In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.
The aim of this study was (i) to produce tissue cultures capable of efficient plant regeneration from European naturally occurring protected and/or pharmacologically important Amaryllidaceae species and (ii) to test them for antioxidant activities in order to select tissue cultures that scavenge efficiently oxygen radicals. Bulb explants were collected from Galanthus woronowii, two Leucojum species, and Sternbergia lutea. Leucojum species were Hungarian isolates. Mostly α-naphthalene acetic acid (NAA) and benzyladenine (BA) were used as growth regulator combinations for the induction and maintenance of tissue cultures and further antioxidant activity studies. Galanthus woronowii and L. vernum cultures produced shoots or whole plants via micropropagation (callus stage was observed only sporadically and callus tissue did not contribute to regeneration), whereas L. aestivum and S. lutea produced efficiently whole plants or multiple shoots via embryogenic calli. Total phenolic content, % inhibition of ABTS radical (ABTS*) cation, and peroxidase activities on native polyacrylamide gels were studied and showed differences between cultures. No relationship could be detected between polyphenol content / radical scavenging capacities and H 2 O 2 reducing enzyme activities. For G. woronowii, S. lutea, and a culture line of L. vernum, polyphenol content and ABTS* cation scavenging activities were high and for G. woronowii, comparable to organs of the native plants used as explant sources. Bulbs of native plants showed low radical scavenging activities in general. For L. vernum and L. aestivum tissue cultures grown in the presence of NAA as the sole growth regulator, ABTS* cation scavenging showed low values. Enzymatic antioxidant (pyrogallol peroxidase) activities were low for all cultures and organs of native plants. This study shows the species conservation value of these cultures and highlights the high antioxidant capacity of G. woronowii and S. lutea, attributed to the presence of non-enzymatic scavengers.
We aimed to produce tissue cultures and plant regeneration from endangered Crocus species: C. scepusiensis, C. tommasinianus, C. vittatus ("Verni" series of the genus) and C. banaticus. For initiation of cultures we used a plant growth regulator (PGR) combination used for in vitro culture of saffron and its relatives: 10 mg L -1 α-naphthaleneacetic acid (NAA) and 1 mg L -1 6-benzyladenine (BA). Shoot tips of young seedlings (C. scepusiensis) and corms (for the rest of species) were used as explants. C. scepusiensis explants developed into organogenic calli. On media with decreased NAA and with or without increased BA concentration, calli produced stigma-like structures and/or shoots and whole plants. In the other species, callus initiation medium induced callus formation with abundant somatic embryos. In C. tommasinianus, embryos developed shoots when auxin content of medium was decreased. In C. banaticus, a decrease of auxin with or without an increase in cytokinin content led to shoot or whole plant regeneration, as in C. scepusiensis. In the case of C. vittatus and C. banaticus, initiation and/or maintenance of cultures on indole-3-butyric acid (IBA) and increased sucrose concentration stimulated whole plant regeneration and in vitro cormlet development. C. scepusiensis and the rest of cultures (organogenic vs. embryogenic) differed at the biochemical level: C. scepusiensis cultures had higher (yet still low) enzymatic antioxidant (catalase, peroxidase) activities. With respect to catalase isoenzyme patterns, C. banaticus was different from the rest of cultures, demonstrating its distinct taxonomical position. Besides germplasm preservation use of the present cultures, they have a potential biotechnological value.
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