high MHP and up to 2.5% of essential oil on twig air-dry weight basis (Masunga, 1998). Based on the average twig dry matter produced (leaf + fine stem) at the 3 field sites (Tables 4, 5, and 10) an oil content of 2.5% would translate to an oil yield of about 230kg/ha. The essential oil is useful in cosmetic industry Essential oil of M. bracteata has recently been found to have very strong miticidal activity against European house-dust mites (Dermatophagoides pteronyssinus) (Yatagai et al. 1998).

We examined the possibility of selecting a superior genotype of M. bracteata which can produce high levels of both MHP and essential oil. A test of 46 families from northern NSW and QLD revealed a 10 fold variation for MHP content, and significant variation in oil concentration and

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composition (Masunga, 1998). Closer examination of MHP levels, oil content and composition revealed that some provenances of M. bracteata contained both high MHP and essential oil and hence combined selection for these two characters should be feasible. Based on the chemistry of the oil, this species may be divided into 5 chemotypes containing elemicin, E-isoelemicin, methyl eugenol, E-methyl isoeugenol, and p-cymene (Brophy and Doran, 1996; Masunga, 1998). Methyl eugenol and E-methyl isoeugenol of M. bracteata are of commercial interest and this species is already being field tested near Maryborough, Qld by Mr. Don Macdonald. Catalogue price of methyl eugenol and E-methyl isoeugenol is in the range of A$10 to 20 (Geoff Davis pers. comm.). We raised seedlings of the above 47 M. bracteata genotypes to field test under common conditions to make a combined selection for high MHP and oil. Continuation of this selection work would require further funding from a commercial partner/RIRDC. We tested the possibility of processing the biomass in sequence to yield essential oil and MHP under laboratory conditions. Harvested biomass could be steam distilled, in a similar way to the distillation used for many essential oil yielding species. The spent biomass could be simply crushed in water to liberate highly water soluble MHP. Well established column chromatography techniques can be used to produce MHP to the desired purity.

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6. Implications Gross return from the cultivation of M. bracteata (Table 14) was estimated to be $13,458/ha while it is only about $9, 000 from the currently cultivated M. alternifolia. The unknown factor in this calculation is the cost of MHP extraction. However, in the early ‘90s Tall Bennett P/L was importing and selling glycinebetaine in Australia from Finnsugar for $5/kg (the current price is about $25/ha). In this case one would reasonably assume that production cost for glycinebetaine would be less than $5/kg. On this basis, the gross profit (after MHP processing cost) from M. bracteata should be around $11, 000. Attempts to seek a commercial partner to take up this technology have been unsuccessful to date.

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7. Recommendations This project has provided with excellent data to support the possibility of developing a new crop to Australia. However, the selection work to identify a dual purpose M. bracteata yielding oil and osmoprotectant should be completed which will make this crop more attractive than M. alternifolia.

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8. Tables Table 2 : Plant height (cm) at Tyagarah

Species March ‘96 June ‘96 August ‘96 January ‘97 April ‘97M. alternifolia 15.6 16.7 28.4 96.4 120.5M. cuticularis 16.5 20.4 26.5 85.7 109.7M. bracteata 18.1 22.1 26.3 103.2 189.9M. glomerata 13.4 16.2 18.5 75.9 99.5M. lanceolata 5.6 6.8 10.5 80.2 110.1M. uncinata 10.1 12.5 16 30.2 80.5LSD (P=0.05) 8.2 7.9 12.2 21.7 20.6

Table 3: Plant height (cm) on Mr. DalSanto’s property near Dimbulah, north Qld

Collection month Species Dec-96 Mar-97 Jun-97 Oct-97 Nov-97

M. alternifolia 21.1 30.1 70.3 120.2 165.2M. cuticularis 19.2 22.9 62.1 109.2 145.3M. bracteata 18.1 22.2 65.3 110.2 141.9M. lanceolata 12.4 22.6 55.8 80.2 90.3M. pauperiflora 12.9 18.3 45.5 60.3 79.8M. uncinata 13.1 15.1 29.9 45.9 85.4LSD (P=0.05) 9.2 12.3 22.1 39.8 38.6

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Table 4: Dry matter production (kg/ha) and its partition at Tyagarah

Collection month August ‘96 January ‘97 April ’97 (Final Harvest) Dry matter proportion(%) Species Total Total Total Leaf Fine stem Thick stemM. alternifolia 1023.3 2633.3 5183.3 21 24 55M. cuticularis 903.3 1866.7 2730.0 18 30 52M. bracteata 2596.7 7406.7 13790.0 31 25 44M. glomerata 196.7 Not

sampled 2363.3 25 35 40

M. lanceolata 670.0 1706.7 3523.3 25 35 40M. uncinata 403.3 630.0 1613.3 30 32 38LSD (P=0.05) 401.7 736.7 1080.0

Table 5: Dry matter production (kg/ha) and its partition on Mr. DalSanto’s property near Dimbulah, north Qld.

Collection month Jun ‘97 Oct ‘97 Nov ’97 (Final harvest) Dry matter proportion(%) Species Total Total Total Leaf Fine stem Thick stemM. alternifolia 3800.0 6733.3 21700.0 20 20 60M. cuticularis 1433.3 2533.3 6000.0 15 35 50M. bracteata 3466.7 6333.3 18333.3 26 27 47M. lanceolata 366.7 966.7 4200.0 25 30 45M. pauperiflora 400.0 1000.0 3366.7 22 28 50M. uncinata 566.7 1733.3 3166.7 20 35 55LSD (P=0.05) 373.3 810.0 1836.7

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Table 6: Concentration of osmoprotectants (% of dry matter) in Melaleuca species at Tyagarah, NSW.

Species Compound June ‘96 August ‘96 January ‘97 April ’97 (Final Harvest) Leaf Leaf Leaf Leaf Fine stem Thick

stem M. alternifolia None N/A N/A N/A N/A N/A N/A M. cuticularis MP 0.1±0.01 0.15±0.01 0.14±0.01 1.5±0.05 1.5±0.06 1.3±0.05M. bracteata MHP 0.5±0.03 0.5±0.02 0.9±0.01 6.1±0.08 4.4+0.06 4.3±0.03M. glomerata MHP 0.1±0.01 0.1±0.01 0.1±0.02 1.0±0.08 1.0+0.05 0.9±0.03

DHP 0.6±0.04 0.9±0.03 0.9±0.06 2.1±0.03 2.2±0.09 1.5±0.08M. lanceolata MHP 0.6±0.02 0.5±0.03 0.9±0.05 9.8±0.08 7.5±0.05 3.7±0.07M. uncinata MHP 0.1±0.05 0.1±0.05 0.1±0.02 2.2±0.09 1.9±0.1 1.0±0.05

DHP 0.2±0.01 1.7±0.03 1.8±0.05 2.5±0.09 3.0±0.08 2.6±0.06

Table 7: Concentration of osmoprotectants (% of dry matter) in Melaleuca species on Mr. DalSanto’s property near Dimbulah, north Qld.

Species Compound Mar-97 Jun-97 Oct-97 Final Harvest Nov-97 Leaf Leaf Leaf Leaf Fine stem Thick

stem M. alternifolia None N/A N/A N/A N/A N/A N/A M. cuticularis MP 0.2±0.03 0.1±0.01 0.6±0.03 1.1±0.01 0.9±0.01 0.8±0.03M. bracteata MHP 0.1±0.01 2.3±0.05 2.2±0.04 2.5±0.05 1.9±0.06 0.9±0.03M. lanceolata MHP 0.6±0.04 8.3±0.06 7.9±0.05 8.6±0.05 7.4±0.06 5.5±0.08M. pauperiflora MHP 0.6±0.02 5.9±0.05 5.2±0.06 5.8±0.02 5.7±0.02 3.7±0.07M. uncinata MHP 0.1±0.05 2.2±0.01 2.3±0.04 2.1±0.03 1.7±0.01 1.0±0.05

DHP 0.2±0.01 5.2±0.1 5.0±0.05 5.3±0.09 4.9±0.07 2.6±0.06

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Table 8: Potential yield (kg/ha) of osmoprotectants at Tyagarah

Species Compound Leaf Fine stem Thick stem Total M. alternifolia None N/A N/A N/A N/A M. cuticularis MP 7.4 12.3 18.5 38.1 M. bracteata MHP 260.8 151.7 260.9 673.4 M. glomerata MHP 5.9 8.3 8.5 22.7

DHP 12.4 18.2 14.2 44.8 M. lanceolata MHP 86.3 92.5 52.1 231.0 M. uncinata MHP 10.6 9.8 6.1 26.6

DHP 12.1 15.5 15.9 43.5

Table 9: Potential yield of osmoprotectants (kg/ha) on Mr. DalSanto’s property near Dimbulah, north Queensland.

Species Compound Leaf Fine stem Thick stem Total M. alternifolia None N/A N/A N/A N/A M. cuticularis MP 9.9 18.9 24.0 52.8 M. bracteata MHP 128.3 101.2 83.5 313.0 M. lanceolata MHP 90.3 93.2 103.9 287.5 M. pauperiflora MHP 43.0 53.7 62.3 159.0 M. uncinata MHP 13.3 18.8 14.2 46.4

DHP 33.6 54.3 37.1 125.0

Table 10: Potential yield of MHP from M. bracteata on Mrs. Villanuva’s property near Dimbulah, northern Qld

Dry matter (kg/ha) MHP (%) MHP Yield (kg/ha)

Leaf 5056 ±123 1.85 ±0.09 93.5 ±.8.5

Fine stem 5263 ±175 1.11 ±0.05 58.4 ±6.7

Thick stem 9433 ±400 0.65 ±0.04 61.3 +7.2

Total 19752 ±592 N/A 212.7 ±9.8

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Table 11: Effect of water stress on the enhancement of betaine analogues in Melaleuca species.

Species Methylated

compound

Control level

(%)

Stress level

(%)

LSD

(P=0.05)

M. alternifolia None None None N/A

M. bracteata MHP 0.01 0.2 0.06

M. cuticularis MP 0.01 0.01 N.S

M .glomerata MHP

DHP

0.1

0.2

0.2

0.9

N.S

0.3

M. lanceolata MHP 0.2 1.0 0.5

M. laterifolia MHP 0.5 1.1 0.4

M. pauperiflora MHP 0.5 0.7 N.S

M. uncinata MHP

DHP

0.2

0.3

0.3

2.1

N.S

0.4

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Table 12: MHP extraction efficiency from biomass:

Method of extraction % Extracted

Experiment 1

% Extracted

Experiment 2

Reflex water 6.8 3.8

Soaking in water (70o C) 46.7 Not performed

Crushing in water 42.4 91.6

Residual in leaf <0.1 <0.1

Residual in fine stem <0.1 <0.1

Residual in thick stem 4.2 4.6

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Table 13: M. bracteata provenances, their collection locations and leaf MHP content.

Seed lot no Location MHP (%) in dry leaf1 DL 2273 Narrabri 0.702 DL 2274 Narrabri 0.103 DL 2275 Narrabri 0.654 DL 2276 Narrabri 2.155 DL 2277 Narrabri 0.546 DL 2285 Mundubbera 1.367 DL 2286 Mundubbera 0.738 DL 2287 Mundubbera 0.879 DL 2288 Mundubbera 0.76

10 DL 2289 Mundubbera 0.6411 DL 2291 Rolleston-Springsure 0.2712 DL 2292 Rolleston-Springsure 0.9413 DL 2293 Rolleston-Springsure 0.6814 DL 2294 Rolleston-Springsure 0.6615 DL 2295 Rolleston-Springsure 0.6516 DL 2296 Clermont-Alpha 0.6317 DL 2302 Capella-Clear Mount 0.7418 DL2306 S of Charters Towers 0.4019 DL2311 E of Cloncurry 0.5620 DL2312 E of Cloncurry 0.3621 DL2313 E of Cloncurry 0.4722 DL2314 E of Cloncurry 0.1623 DL2315 E of Cloncurry 0.5224 DL2316 Mt Isa-Dajarra 0.6725 DL2317 Mt Isa-Dajarra 0.3026 DL2318 Mt Isa-Dajarra 0.4727 DL2319 Mt Isa-Dajarra 0.3128 DL2320 Mt Isa-Dajarra 0.6529 DL2321 Adel's Grove 1.0430 DL2322 Adel's Grove 0.1331 DL2323 Adel's Grove 0.3332 DL2324 W.Gregory Downs 0.3133 DL2326 113 KM N of Hughenden 0.4334 DL2327 113 KM N of Hughenden 0.4235 DL2328 113 KM N of Hughenden 0.3436 DL2329 113 KM N of Hughenden 0.3437 DL2330 113 KM N of Hughenden 0.5538 DL2331 Einasleigh Rd at Lyndhurst 0.1139 DL2332 Forsayth 0.3240 DL2333 Forsayth 0.2241 DL2334 Forsayth 0.2342 DL2335 Forsayth 0.4543 DL2336 Forsayth 0.3044 DL2337 Rookwood 0.1845 DL2338 Rookwood 0.2246 DL2339 Rookwood 0.1047 DL2340 W of Chillagoe-Rookwood 0.1048 DL2341 W of Chillagae-Rookwood 0.6249 DL2342 Lakeland Downs 0.2050 DL2343 Lakeland Downs 0.6251 DL2344 Lakeland Downs 0.15

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Table 14: Average gross returns from M. bracteata plantation

MHP yield (kg/ha) Mean of 3 sites.

Oil yield (kg/ha). Based on mean twig yield and 2.5% oil yield

Gross return M. bracteata

Gross return M. alternifolia

399.7 231 $13, 458 $9, 000

Price of MHP is $25/kg,

Price of oil $15/kg

M. alternifolia yielding 200kg/ha of Tea Tree Oil @ $45/kg

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21

22

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evaluation of the effect of exogenous glycinebetaine on the growth and yield of soybean: timing of application, watering regimes and cultivars. Field Crops Ressearch, 54, 51-64.

Agboma, P.C., Jones, M. G. K., Peltonen-Sainio, P., Rita, H. and Pehu, E. (1997b). Exogenous glycinebetaine enhances grain yield of maize, sorghum and wheat grown under two supplementary watering regimes. Journal of Agronomy and Crop Science, 178, 29-37.

Agboma, P.C., Peltonen-Sainio, P., Hinkkanen, R. and Pehu, E. (1997c). Effect of foliar application of glycinebetaine on yield components of drought-stressed tobacco plants. Experimental Agriculture, 33, 345-352.

Aspinall, D., and Paleg, L.G. (1981). Proline accumulation: Physiological aspects. In The Physiology and Biochemistry of Drought Resistance in Plants (L.G.Paleg and D. Aspinall, eds.), pp.205-241. (Academic Press: Sydney.)

Brophy, J.J., and Doran, J.C. (1996). Essential oils of tropical Asteromyrtus, Callistemon, and Melaleuca species. ACIAR Monograph no. 40.

Campbell, J. A., Naidu, B. P., Weaich, K., and Wilson, J. R. (1996). Preliminary investigation of the effects of foliar application of glycinebetaine on the sucrose content of sugarcane. In ‘Sugarcane: Research towards Efficient and Sustainable production’. (Eds J. R. Wilson, D. M. Hogarth, J. A. Campbell and A. L. Garside) CSIRO Tropical Crops and Pastures, Brisbane pp. 181-182.

Campbell, J. A., Naidu, B. P., and Wilson, J. R. (1999). The effect of glycinebetaine application on germination and early growth of sugarcane. Seed Science and Technology. (in press).

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Hayashi, H., Alia, Mustardy, L., Deshnium, P., Ida, M. and Murata, N. (1997). Transformation of Arabidopsis thaliana with the codeA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. The Plant Journal, 12, 133-142.

Jones, G.P., Naidu, B.P., Starr, R.K., and Paleg, L.G. (1986). Estimates of solutes accumulating in plants by 'H nuclear magnetic resonance spectroscopy. Australian Journal of Plant Physiology 13, 649-658.

Makela, P., Jokinen, K., Kontturi, M., Peltonen-sainio, P., Pehu, E., and Somersalo, S. (1998). Foliar application of glycinebetaine -an approach to increase tomato yield. Industrial Crops and Products 7, 139-148.

Masunga, P. (1998). Analysis of the essential oils of Melaleuca bracteata from eastern Australia. Submitted for a Master of Chemistry degree at the University of New South Wales, Australia.

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Naidu, B. P. (1998). Simultaneous Estimation of Sugars, Polyols, Proline Analogues, and Betaines Accumulating in Stressed Plants by High Performance Liquid Chromatography-Ultra Violet Detection. Australian Journal of Plant Physiology 25, 793-800.

Naidu, B.P., Jones, G.P., Paleg, L.G., and Poljakoff-Mayber, A. (1987). Proline analogues in Melaleuca species: Response of Melaleuca lanceolata and M. uncinata to salinity and water stress. Australian Journal of Physiology 14, 669-677.

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Naidu, B. P. , Walker, M., and Munford, S. (1992) "Foliar application of glycinebetaine increases grain yield of buckwheat under cold stress affected field conditions". Presented in 32nd annual general meeting of Australian Society of Plant Physiologists held in Melbourne from 28th May to 2nd October 1992.

Naidu, B.P., Morris, P.R., and Cameron, D.F. (1996). Treatment with glycinebetaine to increase seed germination, seedling vigour and yield of cotton. Proceedings of 8th Australian Conference, Gold Coast.

Naidu, B. P., Cameron, D. F., and Konduri, S.V. (1998). Improving stress tolerance and productivity of plants by a biochemical approach in agronomy and plant breeding. Proceedings of IX Australian Agronomy Conference, Wagga Wagga 355-358.

Naidu, B. P., Jones, G. P., and Paleg, L. G. (1999). The Accumulation of Proline Analogues and Adaptation of Melaleuca species to Diverse Environments in Australia. Australian Journal of Plant Physiology (Submitted).

Paleg, L.G., Stewart, G.R., and Starr, R. (1985). The effect of compatible solutes on proteins. Plant and Soil 89, 83-94.

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Sakamoto, A., Alia, and Murata, N. (1997). Genetic engineering of glycinebetaine accumulation and stress tolerance in rice. 5th International Congress of molecular biology, Singapore. Abstract No. 84.

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Wyn Jones, R.G., and Storey, R. (1981), Betaines. In The Physiology and Biochemistry of Drought Resistance in Plants (L.G.Paleg and D. Aspinall, eds.), pp.172-204. (Academic Press: Sydney.)

Yang, W.J., Nadolskaorczyk , A., Wood, H.V., Hahn, D.T., Rich, P.J., Wood, A.J., Saneoka, H., Premachandra, G.S., Bonham, C.C., Rhodes, J.C., Joly, R.J., Samaras, Y., Goldsbrough, P.B., and Rhodes, D. (1995). Near-isogenic lines of maize differing for glycinebetaine. Plant Physiology 107, 621-630.

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10. Appendix