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In Vitro Production of Sweet Peas (Lathyrus odoratus L.) via Axillary Shoots
Abstract
The genus Lathyrus is best known because it includes a number of wild relatives of the protein pea which, despite being generally neglected and under-utilised, hold considerable potential as a useful genetic resource for the acquisition of interesting stress resistant traits important for a sustainable agriculture. However, also included in this genus are important commercially produced species with a significant ornamental value, among which the sweet pea (Lathyrus odoratus L.). Surprisingly though, there are no formal reports on the in vitro propagation of this species and, generally, these are scanty for in vitro approaches with all species of Lathyrus. Here, we describe simple, yet reliable strategies for the culture and multiplication of several landraces and species of Lathyrus including sweet peas.
... were also recognized [79][80][81]. There were also a few reports describing tissue culture methodologies for regeneration and genetic transformation of grasspea [82][83][84][85], albeit with limited success. ...
Environmental perturbations are persistent threats to sustainable agriculture, and thus recruitment of resilient crops, especially legumes, exhibiting agronomically important traits has become a priority for plant biologists. It is of utmost importance that the neglected and underutilized legumes (NULs) are identified and utilized as source of germane genes and gene-products, through concerted research platforms. In the present article, we analyzed the current status of NULs with specific emphasis to the potent utility of grasspea owing to its unique characters including stress adaptation, nutritional superiority and ease of cultivation. We have highlighted the landmarks in the history of grasspea, delineating the rapid progress achieved in grasspea biology during the past decades. Despite possession of a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), this neglected legume outshines most food crops with its distinct physicochemical attributes, health and agricultural benefits and resilience to environmental constraints. With the availability of genome sequence, grasspea is now established as an appropriate genetic resource for sustainable agriculture and phytoremediation rendering its genes, proteins and metabolites for targeted genetic manipulation. We conclude that grasspea would serve as a resource for plant translational genomics (TG) research, particularly resilience of legumes to environmental challenges.
... Weber et al. [39] used apical meristems explants of the interspecific hybrids originating from crosses between H. annuus and nine others species of Helianthus, and concluded that a species regeneration capacity was significantly determined by the individual genotype. Variability in the organogenic responses from the axillary buds among genotypes was observed in many other species including Rosa rugosa [72], Vigna unguiculata [73], and Lathyrus sativus [74]. The observed variation in the morphogenetic response among the six tested plants of H. verticillatus warrants future studies for the levels of endogenous PGRs in the context of genotype and stem localization. ...
Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part of the recovery plan for the restoration and protection of H. verticillatus, an efficient micropropagation protocol based on axillary shoot proliferation was developed. Various concentrations of 6-benzylaminopurine (BAP; 0 to 4.44 µM) were examined for their morphogenetic potential in the regeneration of six genotypes of H. verticillatus from the nodal explants derived from greenhouse-grown plants. Both the BAP concentration and genotype had significant effects on the regeneration capacity of H. verticillatus. Although the induced buds were observed on ½-strength Murashige and Skoog medium without plant growth regulators, a higher rate of induction and bud development were achieved on media with either 0.88 or 2.22 µM BAP, regardless of the genotype. Successful rooting of the induced shoots was achieved within four weeks after the transfer from the induction medium to the fresh ½-strength MS medium, but the rooting efficiency was dependent on the plant’s genetic background. Regenerated plantlets, with well-developed shoots and roots, were acclimatized successfully to greenhouse conditions with a 97% survival rate. Simple sequence repeats (SSRs) markers were employed to assess the genetic uniformity of the micropropagated plants of H. verticillatus. No extraneous bands were detected between regenerants and their respective donor plants, confirming the genetic fidelity and stability of regenerated plants. To our knowledge, the protocol developed in this study is the first such report for this endangered species.
... These could be the fi rst steps into the introduction of Ascochyta blight resistance from grass pea into pea (McCutchan et al. 1999;Ochatt et al. 2001Ochatt et al. , 2004Ochatt et al. , 2007, of resistance to rust fungi from L. cicera ), of broomrape resistance from L. ochrus or from L. cicera , or yet of powdery mildew resistance from sweet pea into grass pea (Poulter et al. 2003;Ochatt et al. 2010a). ...
Grass pea (Lathyrus. sativus L.) is an annual legume crop with high protein content and remarkable resistance to extreme environmental conditions, including fl ooding, drought, salinity and low soil fertility, and a signifi cant degree of resistance to biotic stress agents. It is rightly considered 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 and as an interesting alternative for cropping systems diversifi cation in marginal lands in Europe, Australia and America. It is a dual purpose crop with great agronomic potential as a grain and forage legume. Nevertheless, 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. The presence of a neurotoxin proposed to be responsible for lathyrism in humans and animals has also contributed to its abandonment. Recently, there has been renewed interest in the improvement of this high-potential legume species, with the integration of new breeding technology that anticipates great achievements in the development of new varieties.
... Mutagenesis (Biswas 2007) and biotechnology (McCutchan et al. 1999;Ochatt et al. 2001) via in vitro selection (Lambein and Kuo 2004), somatic hybridization (Durieu and Ochatt 2000), gene transfer (Barik et al. 2005) or haplo-diploidization (Ochatt et al. 2009) would permit production of novel grass pea genotypes twinning robustness with better adaptation for human consumption, and introduction of interesting traits from Lathyrus into crossincompatible species such as field pea. On the other hand, Lathyrus odoratus, the sweet pea, is an important ornamental species, but it has seldom been studied in vitro (Razdan et al. 1980;Ochatt et al. 2010). Biswas (2007) has recently reviewed mutation breeding in Lathyrus, where most studies have concerned L. sativus, yielding a wide spectrum of morphological mutations, affecting canopy structure (plant habit, maturity, branching, stem shape, leaf size, stipule shape), flower colour and structure, pod size, and seed size and colour (Nerkar 1976;Biswas 2007;Rybínski 2003). ...
The genus Lathyrus includes a number of neglected wild relatives of pea with potential as genetic resources for acquisition of stress resistance traits, but, due to little breeding, genotypes under culture are mainly landraces and seldom true varieties. Development of in vitro approaches for Lathyrus is also limited, and assessments of nuclear DNA content, for taxonomical or breeding purposes, are sparse. Genome size and AT/GC ratio were determined by flow cytometry, allowing for distinction between protein and forage L. sativus, L. cicera, L. ochrus and L. clymenum and the ornamental sweet pea (L. odoratus), and also between landraces within L. sativus L. and L. cicera L. In addition, explants from in vitro seedlings of eight genotypes from the five Lathyrus species above were cultivated in vitro, plant regeneration was achieved for all landraces and species, and the nuclear DNA content of the regenerants was compared with that of their mother plants, whereby the true-to-typeness of such regenerants was confirmed.
Prod 2018-143 BAP GEAPSI CT1 BAP
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.
Different explants including root, cotyledon (both whole and sectional), hypocotyl, epicotyls, internode, node, axillary buds, petiole, leaf and shoot tip meristem were tested for their callusing response in B5 medium supplemented with 2,4-D (2mg/l) + BAP (0.5mg/l) and B5with NAA (2mg/l) + BAP (0.5mg/l) for callus induction; colour, texture and relative growth of calli. The callus induction medium B5 + 2,4-D(2mg/l) + BAP (0.5mg/l) was better for callus induction (%) than B5+ NAA(2mg/l) + BAP (0.5mg/l) for all explants. Leaf pieces followed by internode explant induced very high callus induction frequency and both produced excellent callus growth. Though leaf explants induced very high callus induction frequency(CIF %), but whitish yellow and soft texture of calli in the former and hard, compact, greenish white calli induced in the latter media; were not supposed to be ideal for regeneration. In this regard, internode explants may be opted for organogenic regeneration due to induction of hard, loose, nodular and dark green calli in B5+ NAA(2mg/l) + BAP (0.5mg/l). Nodal and leaf segments yielded high frequency of direct shoot regeneration(15-20%) in B5+ NAA (2mg/l) + BAP (0.5 mg/l). Induction of highly regenerative calli and the direct regeneration system obtained in this investigation could be amenable for genetic transformation in Lathyrus sativus.
In order to gain a better understanding of the genetical relationship among species of the genus Lathyrus, investigations on the morphological characteristics as well as karyotypes of the plants were carried out. Twenty four strains from twenty annual and perennial species representing the main sections of the genus were used. All the species analyzed had chromosome numbers of 2n=14 except fcr one strain (No.1)of L.pratensis with 2n=28. Karyotypic differences among species involved the shape and size of satellite chromosomes, the length of the arm with satellite, the ratio of the satellite chromosome length to the total length of all the chromosomes, the length and arm ratio on the individual chomosomes. The karyotypes were classified into six types A, B, C, D, E and 4X. The karyo type A was commonly observed in all the sections studied, while the others were all modified karyotypes and detected in a limited number of sections. Morphological characteristics such as configuration of stems, leaves, leaflets, veins, stipules, racemes, styles, pods and sutures were similar to those previously reported by DAVIS (1970) and MEIKLE (1977). Itappears that morphological changes from the primitive to the advanced form, based on the characters of this genus described by BASSLER(1966) and SIMOLA (1968), might correspond to karyotypic changes from the common to the modified type, as suggested by STEBBINS (1971).
Introduction Grasspea (Lathyrus sativus L.) is produced as major crop in Bangladesh, China, India and Pakistan, and to a lesser extent in many countries of Europe, the Middle East, northern Africa, as well as in South America. It is extensively cultivated and naturalised in the Middle East countries of Iraq, Iran, Afghanistan, Syria and Lebanon and in Ethiopia, Egypt, Morocco, Algeria and Libya in northern Africa (1) . In Europe grasspea was cultivated in the Balkans by around 8000 BC (4) . Foods prepared from the seeds have been popular in Europe and are still being used locally in Spain, France, Germany and Bulgaria. According to Milczak (5) grasspea was introduced to Poland (Podlasie Region) in the 17 th century, probably with arrival and settling of the Tatars. Now grasspea is grown on a marginal scale in Eastern Poland, it is cultivated as a vegetable and is locally known as "soczewica podlaska" (Podlaska lentil). The first plant material for breeding purposes in 1991 constituted a local population from the village of Derewiczna. As results of breeding work in the following few years, in cooperation with the Breeding Station for Vegetables in Nochowo, the first Polish cultivars: Derek and Krab were released (5) . The main advantages of grasspea are: resistance to diseases and environmental stress conditions, high yielding potential (at the level of pea) and favourable nutritional composition of seed. Grasspea is very tolerant to drought, due to a very hardy and penetrating root system and can be grown on a wide range of soil types, including very poor soils. Undesirable features such as prostrate plant habit, indeterminate growth, late maturity, pod shattering and neurotoxin content (ODAP) are the most important factors limiting the broader introduction of grasspea for growing in different environmental conditions.
Low ODAP somaclones from root and internode explants were evaluated for physiological and biochemical parameters. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. All the somaclones during development had substantially lower ODAP content in leaves compared to parent P 24. Amongst somaclones, Bio R 202 had the maximum leaf area, biomass as well as photosynthetic rate. Harvest index and seed yield were highest in Biol 222 whereas ODAP content was the least in it.
Somaclones of Lathyrus sativus having low ODAP contents were characterised at molecular level with respect to soluble protein pattern, esterase and ADH isozyme pattern, Southern hybridisation of genomic and mitochondrial DNA with specific probes. In 10-day-old seedling esterase isozyme pattern showed the presence of an unique band of Rm 0.73 in Bio L-12 and 15-8-1 and the absence of a band of Rm 0.80 in 15-8-1, as compared to parent and other somaclones. Southern hybridisation of genomic DNA with cDNA clone 29 showed an extra band of 13.5 kb in L-56 and 15-8-1 only. Hybridisation of mitochondrial DNA with mitochondrial specific ati ATPase probe showed differences with the pattern from Bio R-15 being unique. Some of these differences observed will be useful as marker for their identification.
Soluble proteins in six somaclones and the parent at three different stages of development of Lathyrus sativus seeds showed polypeptides of mol wts ranging from 20–165 kD on SDS-PAGE. Immature seeds showed eight distinct esterase bands and two peroxidase bands. Pattern for both the enzymes among somaclones was almost similar. During maturation a new isoform of peroxidase with high mobility appeared in all the somaclones, while one new isoform of intermediate mobility was visible in somaclones Bio L-08 and Bio I-08. Cytological analysis of all the somaclones confirmed the stability of the chromosome count of 2n=14. At metaphase I seven distinct bivalents and at anaphase I and II univalents showing complements of the seven chromosomes were visible. No structural or chromosomal aberration were observed among somaclones.
Low ODAP somaclones have been evaluated for physiological and biochemical parameters especially in relation to attributes that lead to increased biomass production. All the somaclones during development had substantially lower ODAP content in leaves as compared to parent P24. Considerable variation was observed in relation to leaf width, leaf length, internodal length and leaf area. Somaclone Bio L12 had the highest whereas parent P24 and Bi0164 had the least leaf area. Harvest index was the highest and biomass production was the lowest in the Bio 164. Bio L08 gave the highest seed yield. Photosynthetic rates were also higher in Bio L12, although no significant positive correlation was observed in leaf photosynthesis and seed yield. The differences in physiolpgical and biochemical parameters indicate the possibility of development of high yielding genotypes. The results in present investigation show differences in photosynthetic rate, leaf characteristics, seed yield and ODAP content among somalones and parent. Somaclones with extremely low ODAP content with variability in leaf morphology and photosynthetic rate is indicative of variation induced during plant tissue culture.
Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B5 medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R1 generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R2 generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.
Leaf protoplasts, isolated from three-month old sweet pea plants, entered division, which was sustained with the eventual production of cell colonies. These colonies gave rise to proliferating callus masses.