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The physiological age of mother tubers (Solanum tuberosum L. cv. Kennebec) used as a source of material influenced kinetin-induced in vitro tuberisation. Tuberisation significantly increased with physiological age. Kinetin- or ancymidol-induced tuberisation, plantlet and microtuber dry weight decreased with increasing number of subcultures. Single-node segments obtained from tubers stored for more than 9.5 months at 4 °C showed increased kinetin-induced tuberisation rates and earlier tuberisation than those obtained from younger tubers. For any physiological age, material may be safely multiplied using node propagation until the third subculture and bioassayed for tuberisation without variation in the response.

Histidine-tagged human cardiotrophin-1 (hCT-1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5'-untranslated regions (5'-UTRs) (psbA and phage T7 gene 10). The psbA 5'-UTR promotes recombinant hCT-1 (rhCT-1) accumulation in chloroplasts at higher levels (eight-fold) than those obtained for the phage T7 gene 10 5'-UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT-1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5'-UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (approximately 35%) than that of commercial hCT-1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT-1, suggesting that 32 h of over-lighting affects the biological activity of rhCT-1. Because high levels of rhCT-1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a 'closed system' unit, yielding up to 3.2 kg per year of rhCT-1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT-1 for liver transplantation surgery in a cost-effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant-based biopharmaceuticals.

To optimize minituber production through aeroponics some horticultural management factors should be studied. Potato plantlets, cv Zorba, were grown aeroponically at two different plant densities (60 and 100 plants/m2). Plants showed an extended vegetative cycle of about 5 months after planting. A higher number of stolons was obtained at low plant densities. Tuber formation hastened when supplied N was reduced. Experiments on harvesting intervals (7, 10, and 14 days) indicated that for a density of 60 plants/m2, both number of minitubers and yield increased as harvesting interval decreased. Best results were achieved harvesting every 7 days: a total tuber yield of 118.6 g per plant was obtained (four times higher than for 100 plants/m2). Such a yield was composed, on the average, of 13.4 tubers with a mean tuber weight of 8.1 g. Harvesting intervals did not have an effect on the number of minitubers and yield for a density of 100 plants/m2. The best productivity obtained in this study was 800 minitubers/m2 for weekly harvests and a low plant density (60 plants/m2). We also studied the field performance of aeroponically produced minitubers vs those produced by hydroponics. Minituber behavior under field conditions was independent from the technique used for its production.

The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G(M1)-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (approximately 100 mg for a dose of 500 microg antigen) and would permit encapsulation of freeze-dried material or pill formation.

The gelling agent Phytagel™ was compared with Difco Bacto-agar forin vitro tuberization of six potato cultivars (Jaerla, Blanka, Claustar, Kennebec, Desiree, and Baraka). On a culture medium with 6% sucrose but lacking growth regulators, tuberization was higher when Phytagel™ was used rather than Difco Bacto-agar, regardless of the photoperiodic regime used. Chemical analyses of the gelling agents revealed a higher mineral content and organic impurities in Bacto-agar than in Phytagel™, which is therefore recommended for microtuber production. The same gelling agent should be used in all treatments of microtuberization experiments in order to draw meaningful physiological conclusions.

Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.

SummaryThe maturity of a new potato clone can be determined from physiological measurements in field trials that involve considerable time, space and work. To improve the procedure, an in vitro bioassay based on tuberization behaviour of potato cultivars of different maturity classes was established. Twenty-six sets of temperature, photoperiod and photosynthetic photon flux values were assayed on the eight potato cultivars Jaerla, Zorba, Spunta, Kennebec, Turia, Desiree, Baraka and Fenix. In vitro tuberization was influenced by cultivar, photoperiod and temperature. The environmental condition defined by 12 h photoperiod, 34 μmol m–2 s–1 of photosynthetic photon flux and 25°C temperature produced statistical differences in tuberization among maturity classes. Both the earliness of tuberization and the “degree of tuberization” (based on the type of microtubers developed) permitted assigning a cultivar to a maturity class. Early cultivars showed higher readiness to tuberize and higher values of “degree of t...

A medium containing low amounts of nitrogen (19–23 meq.l−1) produced optimum results in micropropagation as revealed by the number of nodes, internode length, chlorophyll content, and leaf area of four potato cvs. belonging each to four different maturity groups. Decreasing amounts of nitrogen also increased chlorophyll content in all four cultivars tested. The NH 4 + concentration did not have an effect on micropropagation for low nitrogen supplies. In all cvs., except Baraka, there was a “carry over” effect of the nitrogen content in the micropropagation medium onto subsequent tuberization, the lower nitrogen (23 meq.l−1) advancing tuber initiation. Microtuberization of cv. Jaerla was earlier in darkness than under short days regardless of the propagation medium used.

Summary A significant increase in the number of tubers was obtained in the presence of either kinetin (11.6·10 -3 mM), paclobutrazol (8.5·10 -3 mM) or acetate (5.4 mM) for in vitro stolons in darkness, and both single-node sections and intact 3-cm long sprouts were produced after long photoperiod (16 h) subculture. Longitudinal growth decreased whenever tuberization was promoted, especially in the presence of paclobutrazol. Calcium chloride (6 mM) slightly induced tuberization in the dark. Putrescine (0.15 mM) favoured vegetative development in all explants.

14 p., 7 figures and bibliography, We previously showed that recombinant extra domain A from fibronectin (EDA) purified from Escherichia coli was able to bind to toll-like receptor 4 (TLR4) and stimulate production of proinflammatory cytokines by dendritic cells. Because EDA could be used as an adjuvant for vaccine development, we aimed to express it from the tobacco plastome, a promising strategy in molecular farming. To optimize the amount of recombinant EDA (rEDA) in tobacco leaves, different downstream sequences were evaluated as potential fusion tags. Plants generated by tobacco plastid transformation accumulated rEDA at levels up to 2% of the total cellular protein (equivalent to approximately 0. 3 mg/g fresh weight) when translationally fused to the first 15 amino acids of green fluorescence protein (GFP). The recombinant adjuvant could be purified from tobacco leaves using a simple procedure, involving ammonium sulfate precipitation and anion exchange chromatography. Purified protein was able to induce production of tumour necrosis factor-α (TNF-α) either by bone marrow-derived dendritic cells or THP-1 monocytes. The rEDA produced in tobacco leaves was also able to induce upregulation of CD54 and CD86 maturation markers on dendritic cells, suggesting that the rEDA retains the proinflammatory properties of the EDA produced in E. coli and thus could be used as an adjuvant in vaccination against infectious agents and cancer. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of this protein vaccine adjuvant., This work was supported by a grant (Proyecto EUROINNOVA) from the Department of Innovation (Gobierno de Navarra)

Are short-day (SD) conditions inductive or permissive for potato tuberization? This question was addressed using anin vitro culture system. Kinetin induction ofin vitro tuberization in potato stolons cultured in the dark can be partially inhibited by light treatments. Photoperiod required for a major inhibition varied with cultivars: 8 hours for cv. Red Pontiac and 16 hours for cv. Kennebec. Short photoperiods decreased kinetininduced tuberization for all cultivars. Stolons cultured on kinetin-free medium generally did not tuberize, regardless of light treatment. Effects of kinetin and photoperiod on stolon and tuber development are observed. Relationships among kinetin, photoperiod and tuber-inducing stimulus produced under short days are discussed.

Amyloplast division was studied in an in vitro potato (Solanum tuberosum L. cv. Kennebec) stolon tuberizing system which was triggered by the presence of added kinetin. Amyloplast division proceeded by binayr fission of dumbbell shaped amyloplasts. A visible tubular coiled structure from the amylobody appeared to be associated with amyloplast division. Possible ways of daughter amyloplast separation are discussed.

Histidine-tagged human cardiotrophin-1 (hCT-1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5'-untranslated regions (5'-UTRs) (psbA and phage T7 gene 10). The psbA 5'-UTR promotes recombinant hCT-1 (rhCT-1) accumulation in chloroplasts at higher levels (eight-fold) than those obtained for the phage T7 gene 10 5'-UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT-1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5'-UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (approximately 35%) than that of commercial hCT-1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT-1, suggesting that 32 h of over-lighting affects the biological activity of rhCT-1. Because high levels of rhCT-1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a 'closed system' unit, yielding up to 3.2 kg per year of rhCT-1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT-1 for liver transplantation surgery in a cost-effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant-based biopharmaceuticals.

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.

Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.

The aim of the study was to assess the potential in vitro effects of jasmonic acid and kinetin on tuberization of potato (Solanum tuberosum). Of the two, the former was by far the stronger in vitro promoter of stolon tuberization. Number of tubers induced per stolon, tuberization rate, and final tuber weight were higher by factors of 2.8, 2.3, and 6.4, respectively. Bioassay sensitivity of jasmonic acid, measured in terms of the point at which the concentration for inducing tuberization was saturating, was more than 20 times greater than that of kinetin. Tuberization in both cases was associated with a decrease in rooting ability. Jasmonic acid also triggered a general state of induction throughout the stolon.

Ethylene inhibited the tuberization of etiolated potato ( Solanum tuberosum L. var. Red La Soda) sprout sections cultured in vitro . Carbon dioxide did not overcome the C 2 H 4 inhibition but it was required for normal tuberization. Ethylene totally prevented root formation and development. It inhibited stolon elongation, and caused thickening and diageotropical growth of the stolon. In addition, C 2 H 4 prevented the accumulation of both starch and red anthocyanin which are always present in a tuber. Ethylene also inhibited the kinetin-increased tuberization of sprout sections. Three to five days of exposure to CO 2 were required to obtain promotion of tuberization of stolons cultured in vitro . Bicarbonate ion did not affect starch synthetase activity isolated from potato tubers in vitro . The evidence presented suggests that CO 2 gas rather than HCO − 3 or CO 2− 3 ions in equilibrium with dissolved CO 2 was probably responsible for the stimulation. Morphological changes elicited by CO 2 and C 2 H 4 are described and the mechanism of action of both on tuberization is discussed.

Carbon dioxide stimulates tuberization of isolated potato (Solanum tuberosum L.) stolons cultured in vitro. The stimulatory effect is inhibited by C2H4 which is by itself also inhibitory of tuberization. Furthermore, C2H4 inhibits kinetin-induced tuber initiation. Both the formation and elongation of roots are inhibited by C2H4. The antagonistic actions of CO2 and C2H4 on tuberization are discussed.

Phytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.