Auxins increase the occurrence of leaf-colour variants in Caladium regenerated from leaf explants

Leaf explants of Caladium ‘Pink Cloud’ were cultured in vitro on MS medium containing various auxins (NAA, IBA, IAA, 2,4,5-T and 2,4-D) in combination with cytokinin (BA). NAA gave the most vigorous in vitro propagation of this plant, and only 15% of the plants were leaf-colour variants on the medium containing 0.5 μmol NAA. Leaf colour variation was observed in all plants regenerated on the medium containing 2,4-D at 0.5–4.5 μmol. In hormone-free medium, only a few leaf-colour variants (6%) occurred, but the rate of plant regeneration was very low. Application of 0.5 μmol NAA together with 4.5 μmol BA seemed to be the most appropriate for in vitro propagation of Caladium ‘Pink Cloud’ with only a few leaf-colour variants.     

Callus induction and plant regeneration from cotyledonary explants of ash gourd (Benincasa hispida L.)

A protocol for the production of complete plantlets through multiple shoots from the cotyledon-derived calli of ash gourd (Benincasa hispida L.) is described. The embryos were excised from mature seeds and cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurin (BAP, 1–5 μM). After 10 days the well-developed green cotyledons from the growing embryos were isolated and cultured on MS medium fortified with 2,4-D (1–6 μM). The cultured cotyledons gave rise to luxuriantly growing calli after 6 weeks. These calli were subcultured on MS medium supplemented with various concentrations of BAP (1–6 μM) alone or in combination with naphthalene acetic acid (NAA, 0.2 and 0.5 μM) for regeneration. The regenerated shoots were multiplied and rooted on quarter strength MS medium supplemented with indole-3-butyric acid or NAA (1–5 μM). The rooted shoots were transplanted to soil with 90% success.     

Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L.

An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector p^BI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.     

Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis

Clones of Hylocereus and of Selenicereus species were distinguished from each other by the banding pattern generated by one to nine 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. RAPD analysis was also applied to estimate the genetic relationship among five Hylocereus and nine Selenicereus species. A dendrogram was constructed based on a data matrix of 173 polymorphic bands originated by nine primers. Two groups were identified, one consisting of Hylocereus species and the other consisting of Selenicereus species. These results are consistent with the accepted taxonomic classification of the genera studied. The principal coordinate analysis (PCO), i.e. the plot drawn on the basis of the RAPD data, clearly distinguished between three groups, namely, Hylocereus species, S. megalanthus and the rest of the Selenicereus species studied. PCO thus strongly support the notion that the tetraploid S. megalanthus is an exception among the Selenicereus group. The RAPD results support our hypothesis regarding the allopolyploid (rather than autopolyploid) origin of this species.     

Effects of thermoperiodicity and plant population density on stem and flower elongation, leaf development, and specific fresh weight in single stemmed rose (Rosa hybrida L.) plants

Thermoperiodicity, i.e. growth in the alternating temperature regime with the same diurnal mean compared with growth at the constant temperature at which optimal growth occurs, was studied at three plant population densities in four cultivars of Rosa hybrida L. Single-node cuttings with five-leaflet leaves were excised and grown as single-stemmed rose plants at an average photosynthetic photon flux density of about 260 μmol m⁻² s⁻¹ and supplied with carbon dioxide at about 1000 μmol mol⁻¹. The optimal constant temperature regime was 22 °C day (20 h)/22 °C night (4 h); alternating temperatures were 23 °C day (20 h)/18 °C night (4 h). The plant population densities were 100, 131 and 178 plants m⁻² of bench area. Thermoperiodicity was absent, or could not be detected, in the parameters related to the growth period, the formation of fresh biomass, the bloom quality, and most parameters related to shoot elongation. However, classic thermoperiodic effects of alternating regime were significant in the cultivars Red Velvet and Sonia, with shoot elongation promoted (7.1 and 10.5%, respectively) in the growth phase from onset of axillary bud growth until the flower bud became visible. Compared with the other two cultivars, plants of Red Velvet and Sonia tended to develop longer internodes. The results, obtained concurrently at three different plant population densities, suggest that thermoperiodicity can affect (single-stemmed) plant growth and development in R. hybrida. Increased plant population density also increased plant height at visible flower bud, but the bloom quality, expressed as specific fresh weight, and the flower height at anthesis was decreased at the highest density. Increased plant population density increased the number of five-leaflet leaves developed in Red Velvet, but had no effect on leaf number in Texas and Sonia, while, in Lambada the leaf number was decreased at the highest density.     

Transformation of Brassica oleracea var. capitata with bacterial betA gene enhances tolerance to salt stress

The bacterial betA gene for biosynthesis of glycinebetaine was transferred to cabbage (Brassica oleracea var. capitata) cultivar ‘Golden Acre’ through Agrobacterium-mediated transformation of hypocotyl explants. Transgenic status was established through Southern hybridization and mRNA expression in the shoots. The transformants exhibited higher tolerance to NaCl stress compared to untransformed parent plants. In physiological assessment of salinity tolerance, transgenics showed better growth response and greater stability in maintaining plant water relations at increasing levels of salinity. These results demonstrate that engineering glycinebetaine biosynthetic pathway into cabbage can lead to enhanced salt tolerance.     

Role of bioinoculants in development of salt-resistant saplings of Morus alba (var. sujanpuri) in vivo

Pot experiments were conducted for the development of salt-resistant saplings of Morus alba (var. sujanpuri) involving bioinoculants, namely arbuscular mycorrhizal (AM) fungi, Azotobacter and indole butyric acid (IBA). The IBA and sodium chloride (NaCl) concentrations were optimized prior to the experiments. By using both low and high concentrations, 15 ppm IBA and 0.05% NaCl (w/v) were found to be optimum in acting synergistically with AM fungi and Azotobacter and also for increasing all the growth parameters and microbial count in the rhizosphere. For in vivo development of salt-resistant saplings, the optimal concentration of IBA, along with AM fungi and Azotobacter in different combinations, was applied. The saplings were irrigated regularly with 0.05% NaCl water. Although growth parameters such as AM infection percentage, AM spores per 100 g soil and Azotobacter cells/g soil were affected by NaCl watering, the inoculation of both bioinoculants significantly enhanced survival percentage of saplings from 25 to 50% under salt stress. Maximum survival (55%) of saplings was found with IBA (15 ppm)+AM fungi+Azotobacter.     

Aptitude for mycorrhizal root colonization in Prunus rootstocks

Eighteen Prunus rootstock cultivars were inoculated with three arbuscular mycorrhizal fungi under greenhouse conditions in order to evaluate their affinity for mycorrhizal colonization. The rootstocks were peach–almond hybrids, peaches, plums and cherries of Spanish, French and Italian origin. Mycorrhizal colonization was low in plants inoculated with Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappe, and Glomus etunicatum Becker and Gerdemann. In contrast, Glomus intraradices Schenck and Smith, proved to be the most infective endophyte, achieving the highest mycorrhizal colonization rate in most of the rootstocks evaluated. Species of Prunus insititia L. were the only botanical group to show a consistently high affinity for mycorrhizal colonization with G. intraradices.     

Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets

Potato single nodes were cultured in vessels containing MS medium supplemented with 10, 20 and 30 g/l of sucrose. Vessels were closed with a clear polypropylene lid with or without 10 mm microporous polypropylene membrane. Sucrose concentration significantly increased plantlet height, shoot fresh weight and chlorophyll a content. Plantlets grown in ventilated vessels were significantly shorter, had lower shoot fresh weight and higher shoot dry weight than those in non-ventilated vessels. The highest leaf chlorophyll a content (21.83 mg/g fresh weight) was found in plantlets grown in ventilated vessels using MS medium with 20 g/l of sucrose, whereas those grown on medium with 10 g/l of sucrose had the highest chlorophyll b content (24.00 mg/g fresh weight). Total chlorophyll content was significantly higher when plantlets were grown in ventilated vessels containing medium with 10 or 30 g/l sucrose than in non-ventilated vessels. There was no significant difference in total chlorophyll content among plantlets grown in ventilated vessels with different concentrations of sucrose. Stomatal density was significantly lower when plants were grown under ventilated conditions. Leaf replica examination showed that stomata under non-ventilated condition were spherical with wide openings whereas, those in ventilated vessels were elliptical with narrow openings. Plantlets grown in non-ventilated vessels had thinner leaves and failed to build up a distinct defined upper epidermis, palisade parenchyma layer and spongy cells. On the other hand, leaves under ventilated conditions showed comparatively well organized layers with small intercellular space. The vascular system of leaves under the ventilated conditions demonstrated very well developed xylem unlike leaves under non-ventilated conditions. Thus, ventilated vessels with the 20 g/l of sucrose under ambient CO₂ in the growth room could successfully promote photomixotrophic culture and produce healthy plantlets.     

Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.)

A protocol for in vitro propagation of the wild three-lobed sage (Salvia fruticosa Mill.) (Synonym, Salvia triloba L.) was developed. Shoot tips were excised from in vitro seedlings and established on MS, Nitch and Nitch (NN), or B5 medium. For shoot proliferation, in vitro nodal and apical explants were cultured on MS medium containing 0.25–2 μM 6-benzylaminopurine (BA), 6-furfurylaminopurine (kinetin), or thidiazuron (TDZ). Proliferated microshoots were rooted on MS medium supplemented with 2.7–11.4 μM indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), or α-naphthaleneacetic acid (NAA). Results indicated that shoots established at 100% regardless of media type, however, shoot height, nodes per shoot, and leaf number were highest for explants established on MS medium compared to NN or B5. Number and height of proliferated shoots, nodes per shoot, and leaf number were highest for nodal explants cultured on a medium containing 0.75 μM BA. Microshoots cultured on a medium supplemented with 2.7 μM IBA exhibited the highest rooting percentage compared to those cultured with IAA or NAA. Essential oil composition in microshoots and shoots of greenhouse-grown plants was determined by gas chromatography/mass spectrometry. The major essential oils detected in both plant materials were α-pinene, 1,8-cineole, camphor, and borneol. No α-thujone or β-thujone was detected. The content of essential oils, camphor, and borneol were higher in the microshoots than in shoots of greenhouse-grown plants.     

Embryo germination and proliferation in vitro of Juglans regia L.

This paper presents the optimal culture conditions for the in vitro embryo germination and proliferation of Juglans regia L. rootstock cv. Peralta, selected by the IMIDA fruticulture team. J. regia L. rootstock cv. Peralta is characterised by its resistance to salinity, lime and by its vigour. The first experiment determined the best culture medium for in vitro embryo germination. The Murashige and Skoog medium (MS; Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol. Plant. 15, 473–497), the medium developed by our team for walnut (NGE), the Lloyd and McCown medium (WPM; Lloyd, G., McCown, B., 1981. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by the use of shoot tip culture. Proc. Plant Prop. Soc. 30, 421–427) and that of Driver and Kuniyuki (DKW; Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19 (4), 507–509), were compared, all without growth regulators. The best germination percentage was obtained in WPM (81%) with significant differences between the different media. In the second experiment, the optimal benzylaminopurine and indole butyric acid concentrations were determined for the proliferation stage of the explants obtained in the first experiment. The proliferation rates obtained varied from 0 in the medium without cytokinins to 6 in the medium with 0.5 mg l⁻¹ BAP. The cluster proliferation quality and other parameters studied indicate that the optimal treatment was 0.5 mg l⁻¹ BAP.     

Micropropagation of Nolina recurvata Hemsl.: β-Cyclodextrin effects on rooting

Nolina recurvata Hemsl. is a very decorative indoor plant but difficult to micropropagate vegetatively. In vitro cuttings failed to induce adventitious rooting. Investigations for a rapid micropropagation using β-cyclodextrin added to the rooting medium has solved the problem. Rooted N. recurvata plantlets were obtained after successive stages of various culture media.Initiation and in vitro multiplication of this plant was possible with lateral buds cultured in Murashige and Skoog medium supplemented with 4.45 μmol of BA and 0.5 μmol of IBA. The number of axillary shoots by explant obtained was 6.In vitro rooting was obtained in MS medium (1/2 strength of minerals salts) supplemented with β-cyclodextrin. This substance, at 1.76 mmol associated with 5 μmol of IBA, improved quality and the rooting rate by 100%.     

In vitro embryo germination and proliferation of pistachio (Pistacia vera L.)

In vitro development of isolated embryos and axillary bud proliferation were studied in Pistacia vera L. Different regulator-free nutrient media were compared to determine the effects of the mineral solution, agar and sucrose concentrations on seedling development from mature embryos. Nutrient-rich MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15, 473–479] and DKW [Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19, 507–509] mineral solutions were more efficient for the development of aerial parts than nutrient-poor KN [Knop, W., 1884. Bereitung einer concentrierten nährstofflosung für pflanzen. Landwersuhssat 30, 292–294] and WT [Withe, P.R., 1936. Plant tissue cultures. Bot. Rev. 2, 419–437] solutions. Reducing the agar concentration enhanced fresh matter production and balanced seedling development. When tested at different concentrations, sucrose was found to orient mature embryo development, with the best results obtained at concentrations of 2–4%, whereas high concentrations (6 and 12%) mainly inhibited elongation of the aerial parts. Plantlets obtained previously from in vitro cultured embryos were propagated by axillary budding. High bud proliferation (six shoots per explant) was achieved when using 17.8 μM benzyladenine (BA) combined with 0.5 μM indole-3-butyric acid (IBA). The addition of 2.9 μM gibberellic acid (GA₃) to the propagation medium did not improve axillary shoot yields and on average, media with GA₃ produced 2.3–2.6 elongated stems per cultured explant. Shoots were rooted in vitro in half-strength MS medium containing 12.3 μM IBA.     

Effect of substrate,medium composition,irradiance and ventilation on jojoba plantlets at the rooting stage of micropropagation

The effect of substrate, medium composition, irradiance and ventilation on rooting percentage, and root morphology and functioning with respect to mineral uptake was investigated in jojoba [Simmondsia chinensis (Link) Schneider]. Plantlets grown on Sorbarod plugs embedded in perlite accumulated more biomass and showed better rooting than plantlets grown on Sorbarod plugs embedded in agar. In plants grown on the latter substrate, the addition of sucrose to water or salts media promoted rooting and accumulation of shoot and root biomass. On perlite, rooting was high in all treatments, while root growth was enhanced only by 2% sucrose. Sucrose and irradiance had a positive synergistic effect on plantlet growth. Doubling the irradiance from 58 to 116 μE m⁻² s⁻¹ and using high ventilation (22-mm membrane) failed to improve rooting but promoted the ability of the stomata to close, thereby reducing water loss under ambient conditions. High irradiance and ventilation lowered the chlorophyll content, but without impairing biomass accumulation. Plantlets grown under enhanced ventilation and 0.5% sucrose formed fine roots within the cellulose plug and thick roots on the vessel floor. With the 2% sucrose supplementation, the roots were generally thicker, with very few thin roots being formed. ³⁶Cl uptake into plantlets and transport to the leaves decreased in plants exposed to a high sucrose concentration. Acclimatization of jojoba plantlets was improved by enhancing ventilation.     

Reducing oxidative browning during in vitro establishment of Protea cynaroides

Oxidative browning is one of the reasons why Protea cynaroides is not being extensively propagated via tissue culture techniques. The reduction of oxidative browning was best achieved by stirring the explants for 1 h in an antioxidant solution mixture containing 100 mg l⁻¹ ascorbic acid and 1500 mg l⁻¹ citric acid before inserting them into the medium. In combination with this antioxidant solution, in vitro establishment was highly successful where 100% bud growth was achieved by explants growing under 16 h photoperiod.     

Clonal propagation of mulberry (Morus indica L. cultivar M-5) through in vitro culture of nodal explants

A high frequency of sprouting (80.0%) and shoot differentiation was observed in the primary cultures of nodal explants of Morus indica L. cultivar M-5 on MS medium supplemented with 2,4-D (0.3 mg/l). In vitro proliferated shoots were multiplied rapidly by culture of shoot tips on MS medium with BAP (0.5 and 1.0 mg/l) which produced the greatest multiple shoot formation. Multiplication was also achieved by culture of shoot tips on MS medium with BAP (4.0 mg/l) and GA₃ (0.05 mg/l) which facilitated the elongation of shoots followed by sprouting of axillary buds of in vitro grown shoots. A high frequency of rooting (86.7%) with development of healthy roots was observed from shoots cultured on medium with 2,4-D (1.0 mg/l). Plants with well developed roots were transferred to soil with a survival frequency of 80%.     

Kaolin treatment to reduce pomegranate sunburn

The aim of this study was to reduce pomegranate losses due to sunburn in south-western Spain. Summer temperatures may rise above 45 °C, resulting in sunburn damage to the fruits, which may inflict losses of up to 40% of the total yield. Data were obtained in two consecutive years. Surround^® WP, a product comprising processed, refined kaolin, was sprayed over the whole canopy and fruits four times at 2–3-week intervals from mid-June to early August, the first application at 5% and the other three at 2.5%. The resulting white coating significantly reduced fruit and leaf surface temperatures relative to the control by averages of 4.9 and 2.5 °C, respectively. Sunburn damage of fruits was reduced from 21.9% in untreated control to 9.4% in the Surround WP-treated fruits.     

Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Immunolocalization and quantification of IAA after self- and free-pollination in Olea europaea L.

In order to characterise the self-compatibility in ‘Pisa 9’, that is an improved clone of cultivar Leccino (Olea europaea L.), biological, histological and biochemical analyses were carried out. Observations were also focused on ‘Pendolino’, the main pollinator for Italian cultivars considered as being self-incompatible. After self-pollination, in ‘Pisa 9’ the majority of the pollen tubes reached the distal part of the style, entered the ovule leading to a high fruit-set percentage. These same processes were also noted in the Pendolino cultivar, though in a lesser degree. The indole acetic acid (IAA) concentration and its immunocytochemical localization were determined in pollinated flowers after self- and free-pollination and in unpollinated flowers. In both cultivars the highest IAA levels and signal intensity were observed in free-pollinated pistils, whereas after self-pollination a different behaviour emerged. In ‘Pendolino’ the immunostaining was not uniformly distributed and stopped at the style level. This finding sustained the low percentage of fruit-set obtained in the cultivar. In ‘Pisa 9’ the auxin distribution was similar to the free-pollinated pistils where the immunolocalization staining reached the ovary. The correspondence between the IAA signal in the ovary and the high percentage of fruit-set in ‘Pisa 9’ self-pollinated pistils, confirms the role of this hormone in the induction and regulation of the fertilisation process.     

Combining endophytic fungi and bacteria for the biocontrol of Radopholus similis (Cobb) Thorne and for effects on plant growth

Eight endophytic fungal and bacterial isolates with antagonistic activity against Radopholus similis were evaluated in vivo for their individual and combined effects on biocontrol of R. similis and on the growth of “Grand Naine” cultivar banana plantlets in the greenhouse. Penetration efficiency (PE) of R. similis was between 3 and 21% in 29 biological agents (BAs) treatments, less than the 29% of the nematode-alone control (p ≤ 0.0001); 24 of the BAs treatments did not differ from the PE of 5% for a nematicide control. Twenty nine BAs treatments exhibited antagonistic activity against nematodes which reduced final population levels between 18 and 93%, relative to those on nematode-alone control plants (p ≤ 0.0001), and 14 BAs treatments were statistically similar to the nematicide treatment (88% reduction). Twenty four BAs treatments had increments of plant root biomass ranging from 20 to 58%, greater than the control plants; 37% of the treatments with single and combined BAs inoculations had root length increments ranging from 29 to 54% compared with control and chemical treatment. The nematicide, Terbufos 10GR, did not affect plant growth.