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The different acid invertase activity (total, soluble, wall-bound and extracellular) in calli induced on explants (cotyledon, petiole, hypocotyl and leaf) originated from Medicago strasseri seedlings were evaluated. In cultures subjected to 16 h photoperiod, the highest total, soluble and extracellular activities were found in calli from leaves cultured in medium 12 (MS with 0.01 mg·dm⁻³ (0.045 µM) of TDZ), elevated amounts of total and wall-bound invertase being found in calli induced on petioles in 12G medium (MS with 0.01 mg·dm⁻³ (0.045 µM) TDZ and 3.104 mg·dm⁻³ glycerol). In cultures maintained in darkness, the activity detected was lower than that observed in cultures under light conditions. The highest amounts of enzyme was bound in calli cultured on medium 12 (total and extracellular invertase) -leaves- and medium 12D (MS with 0.001 mg·dm⁻³ (0.0045 µM) TDZ) (soluble invertase) -using hypocotyls. In general, the different forms of invertase activity studied seem to appear in greatest amounts in calli induced under light conditions using leaves as explant and TDZ as growth regulator.
The research was conducted on explants of silver fir (Abies alba Mill.) deriving from several forest districts in southern Poland. The study encompassed the influence of the origin of plant material, type of explants, kind of substances used for explants sterilization, PPM and the type of medium on the ability to form embryogenic callus and to develop somatic embryos in silver fir explants. From the plant material collected in three sites, 57 clones were obtained from mature zygotic embryos; this produced an embryogenesis frequency of 6%. Embryogenic callus was obtained with a diameter of 65–70 mm depending on the material origin. The best medium for development of callus inducted on embryos isolated from mature silver fir seeds was the SH medium. Somatic embryos were formed in a globular stadium (24 pieces) on this medium. The 10% solution of NaOCl (used for 15 minutes) turned out to be the most effective substance for seed sterilization.
Under a scanning microscope, leaves from tissue cultures (after 8 weeks in vitro culturing), from 1 year old regenerants and from the 100 years old donor tree were studied. More abnormalities were observed in the tissue culture compared to regenerant and donor. During tissue culture transfer rapid, leaf dehydration occurs due to defective regulation mechanisms making stomata of tissue culture stay open. Epidermis as well as stomata cells were observed already on the leaves in in vitro conditions, but they become physiologically functioning only after transfer into conditions of natural environment.
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.
Sucrose phosphate synthase (SPS) is a key enzyme catalyzing sucrose metabolism in plants. In this study, we isolated the SPS cDNA from Saccharum spontaneum and designated as SsSPS (GenBank accession no. MF398541). The full-length of SsSPS cDNA was 4153-bp with an opening reading frame (ORF) of 3132 nucleotides, which encoded a 1043-amino acid protein. The nucleotide sequences alignment showed that it had 98%, 97% and 87% homology with S. officinarum, Setaria italica and Lolium perenne, respectively. Moreover, the SsSPS was detected to express in leaf and stem tissues of S. spontaneum and exhibited a predominant expression in the stem tissue. However, there was no significant difference in the expression level of SsSPS between young leaves and mature ones. Additionally, we generated transgenic S. spontaneum using Agrobacterium-mediated transformation. Our data will provide a valuable foundation for further study of the potential role of SPS in plants.
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