Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

Rapid shoot regeneration from thin cell layer explants excised from petioles and hypocotyls in four cultivars of <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The present work describes a procedure that allows for the easy and rapid induction of caulogenesis in four cultivars of Brassica napus L. from transversal Thin Cell Layers (tTCLs). In order to investigate the regeneration ability of this crop, the effects of genotype, explant source and culture medium were examined on shoot regeneration. The tTCL explants were excised from hypocotyl and petiole of 2-week-old seedlings and cultured on a solid basal MS medium supplemented with α-naphthaleneacetic acid (NAA: 0.1–0.4 mg l⁻¹), 6-benzylamino-purine (BAP: 1–4 mg l⁻¹) and sucrose (20–40 g l⁻¹). A significant genotypic effect was observed between the four cvs; Jumbo and Drakkar displayed higher capacities to produce shoots than Pactol and Cossair. Regeneration commenced earlier and the percentage of shoot-producing explants as well as the number of shoots per regenerating explant was greater. The comparison between the regeneration ability of different explants showed that the hypocotyls exhibited a high rate of shoot organogenesis when they were cultured on MS medium supplemented with 3 mg l⁻¹ BAP, 0.3 mg l⁻¹ NAA and 30 g l⁻¹ sucrose. Adventitious shoot buds developed from 46% of the tTCLs, with a mean of 7.5 buds. Furthermore, the method was fast with shoot formation occurring by 7 days culture. Plantlets regenerated from all shoots and developed normally. The regenerated plants were fertile and identical to source plants.     

Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting

Many molecular marker techniques are available today. PCR-based approaches are in demand because of their simplicity and requirement for only small quantities of sample DNA. Nonanchored inter simple sequence repeats (ISSRs) are arbitrary multiloci markers produced by PCR amplification with a microsatellite primer. They are advantageous because no prior genomic information is required for their use. We found the technique stable across a wide range of PCR parameters. Polymorphisms were abundant among 7 dicot species tested with 2 tri-nucleotide and 2 tetra-nucleotide primers. Thus, nonanchored ISSR markers are a good choice for DNA fingerprinting.     

Induction of somatic embryogenesis and caulogenesis from cotyledon and leaf protoplast-derived colonies of melon (Cucumis melo L.)

Summary A procedure leading to the regeneration of whole plants from protoplasts of melon is described. Protoplasts were isolated from cotyledons and leaves of plants grown in vitro. After 14 days of culture, average viability and division rates were respectively 60% and 30% for the two organs, considering total initial protoplasts plated. The manipulation of the exogenous auxin / cytokinin balance in regeneration media enabled to direct morphogenesis towards somatic embryogenesis (1 mg·l^–1 2,4-dichlorophenoxyacetic acid and 0.1 mg·l^–1 6-benzylaminopurine) or caulogenesis (0.5 mg·l^–1 6-benzylaminopurine and 0.5 mg·l^–1 kinetin). Contrary to division ability, regeneration capacity was genotype-dependent under our conditions, but the two organs expressed similar division and regeneration capacities. Maltose was superior to sucrose for the development of caulogenic nodules into buds. Some plants were transplanted to soil, where they appeared to be fertile and produced seeds.Abbreviations BAP 6-benzylaminopurine - CPW Cell and Protoplast Washing medium - KIN kinetin - MES 2-(N-morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA 1 — naphthaleneacetic acid - PAS H (staining), Periodic Acid-Schiff / Hematoxylin - 2,4-D 2,4-dichlorophenoxyacetic acid     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Plant regeneration from thin cell layers in Spinacia oleracea

Caulogenesis and somatic embryogenesis were induced from transverse thin cell layers (tTCLs) of two European (Spinacia oleracea L.) spinach genotypes. Regeneration occurred mostly when tTCLs had been excised from seedlings grown on a preconditioning medium consisting of White's macroelements, Nitsch's microelements, Murashige and Skoog's (MS) vitamins, 6 g l^–1 agar and 20 g l^–1 glucose. The explants were cultured on MS medium supplemented with sucrose (10, 30, 50 or 80 g l^–1) or fructose (5, 10 or 30 g l^–1) and several combinations of indole-3-acetic acid (IAA), -naphtalene acetic acid (NAA), 6-benzylaminopurine (BAP) and gibberellic acid (GA₃). Most of the regeneration events were obtained from root explants of the cultivar Carpo. The best result was observed on MS medium supplemented with 50 g l^–1 sucrose, 100 M NAA, 1 M BAP and 10 M GA₃. After an 8-week culture, the calluses were transferred onto MS medium where shoots and somatic embryos appeared 1 week later. The best root development was obtained on MS medium supplemented with 4.9 M indole-3-butyric acid (IBA) and 8 g l^–1 Phytagel. The plantlets were, then, transferred to soil and developed into well-conformed, fertile plants.     

Genetic diversity in european and Argentinian cultivated potatoes (Solanum tuberosum subsp. tuberosum) detected by inter-simple sequence repeats (ISSRs).

In this study, the use of inter-simple sequence repeats (ISSR) to assess genetic diversity between cultivated potatoes (Solanum tuberosum subsp. tuberosum) is reported. ISSR technology rapidly reveals high polymorphic fingerprints and thus determines the genetic diversity among potato cultivars. Nine primers were selected according to the number of amplified markers and the level of polymorphism detected. Three primers (GAG(CAA)5, CTG(AG)8, and (AG)8) were used to cluster the 28 potato accessions and 77 polymorphic markers were sufficient to identify all of the accessions. Among the 15 simple sequence repeat (SSR) motifs tested, the most abundant were CAA and AG. Argentinian- and European-grown potatoes were easily distinguished, with a higher level of genetic diversity among potatoes from Argentina. An ISSR study using a limited number of cultivars and very few primers clearly differentiated between all cultivars, thus ISSR was revealed to be a good tool for the genetic identification of potato and for future germplasm-management programs.