Enhancement of In Vitro Production of Volatile Organic Compounds by Shoot Differentiation in Artemisia spicigera

Callus initiation, shoot formation and plant regeneration were established for Artemisia spicigera, a traditional medicinal plant growing in Armenia, Middle-Anatolia and Iran, and producing valuable volatile organic compounds (VOCs) that are mostly represented by monoterpenoids. Optimal callus initiation and shoot production were obtained by culture of hypocotyl and cotyledon explants on MS medium comprising 0.5 mg L−1 naphthalene acetic acid (NAA) and 0.5 mg L−1 6-benzyladenine (BA). Consequently, the shoots were transferred onto the MS media supplemented with 1 mg L−1 of indole-3-butyric acid (IBA) or 1 mg L−1 of NAA. Both types of auxin induced root formation on the shoots and the resulting plantlets were successfully grown in pots. The production of VOCs in callus tissues and regenerated plantlets was studied by gas chromatography–mass spectrometry (GC-MS) analysis. Although the potential of undifferentiated callus to produce VOCs was very low, an increased content of bioactive volatile components was observed at the beginning of shoot primordia differentiation. Intriguingly, the volatiles obtained from in vitro plantlets showed quantitative and qualitative variation depending on the type of auxins used for the rooting process. The acquired quantities based on total ion current (TIC) showed that the regenerated plantlets using 1 mg L−1 NAA produced higher amounts of oxygenated monoterpenes such as camphor (30.29%), cis-thujone (7.07%), and 1,8-cineole (6.71%) and sesquiterpene derivatives, namely germacrene D (8.75%), bicyclogermacrene (4.0%) and spathulenol (1.49%) compared with the intact plant. According to these findings, in vitro generation of volatile organic compounds in A. spicigera depends on the developmental stages of tissues and may enhance with the formation of shoot primordia and regeneration of plantlets.

Effect of Genotype and Explant Type on Shoot Regeneration in Tomato (Lycopersicon esculentum Mill.) in vitro

The regeneration capacity of six types of explants (segments from hypocotyl, cotyledons, epicotyl, leaf, internodes and petiole) was compared in 13 cultivars of tomato (Lycopersicon esculentum Mill). Explants were cultured on a regeneration medium containing 1 mg/l zeatin and 0.1 mg/l indole-3-acetic acid. The number of shoot primordia and shoots with 1 or more fully developed leaves was evaluated after 6 weeks. The regeneration capacity was significantly influenced by cultivars and explant types. The total number of shoot primordia produced in all types of explants was highest in the cultivars Hana and Premium and lowest in UC 82 and Money Marker. Cv. Hana also produced the highest number of shoots. The most responsive explants in most cultivars were hypocotyls and epicotyls with up to 100% regeneration and mean production of 6.3 and 6.5 shoot primordia per explant, respectively.  

In Vitro Regeneration and Genetic Transformation of Cucumis metuliferus through Cotyledon Organogenesis

This study was undertaken to establish for the first time an efficient regeneration and transformation system for Cucumis metuliferus line PI292190, which is the source of a well-defined resistant gene, Wmv, that provides resistance against Papaya ringspot virus type P (PRSV-P) and PRSV-W (formerly known as Watermelon mosaic virus 1, WMV-1). Different combinations of growth regulators were evaluated for the regeneration of cotyledon explants. Adventitious buds or shoot primordia were obtained within 3 to 4 weeks on regeneration medium. After shoot development, adventitious buds or shoot primordia were transferred to elongation medium for 3 to 4 weeks and these shoots were subcultured onto rooting medium for another 1 to 2 weeks. Under optimal culture conditions, a total of 7 to 10 weeks was necessary to obtain C. metuliferus plantlets from cotyledons. Furthermore, transgenic plants were successfully obtained using an Agrobacterium tumefaciens-mediated transformation method as shown by polymerase chain reaction analysis and histochemical β-glucuronidase (GUS) assay. A total of nine transgenic plants were developed from 360 cotyledon explants, giving a transformation frequency of 2.5%.

In vitro morphogenic events in culture of Lotus corniculatus L. seedling root explants

The experiments were carried out on <em>Lotus corniculatus</em> (L.) seedling root explants of the cultivar varieties Skrzeszowicka, Caroll A10 and strain 175. Callus formation and shoot regeneration were the major explant response depended mainly on of the studied genotype and used plant growth regulators (PGRs). Primary cortex of proximal and distal end of explant was the most active tissue for callus proliferation. For shoot primordia differentiation deeper zones of cortex took a part. The process of meristematic centre initiation was not uniform and various level of shoot differentiation events were observed not earlier than 3 weeks of culture. Usually, the shoot primordia regeneration began on proximal rather than distal end of the explant. BAP rather than urea derivatives stimulated shoot proliferation in extended cultures. Increasing of BAP and TDZ concentrations brought about the explant polarity and expansion of the meristematic zones. The explant position in root did not have significant influence on the number of regenerated shoots. The cultures only had better bud formation by TDZ when compared to BAP. BAP stimulated bud formation and development of the shoots from them. Short term of TDZ treatment of explants stimulated meristem formation which developed into buds and shoots. CPPU stimulated callus proliferation and bud formation when explants pretreatment was prolonged from 12 to 36 hrs.