Assessment of Genetic Stability of Micropropagated Curcuma caesia through Cytophotometric and Molecular Analysis

Abstract

Summary Protocol has been standardized for micropropagation of medicinally important Curcuma caesia using sprouted bud explants from rhizome. Tissue culture raised plantlets of C. caesia could be conserved in vitro through subculturing at an interval of 4 months. The genetic stability of micropropagated plants was studied with an interval of 6 months up to 30 months in culture using cytophotometric, random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. Cytophotometric analysis of 151 plants revealed a unimodal distribution of the DNA content with a peak corresponding to 4C nuclear DNA (3.5 pg). RAPD and ISSR analysis revealed mopnomorphic bands showing the absence of polymorphism in all 73 regenerants analyzed, thus confirming the genetic uniformity among in vitro grown somaclones of Curcuma caesia.

In vitro and ex vitro evaluation of long-term micropropagated turmeric as analyzed through cytophotometry, phytoconstituents, biochemical and molecular markers

“…Cytophotometric analysis of 4C nuclear DNA content of root tips of all regenerants analyzed over 7 years of culture period revealed diploidy in all showing range of variation from 7.63 to 7.70 pg, similar to the value obtained among source explants (Table 1). Cytophotometric analysis has also been used for the assessment of genetic stability among in vitro grown plantlets of Ornithogalum thysoides (Nayak and Sen 1991) Ornithogallum umbellatum (Nayak and Sen 1995) and Zingiber officinale (Mohanty et al , 2010. RAPD and ISSR markers were chosen because of simplicity and cost effectiveness and their efficiency in reliable monitoring of variability of DNA sequences among in vitro conserved plantlets (Zietkiewicz et al 1994;Martins et al 2004;Mohanty et al 2008;Bhatia et al 2009).…”

“…A major problem associated with in vitro culture is possible occurance of somaclonal variation arising in culture regenerated plantlets and their progenies (LarKns and Scowcroft 1981;Gould 1986). Assesment of degree of genetic integrity of micropropagated plants at regular interval can reduce the chances of induction of somaclonal variation that might arise due to length of culture period (Rout and Das 2002;Mohanty et al 2008Mohanty et al , 2010Nayak and Sen 1995). Of several methods phenotypic analysis, chromosome analysis, estimation of nuclear DNA content, analysis of secondary biochemical products are used for detection of somaclonal variation among the regenerants (Potter and Jones 1991;Sen 1991,1997).…”

“…Some of the molecular techniques used to detect genetic variation are polymerase chain reaction (PCR)-based markers such as random amplified DNA polymorphism (RAPD), inter simple sequence repeats (ISSR) and amplified fragent length polymorphism(AFLP), methylation sensitive amplified polymorphism (MSAP) and retrotransposon microsattelite amplified DNA polymorphism (REMAP). These multilocational profiling techniques are powerful tools for assessment of genetic stability of regenerants after micropropagation (Martins et al 2004;Mohanty et al 2008Mohanty et al , 2010Morata-Renau et al 2005;Peredo et al 2009;Smykal et al 2007.…”

Influence of different pathways of regeneration on genetic and phytochemical instability of Curculigo orchioides

“…It was reported that ISSR regions lying within the range of microsatellite repeats have a higher capacity to reveal polymorphism and offer great potential to determine intragenomic and intergenomic diversity as compared to other arbitrary primers such as RAPDs (Zietkiewicz et al 1994). The ISSR primers are now proven to be much more efficient in assessing the genetic integrity among clonally propagated plants as reported by many workers in different species (Zietkiewicz et al 1994, Bhatia et al 2009, Mohanty et al 2010, Bhatia et al 2011. Martins et al (2004) reported genetic homogeneity of almond plantlets regenerated t hrough auxiliary branching after 4 -6 years of in vitro multiplication.…”

High-frequency callus organogenesis, large-scale cultivation and assessment of clonal fidelity of regenerated plants of Curcuma caesia Roxb., an important source of camphor

“…Micropropagations of various Curcuma species, such as C. longa (He and Gang 2014), C. comosa (Loapirukkul et al 2012), C. aromatic (Mohanty et al 2008), C. zedoaria (Loc et al 2005), C. amada (Prakash et al 2004) and C. soloensis (Zhang et al 2011), have been reported. So far, the tissue culture protocol associated with C. caesia has been limited to shoot induction from rhizome bud explants (Bharalee et al 2005;Mohanty et al 2010;Shahinozzaman et al 2013). Although callus induction from C. caesia has been achieved (Mei 2012), shoot organogenesis from callus has not been reported.…”