The effect of auxins in inducing organogenesis or somatic embryogenesis in mature sunflower zygotic embryo derived apex

Induction of shoots or of somatic embryos is the key step for gaining the morphogenetic potential in sunflower (Helianthus annuus L.), species known as recalcitrant to in vitro regeneration. In the immature zygotic embryo derived tissues or in other juvenile tissues resulted from seedlings, the acquisition of the competence for regeneration can be achieved directly by cytokinin treatment or by preconditioning the explants on cytokinin containing medium. In this paper is presented a new type of explant for sunflower in vitro culture, consisting of the apex with primordial leaves, resulted from ungerminated mature zygotic embryo, in which a specific morphogenetic response was triggered by the exogenously applied auxins. Among the auxins tested, indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid are inducers of an organogenetic response, apical/axillary shoots and adventitious buds being regenerated while 2,4-dichlorophenoxyacetic acid, 3,6-dichloro-2-methoxybenzoic acid and 4-amino-3,5,6-trichloropicolinic acid led to somatic embryo formation. Among the auxins tested only 4-amino-3,5,6-trichloropicolinic acid sustains the embryos development up to mature stage. A high amount of sucrose (120 g L-1) supplied during the auxin treatment promotes the maturation of the embryos directly on the induction medium for all tested auxins with embryogenic effect. These findings show that regardless of the type of morphogenetic response aimed in sunflower meristematic tissues resulted from mature embryos, the presence of auxins is mandatory

Biotechnological Strategies for a Resilient Potato Crop

The aim of this chapter is to describe in a synthetic manner the most efficient biotechnological techniques which can be applied in potato breeding with emphasis on multiple resistance traits. To this end, most important results of all biotechnological techniques will be pointed out including new biotechnological tools of genome editing. The somatic hybridization will be the core of the presentation as the only non-GMO strategy with good results in transferring multiple resistances into potato gene pool. The chapter is presenting all data in a synthesized form and made comparisons between the existing techniques and their possible adoption in breeding in different parts of the world, depending on regulations and consumer choice. Moreover, the recently discovered value of potato as a healthy food and its possible applications in cancer treatment will be also discussed with new data on both potato and some of its wild relatives

In vitro culture and medium-term conservation of the rare wild species Gladiolus imbricatus

Gladiolus imbricatus, a rare species spread in the hill region of Europe, is resistant to abiotic and biotic stress being one of the most cold-tolerant in the genus. Moreover it contains high vitamin C and minerals in the leaves and the flowers are considered as edible. The aim of our study was to develop in vitro technologies for micropropagation, multiplication, corm development, somatic embryogenesis and medium-term storage of this endangered species, Red listed in South-Eastern and Central European countries. Initiation of in vitro cultures was started from wounded seeds or excised mature embryos. Micropropagation and multiplication was achieved on Murashige Skoog (MS) solid medium with 30 g L-1 sucrose and 1 mg L-1 N-6-benzyl adenine (BA). The callus developed from wounded seeds on a richer MS medium proved to be embryogenic. Increased concentrations of sucrose promote corm development as also shown for other genotypes of cultivated gladioli. Further on a two-step culture protocol, on solid and liquid shaken MS based media, each for 6 months was done to evaluate the effects of acetic acid and ancymidol on corm development. Acetic acid + sucrose 9%, alone or in combination with ancymidol stimulated corm formation and yield. For medium term conservation maintenance of the in vitro cultures in low temperature in the dark proved to be the best in reducing the growth rate of the shoots after 3, 6 and 12 months. The recovery of plant growth was evaluated after 12 months, by the transfer to normal growth conditions. 25% of the plants were recovered after one year storage. Besides the importance for biotechnology, the in vitro techniques described here might be used, after molecular analysis of genetic stability, for the restoration of natural populations into the habitats where this species became extinct.Keywords: Acetic acid, ancymidol, corm development, somatic embryogenesi

In Vitro Culture as a Stressful Factor Triggers Changes in Polyphenols, Flavonoids and Antioxidant Activity in Somatic Hybrids between Solanum tuberosum and S. bulbocastanum and their Respective Parents

Phenols, important secondary metabolites in plants, are responsible for specific defence mechanisms against abiotic stress, due to their strong antioxidant activity. Flavonoids, as part of phenolic group, are also involved in plant stress responses, being primarily responsible for photo-protection against UV solar radiation. Based on these premises, the plant response to optimized in vitro culture was evaluated, by quantifying the total polyphenolic content, the total flavonoid content and the antioxidant activity, both under in vitro and ex vitro conditions. Four closely related potato genotypes were analysed: the wild species Solanum bulbocatanum, S. tuberosum cv. ‘Rasant’ and two somatic hybrids between them. For all genotypes, both total polyphenolic content and antioxidant activity were increasing under optimized in vitro culture. The shoot responses were genotype dependent and the two somatic hybrids were intermediate between the parents both as morphology and reaction to in vitro stress. The somatic hybrid 1508/5, having morphology similar to potato, was reacting as the wild species, while the somatic hybrid 1508/2, with a similar morphology to the wild species was reacting as potato to in vitro stress. The somatic hybrid 1508/5 being also resistant to late blight is of interest for further use in pre-breeding. Total flavonoid content is decreasing under in vitro as compared to ex vitro conditions, UV-B radiation, the major trigger of flavonoid biosynthesis being absent in the fluorescent light. This study reveals the effect of in vitro culture on flavonoid content and details aspects of the biochemical parameters involved in plant in vitro stress

Biochemical profile, selective cytotoxicity and molecular effects of Calendula officinalis extracts on breast cancer cell lines

Calendula officinalis extracts have been known to possess anti-tumor properties, but questions regarding their mechanisms of action still need to be answered. Therefore, the present study aims to investigate the selective cytotoxicity, the biochemical profile and the corresponding molecular effects of two extracts of C. officinalis: flowers and leaves, against several breast cancer cell lines in vitro. Dry flowers and leaves were subjected to ultrasonication assisted extraction in methanol 70%. The phenolic and volatile profiles of the extracts, determined by HPLC-MS and nontargeted GC-MS, revealed high levels of specific phenolic acids, flavonols and coumarin and several volatile compounds, including mono- and sesquiterpenes, ketones, aldehydes and esters. Both extracts proved to possess selective cytotoxic activities against tumor cells in comparison to healthy endothelial cells, according to the MTT assay. The flower extract was superior in terms of both cytotoxicity and selectivity when compared to the leaf extract, in accordance to their biochemical profiles. The gene expression pattern for 10 genes of interest was evaluated by RT-qPCR. The expression level of several genes involved in apoptosis (BCL2, BAX, BBC3, ZMAT3), and cell cycle progression (NFkB, CCND1, STAT3) was modulated by the treatment with both extracts. Therefore, C. officinalis extracts proved to be rich in compounds characterized by cancer-related cytotoxicity and are capable of inducing selective cytotoxicity on breast cancer cell lines

Drought and saline stress tolerance induced in somatic hybrids of solanum chacoense and potato cultivars by using mismatch repair deficiency

Global climate change, especially when involving drought and salinity, poses a major challenge to sustainable crop production, causing severe yield losses. The environmental conditions are expected to further aggravate crop production in the future as a result of continuous greenhouse gas emissions, causing further temperature rise and leading to increased evapotranspiration, severe drought, soil salinity, as well as insect and disease threats. These suboptimal growth conditions have negative impact on plant growth, survival, and crop yield. Potato is well known as a crop extremely susceptible to drought, which is primarily attributed to its shallow root system. With potato being the fourth major food crop, increasing potato productivity is thus important for food security and for feeding global population. To maintain a sustainable potato production, it is necessary to develop stress tolerant potato cultivars that cope with the already ongoing climate change. The aim of our study is to analyze the response of potato somatic hybrids to drought and salt stress under in vitro conditions; the somatic hybrids studied are the wild relative Solanum chacoense (+) Solanum tuberosum, with or without mismatch repair deficiency (MMR). Upon this selection of drought and salt tolerant genotypes, somatic hybrids and their parents were phenotyped on a semi-automated platform, and lines tolerant to medium water scarcity (20% compared to 60% soil water capacity) were identified. Although none of the parental species were tolerant to drought, some of the MMR-deficient somatic hybrids showed tolerance to drought and salt as a new trait. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

New Phenotypes of Potato Co-induced by Mismatch Repair Deficiency and Somatic Hybridization

As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of MSH2 gene may induce a ‘mutator’ phenotype and microsatellite instability (MSI), as is demonstrated in MSH2 knock-out and silenced lines of Arabidopsis thaliana. The goal of this study was to screen for ‘mutator’ phenotypes in somatic hybrids between potato cvs. ‘Delikat’ and ‘Désirée’ and MMR deficient Solanum chacoense transformed using antisense (AS) or dominant negative mutant (DN) AtMSH2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with ‘mutator’ phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene nptII, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species