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In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with β-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg·g-1 dry weight in tissue and 0.23 mg·dm-3 in medium; modified lines: 4.59 mg·g-1 for the tissue, and 0.48 mg·dm-3 for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.
The specificity of two separate tonoplast permeases transporting oleanolic acid glycosides was investigated in vacuoles isolated from leaf protoplasts of marigold (Calendula officinalis) with the use of chemically synthesized analogs. The results indicate that the proper structure of both parts of oleanolic acid monoglycoside, i.e. aglycon and the sugar moiety, are required for binding to a specific tonoplast carrier.
It has been shown for the first time that [3-3H]oIeanolic acid glycosides formed in the cytosol of C. officinalis leaf cells are transported to the extracellular space in the form of pentaglucoside VI (44%), whereas glucuronides derived from [3-3H]oleanolic acid 3-O-monoglucuronide (29%) as well asa part of glucosides (24%) were transported into the cell walls.
In 2007–2009, a pot experiment was carried out in a greenhouse at the University of Warmia and Mazury in Olsztyn on cv. Promyk pot marigold (Calendula officinalis L.). The aim of the study was to assess the impact of nitrogen and magnesium in plant growth and development and yield of marigold. The experiment included two factors: nitrogen fertilization (g.pot-1): A–0 (control), B–0.3, C–0.6 (0.6+0), D–0.9 (0.6+0.3), E–1.2 (0.6+0.6) and magnesium fertilization (g.pot-1): a–0 (control), b–0.5. The experimental part of the research was designed according to the independent series method and set up in 4 replicates, 4 pots in one replicate, in modified Kick-Brauckmann pots. The experiment demonstrated a significant effect of nitrogen fertilization on the plants hight, number of pot marigold inflorescences, fresh inflorescences weight, fresh and airdry ligulate weight. Magnesium fertilization had a positive effect on all the analyzed traits. No statistical correlation was proven between the nitrogen and magnesium fertilization.
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