Rainer W. Hofmann

Tradeoff between biomass and flavonoid accumulation in white clover reflects contrasting plant strategies

An outdoor study was conducted to examine relationships between plant productivity and stress-protective phenolic plant metabolites. Twenty-two populations of the pasture legume white clover were grown for 4½ months during spring and summer in Palmerston North, New Zealand. The major phenolic compounds identified and quantified by HPLC analysis were glycosides of the flavonoids quercetin and kaempferol. Multivariate analysis revealed a trade-off between flavonoid accumulation and plant productivity attributes. White clover populations with high biomass production, large leaves and thick tap roots showed low levels of quercetin glycoside accumulation and low quercetin:kaempferol ratios, while the opposite was true for less productive populations. The latter included stress-resistant ecotypes from Turkey and China, and the analysis also identified highly significant positive relationships of quercetin glycoside accumulation with plant morphology (root:shoot ratio). Importantly, a high degree of genetic variation was detected for most of the measured traits. These findings suggest merit for considering flavonoids such as quercetin as potential selection criteria in the genetic improvement of white clover and other crops.

Nutrient responses and macronutrient composition of some Trifolium repens×Trifolium uniflorum interspecific hybrids

Abstract. Interspecific hybridisation is being utilised in white clover (Trifolium repens L.) breeding programs to overcome factors currently restricting productivity and persistence. Valuable new traits that may be introduced from the wild relative T. uniflorum include root characteristics and other adaptations to its natural, Mediterranean habitat. This study examined the effect of hybridisation on growth and macronutrient composition of white clover compared with T. uniflorum and T. repens × T. uniflorum backcross 1 (BC1) hybrids in two glasshouse sand culture experiments. Shoot and root dry weights of BC1 hybrids were greater than of white clover in low-concentration nutrient treatments but not in a more concentrated treatment. Decreases in dry weight with decreasing nutrient treatment strength were also smaller for some BC1 hybrids compared with white clover and other hybrid families. Most foliar macronutrient levels were adequate for white clover growth, but mean shoot or leaf phosphorus (P) concentrations were below published critical levels. Higher dry matter production under these low internal P concentrations suggests that some T. repens × T. uniflorum BC1 hybrids may be more tolerant of lower soil P levels than white clover. Such adaptations are likely to have been inherited from T. uniflorum. However, transgressive segregation may also be occurring, as T. uniflorum was larger than white clover in some, but not all, cases of low nutrient supply.

Multivariate associations of flavonoid and biomass accumulation in white clover (Trifolium repens) under drought

White clover (Trifolium repens L.) is an important pasture legume in temperate regions, but growth is often strongly reduced under summer drought. Cloned individuals from a full-sib progeny of a pair cross between two phenotypically distinct white clover populations were exposed to water deficit in pots under outdoor conditions for 9 weeks, while control pots were maintained at field capacity. Water deficit decreased leaf water potential by more than 50% overall, but increased the levels of the flavonol glycosides of quercetin (Q) and the ratio of quercetin and kaempferol glycosides (QKR) by 111% and by 90%, respectively. Water deficit reduced dry matter (DM) by 21%, with the most productive genotypes in the controls showing the greatest proportional reduction. The full-sib progeny displayed a significant increase in the root:shoot ratio by 53% under water deficit. Drought-induced changes in plant morphology were associated with changes in Q, but not kaempferol (K) glycosides. The genotypes with high QKR levels reduced their DM production least under water deficit and increased their Q glycoside levels and QKR most. These data show, at the individual genotype level, that increased Q glycoside accumulation in response to water deficit stress can be positively associated with retaining higher levels of DM production.

Drought resistance of Trifolium repens × Trifolium uniflorum interspecific hybrids

Abstract. White clover (Trifolium repens L.) is a widely used and highly valued temperate legume; however, its productivity and survival are restricted under dryland and drought conditions. This study investigated whether drought resistance of white clover could be improved by interspecific hybridisation with Trifolium uniflorum L. After almost 4 months without irrigation in a rain-shelter facility, shoot dry weight (DW) decreased significantly less in first-generation backcross (BC1) hybrids (–47%) than second-generation backcross (BC2) hybrids (–68%) and white clover (–69%). Stolon morphological parameters such as internode length and leaf lamina area also decreased less under water stress in the BC1 hybrids than in BC2 and white clover. There was also lower senescence in BC1 under water stress than in the other clover types. Genotypes with smaller changes in leaf lamina area, internode length, senescence and lateral spread had smaller changes in shoot DW, and there were significant correlations between constitutive levels of some characteristics and the effect of water stress on shoot DW. Under water stress, the growth form of the BC1 hybrids was compact, dense and prostrate, whereas white clover was more spreading and open. Increased allocation of dry matter to roots under drought, and greater root diameter, may also have influenced the ability of BC1 hybrids to maintain water uptake and key physiological processes. Overall, the data confirm that the drought resistance of white clover can be improved through hybridisation with T. uniflorum.

Effect of hybridisation with Trifolium uniflorum on tap root survival in white clover

A field study was conducted to determine whether tap root survival of white clover could be improved by hybridisation with Trifolium uniflorum. Tap root fragmentation and percentage of surviving tap roots were measured in 13, 16 and 19–20 month old plants. There were no intact healthy tap roots in white clover or second backcross (BC2) hybrids (12.5% T. uniflorum genes) post 13 months, but these were still present in T. uniflorum and first backcross (BC1) hybrids (25% T. uniflorum genes). Survival of T. uniflorum tap roots was higher than BC1, BC2 and white clover—30% of plants had intact, healthy tap roots at 19–20 months. The BC1 generation (31%) also had higher tap root survival than BC2 (13%) and white clover (11%) at 13 months. Although improved survival was not expressed as strongly in older BC1 plants, tap root deterioration was slower than in white clover and BC2. There is potential for targeted selection of specific genotypes and traits to further increase tap root lifespan in BC1 hybrids as there has been no previous selection for root traits in this material. The relationships between root diameter, leaf size and persistence in T. uniflorum and hybrids may differ from those expected for white clover cultivars. Characteristics of nodal rooting would also be expected to play a part in longer-term productivity and persistence.

Intraspecific differences in long-term drought tolerance in perennial ryegrass

Lolium perenne L. (perennial ryegrass) is the most important pasture grass species in temperate regions of the world. However, its growth is restricted in summer dry environments. Germplasm screening can be used to identify accessions or individual plants for incorporation into breeding programs for drought tolerance. We selected nine perennial ryegrass accessions from different global origins and from a range of climatic and environmental conditions. In addition, the perennial ryegrass cultivar ‘Grasslands Impact’ was chosen as a reference. The accessions were grown for 360 days in a controlled environment through six consecutive drought stress and recovery cycles. We observed intraspecific differences in drought stress responsiveness for shoot biomass and survival from the third stress cycle. An accession from Norway had 50% more shoot dry matter than the next best-performing accession after six drought cycles. Compared with the reference cultivar ‘Grasslands Impact’, shoot dry matter of the accession from Norway was more than seven times higher after six drought cycles, indicating superior performance of this ecotype under drought stress. Drought tolerance was characterized by osmotic adjustment and higher relative leaf water content at low soil moisture levels. Furthermore, the findings of this study identify solute potential as an early predictor of drought stress tolerance. These intraspecific differences can be used in breeding programs for the development of drought-tolerant perennial ryegrass cultivars.

Marker-Trait Associations for Flavonoids and Biomass in White Clover (Trifolium repens L.)

White clover, a primary forage legume in temperate permanent pasture, is limited by poor adaptation to abiotic stress factors such as water scarcity. Flavonoids contribute to abiotic stress tolerance in plants. Genetic analysis of flavonoid accumulation may help in understanding its relationship to plant growth and morphology in white clover. The objective of this research was to discover marker-trait associations for biochemical and morphological traits previously identified as associated with drought tolerance, using clonally replicated white clover plants. Parents and 131 progeny of a bi-parental cross between the cultivar ‘Grasslands Kopu II’ (K2) and the ecotype ‘Tienshan’ (T) were genotyped with 104 microsatellite (SSR) markers and a Diversity Array Technology (DArT) assay, revealing 320 polymorphisms segregating from parent K2 and 208 from parent T. Markers on linkage group (LG) 1–2 were significantly (p < 0.005) associated with concentrations of the flavonols quercetin (Q) and kaempferol (K) and the Q:K ratio (QKR). A cluster of linked markers including prs406 accounted for 21 %, 167 % and 53 % change in Q, K, and QKR trait values, respectively. Polymorphic loci on LGs 6–1 and 7–1 in parent K2 influenced shoot and root dry matter. Loci on LGs 7–2 and 8–1 influenced root dry matter but not shoot dry matter. Root to shoot ratio was influenced by loci associated with markers from parent T on LGs 4–1 and 8–1. These results support a hypothesis that flavonoid metabolism is under close genetic control and largely independent of genetic factors influencing growth, suggesting that it might be possible to improve both abiotic stress tolerance and growth potential in white clover. These marker-trait associations revealed deleterious alleles in an elite cultivar, and indicate the potential value of diversity from wild germplasm for white clover improvement.