Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Page 31 Page 32 Page 33 Page 34 Page 35 Page 36 Page 37 Page 38 Page 39 Page 40 Page 41 Page 42 Page 43 Page 44 Page 45 Page 46 Page 47 Page 48 Page 49 Page 50 Page 51 Page 52 Page 53 Page 54 Page 55 Page 56 Page 57 Page 58 Page 59 Page 60 Page 61 Page 62 Page 63 Page 64 Page 65 Page 66 Page 67 Page 68 Page 69 Page 70 Page 71 Page 72 Page 73 Page 74 Page 75 Page 7663 IREC Farmers' Newsletter No. 195 ­ – Rice R&D 2016 This phenomenon, colloquially described as ‘blowing up’ a crop, indicates that an excessive amount of nitrogen at panicle initiation can exacerbate cold damage. The major financial losses incurred with cold susceptible rice varieties means that breeding for cold tolerance at pollen microspore has become a high priority objective in the breeding program, second only to selection for yield. Reliable screening methods to assist the selection of cold tolerant rice varieties are therefore paramount to the breeding program. Why investigate new screening methods? Examining mechanisms of cold- induced sterility and selection methods for determining cold tolerant rice varieties has been a major focus of the rice breeding program for many years. Currently, the program uses whole panicle sterility observations, where one panicle from each plant is tagged at emergence (after a naturally occurring cold event) and harvested at maturity. Spikelets are removed from the panicle and the empty (sterile) and full spikelets are counted as a means to identify genotypes with tolerance to low temperatures. While this process gives accurate data on the level of sterility occurring at crop maturity, it does little to explain the effects at early stages of reproductive development, and therefore alternative screening methods may be more informative. The rice breeding program continues to research improved methods for screening reproductive cold tolerance in Australian rice and the practical application of these methods to select new cold tolerant varieties. More efficient measures of cold tolerance A range of experiments was conducted as part of the cold tolerance research program. The experiment reported in this article investigated pollen germination and pollen viability under cold stress conditions. The variety Reiziq was grown under glasshouse conditions with four treatments consisting of a combination of nitrogen, cold exposure or both. Panicles were tagged (Figure 1) and samples from those panicles were taken for pollen viability counts and in vivo germination (natural within the flower) and in vitro germination (on artificial growth medium). The tagged panicles were harvested at maturity and assessed for sterility. Pollen viability and pollen germination results were compared to observed panicle sterility at maturity. The methods were assessed for possible application to field trials as screening tools for cold tolerance. Pollen viability counts Pollen viability results were obtained by staining pollen grains with iodine, which turns the starch content of pollen black. Pollen grains use starch as ‘fuel’ to grow a pollen tube, the structure that carries sperm cells from the stigma (Figure 2) to the micropyle opening of the ovary for fertilisation, as indicated in Figure 4. Pollen grains that turn black are considered viable/ fertile; grains that turn pale grey or do not change colour are considered infertile, as they contain little or no starch. Cold treated Reiziq showed some correlation between reduced viable pollen numbers and observed panicle sterility and exhibited greater variability between pollen viability and observed panicle sterility than untreated Reiziq (Table 1). However, the numbers of supposedly viable pollen grains appeared adequate for fertilisation to occur in 96% of the cold treated panicles, yet 93.7% showed panicle sterility. Therefore, total viable pollen number does not appear to relate directly to observed panicle sterility. In vitro germination In vitro germination involves collecting pollen grains from mature anthers at the time of flowering and germinating the pollen on artificial growth medium. Pollen grains are considered germinated when their pollen tube length is greater than or equal to pollen diameter. Table 1: Observed % panicle fertility and observed % pollen viability from Reiziq samples with or without cold treatment. Cold treated No cold treatment % panicle % pollen % panicle % panicle fertility^ viability* fertility^ viability* 42 87 83 87 80 73 87 86 62 66 81 85 15 14 73 83 68 82 95 91 58 49 81 81 69 83 87 89 2 54 85 88 81 89 84 87 76 74 93 88 31 52 87 92 80 88 84 89 35 60 92 88 68 89 81 85 * Pollen viability is the average % from five anthers per panicle ^ Panicle fertility is the percent of filled spikelets out of the total number of spikelets on the panicle l Figure 1. Cold treated rice plants in a glasshouse cold tolerance trial. After treatment, panicles were tagged at emergence. Samples from those panicles were taken for pollen viability counts and in vivo and in vitro germination observations. The tagged panicles were harvested at maturity and assessed for sterility.