In vitro-assisted single-seed descent for breeding-cycle compression in subterranean clover (Trifolium subterraneum L.)

Abstract

Subterranean clover (Trifolium subterraneum L.) is widely grown for its forage and ability to fix atmospheric nitrogen. Development of new varieties is constrained by the slow turnover time of generations, with only one generation per year possible under field conditions. We present an in vitro-assisted single-seed descent (IVASSD) technique, which enabled turnover of 2.7–6.1 generations per year across a diverse range of 27 T. subterraneum cultivars encompassing subspecies subterraneum, yanninicum and brachycalycinum. The IVASSD protocol accelerated the generation cycle in two ways: (i) time to floral initiation was minimised by growth under controlled temperature and extended photoperiod; and (ii) the seed-filling period was truncated and embryo and seed-coat dormancy avoided by the in vitro germination of immature seed on B5 medium plus L6KK overlay (0.525 mg gibberellic acid and 1.5 mg indole-butyric acid L–1). For the first time, an IVASSD system was validated on a full-scale breeding population with the production of 175 F7 recombinant inbred lines from an F4 population in less than one year. All F7 plants obtained were morphologically normal and fertile.

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research

“…Even incandescent lighting has been shown to accelerate flowering in clover 6 . However, selection should be based on the space available, plant species and energy resources.…”

“…The generation time of most plant species represents a bottleneck in applied research programs and breeding, creating the need for technologies that accelerate plant development and generation turnover. Recently we reported protocols for 'speed breeding' (SB), which involve extending the photoperiod using supplementary lighting and temperature control, enabling rapid generation advancement in glasshouses with sodium vapour lamps (SVL) or growth chambers fitted with a mixture of metal halide and light-emitting diode (LED) 3,4 , pea (P. sativum), chickpea (C. arietinum), faba bean (Vicia faba), lupin (Lupinus angustifolius) 5 and clover (Trifolium subterraneum) 6 .…”

Gene networks underlying faster flowering induction in response to far-red light

“…Ratio calculations followed the method of Runkle & Heins (2001) where the R: FR ratio was measured as a narrow band (655-665: 725-735 nm). Plants were watered daily and fertilised weekly with a water-soluble NPK fertiliser (15:2.2:12.4) with micronutrients (Peters Excel, Scotts Australia, Bella Vista, New South Wales) at a rate of 2 g per pot, as per Pazos-Navarro et al (2017). Time to flowering was defined as the number of days from sowing to the open floral stage of the first flower and was recorded under the different growth conditions.…”

“…Within this research, we used deep-sequencing technologies to investigate transcriptional activity under different light spectra at three time-points in the long-day plant, subterranean clover. We have previously shown the accelerated onset of flowering in a range of leguminous species under FR-enriched LED light spectra and have thus adopted this wavelength profile and compared it with a blue-enriched LED light spectra (Croser et al 2016; Pazos-Navarro et al 2017; Ribalta et al 2017). We expect that the FR-enriched spectra will accelerate the up-regulation of genes related to floral initiation.…”

Legumes in sustainable agriculture

“…Thus the development of new protocols allowing shorter times and higher selection efficiency are very appreciated by breeders and researchers. The new in vitro-assisted protocol developed by Pazos-Navarro et al (2017) allowed the production of several generations per year (between 2.7 and 6.1) using subterranean clover as a case study. This achievement opens new possibilities for plant breeding and to speed up the development of mapping populations such as Recombinant Inbred Lines for genetic studies.…”