Evidence for quantitative responses during co-culture of Pyrus communis protoplasts and Erwinia amylovora

Summary Mesophyll protoplasts were isolated from axenic shoot cultures of pear cultivars, exhibiting different degrees of susceptibility to fire blight infection at the whole plant level and they were co-cultured with the wild-type strain CFBP 1430 of Erwinia amylovora, and with an avirulent transposon mutant of the former (PMV 6046). Results, as assessed in terms of the effects of bacteria on protoplast viability, the time to the onset of divisions, the percentage of the originally cultivated protoplasts that divided once and of those proliferating to give 10-cell colonies, correlated with field resistance to fire blight of the respective pear genotypes. These results might provide a model for a better understanding of the interaction between pear and E. amylovora.Abbreviations BAP 6-benzylaminopurine - FDA fluorescein diacetate - fwt fresh weight - IAA indole-3-acetic acid - IPE initial plating efficiency - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone (av.molecular weight 10000) - uv ultra-violet     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.)

Summary Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l^-1 6-benzyl-aminopurine and 0.1 mg.l^-1 l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - IBA 4-indole-3yl-butyric acid - IPE initial plating efficiency - f wt fresh weight - KM Kao and Michayluk (1975) - MES 2-N-morpholino ethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA l-naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000)     

In vitro meristem culture of M.4 apple (Malus pumila Mill.). I. Optimal nutrient medium

Shoot tips of M.4 apple clone were excised from actively growing one year-old stoolbed branches, and cultured in order to determine the optimal nutrient medium for each stage of their in vitro culture. The basal medium (BM) used was that described by Murashige and Skoog, supplemented with vitamins, glycine, myoinositol, sucrose, with or without agar, and different combinations of plant growth regulators. Best media for each stage were: BM+0.5 mg 1^-1 indole-3yl-butyric acid (IBA)+0.5 mg 1^-1 6-benzylaminopurine (BAP) for explant establishment (Stage I); BM+0.1 mg 1^-1 IBA+1.0 mg 1^-1 BAP for multiplication and internode enlargement (Stage II); and 2.0 mg 1^-1 IBA+0.1 mg 1^-1 BAP without agar for the rooting of the plantlets (Stage III).     

Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula

Leaf explants from leaflets collected from either in vivo grown or in vitro grown seedlings of Medicago truncatula genotype R108-1 were co-cultivated with bacterial cells of Agrobacterium tumefaciens strains EHA105 or C58pMP90. Each of these strains was carrying the pCambia 1390 plasmid harbouring a hygromycin resistance gene cassette. Explants were then incubated on a medium containing 10 mg/l hygromycin and 800 mg/l augmentin to suppress Agrobacterium growth, and subcultured 4–5 times every 2 weeks for the proliferation of calli. After 8–10 weeks, callusing explants were transferred to hormone-free medium with 10 mg/l hygromycin and 400 mg/l augmentin for shoot regeneration. After rooting, a total of about 300 putative transformants were grown into plantlets, transferred to soil, acclimatized, and then moved to the greenhouse. Of these, a total of 43 independent PCR positive primary transformants and their T1 and T2 progeny were subjected to flow cytometric analysis, to assessing their trueness-to-type, as well as to southern blot analysis.     

Flow cytometry and morphometry of tobacco cells expressing the C-terminal domain of the clathrin heavy chain

Plant Cell, Tissue and Organ Culture (PCTOC) - Transgenic Nicotiana tabacum cells constitutively expressing the C-terminal domain (hub domain) of clathrin heavy chain, as a dominant negative...     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Somatic hybridization of sexually incompatible top-fruit tree rootstocks,wild pear (Pyrus communis var. pyraster L.) and Colt cherry (Prunus avium x pseudocerasus)

Summary Mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L., Pomoideae) were chemically fused with cell suspension protoplasts of cherry rootstock Colt (Prunus avium x pseudocerasus, Prunoideae), following an electroporation treatment of the separate parental protoplast systems. Fusion-treated protoplasts were cultured, on modified K8P medium, where it had been previously established that neither parental protoplasts were capable of division. Somatic hybrid calli were recovered and, following caulogenesis on MS medium with zeatin and after rooting of regenerated shoots, complete trees were obtained and grown in vivo. Hybridity of these trees was confirmed based on morphological characters, chromosome complement and isozyme analysis. Two separate cloned lines of this intersubfamilial rootstock somatic hybrid (wild pear (+) Colt cherry) were produced. This is the first report of the production of somatic hybrid plants of two woody species, of agronomic value, within the order Rosales.