Janine Croser

Low-Temperature Stress: Implications for Chickpea (Cicer arietinum L.) Improvement

Chickpea is the third major cool season grain legume crop in the world after dry bean and field pea. Chilling and freezing range temperatures in many of its production regions adversely affect chickpea production. This review provides a comprehensive account of the current information regarding the tolerance of chickpea to freezing and chilling range temperatures. The effect of freezing and chilling at the major phenological stages of chickpea growth are discussed, and its ability for acclimation and winter hardiness is reviewed. Response mechanisms to chilling and freezing are considered at the molecular, cellular, whole plant, and canopy levels. The genetics of tolerance to freezing in chickpea are outlined. Sources of resistance to both freezing and chilling from within the cultivated and wild Cicer genepools are compared and novel breeding technologies for the improvement of tolerance in chickpea are suggested. We also suggest future research be directed toward understanding the mechanisms involved in cold tolerance of chickpea at the physiological, biochemical, and molecular level. Further screening of both the cultivated and wild Cicer species is required in order to identify superior sources of tolerance, especially to chilling at the reproductive stages.

Toward doubled haploid production in the Fabaceae: progress, constraints, and opportunities

The Fabaceae species have a major role to play in sustainable farming systems, but they have lagged behind other families in respect to the development of doubled haploid protocols for plant improvement. Currently, no plant improvement program uses doubled haploids on a routine basis for any member of the Fabaceae. There has recently been renewed interest in haploid research as the usefulness of doubled haploid material in molecular mapping has become clear. This review provides a comprehensive account of the current information regarding the development of haploid protocols in the Fabaceae. In the Fabaceae crop species there have been isolated reports of haploid plant induction in the phaseoloid clade; soybean, cowpea and pigeonpea, as well as promising progress towards haploidy in peanut and winged bean. As yet there have been no reports of haploid plant production in the galegoid clade, but early stage haploid embryogenesis has been achieved in chickpea, field pea, and lupin. Success in the production of haploid plants has also been reported within the pasture genera Lotus, Medicago, and Trifolium and the arboreal genera Cassia, Peltophorum, and Albizzia. A review of the literature has enabled us to identify some general similarities between the protocols developed for haploid plant induction across the various legumes. These are the culture of intact anthers; use of a cold pretreatment to induce sporophytic development; targeting of microspores at the uninucleate stage of development; and use of MS (Murashige and Skoog, 1962) based nutrient medium with plant growth regulators to encourage continued division following induction. These protocol commonalities will assist researchers to identify approaches suited to their target Fabaceae species. The paucity of research funding for haploid research in most Fabaceae species has highlighted the need for strong collaborative linkages between institutions and researchers. Referees: Professor Laima Kott, Crop Science Department, University of Guelph, Guelph ON N1G 2W1.

Antigibberellin-induced reduction of internode length favors in vitro flowering and seed-set in different pea genotypes

In vitro flowering protocols were developed for a limited number of early flowering pea (Pisum sativum L.) cultivars. This work was undertaken to understand the mechanisms regulating in vitro flowering and seed-set across a range of pea genotypes. Its final goal is to accelerate the generation cycle for faster breeding novel genotypes. We studied the effects of in vivo and in vitro applications of the antigibberellin Flurprimidol together with radiation of different spectral compositions on intact plants, plants with the meristem removed, or excised shoot tip explants. Based on our results, we present a simple and reliable system to reduce generation time in vitro across a range of pea genotypes, including mid and late flowering types. With this protocol, more than five generations per year can be obtained with mid to late flowering genotypes and over six generations per year for early to mid flowering genotypes.

Evaluation and breeding of tedera for Mediterranean climates in southern Australia

Abstract. Tedera (Bituminaria bituminosa C.H. Stirton var. albomarginata and var. crassiuscula) has been identified as one of the most productive and drought-tolerant species of herbaceous perennial legumes based on 6 years of field evaluation in Western Australia in areas with Mediterranean climate and annual rainfall ranging from 200 to 600 mm. Importantly, tedera demonstrated broad adaptation to diverse soils, and some accessions have shown moderate levels of tolerance to waterlogging and salinity. Tedera exhibits minimal leaf shedding during summer and autumn. Economic modelling strongly suggests that giving livestock access to green tedera in summer and autumn will dramatically increase farm profit by reducing supplementary feeding. The breeding program (2006–12) evaluated the available genetic diversity of tedera for its field performance in seven nurseries with 6498 spaced plants in total covering a wide variation in rainfall, soils and seasons. Best overall plants were selected using a multivariate selection index generated with best linear unbiased predictors (BLUPs) of dry matter cuts and leaf retention traits. The breeding program also evaluated tedera for grazing tolerance, grazing preference by livestock, waterlogging tolerance, seed production, cold tolerance, disease susceptibility and presence of secondary compounds. Tedera is a diploid, self-pollinated species. Therefore, 28 elite parents were hand-crossed in several combinations to combine outstanding attributes of parents; F1 hybrids were confirmed with the aid of highly polymorphic, simple sequence repeat markers. The F1s were progressed to F4s by single-seed descent breeding. Elite parent plants were selfed for two generations to be progressed in the breeding program without hybridisation. Over time, selections from the crossing and selfing program will deliver cultivars of three ideotypes: (i) drought-tolerant, (ii) cold- and drought-tolerant, (iii) waterlogging- and drought-tolerant.

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.

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.

In Vitro-Assisted Compression of Breeding Cycles

The compression of breeding cycles to quickly progress segregating material to homozygosity has attracted substantial international research interest for some decades. Modified pedigree breeding methods such as single seed descent (SSD) have enabled faster generation turnover and commercialization of new crop cultivars. Since the latter part of the last century, doubled haploid technology has revolutionized the progression to genome fixation in responsive species. In unresponsive but economically important families, biotechnological tools are being developed to accelerate traditional SSD – either by completing the full plant life cycle in vitro or by coupling controlled environmental conditions in the soil to elicit rapid floral onset with germination of immature seed in vitro to truncate seed filling. Both techniques have resulted in step-change efficiencies in generation turnover with up to fourfold improvements in species such as grain legumes. Such enhanced SSD systems are also valuable for breeding complex traits across a range of species. In this chapter, we explore the recent advances in in vitro-assisted breeding cycle compression in crops, opportunities to combine rapid phenotyping for key traits and the benefits of in vitro life cycle completion when researching under restrictive regulatory frameworks and working with enfeebled or rare material.

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

Light is one of the main signals that regulates flowering. Low red to far-red ratios accelerate flowering in a wide range of species. The central gene pathways controlling flowering time in Arabidopsis, appear to be largely conserved in legumes. However, numerous examples exist of gene duplication and loss. The role of CONSTANS-LIKE genes as integrators of the photoperiod response has been questioned in several dicot species, including legumes. In this study on subterranean clover, using RNA-seq and controlled light spectra, we identified 13 differentially expressed genes related to light signalling, meristem identity and flowering promotion. Of these, we pinpointed genes which seem to link photoperiod and far-red light signalling coding for a With no lysine kinase, a CCT motif related to CONSTANS, a FLOWERING LOCUS T b2 like protein, and their active downstream cascade. The earlier down-regulation of these genes under blue compared to far-red-enriched light may explain their role in floral induction. A second independent approach (qPCR analysis) confirmed our findings. Contrasting responses to light quality related to reproduction and defence mechanisms were also found. These results will contribute to a better understanding of the molecular basis of flowering in response to light quality in long-day plants.

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).