Shoot tips excised from shoot culture of Salvia officinalis were encapsulated in 2% or 3% (w/v) sodium alginate and exposed to 50 mM calcium chloride for complexation. Immediately or after 6, 12 or 24 weeks of storage at 4°C, the synthetic seeds were cultured for 6 weeks on half-strength MS medium supplemented with indole-3-acetic acid (IAA) (0.1 mg/l) and solidified with 0.7% agar. The frequency of shoot and root emergence from encapsulated shoot tips was affected by the concentrations of sodium alginate and additives in the gel matrix (sucrose, gibberellic acid, MS nutrient medium) as well as duration of storage. The frequency of shoot and root induction of non-stored synthetic seeds was highest with shoot tips encapsulated with 2% sodium alginate containing 1.5% sucrose and 0.5 mg/l gibberellic acid (GA3). Shoot tips maintained their viability and ability to develop shoots even after 24 weeks of storage when they were encapsulated in 3% alginate with 1/3 MS medium, sucrose (1.5%) and GA3 (0.25 mg/l). Root formation tended to decrease with storage time. Overall, 90% of the plantlets derived from stored and non-stored synthetic seeds survived in the greenhouse and grew to phenotypically normal plants. This procedure can enable the use of synthetic seed technology for germplasm conservation of S. officinalis, a plant species of high medical and commercial value.
Roots of Codonopsis pilosula (Franch.) Nannf. are among the most popular Chinese herbal medicines, exhibiting various beneficial activities which support immunity and stress resistance. The plant shows high intraspecific genetic variation. There is a need for effective vegetative propagation methods yielding high and sustainable quality. Here we report a micropropagation method using axillary shoot proliferation. Nodal segments from aseptically germinated plants were inoculated on modified MS media enriched with different concentrations of cytokinins: benzyladenine, kinetin (1, 4, 10 or 20 μM) or thidiazuron (1, 4 or 8 μM), with or without the auxin NAA (1 μM). Axillary bud break was initiated most efficiently on media with 1 or 4 μM BA and 1μM NAA. Shoot number increased markedly in subsequent cycles of harvesting and transfer to fresh 1 μM BA and NAA medium, leading to the maximum 69 shoots (mean 38.16±4.35) from a single nodal explant in the fourth harvest. The shoots were successfully (>98% efficiency) rooted in MS medium containing high sucrose (60 g/L) and 5 μM IAA, and acclimatized to soil cultivation with a survival rate of 90%. These results can be used to establish a simple and commercially viable protocol for mass propagation of C. pilosula for plantations or breeding.
This paper is the first published report describing micropropagation of Carlina onopordifolia, using shoot tip and hypocotyl explants. The explants were excised from 10-day-old seedlings and transferred to proliferation medium supplemented with 6-benzylaminopurine (BA; 1.0 or 3.0 mg l-1), kinetin (Kn; 1.0 or 3.0 mg l-1) or zeatin (ZEA; 1.0 or 3.0 mg l-1) in combination with naphthaleneacetic acid (NAA; 0.1 mg l-1). The shoot tips were significantly better than hypocotyls as initial material for shoot regeneration. For shoot multiplication, MS medium supplemented with BA proved superior to the other cytokinins tested. Medium supplemented with 1.0 mg l-1 BA gave the highest shoot propagation frequency (66.9%) and number of shoots per explant (2.5). Single shoots were separated from each other and rooted on MS supplemented with IBA for the whole period of culture, with longor short-pulse IBA application. The highest rooting frequency (84.8%) and root number (18.8) were for shortpulse (1 min) 1000 mg l-1 IBA solution. The higher IBA concentration stimulated callus formation and the development of short roots. The shoots were transferred to MS medium without growth regulators. Survival was highest (54.4%) for the plants from the short-pulse 100 mg l-1 IBA treatment. After 8 weeks of acclimatization the plantlets were removed to field conditions and grew normally.