The effect of low- and high-power microwave irradiation on in vitro grown Sequoia plants and their recovery after cryostorage.

Two distinct microwave power levels and techniques have been studied in two cases: low-power microwave (LPM) irradiation on in vitro Sequoia plants and high-power microwave (HPM) exposure on recovery rates of cryostored (-196°C) Sequoia shoot apices. Experimental variants for LPM exposure included: (a) in vitro plants grown in regular conditions (at 24 ± 1°C during a 16-h light photoperiod with a light intensity of 39.06 µEm-2 s-1 photosynthetically active radiation), (b) in vitro plants grown in the anechoic chamber with controlled environment without microwave irradiation, and (c) in vitro plants grown in the anechoic chamber with LPM irradiation for various times (5, 15, 30, 40 days). In comparison to control plants, significant differences in shoot multiplication and growth parameters (length of shoots and roots) were observed after 40 days of LPM exposure. An opposite effect was achieved regarding the content of total soluble proteins, which decreased with increasing exposure time to LPM. HPM irradiation was tested as a novel rewarming method following storage in liquid nitrogen. To our knowledge, this is the first report using this type of rewarming method. Although, shoot tips subjected to HPM exposure showed 28% recovery following cryostorage compared to 44% for shoot tips rewarmed in liquid medium at 22 ± 1 °C, we consider that the method represent a basis and can be further improved. The results lead to the overall conclusion that LPM had a stimulating effect on growth and multiplication of in vitro Sequoia plants, while the HPM used for rewarming of cryopreserved apices was not effective to achieve high rates of regrowth after liquid nitrogen exposure.

Effects of sucrose preculture on cryopreservation by droplet-vitrification of strawberry cultivars and morphological stability of cryopreserved plants.

The droplet-vitrification method was applied to shoot tips of micropropagated strawberry plants (Fragaria x ananassa DUCH. cvs. 'Senga Sengana', 'Korona' and 'Aroma'). Shoot tips of 2 - 3 mm in length were precultured in sucrose (0.1, 0.25, 0.5, 0.75, 1.0 M) enriched media for 24 and 48 h. Subsequently, they were transferred into 6 microl droplets of PVS2 vitrification solution for 20 min and plunged into liquid nitrogen. Rapid rewarming was done in liquid medium at room temperature. The highest recovery rate in all cultivars (60 percent) was achieved after a preculture with 0.25 M sucrose for 24 h. From the recovered shoot tips not all continued with shoot development and multiplication. The number of off-types observed in the field was affected by the sucrose concentration. After preculture in 0.1 M sucrose no off-types were observed, while after preculture in high sucrose concentrations the number of off-types was much higher reaching 20 percent at the highest sucrose concentration.

Plant regrowth from potato shoot tips cryopreserved by a combined vitrification-droplet method.

Shoot tips obtained from in vitro potato plants (three cultivars) were successfully cryopreserved by the combined vitrification-droplet method and subsequently regenerated shoots. The effect of apex size, sucrose concentration, preculture duration and cold hardening treatments on viability of cryopreserved shoot tips was studied. The excised shoot tips were incubated, precultured and dehydrated with concentrated PVS2 cryoprotective solution at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in Murashige-Skoog (MS) liquid medium, shoot tips were plated on semisolid MS medium (3.5 g/l agar) supplemented with 0.4 mg l(-1) gibberellic acid, 0.5 mg l(-1) zeatin, 0.2 mg l(-1) indole-3-acetic acid and 30 g l(-1) sucrose for regrowth. Cryopreserved shoot tips resumed growth within 20 days and regenerated shoots within 30 days. The highest regrowth levels of apices after cryopreservation were 55% recovery for cv. Désirée, 51% for cv. Ostara and 46% for cv. Santé.

Cryopreservation of Chrysanthemum morifolium (Dendranthema grandiflora Ramat.) using different approaches.

Chrysanthemum species are grown both as ornamentals and for the production of pyrethrum. Recent increased production and breeding efforts have raised the need for the conservation of valuable germplasm. Chrysanthemum has been cryopreserved by controlled-rate-freezing as early as 1990. We report here deep-freezing of shoot tips of C. morifolium var. Escort by different technical procedures: controlled-rate-freezing, encapsulation/dehydration, ultra-rapid-freezing by the droplet method and vitrification. While vitrification yielded the highest shoot regeneration rates, the very simple droplet method was also successful in this respect. Droplet freezing was successfully performed with nine cultivars. Our results open the door to the successful use of alternative methods if one method fails to cryopreserve a variety. Furthermore, it enables comparative investigations of genetic stability and cyro-injury to be carried out.