Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica

Abstract

Crypopreservation methods were firstly developed for root-tips from hairy root cultures of Beta vulgaris, established after transformation by Agrobacterium rhizogenes. The effects of culture age, pre-growth, cryoprotection, freezing rate and post-freeze culture conditions were determined. The resulting freezing protocol was then used to cryopreserve transformed root cultures of Nicotiana rustica. Both species were viable after freezing (ca. 80%), according to fluorescein diacetate vital staining. However, on average the regeneration of proliferating roots from surviving root-tips was low (< 20%). Growth rates, secondary metabolite production and T-DNA structure of a number of hairy root lines were examined and found to be unchanged after cryopreservation.

Morphological and molecular variation in Ri-transformed root lines are stable in long term cultures of Tylophora indica

“…The Ri-transformed root lines of T. indica showed variation in integration and expression of the T-DNA genes and such molecular variation between the root lines were stably maintained in the long term culture. Similar genetic stability of Ri-transformed root lines have been reported in a number of species (Aird et al 1988;Benson and Hamill 1991;Maldonado-Mendoza et al 1993;Lipp Joao and Brown 1994;Baíza et al 1999). It appears that transformed root organ cultures maintained in growth hormone free media are not subjected to factors causing the instability and somaclonal variation associated with in vitro plant cell and organ cultures maintained in hormone supplemented media.…”

“…The presence of morphological, molecular and biochemical variations between Ri-transformed root lines is widely known (Aoki et al 1997;Palazòn et al 1998;Moyano et al 1999;Batra et al 2004;Chaudhuri et al 2005;Bandyopadhyay et al 2007;Alpizar et al 2008;Taneja et al 2010), however, the detailed study on the stability of these different lines with age in long term culture is reported in a very few species (Benson and Hamill 1991;Maldonado-Mendoza et al 1993;Guivarc'h et al 1999). Earlier studies revealed that large variations in morphology, molecular constitution and physiology characterized the carrot as well as potato hairy root lines (De Vries-Uijtewaal et al 1988;Chriqui et al 1996).…”

“…Molecular variation in terms of T-DNA insertion between Ri-transformed root clones are reported and effect of TL-DNA and TR-DNA on morphology, growth and metabolite content has been investigated by many researchers (Palazòn et al 1998;Moyano et al 1999;Batra et al 2004;Bandyopadhyay et al 2007;Alpizar et al 2008;Taneja et al 2010). However, the genetic and phenotypic stability of the variable clones of hairy roots in long term culture have been analysed in very few species (Benson and Hamill 1991;Maldonado-Mendoza et al 1993). Instability in the phenotype and the level of gene expression is reported in longterm cultures of carrot hairy roots (Guivarc'h et al 1999).…”

Cold storage and cryopreservation of hairy root cultures of medicinal plant Eruca sativa Mill., Astragalus membranaceus and Gentiana macrophylla Pall.

“…To date, the HRs of only Beta vulgaris, Nicotiana rustica (Benson and Hamill 1991), Artemisia annua (Teoh et al 1996), Panax (Yoshimatsu et al 1996), Armoracia rusticana (Phunchindawan et al 1997) and Vinca minor (Hirata et al 2002) have been successfully cryopreserved, to our knowledge. It was reported that 60% of Armoracia rusticana HRs was recovered following cryopreservation with EV method (Phunchindawan et al 1997).…”

“…The organs/tissues that were successfully cold stored or cryopreserved mainly included shoot tips (Pennycooke et al 2000;Lambardi et al 2000;Xu et al 2002), callus and somatic embryo (Ford et al 2000;Shiota et al 1999). However, only a few studies have evaluated preservation for HRs (Benson and Hamill 1991;Teoh 1996;Yoshimatsu et al 1996;Phunchindawn et al 1997;Hirata et al 2002), and results varied by plant species and methods used. The purpose of the present work was to develop a protocol for effective, long-term preservation of our HR lines of A. membranaceus, G. macrophylla, and E. sativa.…”