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 7664 IREC Farmers' Newsletter No. 195 ­ – Rice R&D 2016 A number of complications were encountered with this particular screening method. For example, once rice pollen is separated from the anther, pollen viability quickly declines to almost 0% within 10 minutes, leaving a very small window of opportunity to achieve optimum pollen tube growth for each sample collected. Another problem is that rice pollen maintains a high water content after flowering, which makes it highly sensitive to slight changes in temperature and humidity, especially outside of the floral environment. Furthermore, rice pollen is very difficult to germinate after freezing due to its high water content, which makes transporting viable pollen from field trials (often hundreds of kilometres away) a challenging task. Such complications in sampling pollen make germination studies difficult, which is illustrated in Figure 3. When rice pollen was taken from two different flowers on the same plant on the same day, and incubated under similar conditions, they showed very different pollen germination rates, as illustrated in Figure 3. There were no obvious differences between the two flowers to explain such differences in germination. Due to the complications described, in vitro germination of rice pollen on artificial growth medium is therefore an impractical tool for determining pollen viability in field- based experiments. In vivo germination This screening process appears to be more suitable for assessing pollen viability in pollen samples collected from field trials. After pollination occurs naturally, whole flowers can be collected up to 24 hours after anthesis (flowering) and preserved in a fixative for easy transport and storage. Pollen viability is assessed by removing the pistil (female reproductive part) from the flowers and staining the germinated pollen on the pistil with a fluorescent dye. Images of pollen tube growth are then obtained using a fluorescence microscope. Fertilisation is considered successful when pollen tubes can be seen at, or entering, the micropyle opening of the ovary. Preliminary results on in vivo pollen tube observations are promising. Untreated Reiziq from the glasshouse experiment showed excellent pollen germination on the stigma and pollen tube growth reaching all the way to the base of the ovary (Figure 4), while in the majority of cold treated Reiziq plants, pollen tubes seldom reached the embryo sac or micropyle opening to complete fertilisation (Figure 5). Where to from here? Current methods for assessing cold tolerance, although reliable, only supply information about the effects on harvested rice panicles at crop maturity. The above results clearly show that pollen viability counts and in vitro germination are not practical methods to use as screening tools in the breeding program. This leaves the very promising method of in vivo germination and observation as a screening tool. The method is practical and useful as pistils can be preserved in fixative for transport and storage, and are easily stained with fluorescent dye for observations of pollen tube growth. The next phase of our investigation is to look at the combined effect of excessive nitrogen and l Figure 3. In vitro germination of rice pollen taken from different flowers of the same Reiziq rice plant on the same day. Image A shows good pollen germination and image B shows no germination but there appears to be no apparent reason for differences between the two samples. A B l Figure 2. A whole rice flower showing the male and female parts of the flower, i.e. the stamen and pistil, respectively. Stamen: Male part of the flower Anther Filament Pistil: Female part of the flower Stigma Style Ovary