AUSTRALIAN OILSEEDS FEDERATION INCORPORATED FINAL REPORT "Chlorophyll in canola" AOF 15-1 1 May 2000 to 30 June 2002 Dr R.J. Mailer, Agricultural Research Institute, Wagga Wagga (02 69381818) ISBN 0 7347 1421 1 AOF chlorophyll final report 1 Fig 1. (Cover). Samples of canola oil from canola seed samples containing increasing levels of chlorophyll of 6, 23, 35, 53 and 81 mg/kg respectively. AOF chlorophyll final report 2 AUSTRALIAN OILSEEDS FEDERATION INCORPORATED R&D PROJECT 2000/2002 Project Title: Chlorophyll in canola Reference number given by AOF to Preliminary Proposal: AOF 15-1 Organisation: NSW Agriculture Administrative Contact: Title First name Surname Mr Graham Denney Address: NSW Agriculture, Locked Bag 21, ORANGE NSW 2800 Phone: (02) 6391 3219 Fax: (02) 6391 3327 Email: [email protected] Principal Investigator: Address: Title First name Surname Wagga Wagga Agricultural Institute, PMB, Wagga Dr Rodney Mailer Wagga, NSW 2650 Phone: 02 69381818 Fax: 02 69381809 Email: [email protected] Starting date # : 1 / 5 /2000 Finishing date # : 30 / 06 /2002 AOF chlorophyll final report 3 Project Outline and Budget Process Criterion Cost Total 1 Genotype by Analyse canola samples from site x 100 x $31 $3100 environmental cultivar x year. Approx 100 relationship samples. 2 BHC’s study Obtain samples from bulk handlers 100 x $31 $3100 and canola trials/crops with the worst levels of chlorophyll/green seed. Approx 100 samples. 3 Green seed Determine the relationship between 50 x $10 $1500 screening test number of green seeds and spectrophotometric reading. Say 50 samples tested for green seed by dip test and compared with spectrophotometric test. Seed colour photographs, relationship graphs. 4 NIR calibration Develop a calibration for NIR rapid 78 x $31 = $2418 analysis to be used for screening 5 days x future canola trials $0.57/min = $1197 5 Analysis of 5,000 canola trial samples will be Analysis, $2,000 chlorophyll from analysed from 2000 breeding trials reports 2000/01 breeding and results analysed against sites Biometrical $500 trials and cultivars. analysis 6 Travel Meeting with coordinators $1,200 Sample collection 7 TOTAL $15,015 AOF chlorophyll final report 4 Abstract The short growing period for canola in Canada is a problem as incomplete maturity often results in green seed. The chlorophyll in the seed is transferred to the oil during processing, resulting in green oil. The colour is then difficult and expensive to remove. Recent reports suggest that Australian canola may also undergo periodic harvests which result in green seed. Frost damage in particular has been reported as the major cause. This study was designed to determine the degree of the problem, the reasons and possible solutions. Summary 1. Genotype by environmental relationship. Analyse canola samples from site x cultivar x year. Samples from 1997 and 1998 trials were analysed to determine chlorophyll content. The results indicated a wide range of concentrations particularly with some cultivars such as Range (5-27 mg/kg) and Rainbow (3-32 mg/kg). The study was repeated in 1999 and 2000, encompassing seven cultivars from six sites, in replicated trials. The results in 1999 ranged from 0 to 15.1 mg/kg and in 2000 from 0 to 24.5 mg/kg in the seed. No samples exceeded 30 mg/kg. In 1999, samples of frost-damaged seed from WA were tested for chlorophyll with all of the samples being less than 30 mg/kg and only a few above 20 mg/kg. Again in 2000, frost damaged samples were obtained from WA and were also low in chlorophyll with levels below 5 mg/kg. The study indicated that chlorophyll levels in Australian canola were low, with only a few samples exceeding 30 mg/kg over the four years of testing. 2. Bulk Handling Company seed. Obtain samples from bulk handlers and canola trials/crops with the worst levels of chlorophyll/green seed. BHCs throughout the Australian canola growing regions provided 800 samples for chlorophyll analysis. Although generally the concentration was low, 21 samples from the Temora site were consistently high with chlorophyll levels of between 11 to 67 mg/kg and many exceeding 30 mg/kg in the seed. The variety Pinnacle had the highest concentration of chlorophyll. The level of chlorophyll was attributed to plant stress in the region in that year. This study was not unbiased as the samples had been selected as green seed by the receival site. Additionally, the samples were limited in number and many were unknown cultivars. However, the range and intensity of chlorophyll indicated that there is a possibility of high levels of green seed to occur at specific sites. No other seed samples obtained from BHCs in 2000 had significant levels of green seed. 3. Green seed screening test. Determine the relationship between number of green seeds and spectrophotometric reading. Seed samples obtained from Canada and BHCs in 2000 were used to compare the green seed test with the chlorophyll spectrophotometric test described previously. Despite counting either “distinctly dark green seeds” or “partially green seeds” no accurate relationship could be formed between the two. The green seed test was able to identify canola samples with potentially high levels of chlorophyll. Several pale green seeds contributed as much to the chlorophyll content as two distinct green seeds. The test is considered to be limited in usefulness in obtaining an accurate measure of chlorophyll, or for identifying potential problems with green oil, and indicates the need for a rapid test at receival points. AOF chlorophyll final report 5 4. NIR calibration. Develop a calibration for NIR rapid analysis to be used for screening future canola trials A Foss NIR 6500 spectrophotometer was used to develop a calibration for the estimation of chlorophyll in intact canola seed. This has been used by others (Tkachuk et al., 1998; Daun et al., 1994) for ground seed. Calibration samples were obtained from the Canadian Grain Commission's Grain Research Laboratory, Winnipeg, Canada, Australian Breeder's Trials and Australian Bulk Handling Companies. Chlorophyll on the calibration set was determined by AOCS method Ak 2-92. A NIR spectrophotometer with a spinning sample module was used and a calibration established with 97 canola samples ranging from 1 to 66 mg/kg chlorophyll. The instrument was operated in reflectance mode using WINISI software (FOSS Pacific). Partial least squares (PLS) regression was used to develop a calibration from the second derivative of the log 1/R of the spectra. A linear regression (RSQ) of 0.959 was obtained for the set with a standard error of prediction (SEP) of 3.076. The NIR method allowed analysis of 200-300 samples per day. 5. Analysis of chlorophyll from 2000 / 01 breeding trials Breeders' seed was selected from the National Brassica Improvement Project (NBIP) and NSW Agriculture seed trials in 1999/2000. The trials included 8489 samples from 64 individual variety trials and 179 lines from throughout Australia. The trials represented 6 groups including early, mid, and late maturing lines and triazine tolerant types. The study was repeated in 2000/2001 with a further 7220 samples from 75 trials and again incorporating six groups. Samples in both years were analysed by NIR. 1999/2000: Chlorophyll concentration averages exceeded 10 mg/kg at only 5 sites and no site averaged more than 20 mg/kg. There was a wide range of chlorophyll levels within groups. The National early- maturity group, had the greatest range in individual lines with concentration from 0.5 to 51.0 mg/kg. Groups 2 and 4, both NSW Agriculture trial sites, had the least variation from 0.0 to 19.3 and 0.7 to 20.0 mg/kg respectively. Chlorophyll levels for lines in Group 1 (national early maturity trial) and Group 6 (Triazine Tolerant trial) were generally higher than chlorophyll levels for lines in the other groups. Ten sites in the NBIP had lines with chlorophyll levels greater than 20 mg/kg with 91 samples above 20 mg/kg and of these, 19 were above 30 mg/kg. There were some significant cultivar effects with some lines being very variable. PACN175 for example, had a chlorophyll concentration ranging from 2.6 mg/kg at Tamworth to 42.7 mg/kg at Newdegate. Newdegate had the highest concentration of chlorophyll from 4 to 51 mg/kg. 2000/2001: Chlorophyll concentration averages in 2000 exceeded 10 mg/kg at only one site and no site averaged more than 15 mg/kg. Group 6 (triazine tolerant) had the greatest range and highest value from 2.1 to 14.7 mg/kg. The maximum level measured was 43.1 mg/kg chlorophyll at Lameroo in South Australia. 6. Conclusion Generally chlorophyll levels are insignificant in comparison to the levels found in Canada and should not cause refining problems for the industry. Despite this, particular batches of samples obtained from some sites, such as Temora, have shown that regions may produce large quantities of green seed under particular environmental conditions. For this reason, a screening technique for receival sites appears to be justified. The current green seed test does not appear to give an adequate indication of potential problems in canola seed. A more direct test for chlorophyll content, such as NIR, would be more desirable. AOF chlorophyll final report 6 BODY OF THE REPORT Introduction Chlorophyll in canola seed is generally broken down to secondary pigments and by-products during the maturation process. In some cases, canola seed has been shown to retain the chlorophyll intact. In these cases the cotyledons can be seen to be green when the seed coat is removed. As the chlorophyll is oil soluble, when oil is extracted, it retains a green colour, the intensity being proportional to the concentration of chlorophyll in the seed.