Monika Lulsdorf

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.

Endogenous hormone profiles during early seed development of C. arietinum and C. anatolicum

Cicer anatolicum, a perennial species, has ascochyta blight resistance superior to that found in the cultivated chickpea. However, hybridization barriers during early stages of embryo development curtail access to this trait. Since hormones play an essential role in early embryo development, we have determined the hormone profiles of 4-, 8-, and 12-day old seeds from a Canadian chickpea (Cicer arietinum L.) cv. CDC Xena, from Indian cvs. Swetha and Bharati, and from a perennial accession of C. anatolicum (PI 383626). Indole-3-acetic acid content peaked on day 4 in CDC Xena, on day 8 in both Indian cultivars but only on day 12 in C. anatolicum. The cytokinins, isopentenyladenosine (iPA) and trans zeatin riboside (tZR) were predominant in CDC Xena and Swetha seeds on day 4, whereas cis zeatin riboside was the major component in Bharati. In C. anatolicum, iPA maxed out on day 4 and tZR on day 12. The bioactive gibberellin GA1 spiked on day 4 in CDC Xena and Bharati, on day 8 in Swetha but only on day 12 in C. anatolicum. Eight-day old seeds had the highest abscisic acid content in the cultivars but spiked on day 12 in the perennial species. The hormone profiles of the perennial species showed delayed spikes in all four hormone groups indicating that there is a mismatch in the hormone requirements of the different embryos. Improving synchronization of early seed hormone profiles of cultivated and perennial chickpea should improve interspecific hybrid production.

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.

Androgenesis-inducing stress treatments change phytohormone levels in anthers of three legume species (Fabaceae)

AbstractLegumes are recalcitrant to androgenesis and induction protocols were only recently developed for pea (Pisumsativum L.) and chickpea (Cicer arietinum L.), albeit with low regeneration frequencies. Androgenesis is thought to be mediated through abscisic acid (ABA) but other phytohormones, such as auxins, cytokinins, and gibberellins, have also been implicated. In view of improving induction protocols, the hormone content of pea, chickpea, and lentil anthers was measured after exposure to cold, centrifugation, electroporation, sonication, osmotic shock, or various combinations thereof using an analytical mass spectrometer. Indole-3-acetic acid (IAA) had a key function during the induction process. In pea, high concentrations of IAA-asparagine (IAA-Asp), a putative IAA metabolite, accumulated during the application of the different stresses. In chickpea, the IAA-Asp concentration increased 30-fold compared to pea but only during the osmotic shock treatment and likely as a result of the presence of exogenous IAA in the medium. In contrast, no treatment in lentil (Lens culinaris) invoked such an increase in IAA-Asp content. Of the various cytokinins monitored, only cis zeatin riboside increased after centrifugation and electroporation in pea and possibly chickpea. No bioactive gibberellins were detected in any species investigated, indicating that this hormone group is likely not linked to androgenesis in legumes. In contrast to the other stresses, osmotic shock treatment caused a reduction in the levels of all hormones analyzed, with the exception of IAA-Asp in chickpea. A short period of low hormone content might be a necessary transition phase for androgenesis induction of legumes. Key message Five androgenesis-inducing stress treatments changed content of ABA, auxin and cytokinin in anthers of three legumes. Osmotic shock treatment differed because it reduced hormone content to very low levels.

WILD RELATIVES AND BIOTECHNOLOGICAL APPROACHES

Wild species of the genus Lens are an important source of genetic variation for breeding lentil varieties adaptable to new environments and tolerant of biotic and abiotic stresses. The wild species are endemic to a wide range of environments and possess many diverse characteristics. Lens species can be divided into three groups, a primary, secondary and tertiary gene pool, according to their inter-crossability. Crosses between members of the different genepools generally fail because the hybrid embryos abort. However, embryo rescue has been used successfully to obtain viable hybrids between groups. It is possible to intercross most of the wild Lens species with cultivated lentils using plant growth regulators and/or embryo rescue to allow the growth of hybrid plants. Other biotechnology techniques which may impact on lentil breeding include, micropropagation using meristamatic explants, callus culture and regeneration, protoplast culture and doubled haploid production. Micropropagation and regeneration from callus culture are relatively well established techniques with further research required for the development of reliable protoplast regeneration and doubled haploid protocols

Methods and Role of Embryo Rescue Technique in Alien Gene Transfer

Embryo abortion occurs frequently in wide crosses, and thus embryo rescue is required for survival of the next generation. Rescues are performed by either directly transferring the excised embryo to an artificial medium or indirectly through flower (ovary), immature seed (fertilized ovule), or pod (silique) culture. Various techniques used for oil crops, cereals, legumes, and horticultural crops are presented. Altering medium components were the major routes for developing protocols for each species with adaptations to the base medium, sucrose concentration, or vitamin and growth regulator content. Monocot culture tended to be more direct than dicot culture, where many protocols required a multi-step approach from pod to ovule culture to embryo rescue, shoot regeneration, and root induction. Each step required a specific medium and growth conditions. Hybrid embryos as young as 2 days after pollination have been recovered. However, many species such as soybean and chickpea still need procedures for rescue of very young embryos. In other species hurdles such as poor rooting have been overcome by using grafting techniques. Embryo rescue remains a useful component in any breeding program where wide or interspecific crosses are preformed, where rapid cycling through generations is used, and where germplasm preservation is required.

Improvement of embryo rescue technique using 4-chloroindole-3 acetic acid in combination with in vivo grafting to overcome barriers in lentil interspecific crosses

Widening the genetic base of minor grain legume crops such as lentil (Lens culinaris Medik.) is important for achieving future gains in productivity. In order to access genes from wild lentil species embryo rescue techniques are required to overcome reproductive barriers. Removing the seed coat from developing 14-day old immature hybrid seeds and culturing the interspecific zygotic embryos in medium containing auxin 4-chloroindole-3 acetic acid (4-Cl-IAA) improved successful hybrid recovery. Addition of 4-Cl-IAA to media also increased shoot proliferation when combined with a low concentration of zeatin. No significant difference in shoot elongation was observed between 4-Cl-IAA or IAA treatments. Hybrid shoots were then successfully grafted in vivo onto faba bean rootstocks. Hybrids were obtained from crosses of L. culinaris with L. tomentosus Ladiz., L. lamottei Czef., and L. odemensis Ladiz. This efficient and simple embryo rescue protocol resulted in seed production of large interspecific F2 populations from inherently weak zygotic embryos produced from wide hybridization.