Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Significance of explant preparation and sizing in Aloe vera L.—A highly efficient method for in vitro multiple shoot induction

The present study was carried out to assess the effect of explant preparation and sizing for in vitro micropropagation of Aloe vera L. The stem nodal explants and shoot tips were cultured on modified Murashige and Skoog's medium (1962) supplemented with different concentrations of 6-benzylaminopurine (BA), kinetin (KIN), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) either singly or in combination. The best media composition was found to be MS medium supplemented with IAA (11.42 μM), IBA (9.8 μM) and BA (8.88 μM). The explants were divided into 2 sets, with and without ensheathing leaf base. Explant sizing, pruning and retention of mother tissue was highly significant in induction of multiple shoots and roots. The stem nodal explants with leaf base performed much better than those without such covering. A very high number of shoots and roots grew from these explants. The rooted plantlets were successfully acclimatized and transferred to the green house conditions and finally to field conditions.     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

The effects of explant and cytokinin type on regeneration of Prunus microcarpa

We investigated in vitro regeneration ability of Prunus microcarpa subsp. tortusa using various explants (root, cotyledon and hypocotyl pieces) and cytokinins [benzyladenine (BA), meta-Topolin (mT) and thidiazuron (TDZ)]. Sectioned cotyledon, root and hypocotyl pieces of in vitro grown seedlings were cultured on Nas and Read Medium (NRM) containing BA (7.5, 10, 12.5, 15 or 17.5 μM), mT (2.5, 5.0, 7.5, 10 or 12.5 μM) or TDZ (2.5, 5.0, 7.5, 10 or 12.5 μM). As a measurement of morphogenetic reaction, the ratios of regenerating explants and the numbers of primary adventitious shoots per regenerating explant were analyzed. Cotyledon explants exhibited higher regeneration ratios than hypocotyl explants, and the root explants were inappropriate for regeneration. Both BA and mT were effective on shoot regeneration but higher regenerating explant ratios were obtained when BA was used. In comparison with BA and mT, the effect of TDZ on enhancing explant regeneration ability was insignificant. Mean number of adventitious shoot per regenerating explant was between 1 and 4, and regenerating explant ratios were between 0% and 77%. The practical appliacations of the results are discussed.     

Germination of Passiflora mollissima (Kunth) L.H.Bailey, Passiflora tricuspis Mast. and Passiflora nov sp. seeds

Passiflora mollissima, Passiflora tricuspis and Passiflora nov sp. are three passion fruit species occurring in Bolivia. Germination percentages and rates were determined for 11 different treatments. Per species, germination of 100 seeds was monitored every 3 days, during 90 days. Germination started after 9 days and 50% of final germination was reached within a month or less. Successful, recommended methods for P. mollissima are removing the basal point of seeds (27% germination) or removing the basal point in combination with pre-soaking seeds for 48 h in 50 ppm GA₃ (18%). Pre-soaking seeds for 24 h in 400 ppm GA₃ (42%) and removal of the basal point in combination with pre-soaking seeds for 48 h in 50 ppm GA₃ (36%) are suggested methods to improve germination of P. nov sp. Removing the apical point of P. tricuspis seeds resulted in maximal germination (57%). No unique treatment gave satisfactory results for the three species tested. Exogenous dormancy, probably a combination of mechanical and chemical dormancy is present in the three species studied. Presence of physical dormancy was found in P. mollissima.     

Adventitious root formation in Anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as ‘difficult’- or ‘easy-to-root’ when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be ‘difficult’ precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting.     

Commercial production of Cordyline terminalis (L) Kunth. from shoot apex meristem and assessment for genetic stability of somaclones by isozyme markers

Rapid multiplication of Cordyline terminalis (L) Kunth. was achieved from shoot apex explant on Murashige and Skoog's (MS) medium supplemented with 3% (w/v) sucrose and different concentration of growth regulators at various combinations. On MS medium supplemented with BA in combination with Adenine sulphate and IAA, shoot initiation and multiplication were obtained. Best elongations of shoots were found on 1/2 MS basal medium and shoots were rooted on 1/2 MS medium supplemented with IBA. Rooted plants passed through a hardening phase prior to ex vitro transfer. Clonal propagated plants established in garden soil were uniform and identical to mother plant in respect to growth characteristics and morphology. Isozymic profiles of different micropropagated clones were assessed for their genetic stability. Ten clones were tested for six isozymes. Only a few showed variation with respect to the banding pattern in esterase and superoxide dismutase. In superoxide dismutase, the two polymorphic isoforms (Rf 0.06 and 0.45) appeared in the clone C8 of the plants transferred to the field after 15 subcultural passages. Mobility and intensity of bands were monitored in other isozymes. Isozyme markers may be used as a tool for rapid screening of genetic stability in tissue cultured clones of C. terminalis.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.     

Coconut water and BAP successfully replaced zeatin in olive (Olea europaea L.) micropropagation

The data presented report on trials conducted during 24 months using the Portuguese olive cultivar ‘Galega vulgar’. The effectiveness of coconut water, BAP, or kinetin, as possible zeatin substitutes in olive micropropagation protocols, was investigated. In all stages of the micropropagation process, the mineral and vitamin formulation of olive medium (OM) was used. Regarding culture establishment the best results were achieved when 50 ml l⁻¹ coconut water and 2.22 μM BAP were used as medium supplements. For the in vitro multiplication stage, the highest proliferation rates with an average of 3.4 new explants on each 30 days were achieved maintaining the coconut water concentration at 50 ml l⁻¹ and increasing BAP up to 8.87 μM. The effects of IBA and activated charcoal on the in vitro root induction were also studied. Rooting rates of over 85% were obtained by basal immersion of the explants in IBA solution at 3 g l⁻¹ for 10 s, followed by inoculation in the OM culture medium, added with 2 g l⁻¹ of activated charcoal and without growth regulators. All in vitro rooted plants were transferred into Jiffy-Pots filled with vermiculite–perlite 3:1 (v/v) substrate. Those were subsequently wetted with the OM mineral solution, placed into polystyrene plates each one with 100 Jiffy-Pots capacity, which were transferred to traditional rooting mist benches, on a water-cooling equipped greenhouse. Such a simple acclimatization procedure allowed for 95% of plants survival.     

Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange

The present paper demonstrates the potential of nutrient-alginate encapsulation of axenic nodal segments of pomegranate for synthetic seed technology, which could be useful in germplasm distribution and exchange. Nodal segments from in vitro shoot cultures derived from mature nodal explants (source A) or axenic cotyledonary nodes (source B) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog's [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] medium (MS) supplemented with 4.44 μM benzyladenine (BA) and 0.54 μM naphthalene acetic acid (NAA). Of various concentrations of sodium alginate (1–6%) and the complexation solution of calcium chloride (50–125 mM), a combination of 3% sodium alginate and 100 mM calcium chloride was most suitable for formation of ideal synthetic seeds. Morphogenic response of encapsulated nodal segments to seven different planting media was evaluated. Encapsulated nodal segments of both the sources exhibited shoot development only in four selected media. Of the planting media evaluated, % sprouting (shoot development) was the highest in MS medium augmented with 4.44 μM BA and 0.54 μM NAA and lowest in (1/2) MSS medium. One step germination i.e. both shoot and root formation was possible only with encapsulated nodal segments of source B in MS, (1/2) MSS and natural soil + (1/2) MSS, with MS being most effective. Encapsulated nodal segments stored up to 30 days at 4 °C were capable of sprouting. Plants regenerated from the encapsulated nodal segments were hardened off and transferred to soil.     

The effect of gibberellic acid (GA3) on some phenolic substances in globe artichoke (Cynara cardunculus var. scolymus (L.) Fiori)

Globe artichoke has important nutritional values related to its high content of phenolic compounds such as cynarin and chlorogenic acid. Cynarin (1,5-dicaffeoylquinic acid) is a derivative of caffeic acid and has effect on hepatobiliary diseases, hyperlipidaemia, dropsy, rheumatism and cholesterol metabolism. The effect of gibberellic acid (GA₃) on the yield of the phenolics, chlorogenic acid and cynarin, both in leaves and in the edible part of the head of globe artichoke, were studied. Plant samples were collected for chemical analyses at the laboratories of the Chair of Vegetable Science, Department for Plant Sciences, Technische Universität München in Freising-Weihenstephan, Germany. GA₃ at 60 ppm was applied either at 4, 6 or 8 weeks after transplanting date (ATD). When transplant were used, application of GA₃ at 4 weeks ATD significantly increased the content of chlorogenic acid in leaves while the level of cynarin was similar to the untreated control. In contrast when plants were direct seeded into the field, GA₃ treatments had no affect on phenolics in leaves compared to the untreated control. Whereas GA₃ reduced the time to flowering, it did not increase the content of chlorogenic acid or cynarin in leaves or in most of the flower receptacles, except when GA₃ was applied at 6 or 8 weeks. On the other hand, cynarin yield in the 3rd–5th harvested flower heads was higher than controls. The timing of GA₃ application is, thus, critical for decreasing time to flower as well as altering the content of cynarin and chlorogenic acid in globe artichoke.     

Antioxidant potency of white (Brassica oleracea L. var. capitata) and Chinese (Brassica rapa L. var. pekinensis (Lour.)) cabbage: The influence of development stage,cultivar choice and seed selection

The accumulation of total phenols (TP, Folin-Ciocalteu method) and total flavonoids (TF, colorimetric assay with AlCl₃) and the evolution of antioxidant capacity (FRAP assay, DPPH and ABTS radical scavenging assays) have been monitored in juices of Croatian white cabbage (Brassica oleracea var. capitata) cultivars Vara?dinski and Ogulinski, as well as Chinese cabbage (Brassica rapa var. pekinensis), at various developmental stages. Measurements were performed every four weeks, starting from planting to full maturity, throughout the course of eight months. In the first 8–12 weeks, the TP and TF contents as well as antioxidant capacity increased significantly in all analyzed juice samples and in most even doubled. This rapid increase was followed by a gradual decrease in values derived from all assays, over the course of 12–30 weeks, to the final values which were in all cases lower than the values measured at week 4. The results also point to significant variability in TP and TF contents and antioxidant capacity at the fully mature stage between white and Chinese cabbage juices and between juices extracted from cultivars Ogulinski and Vara?dinski.     

The effect of nitrogen source and concentration,medium pH and buffering on in vitro shoot growth and rooting in Eucalyptus marginata

This work examined the effect of nitrogen source and medium buffering on the micropropagation of Eucalyptus marginata Donn ex Sm. The number of shoots was increased when media contained 2-(N-morpholino) ethanesulfonic acid (MES) but this increase was minor and only applied to one of the two clones tested. Highest root production was obtained when the medium contained 7.5 mM nitrogen in a ratio of 2NO₃⁻:1NH₄⁺ and was buffered with 10 mM MES. In the rooting medium the pH was influenced most significantly by the nitrogen source, and then whether the medium was buffered. The media pH remained relatively constant when nitrate was the sole nitrogen source and this was assisted by the addition of 10 mM MES. Lower concentrations (<10 mM) of MES were less effective in buffering media over a four-week culture period in both shoot multiplication and rooting medium.     

Temperature effects on male fertility and flower and fruit development in Capsicum annuum L.

Temperature conditions strongly influenced the development of flowers and fruits of pepper (Capsicum annuum L.) plants. Low temperatures (LTR; 18°C day/15°C night) had much more effect on flowers and fruits than intermediate (ITR; 23°C day/18°C night) or high (HTR; 28°C day/23°C night) temperatures. LTR caused the formation of abnormal petals, stamens and gynoecium in the flowers. Stamens produced were deformed, in some cases partly carpel-like, produced abnormal non-viable pollen, and were thus functionally male-sterile. In the gynoecium, the ovary size of LTR-grown flowers was larger than that of ITR and HTR flowers, but the style elongation was inhibited. Fruits produced under HTR were larger than ITR and were seeded under both temperature regimes. Under LTR, small seedless fruits were produced, but normal seeded fruits were formed if flowers were pollinated with pollen from ITR- or HTR-grown flowers.     

The impact of sodium chloride on root growth,cell division,and interphase silver-stained nucleolar organizer regions (AgNORs) in root tip cells of Allium cepa L.

To evaluate genetic damage, parameters including root growth, chromosomal aberrations, and interphase silver-stained nucleolar organizer regions (AgNORs) in root tip cells of Allium cepa L. were investigated after treatment with NaCl (40–160 mM). The results showed that NaCl caused a decrease in root growth. All concentrations of NaCl showed an inhibitory effect on dividing cells in root tips of A. cepa L. and caused a reduction in mitotic index values. Upon exposure to NaCl, roots exhibited various mitotic abnormalities, including c-mitosis, anaphase bridge, and chromosome stickiness. In addition, interphase cells with micronuclei, budding nuclei, and unequal-sized nuclei were observed. Moreover, total cell aberration increased with increasing NaCl concentration. For AgNOR parameters, the average number of AgNORs per nucleus decreased in roots treated at all NaCl concentrations. The singular AgNOR area and whole AgNOR area in the nucleus containing 1–3 AgNORs were inversely proportional to NaCl concentrations.     

Growing season and hydrogen peroxide effects on root induction and development in Olea europaea L. (cvs ‘Frantoio’ and ‘Gentile di Larino’) cuttings

Semi-hardwood cuttings of ‘Frantoio’ (high rooting ability) and ‘Gentile di Larino’ (low rooting ability) cultivars were obtained from 1-year-old olive shoots sampled in mid-August during the 2000 and 2001 growing season. Semi-hardwood cuttings were dipped in IBA 4000 ppm and H₂O₂ (0%-control or 3.5% w/v) solutions before rooting in greenhouse equipped with an automatic mist system. At 57 and 88 days after the beginning of rooting treatments cuttings were scored for the presence of callus, roots and roots number. In both cultivars and years, the IBA 4000+H₂O₂ treatment significantly modified rooting of cuttings at 57 days in comparison with IBA 4000 treatment. The positive effects of H₂O₂ on rooting were gradually smoothed after 88 days. At the end of the propagation cycle (88th days), cuttings treated with IBA 4000+H₂O₂ had significantly higher root number in comparison with those treated with IBA 4000 alone.     

Relations between time of sprouting of the scion in the nursery,the time of formation and number of basal bottom-breaks,and the number of harvested shoots of glasshouse rose clones on R. canina ‘Inermis’

In 3 successive years, different batches of Hybrid Tea-rose seedlings, selected for cut-rose properties, were bench-grafted on to R. canina ‘Inermis’. Plants were grown in a rose house for 11 months. Clones, the scions of which sprouted early in the nursery, had both earlier and more basal bottom-breaks (BB) — resulting in a higher number of shoots harvested — than late-sprouting clones. Basal BB's emerged over a 22-week period, starting approximately 14 weeks after sprouting of the scion. The value set in practice on a high number of basal BB's as a basis for high flower production was confirmed. In the selection procedure for better yielding cultivars, breeders are recommended to pre-select for a high number of basal BB's in seedlings. The possible use of the time of sprouting of the scion as a parameter for rootstock vigour is discussed.     

Effects of exogenous salicylic acid and nitric oxide on lipid peroxidation and antioxidant enzyme activities in leaves of Brassica napus L. under nickel stress

The effects of nickel in combination with salicylic acid (SA) and sodium nitroprusside (SNP), a donor of nitric oxide (NO) on 21-day-old canola plants were evaluated. Exposure to 0.5 mM NiCl₂·6H₂O for 10 days resulted in toxicity symptoms such as chlorosis and necrosis at leaves. Addition of 0.2 mM SA or 0.2 mM SNP slightly reduced the toxic effects of nickel. After application of both SA and NO, these symptoms considerably decreased. Treatment with Ni resulted in a decrease in dry weight of roots and shoots and chlorophyll content of leaves. In Ni-treated plants, level of lipoxygenase activity and malondialdehyde (MDA), H₂O₂ and proline contents significantly increased, while the activities of the antioxidant enzymes such as catalase, guaiacol peroxidase and ascorbate peroxidase decreased in leaves. The results indicated that Ni caused an oxidative stress in canola plants. The Ni-stressed plants exposed to SA or NO, especially to SA + NO, exhibited an improved growth as compared to Ni-treated plants. SA or NO, especially both together considerably reduced root-to-shoot translocation of Ni and increased the activities of the antioxidant enzymes in leaves of Ni-stressed plants. Interaction of SA and NO improved the chlorophyll content and decreased the level of lipid peroxidation, H₂O₂ and proline accumulation in leaves. These results suggest that SA or NO in particular their combination counteract the negative effects of Ni on canola plants.     

Effect of sampling time and soil type on Mn,Fe, Zn,Ca, Mg,K and P concentrations of olive (Olea europaea L., cv. ‘Koroneiki’) leaves

It has been studied the time course of seven leaf nutrients’ (Mn, Fe, Zn, Ca, Mg, K and P) concentrations when plants of the olive cultivar ‘Koroneiki’ were grown for about 5 months (from the 30th of May till the 17th of October) in three soils from different parent materials (Marl, Gneiss schist and Peridotite), located in the region of Central Macedonia, Northern Greece.