In vitro plantlet regeneration from nodal segments and shoot tips of Capsicum chinense Jacq. cv. Naga King Chili

An in vitro regeneration protocol was developed for Capsicum chinense Jacq. cv. Naga King Chili, a very pungent chili cultivar and an important horticultural crop of Nagaland (Northeast India). Maximum number of shoot (13 ± 0.70) was induced with bud-forming capacity (BFC) index of 10.8, by culturing nodal segments in Murashige and Skoog (MS) medium supplemented with 18.16 μM Thidiazuron (TDZ) followed by 35.52 μM 6-benzylaminopurine (BAP). Using shoot tips as explants, multiple shoot (10 ± 0.37) (BFC 8.3) was also induced in MS medium fortified with either 18.16 μM TDZ or 35.52 μM BAP. Elongated shoots were best rooted in MS medium containing 5.70 μM indole-3-acetic acid (IAA). Rooted plantlets thus developed were hardened in 2–3 weeks time in plastic cups containing potting mixture of a 1:1 mix of soil and cow dung manure and then subsequently transferred to earthen pots. The regenerated plants did not show any variation in the morphology and growth as compared to the parent plant

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis

Background: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results: MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species

Abiotic Stress‐Related Expressed Sequence Tags from the Diploid Strawberry Fragaria vesca

Strawberry ( spp.) is a eudicotyledonous plant that belongs to the Rosaceae family, which includes other agronomically important plants such as raspberry ( L.) and several tree-fruit species. Despite the vital role played by cultivated strawberry in agriculture, few stress-related gene expression characterizations of this crop are available. To increase the diversity of available transcriptome sequence, we produced 41,430 L. expressed sequence tags (ESTs) from plants growing under water-, temperature-, and osmotic-stress conditions as well as a combination of heat and osmotic stresses that is often found in irrigated fields. Clustering and assembling of the ESTs resulted in a total of 11,836 contigs and singletons that were annotated using Gene Ontology (GO) terms. Furthermore, over 1200 sequences with no match to available Rosaceae ESTs were found, including six that were assigned the “response to stress” GO category. Analysis of EST frequency provided an estimate of steady state transcript levels, with 91 sequences exhibiting at least a 20-fold difference between treatments. This EST collection represents a useful resource to advance our understanding of the abiotic stress-response mechanisms in strawberry. The sequence information may be translated to valuable tree crops in the Rosaceae family, where whole-plant treatments are not as simple or practical

The influence of ethylene and ethylene modulators on shoot organogenesis in tomato

[EN] The influence of ethylene and ethylene modulators on the in vitro organogenesis of tomato was studied using a highly regenerating accession of the wild tomato Solanum pennellii and an F1 plant resulting from a cross between Solanum pennellii and Solanum lycopersicum cv. Anl27, which is known to have a low regeneration frequency. Four ethylene-modulating compounds, each at four levels, were used, namely: cobalt chloride (CoCl 2), which inhibits the production of ethylene; AgNO 3 (SN), which inhibits ethylene action; and Ethephon and the precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which both promote ethylene synthesis. Leaf explants of each genotype were incubated on shoot induction medium supplemented with each of these compounds at 0, 10 or 15 days following bud induction. The results obtained in our assays indicate that ethylene has a significant influence on tomato organogenesis. Concentrations of ethylene lower than the optimum (according to genotype) at the beginning of the culture may decrease the percentage of explants with buds (B), produce a delay in their appearance, or indeed inhibit bud formation. This was observed in S. pennellii and the F1 explants cultured on media with SN (5.8-58.0 ¿M) as well as in the F1 explants cultured on medium with 21.0 ¿M CoCl 2. The percentage of explants with shoots (R) and the mean number of shoots per explant with shoots (PR) also diminished in media that contained SN. Shoots isolated from these explants were less developed compared to those isolated from control explants. On the other hand, ethylene supplementation may contribute to enhancing shoot development. The number of isolable shoots from S. pennellii explants doubled in media with ACC (9.8-98.0 ¿M). Shoots isolated from explants treated with ethylene releasing compounds showed a higher number of nodes when ACC and Ethephon were added at 10 days (in F1 explants) or at 15 days (in S. pennellii) after the beginning of culture. Thus, the importance of studying not only the concentration but also the timing of the application of regulators when developing regeneration protocols has been made manifest. An excess of ethylene supplementation may produce an inhibitory effect, as was observed when using Ethephon (17.2-69.0 ¿M). These results show the involvement of ethylene in tomato organogenesis and lead us to believe that ethylene supplementation may contribute to enhancing regeneration and shoot development in tomato. © 2012 Springer Science+Business Media B.V.Carlos Trujillo has a predoctoral fellowship from the Spanish 'Ministerio de Educacion y Ciencia'. This work has been funded by Universitat Politecnica de Valencia (PAID 05-10). The technical assistance of N. Palacios and the revision of the manuscript's English by J. Bergen are gratefully acknowledged.Trujillo Moya, C.; Gisbert Domenech, MC. (2012). The influence of ethylene and ethylene modulators on shoot organogenesis in tomato. Plant Cell, Tissue and Organ Culture. 111(1):141-148. https://doi.org/10.1007/s11240-012-0168-zS1411481111Abeles FB, Morgan PW, Saltveit ME (1992) Ethylene in plant biology. Academic Press, San DiegoBhatia P, Ashwath N, Senaratna T, David M (2004) Tissue culture studies of tomato (Lycopersicon esculentum). Plant Cell Tiss Org Cult 78:1–21Bhatia P, Ashwath N, Midmore DJ (2005) Effects of genotype, explant orientation, and wounding on shoot regeneration in tomato. In Vitro Cell Dev Biol-Plant 41:457–464Biddington NL (1992) The Influence of ethylene in plant-tissue culture. Plant Growth Regul 11:173–187Brown DC, Thorpe TA (1995) Crop improvement through tissue culture. World J Microbiol Biotechnol 11(4):409–415Chraibi KMB, Latche A, Roustan JP, Fallot J (1991) Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors,silver and cobalt. Plant Cell Rep 10:204–207Devi R, Dhaliwal MS, Kaur A, Gosal SS (2008) Effect of growth regulators on in vitro morphogenic response of tomato. Indian J Biotechnol 7:526–530Dias LLC, Santa-Catarina C, Ribeiro DM, Barros RS, Floh EIS, Otoni WC (2009) Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor. Plant Cell Tiss Org Cult 99:199–208Dimasi-Theriou K, Economou AS (1995) Ethylene enhances shoot formation in cultures of the peach rootstock GF-677 (Prunus persica × P. amygdalus). Plant Cell Rep 15:87–90Gisbert C, Arrillaga I, Roig LA, Moreno V (1999) Adquisition of a collection of Lycopersicon pennellii (Corr. D’Arcy) transgenic plants with uidA and nptII marker genes. J Hortic Sci Biotechnol 74:105–109Hughes KW (1981) In vitro ecology: exogenous factors affecting growth and morphogenesis in plant culture systems. Environ Exp Bot 21:281–288Huxter TJ, Thorpe TA, Reid DM (1981) Shoot initiation in light- and darkgrown tobacco callus: the role of ethylene. Physiol Plant 53:319–326Kumar PP, Lakshmanan P, Thorpe TA (1998) Regulation of morphogenesis in plant tissue culture by ethylene. In Vitro Cell Dev Biol Plant 34:94–103Lima JE, Benedito VA, Figueira A, Peres LEP (2009) Callus, shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants. Plant Cell Rep 28:1169–1177Lu J, Vahala J, Pappinen A (2011) Involvement of ethylene in somatic embryogenesis in Scots pine (Pinus sylvestris L.). Plant Cell Tiss Org Cult 107:25–33Mohiuddin AKM, Chowdhury MKU, Abdullah ZC, Napis S (1997) Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell Tiss Org Cult 51:75–78Moshkov IE, Novikova GV, Hall MA, George EF (2008) Plant Growth Regulators III: ethylene. In: George EF, Hall MA, Klerk G-JD (eds) Plant Propaga-tion by Tissue Culture, vol 1. 3rd edn. Springer, The Netherlands, pp 239–248Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Osman MG, Khalafalla MM (2010) Promotion of in vitro shoot formation from shoot tip of tomato (Lycopersicon esculentum Mill. cv. Omdurman) by ethylene inhibitors. Int J Curr Res 4:82–86Ptak A, El Tahchy A, Wyzgolik G, Henry M, Laurain-Mattar D (2010) Effects of ethylene on somatic embryogenesis and galantamine content in Leucojum aestivum L. cultures. Plant Cell Tiss Org Cult 102:61–67Pua EC, Sim GE, Chi GL, Kong LF (1996) Synergistic effects of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro. Plant Cell Rep 15:685–690Reid MS (1995) Ethylene in plant growth, development and senescence. In: Davies PJ (ed) Plant hormones: physiology, biochemistry and molecular biology, 2nd edn. Kluwer Acad Publ, The Netherlands, pp 486–508Trujillo-Moya C, Gisbert C, Vilanova S, Nuez F (2011) Localization of QTLs for in vitro plant regeneration in tomato. BMC Plant Biol 11: art.140Tsuchisaka A, Theologis A (2004) Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family. Proc Natl Acad Sci USA 101:2275–2280Vogel JP, Woeste KE, Theologis A, Kieber JJ (1998) Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively. Proc Natl Acad Sci USA 95:4766–477

Organogenesis and embryogenesis in several hypericum perforatum genotypes

St John’s wort (Hypericum perforatum) is a valuable plant used as a herbal remedy or in phytopharmaceutical drugs to treat a variety of physical ailments. Much research has been performed to study the biochemical production of secondary metabolites of in vitro cultured plants or organs. However, all of these studies have looked at the regeneration of plants from explants in only one genotype. In addition, no study has revealed the mechanism of plant regeneration in H. perforatum, i.e. organogenesis or somatic embryogenesis. We found that different genotypes Helos, Topas, Elixir, and Numi responded similarly to regeneration medium. The regeneration responses (i.e. callus, root, or shoot production) of identical explants from different genotypes were similar. However, the source of explant material (leaves, hypocotyls, and roots) from the same genotype had significant effects on the response to media and plant regeneration frequency. Using scanning electron microscopy and light microscopy, the progress of organogenesis and embryogenesis under similar culture conditions was recorded. Root segments were the most responsive explants, producing the maximum number of shoots per explant of all the genotypes.Fundação para a Ciência e a Tecnologia (FCT) - POCTI/AGR/40 283/2001, SFRH/BPD/17102/2004

Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation

Plant recalcitrance is the major barrier in developing Agrobacterium-mediated transformation protocols for several important plant species. Despite the substantial knowledge of T-DNA transfer process, very little is known about the factors leading to the plant recalcitrance. Here, we analyzed the basis of Hypericum perforatum L. (HP) recalcitrance to Agrobacterium-mediated transformation using cell suspension culture. When challenged with Agrobacterium, HP cells swiftly produced an intense oxidative burst, a typical reaction of plant defense. Agrobacterium viability started to decline and reached 99% mortality within 12 h, while the plant cells did not suffer apoptotic process. This is the first evidence showing that the reduction of Agrobacterium viability during co-cultivation with recalcitrant plant cells can affect transformation