Cryopreservation of in vitro-grown shoot-tips of Rabdosia rubescens by encapsulation-dehydration and evaluation of their genetic stability

Abstract

Shoot-tips of Rabdosia rubescens, excised from in vitro-grown proliferating shoots that were cold-hardened at 5°C for 3 weeks, were encapsulated in alginate beads. Subsequently, these were precultured in a mixture of 0.4 M sucrose and 2 M glycerol for 1 h and then desiccated with silica-gel to about 21% water content prior to freezing in liquid nitrogen. After thawing, about 85% of cryopreserved shoot-tips grew into true-to-type shoots and with enhanced rooting capacity. Eight single-bud sibling lines were used to assess genetic stability of these encapsulated shoot-tips. When the relative DNA content was measured by flow cytometry (FCM), no changes were observed between controls and cryopreserved shoots. Using a sequence-related amplified polymorphism (SRAP) assay, it was observed that seven out of eight cryopreserved lines showed identical banding patterns; while the eighth line displayed an absent band, amounting to a low variance rate of 0.01%. These findings suggested that it was necessary to monitor the genetic stability of recovered cryopreserved R. rubescens shoots.

Morphogenetic response of shoot tips to cryopreservation by encapsulation-dehydration in a solid mutant and periclinal chimeras of Chrysanthemum × grandiflorum /Ramat./Kitam.

“…Ai et al (2012) reported that the Rabdosia rubescens shoots produced after encapsulation-dehydration cryopreservation showed an increased rooting efficiency in comparison to the untreated control. Hao et al (2002) on the other hand, did not observe any influence of storage in LN on citrus rhizogenesis efficiency.…”

<b>Behavior of lateral buds of <i>Hancornia speciosa</i> after cryopreservation by encapsulation-vitrification

“…However, H. speciosa lateral buds seems to be more sensitive to dehydration. Successful cryopreservation was observed in shoot apices of Rabdosia rubescens encapsulated and precultured in MS culture medium with 0.4 M sucrose, in combination with 2.0 M glycerol and dehydrated for 1 hour in silica gel, reaching a water content of 21% and a recovery rate of 85% (Ai, Lu, & Song, 2012). Encapsulationvitrification of apical meristems of date palm (Phoenix dactylifera L) in PVS2 for 15 minutes and immersion in LN, ensured increased survival and recovery of explants (40%) (Fki et al, 2013).…”

“…Moreover, several studies have shown that the encapsulationvitrification technique is quite efficient for different explants and species (Wang et al, 2000;Sakai & Engelmann, 2007: Ai et al, 2012Fki et al, 2012).…”

Analysis of the transcriptome ofIsodon rubescensand key enzymes involved in terpenoid biosynthesis

“…In our study, a total of 50,934,276 clean reads, 101,640 transcripts and 44,626 unigenes were generated through de novo transcriptome assembly. A number of unigenes À 23, 987,10,263, 7359,18,245,17,683,19,485, 9361 À were annotated in the National Center for Biotechnology Information (NCBI) non-redundant protein (Nr), NCBI nucleotide sequences (Nt), Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO), Swiss-Prot, protein family (Pfam), gene ontology (GO), eukaryotic ortholog groups (KOG) databases, respectively. Furthermore, the annotated unigenes were functionally classified according to the GO, KOG and KEGG.…”

“…[17] In order to gain insights into terpenoid biosynthesis, especially that of diterpenoid, characterization of functional genes via genome sequencing of the transcriptome is necessary. RNA sequencing (RNA-seq) has been widely used for de novo transcriptome sequencing to detect gene expression and other useful genomic information in many plant species without prior genomic information.…”