Ping Si

Variation in seed softening patterns and impact of seed production environment on hardseededness in early-maturing genotypes of subterranean clover

This paper describes variation in the dynamics of seed softening (loss of impermeability) in 20 early-maturing genotypes, including 6 cultivars, of subterranean clover (Trifolium subterraneum L.). It reports the effect of 3 sites of seed production in south-western Australia on the pattern of softening in the first summer–autumn and on total softening over the subsequent 2 years. Seeds were softened at a single field location and in a diurnally fluctuating cabinet (60°C/15°C). There was significant variation among genotypes in the pattern of seed softening over the first 5 months after senescence. Cultivars Nungarin, Dwalganup, and Geraldton softened most rapidly in late February, whereas cultivars Dalkeith, Urana, and Izmir softened most rapidly in late March. The duration of field exposure required in order for 50% of the first season’s softening to occur ranged from 44 to 108 days among the 20 genotypes. Persistence of hard seeds into the second and third years also varied among genotypes. Of the cultivars, Nungarin and Izmir had the highest levels of residual hard seed after 30 months (5.3% and 3.9%, respectively), whereas Dalkeith had the lowest (0.9%). Site of seed production had a small but significant effect on both the pattern of softening in the first summer–autumn and the persistence of hard seeds in subsequent years. Seeds produced in a relatively high-rainfall site (768 mm of growing-season rainfall plus supplementary irrigation) had a slower rate of hard seed breakdown than those from either of 2 sites located in the wheatbelt (217 and 423 mm growing-season rainfall). Seed softening through exposure in the field and in a 60°C/15°C fluctuating-temperature cabinet was compared for all genotypes. The cabinet treatment was fairly successful in ranking genotypes for relative between-season hardseededness, although it underestimated total softening by an average of 16%. However, the cabinet treatment was a poor predictor of the within-season pattern of seed softening.

Genotypic variation in metribuzin tolerance in narrow-leafed lupin (Lupinus angustifolius L.)

Tolerance to metribuzin herbicide is an essential agronomic trait for narrow-leafed lupin (L. angustifolius L.) grown in Western Australia (WA), however, metribuzin causes up to 30% yield loss in cv. Tanjil. Tanjil is widely used as a parent in the WA lupin breeding programme to provide anthracnose resistance. Hence, identification of genotypes tolerant to metribuzin and incorporation of this tolerance into the disease-resistant cultivar is necessary for maintaining lupin production. This study identified tolerance to metribuzin among lupin cultivars and advanced breeding lines under both controlled temperature and natural winter conditions. Differences in dose responses between cultivars revealed that cv. Gungurru was tolerant and cv. Tanjil susceptible to metribuzin. Gungurru seedlings survived metribuzin applications of up to 1600 g/ha, whereas Tanjil seedlings exhibited zero survival at 800 g/ha. The rate of herbicide application that caused a 50% growth reduction (GR50, excluding dead plants) for Gungurru was 2 times greater than that for Tanjil. The level of tolerance in Gungurru is adequate to protect plants against metribuzin damage in the field. Large and consistent differences in tolerance between genotypes were identified among cultivars and advanced breeding lines across controlled temperatures (20°C during the day and 12°C at night) and in natural winter conditions. One breeding line (95L208–13–13) showed marginally better tolerance than Gungurru. A number of advanced breeding lines were as susceptible to metribuzin as Tanjil, indicating that it is very important to select for metribuzin tolerance concurrently with disease resistance in the breeding programme. Of the 6 measures of tolerance used in this study, leaf score proved to be the simplest and most effective measure and could be used for the selection of individual tolerant plants in segregating populations. Tolerance was independent of early vigour, suggesting that it is possible to combine both early vigour and tolerance into a cultivar for better weed management. In conclusion, breeding for metribuzin tolerance in lupin is feasible, and the screening method tested here was simple and consistent, which would assist a breeding programme in making rapid progress towards herbicide-tolerant plants.

Non-target site mechanism of metribuzin tolerance in induced tolerant mutants of narrow-leafed lupin (Lupinus angustifolius L.)

Abstract. Narrow-leafed lupin (Lupinus angustifolius L.) is an important grain legume crop in Australia. Metribuzin is an important herbicide used to control weeds in lupin crops. This study investigated metribuzin tolerance mechanism in narrow-leafed lupin by comparing two induced mutants (Tanjil-AZ-33 and Tanjil-AZ-55) of higher metribuzin tolerance with the susceptible wild type. Sequencing of the highly conserved region of the chloroplast psbA gene (target site) revealed that the sequences of the wild type and the mutants were identical and therefore metribuzin tolerance is not target site based. Photosynthetic activity was measured and the leaf photosynthesis of the two tolerant mutants was initially inhibited after metribuzin treatment, but recovered within 2.5 days whereas that of the susceptible plants remained inhibited. The photosynthetic measurements confirmed the target site chloroplast was susceptible and the tolerance mechanism is non-target site based. Investigation with known cytochrome P450 monooxygenase inhibitors (omethoate, malathion and phorate) showed that tolerance could be reversed in both mutants, indicating the tolerance mechanism in two tolerant mutants may involve cytochrome P450 enzymes. Interestingly, the inhibitor tridiphane reversed metribuzin tolerance of only one of the two tolerant mutants, indicating diversity in metribuzin tolerance mechanisms in narrow-leafed lupin. These results signify that further investigation of metribuzin metabolism in these plants is warranted. In conclusion, metribuzin tolerance mechanism in lupin mutants is non-target site based, likely involving P450-mediated metribuzin metabolism.

Coolamon subterranean clover (Trifolium subterraneum L. var. subterraneum)

Izmir is a hardseeded, early flowering, subterranean clover of var. subterraneum (Katz. et Morley) Zohary and Heller collected from Turkey and developed by the collaborating organisations of the National Annual Pasture Legume Improvement Program. It is a more hardseeded replacement for Nungarin and best suited to well-drained, moderately acidic soils in areas with a growing season of less than 4.5 months. Izmir seed production and regeneration densities in 3-year pasture phases were similar to Nungarin in 21 trials across southern Australia, but markedly greater in years following a crop or no seed set. Over all measurements, Izmir produced 10% more winter herbage and 7% more spring herbage than Nungarin. Its greater hardseededness and good seed production, makes it better suited to cropping rotations than Nungarin. Softening of Izmir hard seeds occurs later in the summer–autumn period than Nungarin, giving it slightly greater protection from seed losses following false breaks to the season. Izmir is recommended for sowing in Western Australia, New South Wales, Victoria, South Australia and Queensland. Izmir has been granted Plant Breeders Rights in Australia.

Genetic basis for and inheritance of resistance to Race 1 and Race 2 of Kabatiella caulivora in Trifolium subterraneum ssp. subterraneum and ssp. yanninicum

SummaryA study of a partial diallel involving crosses of seven cultivars of subterranean clover (Trifoliumsubterraneum ssp. subterraneum and ssp. yanninicum) was undertaken to determine the genetic basis of resistance to both Race 1 and Race 2 of Kabatiella caulivora at seedling and adult plant stages. Relationships between variance (Vr) and covariance (Wr) revealed that adult plant resistance to Race 1 and both seedling and adult plant resistance to Race 2 were controlled by simple additive and dominant gene effects, whilst seedling resistance to Race 1 was controlled by epistatic gene effects. Three of the four most resistant parents (cultivars Daliak, Denmark and Meteora) showed a dominant gene effect for adult plant resistance to Race 1. However, the other resistant parent, cv. Goulburn, and the most susceptible parent, cv. Woogenellup, both were controlled by recessive gene effects (i.e, gene effects conferred by recessive genes). Cultivar Denmark again showed dominant gene effects in relation to resistance to Race 2 at both seedling and adult plant stages, whilst recessive gene effects were evident in susceptible adult plants of cv. Daliak in response to Race 2. Estimates of genetic variance components confirmed that dominance variance (H1 and H2) was much greater than additive variance (D) and there were more dominant alleles (i.e., more alleles that had dominant effects) than recessive alleles (i.e., more alleles that had recessive effects) present across all the cultivars. An examination of segregation ratios in three F2 populations revealed that inheritance of adult plant resistance to Race 1 was controlled by a dominant gene in cv. Daliak and cv. Meteora, and by two dominant genes in cv. Denmark. The additional resistance gene present in cv. Denmark may well explain its apparent stable resistance to both races in the field. This study provides an insight into the genetic basis for and inheritance of resistance to this disease that will assist breeding programs aimed at improving resistance.

Coolamon subterranean clover (Trifolium subterraneumL. var.subterraneum)

Coolamon is a mid-season to late-season flowering F4-derived crossbred subterranean clover of var. subterraneum, developed by the collaborating organisations of the National Annual Pasture Legume Improvement Program. It is a replacement for Junee and has been selected for release on the basis of its greater herbage production and persistence, and its resistance to both known races of clover scorch. Coolamon is recommended for sowing in Western Australia, New South Wales, Victoria, South Australia and Queensland. It is best suited to well-drained, moderately acidic soils in areas with a growing season of 6.5–8 months that extends into November. Coolamon is best suited to phase farming and permanent pasture systems. It can also be used in cropping rotations, but at least 2 years of pasture are required between crops. Coolamon has been granted Plant Breeders Rights in Australia.

Napier subterranean clover (Trifolium subterraneum L. var. yanninicum)

Napier is a late flowering F6-derived crossbred subterranean clover of var. yanninicum [(Katz. et Morley) Zohary and Heller] developed by the collaborating organisations of the National Annual Pasture Legume Improvement Program. It is a replacement for both Larisa and Meteora and has been selected for release on the basis of its greater herbage and seed production and disease resistance to both known races of clover scorch and 2 of the common races of Phytophthora root rot. Napier is recommended for sowing in Victoria, Western Australia, New South Wales, and South Australia. It is best suited to moderately acidic soils prone to water-logging and to loamy and clay soils with good water-holding capacity in areas with a minimum growing season length of 7.5 months, which extends into late November. Napier is well adapted to the permanent pasture systems found in the areas in which it will be grown. Its upright, vigorous growth makes it well suited to grazing by cattle or sheep and to fodder conservation. Napier has been granted Plant Breeders Rights in Australia.

Analysis of Nicotiana tabacum PIN genes identifies NtPIN4 as a key regulator of axillary bud growth

The plant-specific PIN-FORMED (PIN) auxin efflux proteins have been well characterized in many plant species, where they are crucial in the regulation of auxin transport in various aspects of plant development. However, little is known about the exact roles of the PIN genes during plant development in Nicotiana species. This study investigated the PIN genes in tobacco (Nicotiana tabacum) and in two ancestral species (Nicotiana sylvestris and Nicotiana tomentosiformis). Genome-wide analysis of the N. tabacum genome identified 20 genes of the PIN family. An in-depth phylogenetic analysis of the PIN genes of N. tabacum, N. sylvestris and N. tomentosiformis was conducted. NtPIN4 expression was strongly induced by the application of exogenous indole-3-acetic acid (IAA), but was downregulated by the application of ABA, a strigolactone analogue, and cytokinin, as well as by decapitation treatments, suggesting that the NtPIN4 expression level is likely positively regulated by auxin. Expression analysis indicated that NtPIN4 was highly expressed in tobacco stems and shoots, which was further validated through analysis of the activity of the NtPIN4 promoter. We used CRISPR-Cas9 technology to generate mutants for NtPIN4 and observed that both T0 and T1 plants had a significantly increased axillary bud growth phenotype, as compared with the wild-type plants. Therefore, NtPIN4 offers an opportunity for studying auxin-dependent branching processes.

A greater relative growth rate of Brassica rapa L. at low temperatures increases biomass at anthesis

0t, n.s.). These relationships suggested that it might be possible to develop early flowering cultivars with increased seed yield by selecting for higher RGR before anthesis. RGR of BC 2 F 3:4 lines in the field during winter was correlated with RGR at low temperatures (13/4°C) in a controlled environment, suggesting that genotypes producing more dry weight at anthesis grew more at the low temperatures normally experienced in the field. This was supported by a significant (P < 0.05 0.05) negative correlation (r = –0.69**) between growth in the field and potassium leakage from leaf tissue at 4

Identification of new metribuzin-tolerant wheat (Triticum spp.) genotypes

Abstract. Herbicide-tolerant wheats are preferred for effective weed management. Rapid phenotyping and effective differential dose are vital for the identification of tolerant genotypes among large quantities of genetic resources. A sand-tray system has been developed to enable rapid assessment of metribuzin damage in wheat seedlings. In total, 946 wheat genotypes were evaluated for metribuzin tolerance by using this system under control and metribuzin-treated conditions. SPAD chlorophyll content index (CCI) offered a non-destructive and rapid analysis of leaf chlorophyll content in wheat seedlings. The application rate for 50% reduction in SPAD CCI (I 50) was 3.2-fold higher in the current tolerant genotype (Eagle Rock) than the susceptible genotype Spear. A confirmed dose of 800 g a.i. ha–1 could differentiate between metribuzin-tolerant and -susceptible lines. The experimental design with two-directional blocking followed by statistical analysis to model the spatial variation was instrumental in selecting potential tolerant or susceptible genotypes. Metribuzin reduced chlorophyll by 51.4% in treated seedlings. The overall adjusted mean SPAD CCI ranged from 13.5 to 42.7 for control (untreated) plants and from 0.1 to 29.9 for treated plants. Through repeated validation, nine genotypes had higher chlorophyll content after metribuzin treatment and significantly (P < 0.05) outperformed the tolerant Eagle Rock, whereas 18 genotypes had significantly (P < 0.05) higher chlorophyll reduction than the susceptible Spear. The top five tolerant and susceptible genotypes were selected for a genetic study of metribuzin tolerance. Domesticated forms of tetraploid and hexaploid wheats had higher tolerance to metribuzin, which suggests that the level of domestication and higher ploidy level contributes to metribuzin tolerance. The new sources of tolerance will accelerate breeding for metribuzin tolerance.