Sergio J. Ochatt

Lathyrus improvement for resistance against biotic and abiotic stresses: From classical breeding to marker assisted selection

SummarySeveral Lathyrus species and in particular Lathyrus sativus (grass pea) have great agronomic potential as grain and forage legume, especially in drought conditions. Grass pea is rightly considered as one of the most promising sources of calories and protein for the vast and expanding populations of drought-prone and marginal areas of Asia and Africa. It is virtually the only species that can yield high protein food and feed under these conditions. It is superior in yield, protein value, nitrogen fixation, and drought, flood and salinity tolerance than other legume crops. Lathyrus species have a considerable potential in crop rotation, improving soil physical conditions; reducing the amount of disease and weed populations, with the overall reduction of production costs. Grass pea was already in use in Neolithic times, and presently is considered as a model crop for sustainable agriculture. As a result of the little breeding effort invested in it compared to other legumes, grass pea cultivation has shown a regressive pattern in many areas in recent decades. This is due to variable yield caused by sensitivity to diseases and stress factors and above all, to the presence of the neurotoxin β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), increasing the danger of genetic erosion. However, both L. sativus and L. cicera are gaining interest as grain legume crops in Mediterranean-type environments and production is increasing in Ethiopia, China, Australia and several European countries.This paper reviews research work on Lathyrus breeding focusing mainly on biotic and abiotic resistance improvement, and lists current developments in biotechnologies to identify challenges for Lathyrus improvement in the future.

Flow Cytometry in Plant Breeding

Since the first report on the flow cytometric study of plant material 35 years ago, analyzing the nuclear DNA content of field bean, an ever increasing number of applications of FCM has been developed and applied in plant science and industry, but a similar length of time elapsed before the appearance of the first complete volume devoted to FCM of plant cells. Most published information on the uses of FCM addresses various aspects of animal (including human) cell biology, thus failing to provide a pertinent substitute. FCM represents an ideal means for the analysis of both cells and subcellular particles, with a potentially large number of parameters analyzed both rapidly, simultaneously, and quantitatively, thereby furnishing statistically exploitable data and allowing for an accurate and facilitated detection of subpopulations. It is, indeed, the summation of these facts that has established FCM as an important, and sometimes essential, tool for the understanding of fundamental mechanisms and processes underlying plant growth, development, and function. In this review, special attention is paid to FCM as applied to plant cells in the context of plant breeding, and some new and less well‐known uses of it for plants will be discussed.

The role of the DNA-binding One Zinc Finger (DOF) transcription factor family in plants.

The DOF (DNA-binding One Zinc Finger) family of transcription factors is involved in many fundamental processes in higher plants, including responses to light and phytohormones as well as roles in seed maturation and germination. DOF transcription factor genes are restricted in their distribution to plants, where they are in many copies in both gymnosperms and angiosperms and also present in lower plants such as the moss Physcomitrella patens and in the alga Chlamydomonas reinhardtii which possesses a single DOF gene. DOF transcription factors bind to their promoter targets at the consensus sequence AAAG. This binding depends upon the presence of the highly conserved DOF domain in the protein. Depending on the target gene, DOF factor binding may activate or repress transcription. DOF factors are expressed in most if not all tissues of higher plants, but frequently appear to be functionally redundant. Recent next-generation sequencing data provide a more comprehensive survey of the distribution of DOF sequence classes among plant species and within tissue types, and clues as to the evolution of functions assumed by this transcription factor family. DOFs do not appear to be implicated in the initial differentiation of the plant body plan into organs via the resolution of meristematic zones, in contrast to MADS-box and homeobox transcription factors, which are found in other non-plant eukaryotes, and this may reflect a more recent evolutionary origin.

Improved microspore culture and doubled-haploid plant regeneration in the brown condiment mustard (Brassica juncea)

Abstract The availability of doubled haploids could greatly contribute to improving seed quality in condiment mustard (Brassica juncea). We have developed an efficient and reliable protocol of microspore culture, modified from that of Baillie et al. (1992), based on a modification of the sucrose concentration of culture media. A comparison of microspore culture media differing in their sucrose content showed that a decrease from 17% (w/v) sucrose during the first 48 h to 10% (w/v) thereafter favoured an increase in the production of embryos whatever the responding genotype tested. Thus, out of the 13 B. juncea genotypes studied, 12 gave rise to embryos, and seven of these embryos could be converted into plants. Doubled-haploid plants were produced after treatment with colchicine.

Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae).

To induce androgenesis in field pea, grass pea and the model legume species Medicago truncatula, isolated microspores of various genotypes of these three species were submitted to a range of abiotic stresses prior to and during their initial culture, in order to stimulate them to divide and form embryos. Some stress agents had a positive effect on androgenesis from the treated microspores. Submission of flower buds to a cold period prior to anther excision or microspore isolation, modifying the osmotic pressure of the medium during initial culture and electroporation of isolated microspores were the three major individual stress agents to have an impact on the efficiency of androgenetic proliferation and subsequent differentiation from the microspores of pea, grass pea and M. truncatula genotypes. A combination of osmotic and electric shocks significantly improved responses from isolated microspores and yielded microcalluses and then calluses, but only few underwent morphogenesis. Further work is under way to improve responses and extend them to other genotypes. The results reported here are, to the best of our knowledge, the first successful results from isolated microspores of these species.

Fertile pea plants regenerate from protoplasts when calluses have not undergone endoreduplication.

Large numbers of viable protoplasts were isolated and cultured from five pea genotypes. Calluses obtained (percent final plating efficiency (% FPE)=0.65-2.82% of initially plated protoplasts) exhibited great differences in proliferation and regeneration competence between and within genotypes. Flow cytometric analyses showed the occurrence of endoreduplication processes correlated with such differences, and could serve as a tool for the early prediction of plant regeneration competence from protoplasts. Fertile plants were produced only from calluses with a normal DNA level.

Ploidy level determination within the context of in vitro breeding

Variations in genome size and chromosome complement of species provide very useful information for biosystematic studies, and also because they influence a range of ecological characteristics. They are also of utmost importance for breeding, especially when in vitro biotechnology tools are used and the need arises to assess the trueness-to-type of regenerated plants. Thus, protocols have existed for a long time for chromosome counting, and more recently also for the determination of the relative nuclear DNA content and genome size. It has also been shown that these latter traits are strongly correlated to regeneration competence and, more generally, to developmental processes in plants. This article will briefly review such approaches from a methodological and breeding-oriented viewpoint.

The growth regulators used for bud regeneration and shoot rooting affect the competence for flowering and seed set in regenerated plants of protein peas

SummaryThe production of whole plants from explants of protein pea (Pisum sativum L.) using an efficient, reliable and rapid strategy, while maintaining trueness to type, will be required before regeneration can be exploited for genetic transformation. Seeds of the pea genotypes Terese, Solara, Frisson and P64 (a hypernodulating mutant line of Frisson) were surface-sterilized and imbibed overnight, whereafter embryo axes were dissected and germinated on hormone-free medium for 7–10 d. Hypocotyl sections lacking pre-existing meristems were harvested and cultured on a range of media with various concentrations and combinations of growth regulators in order to induce either caulogenesis or somatic embryogenesis. Differences in responsiveness were apparent between genotypes, but regeneration via caulogenesis was consistently more reliable than via the induction of somatic embryos. Few explants underwent somatic embryo production and their conversion into plants has remained elusive so far, irrespective of the genotype studied. Conversely, large numbers of buds were produced within 10 d by organogenesis, and healthy, rootable shoots were obtained. A clear relationship was observed between the growth regulators employed for bud regeneration and shoot rooting phases and the subsequent competence of the regenerated plants for flowering, pod formation and viable seed production.

In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plants

A stable salt-tolerant potato cell line, able to grow on media containing 60–450 mM NaCl (i.e. low to high salinity) was selected. Callus grown on 120 or 150 mM NaCl showed higher fresh weights than the rest of the treatments. Replacing NaCl by KCl or Na2SO4 showed that reductions in fresh weight were mainly due to the presence of Na+ ions. When PEG 6000 was added to the medium instead of salt, the salt tolerant cell lines were unable to overcome the PEG-induced water stress. Whole plants, regenerated from salt tolerant callus, exhibited salt stress tolerance as evidenced by their higher fresh and dry weights when watered with 90 mM NaCl, and they also produced more tubers per plant under salt stress. Salt-tolerant plants differed phenotypically from control plants both in terms of leaf shape, tuber flesh and skin colour, which was reddish. In addition, DNA fingerprinting by RAPDs, with 70 different primers, confirmed that the salt tolerant regenerants also differed genotypically from the control, salt sensitive Kennebec potato plants from which they had been selected.

The hyperhydricity ofin vitroregenerants of grass pea (Lathyrus sativus L.) is linked with an abnormal DNA content

Summary The grass pea ( Lathyrus sativus ) is a wild relative of the protein pea which may be a useful genetic resource for the acquisition of interesting stress resistance traits. However, grass pea is cross incompatible with pea, leaving protoplast fusion as the only alternative to produce interspecific hybrids of grass pea and pea. In addition, as all grass pea seeds contain a toxic aminoacid, low-toxin containing genotypes will have to be produced by gene transfer. In this context, it is therefore essential that regenerated plants are fertile, true-to-type and not chimaeric in nature when they have been obtained in absence of any selection treatment. In the present study, shoot buds were regenerated from hypocotyls of three grass pea genotypes, and flow cytometry permitted us to characterise them in terms of nuclear DNA content. Plant regeneration competence was genotype-dependent and strongly also was correlated with a normal DNA content. The auxin/cytokinin balance of regeneration media affected the DNA level of regenerants. In turn, an abnormal DNA content was systematically associated with severe hyperhydricity symptoms, which hampered the regeneration of rooted, fertile plants.