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An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5-6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp. [ABSTRACT FROM AUTHOR]

Grapevine ( Vitis genus) is one of the economically most important fruits worldwide. Some species and cultivars are rare and have only a few vines, but represent national heritages with a strong need for preservation. Field collections are labor intensive, and expensive to maintain, and are exposed to natural disasters. In addition, infection with pathogens, especially viruses, is common in grapevine because of vegetative propagation, which is conventionally used for this genus. Cryopreservation provides an alternative and ideal means for the long-term preservation of Vitis germplasm, which can be used as a backup to field collections for important autochthonous cultivars or only as cryo-banks for rare, native cultivars that are worthy of preservation. Cryotherapy, based on cryopreservation protocols, provides an efficient method for the eradication of grapevine viruses. This review provides comprehensive and updated information on cryopreservation for long-term preservation of genetic resources and cryotherapy for virus eradication in Vitis. Additional research in grapevine cryopreservation and cryotherapy is needed. [ABSTRACT FROM AUTHOR]

Field performance evaluation and genetic integrity assessment were conducted in Argyranthemum plants derived from cryopreserved shoot tips. Some variations in root formation and vegetative growth were found in the plants following cryopreservation, but morphologies of the leaves and flowers, and color, number and size of the flowers remained unchanged in the plants recovered from cryopreservation, compared with the control. Assessments of genetic integrity by the two molecular markers: inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands across the plants tested following cryopreservation. These data indicate that cryopreservation reduces, to a certain degree, root formation and vegetative growth, but it does not alter morphologies of leaves and flowers, may not cause any genetic alternations, and has no adverse effects on quantity and quality of the flowers. Therefore, the droplet vitrification cryopreservation can be considered promising for long-term preservation of Argyranthemum germplasm. [ABSTRACT FROM AUTHOR]

Plant regeneration via both shoot regrowth and de novo embryo-like structures was obtained from cryopreserved shoot tips of Lilium spp. Shoot tips (1.0-3.0 mm in length) of Lilium Oriental hybrid 'Siberia' containing two- to four-leaf primordia were excised from 4-wk-old adventitious shoots and cryopreserved by droplet vitrification. When post-cultured for 6 wk on recovery medium containing 0.1 mg/l α-naphthaleneacetic acid (NAA) in combination with different kinetin (KT) concentrations (0-0.4 mg/l), three types of regenerants were produced: (1) only embryo-like structures, (2) shoot regrowth with embryo-like structures, and (3) only shoot regrowth. The highest frequencies (≥75.0%) of total embryo-like structures were obtained from cryopreserved shoot tips post-cultured on recovery medium containing 0.1 mg/l NAA and 0.1-0.2 mg/l KT. Embryo-like structures proliferated within 4 wk of culture on medium containing 1.0 mg/l NAA and 0.05 mg/l thidiazuron (TDZ). Somatic embryo-like structures matured and converted into whole plantlets after 8 wk of culture on Murashige and Skoog (MS) medium without any plant growth regulators. The highest frequencies (≥25.0%) of total shoot regrowth were produced from cryopreserved shoot tips post-cultured on recovery medium containing 0.1 mg/l NAA and 0.05-0.4 mg/l KT. With the protocols reported here, frequencies of total embryo-like structures and shoot regrowth ranged from 45.0 to 90.0% and 25.0 to 52.5%, respectively, from six lily varieties belonging to five Lilium species or hybrids, representing diverse Lilium germplasm. The results reported here have potential applications to long-term conservation of genetic resources and genetic transformation in Lilium spp. [ABSTRACT FROM AUTHOR]

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation-dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2-3 mg L thidiazuron, depending on genotype, and 0.5 mg L indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of 'Gala'. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus. [ABSTRACT FROM AUTHOR]

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8-1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH 5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23 ± 2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92-100% and mean number of shoots of 4.7-7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid 'Siberia' revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of 'Siberia', no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium. [ABSTRACT FROM AUTHOR]