1NSW Department of Primary Industries, Yanco Agricultural Institute, Yanco, NSW 2703, AUSTRALIA
2NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, Wagga Wagga, NSW 2650, AUSTRALIA
Fragrant rice varieties have a special niche market and secure premium price in the export market. The major aromatic compound contributing to the ‘nutty–popcorn’ flavour and aroma found in fragrant rices is thought to be 2-acetyl-1-pyrroline (2AP) (Buttery et al, 1982, 83). Although the amount of 2AP present in a fragrant rice grain can be greatly influenced by the growing season (Itani et al, 2004) and storage conditions (Widjaja et al, 1996; Wongpornchai et al, 2004), it is mainly dependant on the genetic make up of the rice line. For breeding fragrant rice varieties, 2AP is highly sought after in potential breeding lines. In the past, fragrant crossbreds of rice were mainly screened by taste panels, which is a subjective and non-quantitative test. In recent years, identification of fragrant rice lines has been performed by more precise tests where aromatic compounds are extracted from rice flour and analysed by gas chromatography (Bergman et al, 2000). The entire process requires the rice sample to be harvested, dried, cleaned through an aspirator (kice), de-hulled, milled and finely ground prior to 2AP extraction (Bergman et al, 2000). This can mean it is many weeks before rice breeders have any information regarding the aroma content of fragrant crossbreds, and often requires the consumption of large quantities of limited seed material from breeding lines.
The aim of this project was to estimate the amount of 2AP in different parts of rice paddy in order to identify a suitable tissue for rapid screening of aromatic rices on a large scale, and to obtain comparative results for a follow up study aiming at extracting 2AP from vegetative tissues in the field at pre-harvest.
Materials and methods
Two Australian cultivars, Langi (non-fragrant) and Kyeema (fragrant) were utilised in this study. Paddy from both varieties, harvested in 2004, was dehulled, milled and ground in a Willey mill. Different tissues samples ie. Hull, bran, brown grain flour, white grain flour, brown grain and white grain were separated for extraction of 2AP and other aromatic compounds. For 2AP extractions, a procedure based on dichloromethane (DCM) incubation (Bergman et al 2000) was used. Various rice samples (300mg, in triplicate) were weighed out in 2ml glass vials and 750ul of a DCM stock solution containing 1ug/ml of Collidine (2, 4, 6-trimethylpyridine (TMP)) as an internal standard was added to each vial. The vials were sealed with crimp caps and incubated for 150min in a water-bath at 85oC. After incubation, vials were centrifuged at 3000 rpm for 5min and clear supernatant analysed on an Agilent 6890 GC fitted with a flame ionisation detector and an HP-5 wide bore column (30m, id 0.32, film coating 0.25um). Helium was the carrier gas and a 2ul sample was injected, using a split-less injection.
Results an discussion
Relative amounts of 2AP extracted from various parts of Kyeema and Langi paddy was estimated in relation to the known concentration of TMP (1ug/ml) using the equation: [peak area of 2AP/ peak area of TMP]. In contrast to the fragrant line Kyeema, the 2AP peak was absent in all the tissue samples of the non-fragrant line Langi (data not shown). The relative amount of 2AP extracted from different tissue samples of Kyeema is shown in Figure 1. For grain tissue, the DCM extracts from flour meal samples (both brown and white) showed higher amounts of 2AP than the intact grains. This is mainly because of the more surface area accessible to the solvent in flour samples, as compared to the intact grains. DCM seems to be impermeable through the bran layer and may not be an ideal solvent for 2AP extraction from bran and brown grain.
Figure 1. Relative amount of 2AP extracted with DCM
Figure 2. Relative amount of 2AP in husk tissue of Langi and Kyeema
It was interesting to note that the hull tissues contained an appreciable amount of 2AP (~640ng/g tissue) in Kyeema, while it was extremely low in the non-fragrant rice Langi (Figure 2). Thus hull tissues, which are generally discarded, could be used for screening of fragrant rice crossbreds and the limited seed can be saved for further generations.
This preliminary study has shown that in fragrant rice lines 2AP is present in all parts of the paddy rice. The amount of 2AP even in the hulls was noticeable (especially when compared to non-fragrant Langi) making them suitable tissue for analysis of fragrant rice lines. Use of hull tissues, for screening of crossbreds, would eliminate the preparation of flour meal. Moreover, as hull tissues are normally discarded, the precious seed material can be retained in the breeding program.
Authors would like to thank the RIRDC for providing funds (DAN183 & 238A) to carry out this research. Provision of paddy samples from the rice breeders at Yanco is duly acknowledged.
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