Bracale, M.

Croce, P.

Vannini, C.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 44 (4), 197–198 (2005)Research NoteAn easy and convenient method formaintenance of embryogenic culturesof Vitis viniferaP. CROCE, C. VANNINI and M. BRACALEDASS Dipartimento di Ambiente, Salute e Sicurezza, Universitàdell’Insubria, Varese, ItalyK e y    w o r d s :  in vitro culture, embryo.Introduction: Regeneration of Vitis vinifera cultivars isgenerally achieved via somatic embryogenesis. Since thisprocess is genotype- and explant type-dependent there isno general protocol for regeneration to grape. Variousexplants including anthers, ovaries (MARTINELLI et al. 2001 a),leaves (SCORZA et al. 1996), petioles and tendrils (SALUNKHEet al. 1997) have been tested to establish an embryogenicculture.Grapevine embryo cultures have been used as a tool forthe selection of toxin resistant strains (JAYASANKAR et al.2000) and for transformation, where the availability of a ho-mogeneous and constant supply of embryogenic explantsis an important prerequisite (SCORZA et al. 1996).The maintenance of embryogenic competence is geno-type dependent, too. Embryogenic callus cultures of cvsChardonnay and Brachetto have been maintained for morethan three years, alternating a 2,4-D containing medium (non-differentiation) and a differentiation medium every twomonths (MARTINELLI et al. 2001 a). Perennial embryo cul-tures of Thompson seedless have been maintained bysubculturing the proembryogenic masses produced by theculture itself on a hormone-free medium (SCORZA et al. 1996,LI et al. 2001), or by recurrent cycles of induction of second-ary embryogenesis in Vitis rupestris (MARTINELLI et al.2001b). The embryogenic competence of the primary explantscan also be preserved by culturing the embryogenic mate-rial obtained from the primary explants (PERRIN et al. 2001).In this paper, a convenient method for the cyclic induc-tion and proliferation of embryogenic cell culture is presented.Material and Methods: Inflorescences from Vitis viniferacvs Trebbiano d’Abruzzo, Trebbiano toscano 3D, Trebbianotoscano R4, Sangiovese, Bombino bianco, Regina andMontepulciano were harvested two weeks prior to bloom.The anthers were immature, green or only partially yel-low in colour. Inflorescences were sterilized with a dilutedcommercial sodium hypochlorite solution (2 % active chlo-rine) and 0.05 % v/v Tween 20 for 15 min. Flowers wereexcised from the inflorescence and dissected in order to sepa-rate anthers from ovaries.Sterile anthers and ovaries were placed on initiation me-dium (MARTINELLI et al. 2001 a). Briefly, initiation mediumconsisted of NN salts (NITSCH 1969) with 20 g·l-1 sucrose,2.5 g Gelrite (Duchefa), 9 µM 2,4-D, and 4.4 µM BAP. The pHwas adjusted to 5.8 with KOH before autoclaving .Petri dishes were incubated at 24 °C in the dark. Cul-tures were visually screened for the formation of embryo-genic callus after 30 and 50 d.Embryogenic calli were identified by visual screening(PERRIN et al. 2004) and transferred after 30 or 50 d from theinitiation to the proliferation medium consisting of modifiedGS1CA (FRANKS et al. 1998) containing NN salt and vita-mins, with the exception of ammonium nitrate 60 g·l-1 su-crose. 1 µM BAP was added before autoclaving, while 20 µMIAA and 10 µM NOA were added after autoclaving. Themedium was solidified with 2.5 g·l-1 Gelrite (Duchefa) and pHwas adjusted to 5.8 with KOH before autoclaving.Culture maintenance is based on a two-step cyclic pro-cedure. The first step involves the induction of embryogen-esis from somatic embryos: single somatic embryos wereplated on the induction medium, modified PIV (FRANKS et al.1998), (NN salt and vitamins, 60 g·l-1 sucrose, 8.9 µM BAP,4.5 µM 2,4-D, 2,5 g·l-1 Gelrite, pH adjusted to 5.8 beforeautoclaving), and incubated for 30 d in the dark at 24 °C. Thesecond step involves the proliferation of newly formed andsynchronised somatic embryos: calli produced by the so-matic embryos cultured on the induction medium were trans-ferred onto the proliferation medium (composition as be-fore, but with addition of 720 mg·l-1 ammonium nitrate) for30 d in order to produce secondary embryos. Embryos pro-duced on proliferation medium were suitable material for theinitiation of another inductive cycle if transferred onto theinduction medium.For the production of plants, embryos were placed onWPM medium with 20 g·l-1 sucrose, 2,5 µM BAP, 3 g·l-1Gelrite, pH 5,8 at 24 °C, photoperiod 16 h light, until germina-tion and the emission of 3 leaves. Plantlets were then trans-ferred to pots and acclimated in the greenhouse.Results and Discussion:  E m b r y o g e n e s i s   i n d u c-t i o n :  Most of the explants produced callus after 30 d ofculture on the initiation medium. Embryogenic calli were whiteto yellow and were friable and compact, while non-embryo-genic calli were white, soft and spongy or else yellow tobrown and wet. All of the tested cultivars formed embryo-genic callus at least from the ovaries.The rate of formation of the proembryogenic callus washighly dependant on the type of the explants and thecultivars tested (Table).The percentage of embryogenic callus induction washigher for the ovaries and lower for the anthers, confirmingthe high susceptibility of ovaries in producing these par-ticular cells (MARTINELLI et al. 2001 a).E m b r y o   p r o d u c t i o n :  All of the embryogenic calliproduced in the induction phase were transferred onto theproliferation medium. In 4 cultivars out of the 7 tested, thecallus produced somatic embryos that enlarged, turned greenand emitted the radicle. In these cases, the green embryosformed new embryos at the root-shoot transition zone thatcould be sub-cultured on the same medium for an indefinitenumber of cycles. The embryos produced were asynchro-nous, heterogeneous and small in quantity making this pro-Correspondence to: Dr. P. CROCE, DASS Dipartimento di Ambiente,Salute e Sicurezza, Università dell'Insubria, Via Peschiera 20, 21100Varese, Italy. E-mail: p.croce@infinito.it198 P. CROCE et al.cedure not suitable to produce embryos amenable for trans-formation.C u l t u r e   m a i n t e n a n c e :  In order to establish asynchronous embryo culture, avoiding embryo germination,we decided to establish a two-step procedure. The first stepwas the  subculture of single somatic embryos on a 2,4-Dcontaining medium in order to promote the formation of em-bryogenic material. The synthetic auxin 2,4-D is known toinhibit embryo germination, stimulating somatic embryogen-esis.When cultured on the induction medium, the embryosformed calli that, in some cases, were of the embryogenictype. Alternatively Regina and Sangiovese also producedmasses of translucent proembryos able to convert into so-matic embryos once placed on the proliferation medium.The second step was the subculture of the calli on theproliferation medium. When cultured on the differentiationmedium, the embryogenic-type calli were able to generatelarge amounts of somatic embryos with a synchronous pat-tern of growth and which did not germinate on the medium(Figure). Once on the proliferation medium, all of the em-bryogenic calli produced in the induction medium prolifer-ated into somatic embryos but, in contrast to the report ofMARTINELLI et al. (2001a) for Chardonnay and Brachetto, noformation of callus of any sort was observed. This confirmsthe difficulty of establishing a general method for the main-tenance of perennial embryogenic cultures of Vitis sp.White somatic embryos produced on the proliferationmedium were used to induce somatic embryogenesis for apotentially indefinite number of cycles. Embryogenic cul-tures have been maintained with this method for more thantwo years. Interestingly, the embryos produced by second-ary embryogenesis were more susceptible to producing mor-phogenetic material than primary embryos. A side from be-ing synchronised, the formed embryos also had the advan-tage of a massive production of embryos amenable to trans-formation.FRANKS, T.; HE, D. G.; THOMAS, M.; 1998: Regeneration of transgenicVitis vinifera L. Sultana plants: Genotypic and phenotypic analy-sis. Mol. Breed. 4, 321-333.JAYASANKAR, S.; LI, Z.; GRAY, D. J.; 2000: In vitro selection of Vitisvinifera ‘Chardonnay’ with Elsinoe ampelina culture filtrate isaccompanied by fungal resistance and enhanced secretion ofchitinase. Planta. 211,  200-208.LI, Z.; JAYASANKAR, S.; GRAY, D. J.; 2001: Expression of a bifunctionalgreen fluorescent protein (GFP) fusion marker under the controlof three constitutive promoters and enhanced derivatives intransgenic grape (Vitis vinifera). Plant Sci. 160, 877-887.MARTINELLI, L.; GRIBAUDO, I.; BERTOLDI, D.; CANDIOLI, E.; POLETTI, V.; 2001 a:High efficiency somatic embryogenesis and plant germinationin grapevine cultivars Chardonnay and Brachetto a grappololungo. Vitis, 40, 111-115.MARTINELLI, L.; CANDIOLI, E.; COSTA, D.; POLETTI, V.; 2001 b: Morpho-genic competence of Vitis rupestris S. secondary somatic em-bryos with a long culture history. Plant Cell Rep. 20, 279-284.NITSCH, J. P.; 1969: Experimental androgenesis in Nicotiana.Phytomorphology 19, 389-404.PERRIN M.; MARTIN D.; JOLY D.; DEMANGEAT G.; THIS P.; MASSON J. E.;2001: Medium-dependent response of grapevine somatic em-bryogenic cells. Plant Sci. 161, 107-116.PERRIN, M.; GERTZ, C.; MASSON, J. E.; 2004: High efficiency initiationof regenerable embryonic callus from anther filaments of 19grapevine genotypes grown worldwide. Plant Sci. 167, 1143-1149SCORZA, R.; CORDTS, J. M.; GRAY, D. J. ; GONSALVES, D.; EMERSHAD, R. L.;RAMMING, D. W.; 1996: Producing transgenic “Thompson seed-less” grape (Vitis vinifera) plants.. J. Am. Soc. Hort. Sci. 121,616-619.SALUNKHE, C. K.; RAO, P. S.; MHATRE, M.; 1997: Induction of somaticembryogenesis and plantlets in tendrils of Vitis vinifera. PlantCell Rep. 17, 65-67.Received April 13, 2005T a b l e   1Number of explants cultured for each cultivar and percentage of the explant producing proembryonic callusCultivars tested Explant type Efficiency of embryogenicand number* callus induction (%)aAnthers Ovaries Anthers OvariesTrebbiano d'Abruzzo 600 118 0 14.40 ± 10.0Trebbiano Toscano 3D 450 96 0 5.20 ± 0.88Trebbiano Toscano R4 350 70 0 12.86 ± 3.53Sangiovese 900 112 2.70 ± 0.8 5.36 ± 2.33Bombino blanco 850 130 0.24 ± 1.4 6.92 ± 5.4Regina 550 120 1.82 ± 1.7 7.50 ± 5.3Montepulciano 400 70 1 ± 1.13 34.29 ± 19.44*Sum of the explant cultured in three different repetitions.aData from all experimental repetitions were used for means and standard deviation calculation.Figure:  Embryos produced on the proliferation medium af-ter one month from subculture.

An easy and convenient method for maintenance of embryogenic cultures of Vitis vinifera

https://ojs.openagrar.de/index.php/VITIS/article/download/4336/4287

An easy and convenient method for maintenance of embryogenic cultures of Vitis vinifera

Abstract

Similar works

Full text

Available Versions

JKI Open Journal Systems (Julius Kühn-Institut)