Maria Pazos-Navarro

Time to flowering of temperate pulses in vivo and generation turnover in vivo–in vitro of narrow-leaf lupin accelerated by low red to far-red ratio and high intensity in the far-red region

Understanding the role light quality plays on floral initiation is key to a range of pre-breeding tools, such as accelerated single-seed-descent. We have elucidated the effect of light quality on early flowering onset in cool-season grain legumes and developed predictive models for time to flowering under the optimised light conditions. Early and late flowering genotypes of pea, chickpea, faba bean, lentil and lupin were grown in controlled environments under different light spectra (blue and far red-enriched LED lights and metal halide). All species and genotypes showed a positive response to a decreasing red to far-red ratio (R:FR). In general, ratios above 3.5 resulted in the longest time to flowering. In environments with R:FR below 3.5, light with the highest intensity in the FR region was the most inductive. We demonstrate the importance of considering both relative (R:FR) and absolute (FR photons) light values for flower induction in grain legumes. Greater response to light spectra was observed in the later flowering genotypes, enabling a drastic compression of time to flowering between phenologically diverse genotypes. A novel protocol for robust in vitro germination of immature seeds was developed for lupin, a species known for its recalcitrance to in vitro manipulation. We show how combining this protocol with growth under conditions optimized for early flowering drastically speeds generation turnover. The improved understanding of the effect of light on flowering regulation and the development of robust in vitro culture protocols will assist the development and exploitation of biotechnological tools for legume breeding.

Regulation of the drought response of sweet pepper (Capsicum annuum L.) by foliar-applied hormones, in Mediterranean-climate greenhouse conditions

Plant hormones play an important role in regulating stress responses and signaling in plants; many of them act to alleviate environmental stresses. However, the specific effects and physiological changes could be significantly altered according to the crop species, application concentration and frequency, and cultivation conditions. In this study, we investigated the effect of leaf-applied abscisic acid (ABA), gibberellic acid (GA3), and indole-3-acetic acid (IAA) on plant growth before, during, and after water stress. The objective was to determine their effects on pepper plants (Capsicum annuum L.) in commercial greenhouse conditions, specifically their ability to mitigate water stress, through the study of different stress traits—such as plant growth, gas exchange parameters, chlorophyll content and fluorescence, ascorbate peroxidase activity, total phenolic compounds, and lipid peroxidation. While ABA and IAA heightened the water shortage in the leaves along the experiment, GA3 diminished it. The effects of ABA involved short-term responses, such as stomatal closure and decreased transpiration, and long-term changes, affecting the ratios and concentrations of chlorophylls. Moreover, GA3 complicated the crop management since the plants suffered high stress when treated with this hormone. The results obtained represent a first approach to studying the effect of foliar hormone application in sweet pepper and its ability to regulate (mitigate or amplify) the water stress suffered by the plant under greenhouse conditions.

Micropropagation from apical and nodal segments of Bituminaria bituminosa and the furanocoumarin content of propagated plants

Summary An efficient micropropagation protocol has been developed for Bituminaria bituminosa, a species considered to be of pharmaceutical interest for its furanocoumarin (FC) content. Four accessions of B. bituminosa with different FC contents were used: variety albomarginata (“albo-tedera”), variety bituminosa (“bitu-Calnegre”), var. albomarginata × var. bituminosa (“albo-hybrid”), and var. bituminosa × var. albomarginata (“bitu-hybrid”). Different concentrations of sodium troclosene solution were evaluated for in vitro establishment of apical and nodal explants cultured on modified Murashige and Skoog (MS) medium. The effects of the type of agar and the addition of activated charcoal (AC) were also evaluated. The best treatment for surface sterilising the explants was 3.5 g l–1 sodium troclosene. The lowest frequency of hyperhydrated shoots was achieved using 0.8% (w/v) American bacteriological agar. Apical explants resulted in greater shoot proliferation than nodal explants. The addition of AC increased the quality and development of regenerated shoots. The highest average numbers of shoots ≥ 5 mm per explant were obtained from apical explants of “albo-tedera” (7.8 shoots) and “albo-hybrid” (7.6 shoots) on a modified MS medium supplemented with plant growth regulators (T2 media) and for “bitu-Calnegre” (6.3 shoots) and “bitu-hybrid” (5.3 shoots) on a modified MS medium without plant growth regulators (T1 media). The regenerated shoots rooted efficiently on different media depending on the plant material. Plantlets were acclimatised and maintained in a greenhouse until they were transferred to the experimental field. The total FC contents in micropropagated field grown plants were found to be higher or equal to those in the non-micropropagated mother plants. The accession “bitu-Calnegre” was found to have higher, or equivalent FC levels to those in other species such as Psoralea corylifolia and Ruta graveolens, reported as sources of FC. All micropropagated plants maintained the characteristics of the non-micropropagated mother plants.

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.

Embryogenesis and plant regeneration of the perennial pasture and medicinal legume Bituminaria bituminosa (L.) C.H. Stirton

Abstract. Bituminaria bituminosa (common name tedera) is a drought-tolerant perennial pasture species of agronomic and pharmaceutical interest for Mediterranean climates. Considering the importance of this legume, in vitro experiments were conducted to develop protocols for plant regeneration from embryogenic calli of leaves, petioles and anthers to efficiently exploit and maintain selected important clones from the tedera breeding program. The type of explant was a key factor in the frequency of embryogenesis and the number of embryos per callus. For plant regeneration from cultured anthers, appropriate anther physiological state (uninucleate stage of microsporogenesis), stress treatments (electroporation, 25 Ω, 25 µF, 1500 V) and culture conditions were determined. A robust flow-cytometry method was developed to analyse the ploidy status of callus, in vitro shoots and in vivo acclimatised plants derived from anther and leaf explants.

Erratum to: Time to flowering of temperate pulses in vivo and generation turnover in vivo–in vitro of narrow-leaf lupin accelerated by low red to far-red ratio and high intensity in the far-red region (Plant Cell, Tissue and Organ Culture (PCTOC), (2016), 127, 3, (591-599), 10.1007/s11240-016-1092-4)

In the original publication, the name of the model/manufac-turer of the blue LED light used in Environments 1 and 2 is not correct. Environment 1 contained blue LED light from 18w-T8 LED tubes (model 108D18-V12; S-Tech Lighting, Australia) and Environment 2 contained a combination of blue LED light used in E1 plus far red-enriched LED light from AP67 Valoya L series (Helsinki, Finland).