Development of an encapsulation-vitrification protocol for Rubia akane (nakai) hairy roots: a comparison with non-encapsulation.

BACKGROUND: A comparison of different cryopreservation techniques should be based on the characteristics of both the methodology and the material in question using an optimized procedure. OBJECTIVE: This study aimed at developing an encapsulation-vitrification procedure for hairy roots of Rubia akane using alternative loading and vitrification solutions, based on the existing optimized droplet-vitrification procedure. MATERIALS AND METHODS: Encapsulated roots were first precultured in liquid medium with 10% sucrose for 3 days, then with 17.5 % sucrose for 1 day, after which they were osmoprotected with solution C6-40 % (20 % glycerol + 20 % sucrose) for 50 min, cryoprotected with solution A3-90 % (37.5 % glycerol + 15 % DMSO + 15 % EG + 22.5 % sucrose, w/v) on ice for 40 min, cooled and warmed in 2 ml cryovials, and unloaded in 35% sucrose solution for 60 min. RESULTS: Through the application of this procedure to aged-clustered roots, up to 97.5 % post-cryopreservation regeneration was observed. In our previous study, droplet-vitrification of hairy roots of R. akane resulted in 83.8 % post-rewarming regeneration following preculture with 10 % sucrose for 2 days and 17.5 % sucrose for 4-5 h, and osmoprotection with solution C4-35 % (17.5 % glycerol + 17.5 % sucrose) for 30 min, and cryoprotection with solution A3-70 % (29.2 % glycerol + 11.7 % DMSO + 11.7% EG + 17.4% sucrose, w/v) on ice for 20 min. In the present study, higher post-cryopreservation regeneration was observed by using a higher concentration of vitrification solution (A3-70 % → A3-90 %, B5-80 % → B1-100 %) and/or a longer cryoprotection duration (A3-70 % at room temperature (RT) for 8 min → 15-30 min, on ice for 20 min → 40-80 min; B5-80 % for 15 min → 30-60 min). CONCLUSION: Even though encapsulation provided some degree of protection from the cytotoxicity of vitrification solutions to cytotoxicity-sensitive R. akane hairy roots, an overall higher post-cryopreservation regrowth was obtained using the droplet-vitrification procedure under optimized conditions. This result implies that this sensitive material was not sufficiently cryoprotected, and thus, rapid cooling and warming using foil strips was more efficient than cryopreservation of encapsulated samples.

Development of vitrification protocol in Rubia akane (nakai) hairy roots using a systematic approach.

BACKGROUND: A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions. OBJECTIVE: The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions. METHODS: Due to the poor response of 10-year in vitro maintained R. akane hairy roots to already established cryopreservation protocols, the following sets of experiments were designed: 1) combinational effect of preculture, osmoprotection and cryoprotection with PVS2-based (A3-70%) and PVS3-based (B5-80%) vitrification solutions; 2) different cooling/warming rates and warming temperature; 3) varying unloading solutions (25%, 35%and 45% sucrose) and durations (7 min and 30 min) with or without changing the unloading solutions. RESULTS: Preculture and osmoprotection treatments were necessary to acquire cytotoxicity tolerance in both vitrification solutions tested and osmoprotection treatment was more critical, especially in B5-80%. A sequential osmoprotection treatment (C10-50%) following conventional osmoprotection (C4-35%) was needed to increase the post-cryopreservation regrowth. Aluminum foil strips were superior to cryovials, but the warming temperature tested (20 degree C and 40 degree C) did not affect post-cryopreservation recovery. In the unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to osmotic stress. CONCLUSION: R. akane hairy roots are very sensitive to cytotoxicity (both osmotic stress and chemical toxicity) and thus a proper process (preculture, osmoprotection, cryoprotection and unloading) is necessary for higher post-cryopreservation recovery.

Contrasting selective pressures on seed traits of two congeneric species by their main native guilds of dispersers on islands.

Many fleshy-fruited plants from the Mediterranean and Macaronesian islands are dispersed through endozoochory. In mainland Mediterranean areas, reciprocal adaptations have been found between plants and animals, although evidence is scarce. On small isolated oceanic islands, such reciprocal adaptations might well be more prevalent due to intrinsic island traits. Here we evaluate the existence of selective pressures exerted by two different disperser guilds (lizards and birds) on two seed traits (seed coat thickness and seed germination pattern) of two congeneric species present on Mediterranean and Macaronesian islands. In the continental Balearic Islands, Rubia peregrina has evolved mostly with birds, although frugivorous lizards are present in some of these islands and are known to eventually consume its fruits. By contrast, R. fruticosa, endemic to the Macaronesian archipelago, has evolved mostly interacting with lizards and only recently with birds. We hypothesized that R. fruticosa would be especially adapted to saurochory, with thicker seed coats and higher germination proportion, whereas R. peregrina would be more adapted to ornithocory, with thinner seed coats and showing a lower germination percentage after being ingested by lizards. Captivity experiments of seed ingestions by natural and non-natural dispersers (i.e., frugivores that have not evolved with those plants) were conducted. Results suggest that dispersers did not exert any strong enough selective pressure to induce changes in germination patterns. We attribute this to the fact that the Rubiaceae is an ancestral family in the Mediterranean (both on continent and islands) and thus probably interacted with lizards in the past. Lastly, although we hold that the seed coat structure of R. fruticosa is probably associated with its evolutionary success after a long interaction with insular lizards, our findings support the idea that the relationship between endozoochorous plants and the guild of dispersers with whom they evolved is rather unspecific.

Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (Nakai) hairy roots using a systematic approach.

A systematic approach using a set of 13 treatments was applied to develop a droplet-vitrification protocol for Rubia akane hairy roots, based on their responses to preculture, loading, dehydration and cooling/rewarming steps. The roots were very sensitive to osmotic stress induced by both preculture in liquid sucrose-enriched medium (up to 0.5 M sucrose) and by dehydration with highly concentrated vitrification solutions (VSs). Loading was necessary before dehydration of explants with VS, and the composition of the loading solution (LS) significantly affected their post-cryopreservation regeneration. Due to high sensitivity of roots to both chemical cytotoxicity and osmotic stress produced by VSs, cryoprotection with alternative VSs, i.e. B5-80 percent (40 percent glycerol + 40 percent sucrose, w/v) at room temperature for 15 min or with A3-70 percent (29.2 percent glycerol + 11.7 percent DMSO + 11.7 percent EG + 17.4 percent sucrose, w/v) at 0 degree C for 20 min ensured the highest post-cryopreservation regeneration. However, when using these solutions, endothermic peaks (enthalpies) with -2.9 and -5.8 J per gram fresh weight, respectively, were recorded by differential scanning calorimetry (DSC) during the rewarming phase. Droplet-vitrification using foil strips showed higher post-cryopreservation regeneration (86 percent) compared with vitrification in cryovials (59 percent), possibly due to the higher cooling and rewarming rates achieved with droplet-vitrification. The developed protocol was applied to hairy roots of five other species with minor modifications in explant type, the duration of the last subculture before explant excision, and the dehydration duration with VS B5-80 percent.

'Personalisation' of droplet-vitrification protocols for plant cells: a systematic approach to optimising chemical and osmotic effects.

Although an appropriate cryopreservation protocol is a prerequisite for basic studies and large-scale implementation as well as further cryopreservation studies, the process relies on trial and error. Among the vitrification-based cryopreservation techniques, droplet-vitrification produces high post-cryopreservation recovery. However, the protocol itself cannot solve the problems engaged in plant cryopreservation, prominently due to dehydration with cytotoxic vitrification solutions. This paper proposes a set of treatments to develop droplet-vitrification using a standard procedure associated with additional treatments and alternative vitrification solutions. The proposed standard protocol consists of a progressive preculture with 0.3 M sucrose for 31 h and with 0.7 M for 17 h, loading with vitrification solution C4-35% (17.5 percent glycerol + 17.5 percent sucrose, w/v) for 20 to 40 min, dehydration with vitrification solutions A3-90 percent (37.5 percent glycerol + 15% DMSO + 15 percent EG + 22.5 percent sucrose) for 10 to 30 min or B1-100 percent (PVS3) for 40 to 120 min at room temperature, cooling the samples using aluminum foil strips, rewarming by plunging into pre-heated (40 degree C) unloading solution (0.8 M sucrose) and further unloading for 20 to 60 min, depending on size and permeability of the materials. Using this systematic approach we can identify whether the material is tolerant or sensitive to chemical toxicity and to the osmotic stress of dehydration with vitrification solutions, thus revealing which is the main barrier in solution-based vitrification methods. Based on the sensitivity of samples we can design a droplet-vitrification procedure, i.e. preculture, loading, dehydration with vitrification solutions, cooling and rewarming. Using this approach, the development of appropriate droplet-vitrification protocol is facilitated.

Can plant oncogenes inhibit programmed cell death? The rolB oncogene reduces apoptosis-like symptoms in transformed plant cells.

The rolB oncogene was previously identified as an important player in ROS metabolism in transformed plant cells. Numerous reports indicate a crucial role for animal oncogenes in apoptotic cell death. Whether plant oncogenes such as rolB can induce programmed cell death (PCD) in transformed plant cells is of particular importance. In this investigation, we used a single-cell assay based on confocal microscopy and fluorescent dyes capable of discriminating between apoptotic and necrotic cells. Our results indicate that the expression of rolB in plant cells was sufficient to decrease the proportion of apoptotic cells in steady-state conditions and diminish the rate of apoptotic cells during induced PCD. These data suggest that plant oncogenes, like animal oncogenes, may be involved in the processes mediating PCD.

Individual and combined effects of the rolA, B, and C genes on anthraquinone production in Rubia cordifolia transformed calli.

It is known that the rolA, rolB, and rolC genes of Agrobacterium rhizogenes T-DNA affect processes of plant development and activate the synthesis of secondary metabolites in transformed plant cells. Although a synergistic activity of the rol genes on root formation is well-documented, little is known about their individual and combined action on secondary metabolism. In the present investigation, we provide evidence indicating that individual rolA, rolB, and rolC genes are capable of increasing biosynthesis of anthraquinones (AQs) in transformed calli of Rubia cordifolia. The stimulatory effect was due to the increased transcription of a key gene of AQ biosynthesis, the isochorismate synthase (ICS) gene. The strongest AQ-stimulating activity was shown for an R. cordifolia culture expressing rolB at high levels, where rolB ensured a 15-fold increase of AQ accumulation compared with the control, non-transformed calli. A tyrosine phosphatase inhibitor abolished the rolB-induced increase of AQ production, thus indicating the involvement of tyrosine (de)phosphorylation in the rolB-mediated AQ stimulation. The rolA- and rolC-expressing cultures produced 2.8- and 4.3-fold higher levels of AQs, respectively, when compared with the control calli. However, the effect of rolA, rolB, and rolC on AQ biosynthesis was not synergistic because rolA and rolC apparently attenuated the stimulatory effect of rolB on AQ biosynthesis. Therefore, the rol-gene-mediated signals that promote root formation and those which activate biosynthesis of secondary metabolites seem to have a point of divergence.