Abstract
The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.
Highlights
V. dentatum, commonly called arrowwood viburnum, is a multi-stemmed deciduous shrub with glossy leaves bearing clusters of white flowers in spring which turn into bluish berries in autumn [1].The plant, which is adapted to low and high temperatures, as well as drought and alkaline soil, is used in ornamental horticulture, as a landscaping edging plant and as a gardening container-grown plant [2]
Artificial seeds are defined as artificially encapsulated somatic embryos, shoot tips, shoot buds, nodal segments, cell aggregates or any tissue that can be used for sowing as a seed and that possess the ability to convert into a plant under in vitro or in vivo conditions [8,9,10]
Shoot tip and first-node explants, excised from in vitro-derived microshoots that were produced in viburnum subcultures, formed new microshoots at frequencies of 100% (Table 1)
Summary
V. dentatum, commonly called arrowwood viburnum, is a multi-stemmed deciduous shrub with glossy leaves bearing clusters of white flowers in spring which turn into bluish berries in autumn [1].The plant, which is adapted to low and high temperatures, as well as drought and alkaline soil, is used in ornamental horticulture, as a landscaping edging plant and as a gardening container-grown plant [2]. Artificial seed technology mimics that of the conventional seed technology and combines the advantages of seed (sexual) propagation with those of vegetative (asexual) propagation [11,12,13]. By this method, the genetic uniformity among regenerated plantlets, in vitro mother culture and in the field-grown or in the greenhouse-grown mother plant is maintained in most cases, while elite genotypes can be stored for a short to long term and can be regenerated later upon demand for plant propagation material [14,15]. The method offers the easy handling of artificial seeds during storage, unhindered transportation among countries from customs barriers and/or quarantines and cost efficiency in comparison with the traditional method of in vitro culture, which requires frequent and costly subcultures [16,17,18]
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