Somatic embryo germination and plant regeneration of three grapevine cvs: Effect of IAA, GA3 and embryo morphology

Plant regeneration was achieved using somatic embryos obtained from the Vitis vinifera cvs Sugraone, Crimson Seedless and Don Mariano. White somatic embryos, 1.5-3 mm in length, were cultured on embryo germination medium with or without IAA (10 μM) + GA3 (1 μM). Germinated embryos showed 5 different morphologies, (1) one cotyledon, (2) two cotyledons, (3) three cotyledons, (4) trumpet-like and (5) fused cotyledons before transfer to half strength MS medium to obtain plants. The conversion rate was higher when somatic embryos were germinated in culture medium with IAA and GA3. Embryo morphology also had an effect on plant regeneration, somatic embryos with developed cotyledons (one, two or three) showing a higher conversion rate (57.8-70.2 %) than those with abnormal or no cotyledons (36.2-36.3 %).

High embryogenic ability and plant regeneration of table grapevine cultivars (Vitis vinifera L.) induced by activated charcoal

Somatic embryos and plants from immature anthers or ovaries were obtained from the Vitis vinifera cvs Sugraone, Crimson Seedless, Italia and Don Mariano. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4, 7 or 10 μM) and 6-benzyladenine (0.7, 1 or 1.3 μM) for callus induction. Callus frequency depended on genotype, explant type and the culture medium used. Calli were transferred to half strength MS without growth regulators for embryo differentiation. The presence of activated charcoal (AC, 0.25 %) in this medium was essential to obtain somatic embryos in the case of Crimson Seedless, Italia and Don Mariano and to increase the frequency of embryogenic calli in Sugraone (from 5.8 % without AC to 99.5 % with AC). Ovaries and anthers showed different degrees of embryogenic competence. When somatic embryos were placed in a medium with indole-3-acetic acid (10 μM), gibberellic acid (1 μM) and 0.25 % AC, embryo germination was normal, i.e. embryos turned green, the hypocotyls and cotyledons started to grow and the apical root axis developed. The percentage of germinated embryos was 100 % for Sugraone and 27.5 %, 38.1 % and 54.5 % for Don Mariano, Italia and Crimson Seedless, respectively. When the germinated embryos were transferred to half strength MS medium in test tubes, 100 % of Crimson, Italia and Don Mariano and 68.3 % of Sugraone embryos developed into normal plants.

Improvement of regeneration in pepper: a recalcitrant species

[EN] Organogenesis is influenced by factors like genotype, type of explant, culture medium components, and incubation conditions. The influence of ethylene, which can be produced in the culture process, can also be a limiting factor in recalcitrant species like pepper. In this work, bud induction was achieved from cotyledons and hypocotyls-from eight pepper cultivars-on Murashige and Skoog (MS) medium supplemented with 22.2 mu M 6-benzyladenine (6BA) and 5.71 mu M indole-3-acetic acid (IAA), in media with or without silver nitrate (SN) (58.86 mu M), a suppressor of ethylene action. In the SN-supplemented medium, the frequencies of explants with buds and with callus formation were lower in both kinds of explant, but higher numbers of developed shoots were isolated from explants cultured on SN. Bud elongation was better in medium with gibberellic acid (GA(3)) (2.88 mu M) than in medium free of growth regulators or supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) at 34.5 mu M. However, isolation of shoots was difficult and few plants were recovered. The effect of adding SN following bud induction (at 7 d) and that of dark incubation (the first 7 d of culture) was also assessed in order to improve the previous results. When SN was added after bud induction, similar percentages of bud induction were found for cotyledons (average frequency 89.37% without SN and 94.37% with SN) whereas they doubled in hypocotyls (50% without SN and 87.7% with SN). In addition, in both kinds of explant, the number of developed plants able to be transferred to soil (developed and rooted) was greatly increased by SN. Dark incubation does not seem to improve organogenesis in pepper, and hypocotyl explants clearly represent a better explant choice-with respect to cotyledonary explants-for the pepper cultivars assayed.We thank the COMAV germplasm bank at Universitat Politecnica de Valencia and the Arid Lands Institute for pepper seeds and the Tunisian Ministry of Higher Education and Scientific Research who fund N. Gammoudi's stay.Gammoudi, N.; San Pedro-Galan, T.; Ferchichi, A.; Gisbert Domenech, MC. (2018). Improvement of regeneration in pepper: a recalcitrant species. In Vitro Cellular & Developmental Biology - Plant. 54(2):145-153. https://doi.org/10.1007/s11627-017-9838-1S145153542Ashrafuzzaman M, Hossain MM, Razi Ismail M, Shahidul Haque M, Shahidullah SM, Uz Zaman S (2009) Regeneration potential of seedling explants of chilli (Capsicum annuum). Afr J Biotechnol 8:591–596Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297Brown DC, Thorpe TA (1995) Crop improvement through tissue culture. World J Microbiol Biotechnol 11:409–415Carvalho MAF, Paiva R, Stein VC, Herrera RC, Porto JMP, Vargas DP, Alves E (2014) Induction and morpho-ultrastructural analysis of organogenic calli of a wild passion fruit. Braz Arch Biol Technol 57:581–859Christopher T, Rajam MV (1996) Effect of genotype, explant and medium on in vitro regeneration of red pepper. Plant CellTiss Org Cult 46:245–250Dabauza M, Peña L (2001) High efficiency organogenesis in sweet pepper (Capsicum annuum L.) tissues from different seedling explants. Plant Growth Regul 33:221–229De Filippis LF (2014) Crop improvement through tissue culture. In: Ahmad P, Wani MR, Azooz MM, Tran LSP (eds) Improvement of crops in the era of climate changes, vol 1. Springer, New York, pp 289–346Gammoudi N, Ben Yahia L, Lachiheb B, Ferchichi A (2016) Salt response in pepper (Capsicum annuum L.): components of photosynthesis inhibition, proline accumulation and K+/Na+ selectivity. JJ Aridland Agri 2:1–12González A, Arigita L, Majada J, Sánchez Tamés R (1997) Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L. Plant Growth Regul 22:1–6Grozeva S, Rodeva V, Todorova V (2012) In vitro shoot organogenesis in Bulgarian sweet pepper (Capsicum annuum L.) varieties. EJBio 8:39–44Gunay AL, Rao PS (1978) In vitro plant regeneration from hypocotyls and cotyledon explants of red pepper (Capsicum). Plant Sci Lett 11:365–372Huxter TJ, Thorpe TA, Reid DM (1981) Shoot initiation in light- and dark-grown tobacco callus: the role of ethylene. Physiol Plant 53:319–326Hyde CL, Phillips GC (1996) Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis. In Vitro-Plant 32:72–80Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli peppers—a review on tissue culture and transgenesis. Biotechnol Adv 28:35–48Kumar AO, Rupavathi T, Tata SS (2012) Adventitious shoot bud induction in chili pepper (Capsicum annuum L. cv. X-235). In J Sci Nat 3:192–196Kumar PP, Lakshmanan P, Thorpe TA (1998) Regulation of morphogenesis in plant tissue culture by ethylene. In Vitro Cell Dev Biol Plant 34:94–103Liu W, Parrott WA, Hildebrand DF, Collins GB, Williams EG (1990) Agrobacterium induced gall formation in bell pepper (Capsicum annuum L.) and formation of shoot-like structures expressing introduced genes. Plant Cell Rep 9:360–364Maligeppagol M, Manjula R, Navale PM, Babu KP, Kumbar BM, Laxman RH (2016) Genetic transformation of chilli (Capsicum annuum L.) with Dreb1A transcription factor known to impart drought tolerance. Indian J Biotechnol 15:17–24Mantiri FR, Kurdyukov S, Chen SK, Rose RJ (2008) The transcription factor MtSERF1 may function as a nexus between stress and development in somatic embryogenesis in Medicago truncatula. Plant Signal Behav 3:498–500Mezghani N, Jemmali A, Elloumi N, Gargouri-Bouzid R, Kintzios S (2007) Morpho-histological study on shoot bud regeneration in cotyledon cultures of pepper (Capsicum annuum). Biologia 62:704–710Mohamed-Yasseen Y (2001) Influence of agar and activated charcoal on uptake of gibberellin and plant morphogenesis in vitro. In Vitro Cell Dev Biol - Plant 37:204–205Moshkov IE, Novikova GV, Hall MA, George EF (2008) Plant growth regulators III: ethylene. In: George EF, Hall MA, Klerk G-JD (eds) Plant propagation by tissue culture, vol 1, 3rdedn. Springer, Dordrecht, The Netherlands, pp 239–248Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Nogueira RC, Paiva R, de Oliveira LM, Soares GA, Soares FP, Castro AHF, Paiva PDO (2007) Calli induction from leaf explants of murici-pequeno (Byrsonima intermedia A. Juss.) Ciênc Agrotec 31:366–370Ochoa-Alejo N, Ramirez-Malagon R (2001) In vitro chili pepper biotechnology. In Vitro Cell Devl Biol Plant 37:701–729Orlińska M, Nowaczy P (2015) In vitro plant regeneration of 4 Capsicum spp. genotypes using different explant types. Turk J Biol 39:60–68Reid 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, Dordrecht, The Netherlands, pp 486–508Sanatombi K, Sharma GJ (2008) In vitro plant regeneration in six cultivars of Capsicum spp. using different explants. Biol Plant 52:141–145Santana-Buzzy N, Canto-Flick A, Barahona-Pérez F, Montalvo-Peniche MC, Zapata-Castillo PY, Solís-Ruiz A, Zaldívar-Collí A, Gutiérrez-Alonso O, Miranda-Ham ML (2005) Regeneration of habanero pepper (Capsicum chinense Jacq.) via organogenesis. Hortscience 40:1829–1831Santana-Buzzy N, Canto-Flick A, Iglesias-Andreu LG, Montalvo-Peniche MC, López-Puc G, Barahona-Pérez F (2006) Improvement of in vitro culturing of habanero pepper by inhibition of ethylene effects. Hortscience 41:405–409Sawai S, Ohyama K, Yasumoto S, Seki H, Sakuma T, Yamamoto T, Takebayashi Y, Kojima M, Sakakibara H, Aoki T, Muranaka T, Saito K, Umemoto N (2014) Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato. Plant Cell 26:3763–3774Shah SH, Ali S, Jan SA, Din J, Ali GM (2014) Assessment of silver nitrate on callus induction and in vitro shoot regeneration in tomato (Solanum lycopersicum Mill.) Pakistan J Bot 46:2163–2172Steinitz B, Wolf D, Matzevitch-Josef T, Zelcer A (1999) Regeneration in vitro and genetic transformation of pepper (Capsicum spp.): the current state of the art. Capsicum Eggplant Plant Newsletter 18:9–15Tamimi SM (2015) Effects of ethylene inhibitors, silver nitrate (AgNO3), cobalt chloride (CoCl2) and aminooxyacetic acid (AOA), on in vitro shoot induction and rooting of banana (Musa acuminata L.) Afr J Biotechnol 14:2510–2516Trujillo-Moya C, Gisbert C (2012) The influence of ethylene and ethylene modulators on shoot organogenesis in tomato. Plant Cell Tissue Organ Cult 111:41–48Yasmin S, Mensuali-Sodi A, Perata P, Pucciariello C (2014) Ethylene influences in vitro regeneration frequency in the FR13A rice harbouring the SUB1A gene. Plant Growth Reg 72:97–103Zhao Y, Stiles AR, Saxena PK, Liu CZ (2013) Dark preincubation improves shoot organogenesis from Rhodiola crenulata leaf explants. Biol Plant 57:189–19

Pepper, Sweet (Capsicum annuum)

Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium ( Agrobacterium tumefaciens ). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper transformation is the low frequency of cells that are both susceptible for Agrobacterium infection and have the ability to regenerate. Here, we describe a protocol for the effi cient regeneration of transgenic sweet pepper ( C. annuum ) through inducible activation of the BABY BOOM (BBM) AP2/ERF transcription factor. Using this approach, we can routinely achieve a transformation effi ciency of at least 0.6 %. The main improvements in this protocol are the reproducibility in transforming different genotypes and the ability to produce fertile shoots. An added advantage of this protocol is that BBM activity can be induced subsequently in stable transgenic lines, providing a novel regeneration system for clonal propagation through somatic embryogenesis

In vitro organogenesis in watermelon cotyledons Organogênese in vitro em cotilédones de melancia

The objective of this work was to study the in vitro organogenesis of Citrullus lanatus, by the induction of adventitious buds in cotyledon segments cultured in medium supplemented with cytokinin. Explants were collected from one, three and five-day-old in vitro germinated seedlings, considering the distal and proximal cotyledon regions. The data obtained showed that in vitro organogenesis of watermelon occurred with higher efficiency, when cotyledon segments from the proximal region collected from three-day-old seedlings were cultivated in medium MS, supplemented with BAP (1 mg L-1) and coconut water (10%). The histological study showed that the organogenesis occurs directly, without callus formation, on epidermal and subepidermal layers of the explants. Adventitious shoots were characterized by the development of shoot apical meristem and leaf primordia. The formation of protuberances, that do not develop into adventitious buds, was also observed.<br>O objetivo do trabalho foi estudar a organogênese in vitro de C. lanatus, pela indução de gemas adventícias, em segmentos de cotilédones, cultivados em meio de cultura suplementado com citocinina. Os explantes consistiram de segmentos das regiões distal e proximal de cotilédones, coletados de plantas germinadas in vitro com um, três e cinco dias de idade. Os dados obtidos mostram que a organogênese de melancia, in vitro, ocorre com maior eficiência em segmentos da região proximal dos cotilédones, coletados de plântulas com três dias de idade e cultivados em meio de cultura MS, suplementado com a combinação BAP (1 mg L-1) e água de coco (10%). Pelo estudo histológico, verificou-se que a organogênese ocorre diretamente, sem a formação de calo, na epiderme e subepiderme do explante. As gemas adventícias foram caracterizadas pela presença de meristema apical e primórdios foliares. Observou-se, também, o desenvolvimento de protuberâncias que não se desenvolvem em gemas adventícias

Enhanced resistance to Botrytis cinerea in genetically-modified Vitis vinifera L. plants over-expressing the grapevine stilbene synthase gene

Grapevine (Vitis vinifera L.) was genetically modified with a construct containing a cDNA insert encoding the stilbene synthase gene (Vst1) from grapevine, under the control of the cauliflower mosaic virus 35S promoter in order to test the potential of over-production of resveratrol to protect plants from fungal attack. Southern blot hybridization and quantitative real-time PCR analysis demonstrated the presence and integration of one copy of exogenous DNA sequences in two grapevine-modified lines. Relative expression of the Vst1 gene in different modified lines was confirmed by using gene-specific quantitative real-time PCR. Compared to the control, the concentration of trans-resveratrol quantified by HPLC was up to 7.5 fold higher in the modified plants. The necrotic lesion size of leaves of intact modified plants inoculated by Botrytis cinerea B05.10 strain was consistently smaller and significantly different (p B 0.05) than in control plants, showing that modified grapevine plants were more resistant to the pathogen than the control plants