Chloroplast Proteases

J.S. Nair, N.K. Ramaswamy

Biologia plantarum 48:321-326, 2004 | DOI: 10.1023/B:BIOP.0000041081.00086.85

The chloroplast within the plant cell has a dynamic environment where proteases play an important role in processing of precursor proteins, degradation of incomplete proteins lacking cofactors, stress-induced degradation and removal of damaged proteins. A number of proteases in the chloroplast are well characterized and found to be localized within different compartments such as stroma, thylakoids and lumen. In recent years, an increasing number of proteases in chloroplasts have been discovered and identified as bacterial protease homologues. These include the stromal Clp, thylakoidal FtsH and lumenal DegP. The current focus is to understand their...

Imaging of Calcium Channels During Polarity Induction in Plant Cells

S.C. Bhatla, G. Kalra

Biologia plantarum 48:327-332, 2004 | DOI: 10.1023/B:BIOP.0000041082.37723.c7

Understanding the molecular basis of polarity induction in plant cells is a research aspect that extends from signal perception and transduction to morphogenesis. A gradient of cytoplasmic ion fluxes generated through ion channels plays a crucial role in subsequent events leading to polar growth. Convincing evidence is now available implicating temporal and spatial distribution of Ca²⁺ in cytoplasm, generated by localized activity of calcium channels, as the early biochemical events associated with polarity induction. Ion channel antagonists are common tools for studying ion channel structure and function. Coupled with a fluorescent dyes,...

In vitro Regeneration and Transformation of Blackstonia perfoliata

A. Bijelović, N. Rosić, J. Miljuš-Djukić, S. Ninković, D. Grubišić

Biologia plantarum 48:333-338, 2004 | DOI: 10.1023/B:BIOP.0000041083.41294.31

In vitro root culture of yellow wort (Blackstonia perfoliata (L.) Huds.) was initiated on Murashige and Skoog (MS) medium. In the presence of benzylaminopurine (BAP) numerous adventitious buds formed, which developed into shoots. Presence of indole-3-butyric acid (IBA) in media significantly decreased number of buds, but increased development of lateral roots. On hormone-free medium shoots successfully rooted and developed flowers and viable seeds that formed another generation. Shoot cultures of B. perfoliata inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS developed hairy roots at 3 weeks and they...

Micropropagation of an Endangered Orchid Anoectochilus formosanus

N.V. Ket, E.J. Hahn, S.Y. Park, D. Chakrabarty, K.Y. Paek

Biologia plantarum 48:339-344, 2004 | DOI: 10.1023/B:BIOP.0000041084.77832.11

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^-3 benzyladenine or 1 - 2 mg dm^-3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^-3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented...

Cloning of a Gene Encoding a Putative Ethylene Receptor in Catharanthus roseus and its Expression in Plant and Cell Cultures

N. Papon, M.M. Senoussi, F. Andreu, M. Rideau, J.-C. Chenieux, J. Creche

Biologia plantarum 48:345-350, 2004 | DOI: 10.1023/B:BIOP.0000041085.82296.9c

A full length cDNA (designated CrETR1) was isolated by polymerase chain reaction amplification of a cDNA library from periwinkle (Catharanthus roseus) cell cultures. CrETR1 cDNA encodes a polypeptide of 740 amino acids with a predicted molecular mass of 82 kDa. The deduced protein contains a hydrophobic ethylene-binding transmembrane region, a GAF domain, a third domain homologous to the histidine protein kinase domain of the prokaryotic two-component systems, and a fourth carboxyl-terminal domain homologous to the receiver domain of the response regulators, as found in the A. thaliana ethylene receptor ETR1. CrETR1...

Protein Phosphorylation During the Transitions from G1 Arrest Point to S and G2 Arrest Point to M in Vicia faba Root Meristem

J. Polit, J. Maszewski

Biologia plantarum 48:351-359, 2004 | DOI: 10.1023/B:BIOP.0000041086.68286.b1

Using monoclonal FITC conjugated antibodies that specifically recognize phosphorylated form of threonine (α-T^P-FITC) it was shown that in excised, sugar-starved root meristems of Vicia faba subsp. minor the expression of two principal control points (PCPs) is correlated with a marked decrease in nuclear and nucleolar phosphorylation of proteins. When stationary phase meristems are supplied with 2 % sucrose, the G1-arrested cells start out DNA replication (assessed by pulse ³H-thymidine labeling), while the cells blocked in G2 phase initiate mitotic divisions. Using this model, we have found that depending on...

Khush, G.S., Brar, D.S., Hardy, B. (ed.): Rice Genetics IV

J. Doležel

Biologia plantarum 48:360, 2004 | DOI: 10.1023/B:BIOP.0000041123.29979.b7

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

J. Song, D.E. Lee, S. Jung, H. Kim, O. Han, B.H. Cho, I.J. Lee, K. Back

Biologia plantarum 48:361-366, 2004 | DOI: 10.1023/B:BIOP.0000041087.17353.d8

Fatty acid ω-3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 - 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic...

Production of Transgenic Soybean Plants with Two Anti-Fungal Protein Genes Via Agrobacterium and Particle Bombardment

H.Y. Li, Y.M. Zhu, Q. Chen, R.L. Conner, X.D. Ding, J. Li, B.B. Zhang

Biologia plantarum 48:367-374, 2004 | DOI: 10.1023/B:BIOP.0000041088.62614.76

Utilizing either Agrobacterium-mediated transformation or particle bombardment we obtained transgenic soybean [Glycine max (L.) Merr.] plants expressing the chitinase gene (chi) and the barley ribosome-inactivating protein gene (rip). Six regenerated plants were grown to maturity and set seed. The identification of transgenic soybean plants that co-integrated the two anti-fungal protein genes was determined by polymerase chain reaction (PCR) and Southern blot analysis. Protein detection from the soybean leaves demonstrated the expression of the chitinase (CHI) and the ribosome-inactivating protein (RIP) in the six R₀...

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

M. Grilli Caiola, P. Caputo, R. Zanier

Biologia plantarum 48:375-380, 2004 | DOI: 10.1023/B:BIOP.0000041089.92559.84

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The...

Senescence of Unfertilised Flowers in Epiphyllum Hybrids

O. Erdelská, M. Ovečka

Biologia plantarum 48:381-388, 2004 | DOI: 10.1023/B:BIOP.0000041090.36790.86

Epiphyllum hybrids served as a model for the study of reserve remobilisation from unfertilised flowers to the mother stem tissues. Early phases of the tube senescence characterised by wilting were connected with degradation and transfer of reserve substances to the somatic organs of the mother plant. The degradation process began in perianth and stamens and continued through the successive zones (receptacular, pericarpellar and pedicellar) of the flower tube. The phloem-mediated backward substance transport was naturally indicated by the red pigment of the perianth -- cactorubin, while integrity of cells and tissues and green colour of the flower...

Heat Shock Induces Programmed Cell Death in Wheat Leaves

T. Fan, T. Xing

Biologia plantarum 48:389-394, 2004 | DOI: 10.1023/B:BIOP.0000041091.03068.ee

Programmed cell death (PCD) was triggered in wheat leaves by a heat treatment (42 °C). This treatment caused DNA fragmentation as shown in DNA laddering analysis and in terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate (dUTP) nick end labeling (TUNEL) analysis. Methanol and acetone treatment of leaves significantly blocked PCD. Western analysis indicated that a 65 kDa poly(ADP-ribose) polymerase-like protein was degraded during the treatment. However, high temperature (80 °C) treatment caused necrosis but not PCD.

Effects of Pre-Treatments with Abscisic Acid and/or Benzyladenine on Gas Exchange of French Bean, Sugar Beet, and Maize Leaves During Water Stress and After Rehydration

J. Pospíšilová, P. Baťková

Biologia plantarum 48:395-399, 2004 | DOI: 10.1023/B:BIOP.0000041092.40705.6b

Net photosynthetic rate (P_N), transpiration rate (E), and stomatal conductance (g_s) during water stress and after rehydration were measured in Phaseolus vulgaris, Beta vulgaris, and Zea mays. Immediately before imposition of water stress by cessation of watering, plants were irrigated with water (control), 100 μM abscisic acid (ABA), and/or 10 μM N⁶-benzyladenine (BA). In all three species, application of ABA decreased g_s, E, and P_N already 1 h after application. However, during water stress g_s, E, and P_N in plants pre-treated with ABA remained higher than...

Davidian, J.-C., Grill, D., De Kok, L.J., Stulen, I., Hawkesford, M.J., Schnug, E., Rennenberg, H. (ed.): Sulfur Transport and Assimilation in Plants. Regulation, Interaction and Signaling

O. Gašparíková

Biologia plantarum 48:400, 2004 | DOI: 10.1023/B:BIOP.0000041124.30253.21

Changes Induced by Low Oxygen Concentration in Photosynthetic and Respiratory CO₂ Exchange in Phosphate-Deficient Bean Leaves

S. Maleszewski, I. Ciereszko, A. Skowrońska, E. Mieczejko, B. Kozłowska-Szerenos

Biologia plantarum 48:401-405, 2004 | DOI: 10.1023/B:BIOP.0000041093.46102.0d

Effect of phosphorus deficiency on photosynthetic and respiratory CO₂ exchanges were analysed in primary leaves of 2-week-old bean (Phaseolus vulgaris L. cv. Golden Saxa) plants under non-photorespiratory (2 % O₂) and photorespiratory (21 % O₂) conditions. Low P decreased maximum net photosynthetic rate (P_Nmax) and increased the time necessary to reach it. In the leaves of P-deficient plants the relative decrease of P_Nmax at 2 % O₂ was larger than at 21 % O₂. The results suggested the influence of photorespiration in the cellular turnover of phosphates.

Delmer, D.P., Bohnert, H.J., Merchant, S. (ed.): Annual Review of Plant Biology. Volume 54, 2003

Z. Šesták

Biologia plantarum 48:406, 2004 | DOI: 10.1023/B:BIOP.0000041125.66998.3b

Effects of Brassmosteroids on Growth, Nodulation, Phytohormone Content and Nitrogenase Activity in French Bean Under Water Stress

K.K. Upreti, G.S.R. Murti

Biologia plantarum 48:407-411, 2004 | DOI: 10.1023/B:BIOP.0000041094.13342.1b

Effect of pre-treatments of 1 and 5 μM epibrassinolide or homobrassinolide prior to water stress induction on changes in root nodulation and contents of endogenous abscisic acid (ABA) and cytokinin trans-zeatin riboside (ZR), and nitrogenase activity was investigated in the nodulated roots of Phaseolus vulgaris L. cv. Arka Suvidha. Brassinosteroids in the unstressed plants increased root nodulation, ZR content and nitrogenase activity, and also ameliorated their stress-induced decline in the nodulated roots. The ABA contents in the nodules of control or stressed plants were not altered by brassinosteroids treatment. There was an increase...

Effects of Cadmium on the Metabolic Activity of Avena sativa Plants Grown in Soil or Hydroponic Culture

S. Astolfi, S. Zuchi, C. Passera

Biologia plantarum 48:413-418, 2004 | DOI: 10.1023/B:BIOP.0000041095.50979.b0

Oat (Avena sativa L.) plants cultured in soil and hydroponic culture were treated with cadmium [0.154 mg g^-1 (dry soil) and 100 μM CdSO₄, respectively] for 21 d and growth rate and various biochemical processes were studied. Applied cadmium reduced plant growth and chlorophyll content. Changes in activity of enzymes involved in C, N and S metabolism and in guaiacol peroxidase activity were observed. In particular, O-acetylserine sulphydrylase (OASS; EC 4.2.99.8) activity was increased by Cd exposure in both growth conditions, probably as a resistance mechanism to cadmium based on the production of phytochelatins....

TMV-RNA Biosynthesis in the Light-Green and Dark-Green Regions of Tobacco Leaves

M. Šindelářová, L. Šindelář

Biologia plantarum 48:419-423, 2004 | DOI: 10.1023/B:BIOP.0000041096.21583.61

Changes in the proteins, chlorophyll, virus content and activity of key enzymes of viral RNA biosynthesis were investigated in the light- and dark-green regions of tobacco leaves systemically infected with tobacco mosaic virus. The protein content was increased to 118 % in the dark-green islands in contrast to 60 % in the light-green regions when compared with the control healthy leaves. The comparative analysis of soluble proteins from healthy and light- or dark-green regions of leaves by means of SDS-PAGE revealed that the main soluble proteins are equal in pattern but differ in quantity. The contents of chlorophylls did not differ from healthy tissues...

Jasmonic Acid and Salicylic Acid Induce Accumulation of β-1,3-Glucanase and Thaumatin-Like Proteins in Wheat and Enhance Resistance Against Stagonospora nodorum

J. Jayaraj, S. Muthukrishnan, G.H. Liang, R. Velazhahan

Biologia plantarum 48:425-430, 2004 | DOI: 10.1023/B:BIOP.0000041097.03177.2d

The effect of application of jasmonic acid (JA) and salicylic acid (SA) on the induction of resistance in wheat to Stagonospora nodorum and on the induction of μ-1,3-glucanase and thaumatin-like proteins (TLPs) was studied. Western blot analysis revealed that two β-1,3-glucanases with apparent molecular masses of 31 and 33 kDa that cross-reacted with a barley glucanase antiserum were induced in wheat leaves after treatment with JA and SA. When wheat plants were treated with SA and JA, a TLP with an apparent molecular mass of 25 kDa and several other isoforms of TLP were induced. Pre-treatment of wheat plants with SA and JA significantly...

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

T. Sretenović-Rajičić, S. Ninković, B. Vinterhalter, J. Miljuš-Djukić, D. Vinterhalter

Biologia plantarum 48:431-436, 2004 | DOI: 10.1023/B:BIOP.0000041098.18836.be

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm^-3 6-benzyladenine and 0.5 mg dm^-3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm^-3 phosphinothricin (L-PPT) supplemented medium...

Esterase Isoenzymes as Markers for the VA 1 Gene of Zea mays and for the B Linkage Group of Tripsacum dactyloides

V. Tsanev, R. Vladova, K. Petkolicheva, B. Kraptchev, C. Milanov

Biologia plantarum 48:437-439, 2004 | DOI: 10.1023/B:BIOP.0000041099.33602.7f

It is suggested that cathodal esterase isoenzyme (E₁) might be used as a marker for the VA 1 gene on 7 S maize chromosome and for the "B" linkage group of Tripsacum dactyloides in Zea mays L. ×T. dactyloides L. hybrids. The latter genic zones have a regulatory effect on fertility and on the apomictic mode of reproduction.

In vitro Crown Galls Induced by Agrobacterium tumefaciens Strain A281 (pTiBo542) in Trigonella foenum-graecum

K.M. Khawar, S. Gulbitti-Onarici, S. Çöçü, S. Erisen, C. Sancak, S. Özcan

Biologia plantarum 48:441-444, 2004 | DOI: 10.1023/B:BIOP.0000041100.94688.2d

Transformation of fenugreek (Trigonella foenumgraecum) was carried out with A281 oncogenic strain of Agrobacterium tumefaciens using root, cotyledon and hypocotyl explants excised from 1-week-old seedlings, which showed that the plant was highly susceptible to transformation. Tumors (calli) were selected on 50 mg dm^-3 kanamycin. They were analyzed for β-glucuronidase (GUS) expression. Presence of uidA (gus) gene, was confirmed by polymerase chain reaction (PCR) amplification.

Influence of Phosphorus Application on Water Relations, Biochemical Parameters and Gum Content in Cluster Bean Under Water Deficit

Shubhra, J. Dayal, C.L. Goswami, R. Munjal

Biologia plantarum 48:445-448, 2004 | DOI: 10.1023/B:BIOP.0000041101.87065.c9

Relative water content (RWC), leaf water potential (Ψ_w) and osmotic potential (Ψ_s), contents of chlorophyll (Chl) a, Chl b, soluble sugars, and seed quality (gum content) were used to evaluate the role of phosphorus in alleviation of the deleterious effect of water deficit in clusterbean (Cyamopsis tetragonoloba L. Taub). Under water stress, Ψ_w, Ψ_s, and Chl and gum contents decreased and soluble sugar contents increased. Phosphorus application increased Chl and sugar contents in control plants and ameliorated negative effects of water stress.

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

S. Çöçü, S. Uranbey, A. İpek, K.M. Khawar, E.O. Sarihan, M.D. Kaya, İ. Parmaksiz, S. Özcan

Biologia plantarum 48:449-451, 2004 | DOI: 10.1023/B:BIOP.0000041102.79647.b6

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^-3 TDZ and either 0.25 or 0.50 mg dm^-3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range...

Alleviation of Cadmium Toxicity by Naphthenate Treatment

S. Kevrešan, V. Ćirin-Novta, K. Kuhajda, J. Kandrač, N. Petrović, Lj. Grbović, Ž. Kevrešan

Biologia plantarum 48:453-455, 2004 | DOI: 10.1023/B:BIOP.0000041103.17285.26

The work is concerned with the effect of low concentrations (10^-7 mol dm^-3) of sodium naphthenate on total content of Cd and its particular forms in the intercellular space and inside cells, as well as on some physiological and biochemical parameters of young soybean plants grown in the presence of 1 mmol dm^-3 solution of cadmium chloride. Presence of naphthenate reduced in average by 40 % content total and intracellular Cd in root, stem and leaves and alleviated the harmful effect of Cd on activity of nitrate reductase and content of photosynthetic pigments.

Variation in Drought Tolerance of Different Stylosanthes Accessions

A. Chandra, P.S. Pathak, R.K. Bhatt, A. Dubey

Biologia plantarum 48:457-460, 2004 | DOI: 10.1023/B:BIOP.0000041104.54922.31

Twenty genotypes of Stylosanthes consisting four species were evaluated under rain fed condition employing biochemical and physiological attributes to select drought tolerant lines. Relative water content measured at 50 % flowering stage of the plants showed significant variations among the lines which ranged from 32.11 in S. scabra RRR94-86 to 83.33 % in S. seabrana 2539. The results indicated that S. scabra genotypes were more tolerant to drought over other lines as evidenced by high leaf thickness, proline accumulation, content of sugars and chlorophyll, and nitrate reductase activity.

Influence of Salt Stress on Growth, Ion Accumulation and Seed Oil Content in Sweet Fennel

M. Ashraf, N. Akhtar

Biologia plantarum 48:461-464, 2004 | DOI: 10.1023/B:BIOP.0000041105.89674.d1

A greenhouse experiment was conducted to assess the effect of 25, 50, 75, and 100 mM NaCl on growth, ion accumulation, seed yield, and seed oil content in 67-d-old plants of Foeniculum vulgare Mill. Increasing NaCl concentration caused a significant reduction in fresh and dry masses of both shoots and roots as well as seed yield. Na⁺ and Cl^- in both shoots and roots increased, whereas K⁺ and Ca²⁺ decreased consistently with the increase in NaCl concentration. Plants maintained markedly higher Ca²⁺/Na⁺ ratios in the shoots than those in the roots, whereas that of K⁺...

Responses of Vigna radiata to Foliar Application of 28-Homobrassinolide and Kinetin

Q. Fariduddin, A. Ahmad, S. Hayat

Biologia plantarum 48:465-468, 2004 | DOI: 10.1023/B:BIOP.0000041106.77930.d6

Effects of 28-homobrassinolide (HBR) and kinetin (KIN) on photosynthesis, nitrogen metabolism, and the seed yield were studied. The leaves of 25-d-old plants of Vigna radiata (L.) Wilczek were sprayed with 0.01, 1.0 or 100 μM aqueous solution of KIN, or 0.0001, 0.01 or 1.0 μM that of HBR. KIN and especially HBR increased the activities of nitrate reductase and carbonic anhydrase, chlorophyll and total protein contents and net photosynthetic rate in the leaves, and pod number and seed yield, at harvest.

Influence of Sugars on in vitro Rooting and Acclimatization of Carob Tree

L. Custódio, M.A. Martins-Loução, A. Romano

Biologia plantarum 48:469-472, 2004 | DOI: 10.1023/B:BIOP.0000041107.23191.8c

Carob tree (Cerafoma siliqua L.) micropropagated shoots were rooted on half-strength Murashige and Skoog medium, supplemented with different types and concentrations of sugars, in order to determine the effects of sugar composition and concentration on in vitro rooting and in vivo establishment of the plantlets. Among the various sugars tested, the best rooting response was obtained with 145 mM sucrose, both in terms of rooting frequency and index of rooting. The use of filter-sterilized rather that autoclaved fructose increased root number and root length. Sugar treatment during rooting slightly influenced plantlet survival and...

Callus Induction and in vitro Regeneration from Barley Mature Embryos

J.M. Zapata, B. Sabater, M. Martín

Biologia plantarum 48:473-476, 2004 | DOI: 10.1023/B:BIOP.0000041108.89399.85

We have assayed different combinations of nutrient media and growth regulators to induce callus and plant regeneration from explants of root, shoot and leaf, complete seed, and isolated mature embryo of barley (Hordeum vulgare L. cv. Hassan). The best results were obtained with mature embryo in J25-8 medium supplemented with 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid where about 75 % developed friable calli. Some 80 - 85 % of these calli regenerated barley plants in the same J25-8 medium supplemented with 1.0 mg dm^-3 indole-3-butyric acid and 0.1 mg dm^-3 kinetin.

Cytokinin-Mediated Axillary Shoot Formation in Pinus heldreichii

D. Stojičić, S. Budimir

Biologia plantarum 48:477-479, 2004 | DOI: 10.1023/B:BIOP.0000041109.27037.21

Shoot formation from seedling explants of Pinus heldreichii was induced by pulse treatment with benzyladenine at different concentration, followed by culture on medium lacking plant growth regulators. After treatment with 222 μM benzyladenine (BA) an average of 4.6 shoots per explant was obtained. Shoots, detached from explants, rooted with a frequency of about 10 %, and rooted plantlets were successfully transferred to ex vitro conditions.