Rapid micropropagation of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> using shoot tip explants pre-cultured in thidiazuron supplemented liquid medium

An efficient protocol has been developed for rapid micropropagation of Ocimum basilicum. Multiple shoots were induced by culturing shoot tip explants excised from mature plants on a liquid Murashige and Skoog (MS) medium supplemented with 5–100 μM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplementation to the culture medium was 50 μM for 8 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (78 %), mean number of shoots (11.6 ± 1.16) and shoot length (4.8 ± 0.43 cm) per explant. A culture period longer than 8 d with TDZ resulted in the formation of fasciated or distorted shoots. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA). The micropropagated shoots with well developed roots were successfully established in pots containing garden soil and grown in greenhouse with 95 % survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

Plant regeneration of three<Emphasis Type="Italic"> Opuntia</Emphasis> genotypes used as human food

In vitro micropropagation Opuntia cactus plants, whose young stems are consumed as vegetables, is reported. Cladode explants from three Opuntia genotypes, were cultivated in MS medium containing BA and GA₃. Shoots produced were used as secondary explants and BA added at different concentrations to induce shoot development. 0.5 M BA was the best for bud formation. Satisfactory rooting occurred when IBA was added to the medium, and plants were successfully established in soil and adapted to greenhouse conditions. The protocols developed in this work provide a basis for the establishment of a genetic transformation system for Opuntia spp.     

Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber <Emphasis Type="Italic">in vitro</Emphasis>

The relationship between polyamines (PAs) metabolism and adventitious shoot morphogenesis from cotyledons of cucumber was investigated in vitro. The endogenous levels of free putrescine (Put) and spermidine (Spd) in the explants decreased sharply, whereas endogenous spermine (Spm) increased during adventitious shoot morphogenesis. The presence of 1–15 mM Put, 1–2 mM Spd, 0.05–1 mM Spm, 5–10 M aminoethoxyvinylglycine (AVG) or 5 M AVG together with 50 M 1-aminocyclopropane-1-carboxylic acid (ACC) in the regeneration medium could promote adventitious shoot formation. Conversely, 1–5 mM D-arginine (D-Arg) or 0.01–0.1 mM methylglyoxal bis-guganylhydrazone (MGBG) inhibited regeneration; and 0.005–0.05 mM ACC displayed little or no evident effects. The explants growing on medium containing 5 M AVG produced higher levels of free Put and Spm, and on medium containing 5 mM Put the explants responded similarly to the AVG-treated explants. However, the exogenous use of 1 mM D-Arg reduced the levels of Put, Spd and Spm, and 0.1 mM MGBG reduced the levels of free Spd and Spm. Moreover, although the explants cultured on medium containing Put and MGBG enhanced ethylene production, AVG and D-Arg inhibited ethylene biosynthesis. This study shows the PAs requirement for the formation of adventitious shoot from cotyledons of cucumber in vitro and the enhanced adventitious shoot morphogenesis may be associated with the elevated level of endogenous free Spm, albeit the promotive effect of PAs on adventitious shoot morphogenesis may not be related to ethylene metabolism.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

Influence of different co-cultivation temperatures,periods and media on <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Tobacco leaf disc explants were inoculated with Agrobacterum tumefaciens strain GV2260 carrying p35S GUS-INT to determine the influence of different co-cultivation temperatures (18 – 26 °C), periods (24 – 96 h) and media (solid and liquid) on transformation efficiency. Kanamycin-resistant shoots developed on leaf discs inoculated with Agrobacterium after 4 weeks of culture initiation. Regenerated shoots were excised and rooted in the basal medium supplemented with 100 mg dm ^–3 kanamycin and 250 mg dm ^–3 augmentin. The rooted plantlets were finally transferred to compost and confirmed by GUS assay and PCR analysis. The highest transformation frequency was achieved from the explants co-cultivated with A. tumefaciens in liquid medium for 48 h at 22 or 24 °C.     

Seasonal variation of organogenetic activity and reserves allocation in the shoot apex of Pinus pinaster Ait

BACKGROUND AND AIMS: To understand better the basic growth characteristics of pines and the fundamental properties of the shoot apical meristem (SAM), variations within the shoot apex of buds were studied. METHODS: A detailed structural comparison of meristem dimensions, organogenetic activity, and the presence of lipids, starch grains and tannins was performed on shoot apices of juvenile, and male and female adult Pinus pinaster at five different times in the annual growth cycle. KEY RESULTS: There were significant correlations among traits and differences in the pattern for juvenile and adult shoots. In juvenile shoots, peaks of organogenesis were present in spring and autumn, but not in summer. In adult shoots, one peak, characterized by an increase in meristem dimensions, was present in summer. The accumulation of starch grains beneath the SAM and of tannin in sub-apical pith parenchyma were at their maximum when organogenetic activity was high in spring and autumn in juvenile plants, and in summer in adult plants. In juvenile and adult plants, lipids were stored within the SAM in autumn, filling a large part of the bud in winter, and were depleted in the cortical parenchyma and then in the pith during shoot elongation. CONCLUSIONS: Depending on the sites of accumulation within the SAM and on the stage of the annual growth cycle, lipids, starch and tannins may be involved in different processes. In spring, energy and structural materials released by lipid hydrolysis may contribute to stem elongation and/or cell-to-cell communication. During organogenesis, energy and structural materials released by starch hydrolysis may influence developmental programmes in the SAM and adjacent cells. Tannins may be involved in cellular detoxification. At the end of the growing season, accumulation of lipid and starch is positively correlated with the onset of dormancy.     

Rescue of shoot apical meristems of chrysanthemum by culturing on root tips

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum Piato attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.Communicated by K.K. Kamo     

High fertigation frequency and phosphorus level: Effects on summer-grown bell pepper growth and blossom-end rot incidence

The objective was to examine the effects of fertigation frequency and P application rate on bell pepper growth and blossom-end rot (BER) incidence, under hot conditions. The experiment comprised six treatments: two concentrations of phosphorus (3 and 30 mg L^–1) combined with three fertigation frequencies (two and eight events per day, and for 1.5 min every 25 min throughout the day). Increasing the fertigation frequency significantly increased the plants acquisition of nutrients, especially phosphorus and manganese. A significant linear regression was obtained between aboveground biomass, and leaf P concentration in the early vegetative stage. Based on the linear regression, 96% of the dry weight variations could be explained by differences in leaf P concentration, indicating that the main effect of fertigation frequency was related to improved P mobilization and uptake. Increasing the daily fertigation frequency from two to eight and to 30 applications reduced the number of BER fruits from 7 to 3 and to 2 per plant, respectively, and accordingly, increased the yield of export-quality fruits from 6.5 to 10 and to 10.5 per plant, respectively. The Mn concentration in plants exposed to low fertigation frequency were low, probably in the deficiency range, but they increased with increasing fertigation frequency. A negative correlation was found between the accumulated number of BER-affected fruits throughout the experiment and fruit-Mn concentrations. In light of recent findings that BER effects in the fruit tissue include the production of oxygen free-radicals and diminution of anti-oxidative compounds and enzymatic activities, and the known crucial role of manganese in enzyme activities and in detoxification of oxygen free-radicals, the relationships between BER incidence and fruit-Mn concentration may indicate that BER is related to Mn deficiency. Future researches are needed to validate this hypothesis.     

Biomass allocation and leaf chemical defence in defoliated seedlings of Quercus serrata with respect to carbon-nitrogen balance

BACKGROUND AND AIMS: Both nutrient availability and defoliation affect the carbon-nutrient balance in plants, which in turn influences biomass allocation (e.g. shoot-to-root ratio) and leaf chemical composition (concentration of nitrogen and secondary compounds). In this study it is questioned whether defoliation alters biomass allocation and chemical defence in a similar fashion to the response to nutrient deficiency. METHODS: Current-year seedlings of Quercus serrata were grown with or without removal of all leaves at three levels of nutrient availability. KEY RESULTS: Plant nitrogen concentration (PNC), a measure of the carbon-nutrient balance in the plant, significantly decreased immediately after defoliation because leaves had higher nitrogen concentrations than stems and roots. However, PNC recovered to levels similar to or higher than that of control plants in 3 or 6 weeks after the defoliation. Nitrogen concentration of leaves produced after defoliation was significantly higher than leaf nitrogen concentration of control leaves. Leaf mass per plant mass (leaf mass ratio, LMR) was positively correlated with PNC but the relationship was significantly different between defoliated and control plants. When compared at the same PNC, defoliated plants had a lower LMR. However, the ratio of the leaf to root tissues that were newly produced after defoliation as a function of PNC did not differ between defoliated and control plants. Defoliated plants had a significantly lower concentration of total phenolics and condensed tannins. Across defoliated and control plants, the leaf tannin concentration was negatively correlated with the leaf nitrogen concentration, suggesting that the amount of carbon-based defensive compounds was controlled by the carbon-nutrient balance at the leaf level. CONCLUSIONS: Defoliation alters biomass allocation and chemical defence through the carbon-nutrient balance at the plant and at the leaf level, respectively.