The effect of the type of closure on the gas composition of the headspace and the growth of GF 677 peach × almond rootstock cell suspension cultures

Summary The influence of culture flask closures, i.e., cotton plugs and rubber and aluminum-foil caps, on headspace gas composition and growth of leaf petiole callus-derived GF 677 cell suspensions was comparatively tested. Oxygen concentration always remained comparable to that of the lab atmosphere and CO₂ and C₂H₄ levels were slightly higher when flasks were closed with cotton plugs. By contrast, the gas environment markedly changed within 72 to 96 h in culture inside rubber and aluminum-capped flasks. Under rubber caps, CO₂ increased up to 18%, with a net production of about 0.8 mmol CO₂, oxygen decreased to 6% within 72 h, and ethylene accumulated up to 9 μl liter⁻¹ after 96 h. When aluminum foil closures were used, C₂H₄ and CO₂ increased over the first 72 h, reaching concentrations of about 6 μl liter⁻¹ and 7% (0.3 mmol produced), respectively, and decreasing after 192 h to 0.1 μl⁻¹ and 2%, respectively. The gaseous environment markedly affected cell growth. The exponential growth period of suspensions cultured in flasks closed with cotton plugs and aluminum foil caps started after about 72 h and the stationary phase after 240 h, the cell dry weight reaching its maximum at about threefold the initial weight. Large cell aggregates were found in flasks closed with cotton plugs, slight aggregation with aluminum closures, and no aggregates with rubber caps. However, hardly any growth, progressive browning of the suspensions, and the death of most cells occurred in rubber-capped flasks. A type of closure allowing low gas exchange rates, like aluminum caps, and frequent subcultures thus seems most conducive to rapid growing, more homogeneous GF 677 cell suspension cultures, and the prevention of aggregates, if undesired.     

The influence of ethylene on in vitro rooting of GF 677 (Prunus persica × Prunus amygdalus) hybrid peach rootstock

Summary Treatments differing from each other for the type of tube closure (i.e., cotton plug for free gas exchange, airtight rubber cap, and rubber cap with ethysorb) and/or rooting culture medium (i.e., enriched or not by 25 to 100 μM acetylsalicylic acid) were compared for their effects on gaseous composition of the culture atmosphere and microcutting rooting of the GF 677 (Prunus persica × Prunus amygdalus) hybrid. Rubber capping, which leads to rapid ethylene accumulation inside tubes, strongly reduced rooting time and in some cases enhanced final rooting percentage over that of cotton plugs. Ethysorb almost completely absorbed ethylene produced by shoots, which showed lower rooting percentages within 9 d than microcuttings cultured in the absence of ethysorb. In contrast, no significant difference in rooting was found between the two treatments after 14 d. Carbon dioxide concentration was similar in all treatments within 5 to 9 d and seemed to be ineffective for rooting. The influence of acetylsalicylic acid on rooting was unclear. Root number and length were not significantly influenced by the treatments. These results demonstrate that the use of airtight closures, leading to rapid ethylene accumulation, can reduce time of rooting expression for GF 677 microcuttings. However, free gas exchange towards the end of the rooting period (from Day 9 to Day 14) is advisable to prevent leaf yellowing. No significant difference in plantlet survival and growth after transfer ex vitro was found among treatments.     

Exploring almond genetic variability useful for peach improvement: mapping major genes and QTLs in two interspecific almond × peach populations

Genetic analysis of a diverse set of 42 traits for flower (5), phenology (9), fruit quality (19), leaf (8) and disease resistance (1) was carried out in two interspecific almond × peach populations, an F₂ (T × E) and a BC₁ (T1E), from the cross between ‘Texas’ almond and ‘Earlygold’ peach. Traits related to flower, phenology, fruit quality, leaf morphology and resistance to powdery mildew were phenotyped over 3 years in two locations and studied for co-segregation with a large set of SNP and SSR markers. Three maps were used, one for the T × E and two for the T1E (T1E and E) population. Nine major genes were identified and mapped: anther color (Ag/ag and Ag2/ag2), flower color (Fc2/fc2), maturity date (MD/md), almond fruit type (almond vs. peach; Alf/alf), juiciness (Jui/jui), blood flesh (DBF2/dbf2), powdery mildew resistance (Vr3) and flower type (showy/non-showy; Sh/sh). These genes were often located in genome positions different from those for major genes for similar traits mapped before. Two of them explain fundamental aspects that define the fruit of peach with respect to that of almond: Alf and Jui, for its thick and juicy mesocarp, respectively. The genetics of quantitative traits was studied, and 32 QTLs were detected, with consistent behavior over the years. New alleles identified from almond for important traits such as red skin color, blood flesh, fruit weight and powdery mildew resistance may prove useful for the introduction of new variability into the peach gene pool used in commercial breeding programs.     

Construction of a saturated linkage map for Prunus using an almond×peach F2 progeny

 A map with 246 markers (11 isozymes and 235 RFLPs) was constructed using an interspecific F₂ population between almond (cv Texas) and peach (cv Earlygold). RFLPs were obtained using 213 probes from the genomic and cDNA libraries of different species (almond, peach, P. ferganensis, cherry, plum and apple), including 16 almond probes which correspond to known genes. All markers were distributed in eight linkage groups, the same as the basic chromosome number of the genus, covering a total distance of 491 cM. The average map density was 2.0 cM/marker and only four gaps of 10 cM or more were found; the two largest gaps were 12cM each. This map was compared with one constructed previously with an intraspecific almond population sharing 67 anchor loci. Locus order was nearly identical and distances were not significantly different. A large proportion of the mapped loci (46%) had skewed segregations; in approximately half of them, the distortion was due to an excess of heterozygotes. One of the distorted regions could be associated with the position of the self-incompatibility gene of almond. Received: 6 November 1997 / Accepted: 26 May 1998     

Effects of antimicrobial activity of silver nanoparticles on in vitro establishment of G × N15 (hybrid of almond × peach) rootstock

In the present investigation were evaluated the antifungal and antibacterial activities of Nano-silver (NS). Two separate experiments were done to evaluate the potential of silver nanoparticles in controlling the contamination of G × N15 micro-propagation. In the first experiment, a factorial experiment based on a completely randomized design with 15 treatments including five different NS concentrations (0, 50, 100, 150 and 200 ppm) and three soaking time of explants (3, 5 and 7 min) with five replications was conducted. In the other experiment, NS was added to Murashige and Skoog (MS) medium with concentrations of 0, 50, 100, 150 and 200 ppm in a completely randomized design. Results showed that the application of 100 and 150 ppm NS both as an immersion and as added directly to the culture medium significantly reduces internal and external contaminations compared with the control group. Using NS in culture medium was more efficient to reduce fungal and bacterial contamination than immersion. High concentrations of NS had an adverse effect on the viability of G × N15 single nodes and this effect was more serious in immersed explants in NS than directly added NS ones regarding the viability of buds and plantlet regeneration. In conclusion, due to high contamination of woody plants especially fruit trees and also adverse environmental effects of mercury chloride, the NS solution can be used as a low risk bactericide in micro-propagation of G × N15 and can be an appropriate alternative to mercury chloride in the future.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Effects of nutrient media,different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond × peach) vegetative rootstock

The objective of this research was to assess the effects of different media i.e. Murashige and Skoog (MS) and Quoirin and Lepoivre (QL), cytokinin type i.e. 6-Benzyladenin (BA) and 6-Benzylaminopurine (BAP) and cytokinin concentration on in vitro proliferation of the G × N15 rootstock. To evaluate the effects of different media and cytokinin type, two separate experiments were conducted as factorial based on completely randomized design, and single nodes were used as explants. The results showed that MS nutrient medium was found to be superior to QL nutrient medium. Regarding the interaction between media and growth regulators, the best interaction was found in MS medium supplemented with 1 mg l⁻¹ BAP resulting in 8.5 new micro shoots/explant while 7.75 shoots were observed in MS medium containing 1.25 mg l⁻¹ BA. The longest length of new micro-shoots (2.10 cm) was obtained in hormone-free MS medium. Findings of this study showed that there is a significant correlation between the hormone level and plantlet height and formed callus weight so that an increase in BAP and BA levels in both of MS and QL media resulted significantly in height decrease and callus weight increase. The results also suggest that the best and the worst plantlets in terms of quality were observed in hormone-free QL medium and MS medium supplemented with 1.25 mg l⁻¹, respectively. These results reflect the fact that the presence of high amounts of NH₄NO₃ and cytokinin especially BAP in culture medium triggered inhibitory effect on shoot growth.     

Protoplast isolation and culture from Kalanchoë species: optimization of plant growth regulator concentration for efficient callus production

Plant Cell, Tissue and Organ Culture (PCTOC) - A high yield of isolated protoplasts and efficient regeneration protocols are prerequisites for successful development of somatic hybrids. In the...     

Short Communication—Effects of oligosaccharins on callus growth and saponin content of Panax notoginseng

This work provides some evidences for the saponin production of Panax notoginseng callus by using biologically active,wall-related oligosaccharins.In an appropriate concentration,three kinds of oligosaccharins stimulated saponin formation of callus growth.The concentration of DO,GO and CO for saponin production of Panax notoqinseng callus culture were 15ppm,15ppm aud 20ppm respectively by comparing saponin yield,It was very obvious for DO to increase saponin content when the concentration was 10ppm,and for GO to stimulate callus growth when the concentration was 20ppm,It would be a good way to produce sapoin by using oligosaccharins in large scale culture in the future.     

Induction of petal-bearing embryos from root-derived callus of Oncidium ‘Gower Ramsey’

A repeatable in vitro culture method was established and it could induce a series of abnormal embryos by which their scale leaves were substituted by petals. These petal-bearing embryos were derived from long-term root calli of an orchid cultivar, Oncidium ??Gower Ramsey??. The calli were induced and subcultured on a modified 1/2MS medium supplemented with five combinations of TDZ and dicamba. When 1-year-old callus, which induced and subcultured at 3?mg/l TDZ and 5?mg/l dicamba (line 13 callus), was transferred onto 1/2MS medium supplemented with 0.1?ml/l NAA and 3?mg/l TDZ, it gave the highest number of petal-bearing embryos. However, line 13 root explants gave one of the lowest percentage of callus formation (12.5%) and the number of somatic embryos per callus was also one of the lowest (along with lines 10, 11 and 12). The efficiency of embryogenesis decreased with age of callus and the significant decrease was started from the third year of culturing. Flowering characteristics of plantlets from normal embryos and petal-bearing embryos were both evaluated after 3?years of culture in the greenhouse. Parameters including length of the longest inflorescence, numbers of flowers per plant, length of flowers and width of flowers were all not significantly different between abnormal embryo-derived plants and normal embryo-derived plants.     

Biosynthesis of cyclopentenylglycine from α-ketopimelate in Idesia polycarpa callus cultures

The nonproteinogenic amino acid, cyclopentenylglycine, is found in certain Flacourtiaceae. This compound may be synthesized by two C₁-chain elongations of -ketoglutarate via -ketopimelate (C₅+2C₁) or by condensation of C₄ and C₃ units (C₄+C₃), a pathway not involving -ketopimelate. The following experimental design elucidated the biosynthetic pathway: Idesia polycarpa callus cultures were freshly established from leaf petioles; synthetic -[1,2-¹⁴C]ketopimelate was added to the medium and cultures were incubated for 3 weeks. After isolation and separation of free amino acids from the tissues, the radioactivity incorporated into cyclopentenylglycine was determined. The results establish -ketopimelate as a precursor for cyclopentenylglycine, thus providing evidence for the C₅+2C₁ biosynthetic path.     

Induction and growth of callus from immature inflorescences of “Zebra” bermudagrass as affected by casein hydrolysate and 2,4-D concentration

Summary Regeneration of grass plants through tissue culture is affected by many factors including genotype and type and concentration of medium components, particularly growth regulators. Objectives of this research were to characterize differentiation and growth of callus from immature inflorescence-explants of a variegated bermudagrass,Cynodon dactylon (L.) Pers. cv. “Zebra”, and to assess the effects of casein hydrolysate (CH) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations on development of total callus mass and the embryogenic (E) portion. Immature inflorescences about 6 mm in length were inoculated in petri dishes containing modified Murashige and Skoog (MS) agar medium. Two levels of CH (0, 200 mg L⁻¹) and four 2,4-D concentrations (1,3,5,7 mg L⁻¹) in subplots were tested. Regeneration of 57 plants via somatic embryogenesis was achieved. Both fresh callus weight and E callus, expressed as a percentage of total callus, were significantly affected by CH and 2,4-D treatments. Maximum callus fresh weight and E callus concentration were attained in medium with 3 mg L⁻¹ 2,4-D. Addition of CH to the medium increased fresh weight and E callus within each level of 2,4-D, but not to the same extent as indicated by a significant (P<.01) 2,4-D X CH interaction. Presence of CH and 3 mg L⁻¹ of 2,4-D maximized both fresh weight and percentage of E callus. Journal Article 5331 of the Agric. Exp. Stn., Oklahoma State University, Stillwater, OK. I. R. A. and C. M. T. are former Graduate Research Assistant and Professor, respectively, Department of Agronomy; and B. B. J. is Professor, Department of Botany and Microbiology, Oklahoma State University, Stillwater, OK 74078.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

In vitro shoot growth of Brugmansia × candida Pers

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida ‘Creamsickle’. Several mineral nutrient experiments were conducted to determine the effect of NH₄⁺, NO₃⁻, K⁺, FeSO₄/EDTA, ZnSO₄, MnSO₄, and CuSO₄ on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH₄⁺, NO₃⁻, and K⁺, was conducted as a 2-component NH₄⁺: K⁺ mixture crossed by [NO₃⁻] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH₄⁺-K⁺-NO₃⁻ experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH₄⁺:K⁺ ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO₄ had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO₄ had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH₄⁺-K⁺-NO₃⁻, FeSO₄/EDTA, and ZnSO₄ was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO₄ and CuSO₄, these formulations were characterized by lower levels of NH₄⁺, K⁺, NO₃⁻ and Zn, and higher levels of FeSO₄/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. ‘Creamsickle’.     

Estimation of psychomotor delay from the Fitts’ law coefficients

An intrinsic property of human motor behavior is a trade-off between speed and accuracy. This is classically described by Fitts’ law, a model derived by assuming that the human body has a limited capacity to transmit information in organizing motor behavior. However, Fitts’ law can also be realized as an emergent property of movements generated by delayed feedback. In this article, we describe the relationship between the Fitts’ law coefficients and the physiological parameters of the underlying delayed feedback circuit: the relaxation rate or time constant, and the psychomotor delay of the feedback process. This relationship is then used to estimate the motor circuit delay of several tasks for which Fitts’ law data are available in the literature. We consistently estimate the delay to be between 0 and 112 ms. A further consequence of this model is that not all combinations of slope and Y-intercept in Fitts’ law are possible when movements are generated by delayed feedback. In fact, it is only possible for an observed speed–accuracy trade-off to be generated by delayed feedback if the Fitts’ law coefficients satisfy ?0.482 ≤ a/b ≤ 3.343 [bits] where b represents the slope in bits per second and a represents the Y-intercept in seconds. If we assume human movement is generated by delayed feedback, then the Fitts’ law coefficients should always be restricted to this range of values.     

The influence of photoperiodic growth condition on isolation of RNA from strawberry (Fragaria × ananassa duch.) tissue

Purification of high-quality RNA from different strawberry tissues is often affected by the presence of high levels of contamination by polysaccharides and phenolic compounds. With the protocol detailed here we describe for the first time total RNA purification from petiole tissue. Treating the plants used as source of material with short-day light regime prior the extraction we are able to obtain RNA suitable for further applications such as in vitro translation, RT-PCR, and RNA blot analysis. The yield of total RNA extraction is significantly enhanced when tissue from plants grown under short-day photoperiodic condition is used compared with that taken from plants grown under long day photoperiod.     

Effect of phytohormones on plant regeneration from callus ofGladiolus cultivar “Jenny Lee”

Summary Callus capable of plant regeneration was initiated at a higher frequency from the basal leaves of in vitro plants (70% explants) as compared to cormel slices (30% explants) when cultured on medium containing various concentrations of auxin. The greatest number of plants were regenerated from 4-mo.-old callus (112 plants/g fresh weight callus) cultured on medium containing 10 mg/liter (53.8μM) 1-napthaleneacetic acid. The addition of 2 mg/liter (9.3μM) kinetin to a Murashige and Skoog’s basal salts regeneration medium resulted in an average two- to three-fold increase in the number of plants regenerated compared to regeneration on medium without hormones. Ten months after callus initiation, all callus maintained on auxin-supplemented media showed a drastic reduction in its capacity to regenerate plants. Ten-month-old callus maintained on dicamba regenerated the greatest number of plants (14 to 23 plants regenerated per gram fresh weight callus) as compared to callus maintained 10 mo. on medium containing 1-napthaleneacetic acid or 2,4-dichlorophenoxyacetic acid. Cormel slices cultured on cytokinin-supplemented media formed small amounts of callus which regenerated up to 19 plants per cormel slice within 1 to 2 mo. after the cormel slice had been placed on either 10 mg/liter (49.2μM) N⁶-2-isopentenyladenosine or 1 mg/liter (4.4μM) 6-benzylaminopurine.