Cryopreservation of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens immobilized on Ca2+ alginate beads and under resting cell condition

Pseudomonas nitroreducens MILB-8054A isolated from petroleum-contaminated soil, immobilized on calcium alginate beads, and under resting cell condition, produced biosurfactants. Immobilized cells gave a best yield of 5.6 g rhamnolipid l^?1 using sucrose as carbon source. Time course study using resting cells showed that 2 % v/v of palm oil (preculture carbon source) and 10 % diesel (carbon source) gave the best rhamnolipid yield of 5.1 g l^?1 at pH 8 and temperature of 30 °C. Carbon utilization by resting cells was compared with that of growing cells. The best biosurfactant recovery procedure was acetone extraction.     

A simple method for cryopreservation of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> callus

Two-years-old Ginkgo biloba cell culture initiated from cotyledonary explants was cryopreserved by a simple desiccation method. Preliminary incubation of callus clumps on MS preculture medium supplemented with 100 g l⁻¹ sucrose and 2 mg l⁻¹ ABA for 7 and 14 days resulted in accumulation of endogenous soluble sugars and was essential for cell culture post-cryopreservation survival. The optimal time for the preculture on sucrose-and-ABA containing medium was found to be 14 days. The sufficient desiccation duration was determined as 150 min. FCM profiles of calli maintained for 2 years remained stable and were not affected by cryopreservation.     

Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

The association between DNA methylation status and embryogenic competency in oil palm tissue culture was examined through Representational Difference Analysis (RDA) approach, using methylation-sensitive restriction endonucleases. “Difference Products” (DPs) of RDA derived from palms of similar genetic backgrounds but exhibiting different embryogenesis rates during the regeneration process were isolated. The DPs were sequenced using a pyrosequencing platform. To our knowledge, this is the first study profiling partial HpaII methylation sites in oil palm young leaf tissues which are potentially associated with embryogenic amenability through a genomic subtractive approach. Quantitative real-time PCR analysis demonstrated that the methylation status of a novel fragment, EgNB3, was higher in highly embryogenic leaf explants compared to low embryogenesis rate materials. These differences are likely to be contributed by the 5′-^mCCGG-3′ and/or 5′-^mC^mCGG-3′ methylation patterns. Our data suggest that the differentially methylated site in EgNB3 has potential as a molecular biomarker for the screening of oil palm leaf explants for their embryogenic potentials.     

In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species

Efficient micropropagation and cryopreservation of Hypericum richeri ssp. transsilvanicum, an endemic species in Romania, and Hypericum umbellatum, a rare and endangered Daco-Balkan species, was achieved. The effects of type of explant and cytokinin on in vitro plant regeneration were investigated. Shoot organogenesis was achieved in all explants, but stem nodes regenerated best. Organogenesis from nodal segments was promoted by incubating these explants on Murashige and Skoog (MS) medium in the presence of cytokinins (6-benzyladenine, thidiazuron, kinetin or 6-??,??-dimethylallylaminopurine), each tested at four concentrations. The best morphogenic response for both Hypericum species (number of shoots per explant, shoot length, axillary branching of shoot, and frequency of shoot organogenesis) was observed when explants were incubated on MS medium containing 0.44 or 1.11???M 6-benzyladenine. Root induction was achieved only when regenerated shoots were transferred to fresh medium with or without auxin. Maximum rooting was recorded on MS medium supplemented with 2.45???M indole-3-butyric acid. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development. Shoot tips and axillary buds excised from the in vitro regenerated plants were successfully cryopreserved in liquid nitrogen by the droplet-vitrification method. Following preculture in 0.25?M sucrose, dehydration and cryopreservation, the highest regeneration rates were obtained in both species by using axillary buds (68?% for H. richeri ssp. transsilvanicum and 71?% for H. umbellatum).     

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.     

Studies on the in vitro regenerative competence of aerial roots of two horticultural important Cymbidium species

In the present study aerial roots were successfully used as explants source for in vitro propagation of Cymbidium aloifolium and Cymbidium iridioides. Aerial roots of ~5?C6?week old from axenic cultures were cultured on MS medium adjuncts with different additives. In C. aloifolium within 20?days of culture ~60% of explants responded positively on MS medium containing sucrose (3%, w/v) and Kn (3???M) and formed PLBs. While in C. iridioides ~50% root explants responded positively on medium enriched with sucrose (3%), AC (0.1%) and IAA (3???M) after 40?days of culture. The shoot buds/PLBs converted into plantlets when maintained on regeneration medium. Of the three basal media tested, MS medium supported optimum regeneration and culture proliferation in both the species. In C. aloifolium ~12 shoot buds developed on medium nourished with sucrose (3%) and BA (3???M) but in C. iridioides optimum regeneration was achieved when medium supplemented with sucrose (3%), CW (15%), CH (100?mg?L^?1) and ~20 shoot buds formed per subculture. The well rooted plantlets were acclimatized for 3?C4?week in 1/10th MS salt solution containing sucrose (1%), charcoal pieces, brick pieces and chopped mosses as support under normal laboratory conditions. The hardened plants were transferred to potting mix where 80 and 75% of transplants survived in C. aloifolium and C. iridioides respectively after 2?months of transfer.     

Enhancement of in vitro shoot regeneration from leaf explants of apple rootstock G.41

Apple (Malus domestica) rootstock G.41 is an excellent member of the Geneva series because it has traits for resistance to abiotic and biotic stresses. A simple and efficient protocol for obtaining shoots from leaf explants was established by optimizing the combinations of plant growth regulators, mode of wounding, and explant orientation on the culture medium. The best shoot multiplication index (2.58) was obtained from successful subculture medium that was the standard Murashige and Skoog (MS) medium supplemented with 7.5 g L^?1 agar, 3.55 μM N ⁶-benzyladenine, 0.16 μM indole-3-butyric acid, and 30 g L^?1 sucrose. Regeneration rates were highest (99%) when MS medium was supplemented with 2.7 μM thidiazuron and 0.9 μM 1-naphthaleneacetic acid, and cut-wounding explants before placing the abaxial surface in contact with the medium. The best rooting percentage (80%) was obtained on MS medium supplemented with 4.92 μM indole-3-butyric acid. Plantlets were rooted in vitro and survived acclimatization in the laboratory and greenhouse.     

Induction and identification of tetraploid <Emphasis Type="Italic">Hedychium coronarium</Emphasis> through thin cell layer culture

We describe an encapsulation–dehydration procedure with prefreezing steps for the cryopreservation of rhizome bud explants of Asparagus officinalis L. cv. Morado de Huétor. With this procedure, survival of Rhizome buds was at least 84 and 42% developed to complete plantlets at 8 weeks. Flow cytometry and EST-SSR molecular markers were used to assess genetic stability of the regenerated material. Effects of preculture time in a medium rich in sucrose and prefreezing treatments (0 °C or/and ??20 °C) on plant recovery were evaluated. Rhizome Buds of the “Morado de Huétor” landrace were incubated in preculture medium (MS?+?0.3 M sucrose) for 48 h, encapsulated in alginate beads and desiccated until a water content of 35%, prefrozen for one hour at 0 °C plus one hour at ? 20 °C, followed by cryopreservation in liquid nitrogen, and then were rewarmed and recovered in ARBM medium for 6 weeks and finally incubated in ARBM-0 for 4 weeks. Analyses of ploidy and molecular stability of plantlets recovered from cryopreserved rhizome buds of two selected genotypes showed no differences compared with the mother plants. Cryopreservation of RB explants of A. officinalis with this Encapsulation–Dehydration procedure will be useful in long-term preservation programs.     

Effect of Explant Source on Axillary Shoot Multiplication During Micropropagation of a Rare Medicinal Plant — Wattakaka volubilis (L f) Stapf

Nodal explants of in vivo plants and in vitro seedlings of Wattakaka volubilis were cultured on Murashige and Skoog medium fortified with various concentrations of cytokinins — BA (0.5–5 mg l^?1), KN (0.5–10 mg l^?1),TDZ (0.05–1 mg l^?1) either singly or in combination with NAA (0.1 mg l^?1). KN proved best for inducing healthy shoots in both in vitro and in vivo derived explants. Maximum number of shoots (14.1±0.84) with 80% regeneration frequency was obtained from nodal explants of seedlings cultured on 5 mg 1^?1 KN + 0.1 mg l^?1 NAA. In vivo nodal explants produced a maximum of 4.2 shoots on MS medium fortified with 2 mg l^?1 BA+0.1 mg l^?1 NAA. The differentiated shoots from both could be rooted with 85% frequency on 1/2 strength MS medium (1% sucrose) with 0.6% agar + 1 mg l^?1 IBA + 0.2 mg l^?1 KN. Rooted shoots were transplanted to vermiculite-soil (3:1) mixture in polyethylene covered pots with 45% transplantation success. Peroxidase isozymes (native PAGE) analysis helped to verify the variation in regenerated plants.     

Micropropagation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) and application of mycorrhiza to improve plantlet establishment

Micropropagation methods were developed for the three French varieties of olive (Olea europaea L.), Aglandau and Tanche, that have the Appelation d’Origine Contrôlée (AOC) status and one ecotype (05300, Laragne, France). Explants consisting of partially lignified nodal segments were collected from rejuvenated glasshouse-grown plant material. Nodal explants with axillary buds were cultured on different media. For AOC varieties, olive medium modified (OM mod) to contain half the concentration of macroelements was the most efficient in inducing bud break and growth when supplemented with 30 g l^?1sucrose and 4 mg l^?1 zeatin. The resulting shoot buds were further multiplied and maintained on OM mod medium. Rooting was best achieved on OM supplemented with 4 mg l^?1 indole-3-butyric acid (IBA). For the Laragne ecotype, maximal shoot proliferation occurred when explants were cultured on woody plant medium supplemented with 15 g l^?1 sucrose and 0.1 mg l^?1 zeatin. Efficient rooting was achieved with 1 mg l^?1 IBA combined with 0.75 mg l^?1 naphthaleneacetic acid. After acclimatization in the glasshouse, survival rates ranged from 57 to 92%, depending on the genotype. Inoculation of Laragne ecotype microplantlets with the arbuscular mycorrhizal fungus Glomus mosseae significantly improved plant survival and subsequent plant development and growth.     

An improved protocol for micropropagation of Mallotus repandus (Willd.) Müll.Arg.

Node and internode explants of Mallotus repandus were precultured on basal medium (BM: Murashige and Skoog (MS) medium with 3% sucrose and 0.55% Agargel) for 0–18 d before culture on shoot induction Medium (SIM: BM added with 4.44 μM of benzylaminopurine) for 4 wk. The cultures were subsequently transferred to BM for 4 wk for shoot elongation. Node explants precultured on BM for 14 d before incubation on SIM were at an optimum for shoot regeneration with the response rate of 95%, compared to a 21% response for the control without preculture. Internode explants precultured on BM for 16 d responded with an optimal shoot formation response rate of 69%, whereas the control response rate was 6%. The maximum shoot regeneration rates were 3.1 ± 0.3 and 2.7 ± 0.4 shoots/responding explant in node and internode explants, respectively. This study demonstrates for the first time that shoot organogenesis can be induced from internode explants of M. repandus. Furthermore, the results suggest that the explants need to acquire competence before shoot organogenesis. Rooting was obtained by incubation of regenerated shoots on half-strength MS with 10.74 μM of 1-naphthylacetic acid for a week before culture on half-strength MS for 4 wk. Regenerated plants were successfully transferred to soil.     

Carbohydrates Modulate the In Vitro Growth of Olive Microshoots. I. The Analysis of Shoot Growth and Branching Patterns

The rate of microshoot proliferation during the micropropagation of olive (Olea europaea L.) plants is limited by the low rates of both bud sprouting and growth of secondary shoots following subculturing. The aim of this study was to determine (1) the effects of sucrose and mannitol on shoot growth, (2) whether either of these sugars modifies the pattern of shoot development of the explants, and (3) the influence of apical dominance on explant development. Working with single-node microcuttings of Olea europaea L. cv. Maurino with two opposite axillary buds, we added 17, 34, or 68 g L^?1 of sucrose or mannitol to the medium as the primary carbon source. Shoot development was classified as either (a) an outgrowth of the first bud on an explant (shoot-type A), (b) an outgrowth of the second bud (shoot-type B), or (c) an outgrowth of an axillary bud on either an A- or B-type shoot (shoot-type C). Explant survival, fresh-mass production, and patterns of shoot development were influenced by the type and concentration of sugar used. Mannitol promoted the sprouting and growth of A-, B-, and C-type shoots more than sucrose. The developmental responses observed indicate that the growth of axillary meristems of in vitro olive explants is not regulated by apical dominance. The results demonstrate that the sugar alcohol plays an important role in the developmental regulation of olive explants. Mannitol may also protect against detrimental effects associated with in vitro growth conditions.     

Indirect somatic embryogenesis and plant regeneration from leaf and internode explants of Paulownia elongata

Several plant growth regulators BA, TDZ, 2,4- and Kn were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf and internode explants of Paulownia elongata. Calli were produced when leaf explants were cultured on Murashige and Skoog (MS) medium containing 3 % sucrose, 0.4 % phytagel, 4 mg l^-1 TDZ and 0.1 mg l^-1 Kn after 3 weeks and the initiation rate was 54.1%. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto MS medium supplemented with 3 % sucrose, 0.4 % phytagel, 0.1 mg l^-1 TDZ, 1 mg l^-1 Kn and 2 mM glutamine. An average of 50.7 somatic embryos were obtained from 100 mg of embryogenic callus after 4 weeks at high frequency (64.7 %). Afterward, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination (80 %) and development into plantlets. Plantlets were transferred to pots with a mixture of peat and perlite in a 3:1 ratio and showed a survival rate of 70–80 %. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in the greenhouse.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Germplasm conservation of Guazuma crinita,a useful tree in the Peru-Amazon,by the cryopreservation of in vitro-cultured multiple bud clusters

In vitro-cultured adventitious bud clusters of Guazuma crinita Mart. were successfully cryopreserved by the one-step vitrification method. Small segments (1.0-1.5 mm³) cut from adventitious bud clusters were exposed to a cryoprotectant mix solution containing (w/v), 25 glycerol, 15 sucrose, 15 ethylene glycol, 13 dimethyl sulfoxide, and 2 polyethylene glycol, at 25 °C for 15-60 min prior to storage in liquid nitrogen. After rapid warming (37 °C), the segments were treated with woody plant medium containing 40 (w/v) sucrose for 20 min at 25 °C, and then transferred to recovery-growth medium. High survival rates (about 80) were achieved without any cold hardening and/or pre-culturing treatments, and about 30 of the surviving cryopreserved explants regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomoea batatas)

Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Propagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. (holm oak) trees by somatic embryogenesis

Somatic embryogenesis from in vitro leaf and shoot apex explants excised from axillary shoot cultures established from two mature Quercus ilex trees has been developed. Somatic embryos (SE) were obtained from both explant types and genotypes evaluated, although embryogenic frequencies were influenced by the genotype, auxin concentration, and explant type. The explants were cultured on Murashige and Skoog salts and vitamins, supplemented with 500 mg L^?1 casein hydrolysate (CH) and different concentrations of indole-3-acetic acid or α-naphthalene acetic acid (NAA) in combination with 2.22 µM 6-benzylaminopurine (BA). In both genotypes, shoot apex explants were more responsive than leaf explants. The best results were obtained with apex explants of clone Q3 (11%) cultured on medium with 21.48 µM NAA plus 2.22 µM BA. This combination was also effective for initiating SE from leaf explants, although the induction rates were lower (1–3%). Embryogenic lines were maintained by repetitive embryogenesis following culture of nodular embryogenic structures on Schenk and Hildebrand medium without plant growth regulators. Low embryo multiplication rates were obtained when torpedo or early cotyledonary SE were used as initial explant for embryo proliferation, or when glutamine or CH (500 mg L^?1) was added to proliferation medium. For germination, cotyledonary-stage SE were isolated and stored at 4 °C for 2 months. After cold storage, SE were cultured on germination medium consisting of Gresshoff and Doy medium, supplemented with 0.44 μM BA and 20 μM silver thiosulphate. Under these conditions, plantlets were regenerated from 21 to 66.7% of the SE generated for both genotypes.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.