Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)

Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 μM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 μM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 μM 2,4-D and 1 μM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 μM 2,4-D and 1 μM BA and subsequently transferred to media containing 2–4 μM 2,4-D and 0 or 0.5 μM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 μM 2,4-D and 1 μM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 μM 2,4-D and 0.5 μM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks.     

Optimizing shoot regeneration and transient expression factors for Agrobacterium tumefaciens transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency

An efficient, adventitious shoot regeneration protocol was devised, and transient expression studies were carried out to enable Agrobacterium-mediated stable transformation of sour cherry (Prunus cerasus L.) cultivar Montmorency. Leaves, from in vitro stock cultures, with the petiole removed and four partial cuts made transversely and equidistant through the midrib area were found to be the optimum explant type. A 24 h liquid TDZ-pretreatment (0.05, 0.10 or 0.25 mg/l) in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.] of leaf explants stimulated shoot formation upon subsequent culture on QL medium [Quoirin, M., Lepoivre, P., 1977. Improved media for in vitro culture of Prunus sp. Acta Hort. 78, 437–442.] supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA. A frequency of 38.9–54.4% of the explants produced at least one shoot with the maximum mean number of shoots, 4.5 per explant with the 0.10 mg/l TDZ pretreatment. The shoot regeneration scheme was subsequently linked with inoculation with Agrobacterium tumefaciens strains EHA105, GV3101 or LBA4404, each harboring the binary plasmid pBISN1. PBISN1 contains an intron interrupted ß-glucuronidase (GUS) gene (gusA) under control of the chimeric super promoter (Aocs)₃AmasPmas. Blue stained leaf cells were observed after co-cultivation with all three strains. Co-cultivation for 4 days with 19.6 mg/l acetosyringone (AS) and assay by GUS indicated over 90% of the leaf explants were infected with an average 7.5–8.8 blue foci per explant. No differences were observed in regard to A. tumefaciens strain used.     

De novo differentiation of shoot buds from leaf-callus of Dianthus caryophyllus L. and control of hyperhydricity

A method is described for producing de novo shoots from leaf derived callus of carnation (Dianthus caryophyllus L.). Plants were regenerated in four steps, viz. callus induction, shoot regeneration, removal of hyperhydricity from regenerated shoots and root development. Callus induction medium contained 2,4-D and BAP. Shoot buds were formed when the callus was further subcultured on 2,4-D- and BAP-containing medium, or MS medium without any growth regulators. The shoots so formed were hyperhydric, bushy in appearance with reduced stem length and watery leaves. The normal conformation of shoots was restored by culturing the hyperhydric shoots onto medium supplemented with GA₃ and bactopeptone. The recovered shoots were rooted on MS medium added with NAA (1 mg/l) or IBA (2 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions after initial acclimation.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.     

Somatic embryogenesis and plant recovery from callus of ‘Nabali’ Olive (Olea europea L.)

Regeneration via somatic embryogenesis from callus was studied in ‘Nabali’ olive (Olea europea L.). Among different explant sources (leaf blades, leaf petioles, hypocotyls of germinated seeds and roots of germinated seeds), roots gave the highest (46%) callus induction. Somatic embryogenesis was induced from root callus on embryogenesis induction medium (EIM) containing 5.0 μM 2,4-D, 0.5 μM kinetin and 5.0 μM NAA in darkness. Embryo regeneration was studied by transferring the callus from EIM to embryogenesis expression medium (EEM) containing different concentrations (0.0, 5.0, 10.0 and 15.0 μM) of 2-isopentenyladenine (2iP), BA, thiadiazuron (TDZ), zeatin or kinetin. Among the tested concentrations, 2iP at 10.0 μM outperformed the other growth regulators. 2,4-D at 5.0 μM in the EIM was satisfactory for embryogenesis induction. Sucrose at 0.2 M evoked higher embryogenesis than any other concentration of fructose and glucose in EIM, while sorbitol and mannitol at 0.1, 0.2 or 0.3 M reduced embryogenesis significantly and inhibited it totally at 0.4 M. Somatic embryos were rooted by transferring them to hormone-free medium (HFM). About 85% of embryos converted to rooted plantlets, 5% showed secondary embryogenesis and 10% were not developed and died. Rooted plantlets gave 95% survival when acclimatized ex vitro. Acclimatized plantlets developed into whole plants in the greenhouse and they were phenotypically similar.     

Micropropagation of Vitis thunbergii Sieb. et Zucc., a medicinal herb,through high-frequency shoot tip culture

This study describes a protocol for rapid and large-scale in vitro propagation of the valuable medicinal herb Vitis thunbergii Sieb. et Zucc.. Culture conditions influencing shoot proliferation and rooting of the two clones (three- and five-lobed) were examined. Three medium formulations, Murashige-Skoog (MS), Woody Plant Medium (WPM) and Nitsch and Nitsch (NN) medium, were tested for growth of shoot tip culture, and WPM was found to have a superior proliferation rate. The chlorophyll content of leaves was highest in those cultured on NN, followed by those on MS and WPM medium. WPM medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BA) displayed the highest proliferation rate (15–19 nodes or 3–4 shoots per explant). The rooting was optimized using MS medium supplemented with 0.5 mg l⁻¹ napthaleneacetic (NAA) with eight roots, 3 cm long after 1 month of culture. High frequency callus formation was observed in the basal end of explants cultured on NAA-containing medium. Following acclimatization, rooting plantlets were transferred to the plastic house with a 95% survival rate.     

In vitro induction of tetraploid ginger (Zingiber officinale Roscoe) and its pollen fertility and germinability

In vitro induction of tetraploid ginger (Zingiber officinale Roscoe) and its pollen fertility and germinability were investigated. The growth of shoot tip cultures on agar MS medium containing 2.0 mg l⁻¹ BA was greater than that of similar cultures in liquid MS medium with the same BA concentration. In liquid medium, the combinations of 0.5, 1.0, or 2.0 mg l⁻¹ BA with 0.05 mg l⁻¹ NAA tended to enhance the growth of the cultures. The efficiency of tetraploid induction was assessed by treating shoot tip explants on agar or in liquid MS medium containing 2.0 mg l⁻¹ BA, 0.05 mg l⁻¹ NAA, and 0.2% (w/v) colchicine for 4, 8, 12, and 14 days. The total number of tetraploids induced on solid medium was 18, but only five in liquid medium. On both media, the colchicine treatment for 8 days gave the maximum level of tetraploid induction. Therefore, it was found that the treatment of shoot tip explants on agar medium containing 2.0 mg l⁻¹ BA, 0.05 mg l⁻¹ NAA, and 0.2% (w/v) colchicine for 8 days was the most efficient way of inducing tetraploid ginger. Induced tetraploid strains, ‘4× Kintoki’, ‘4× Sanshu’, and ‘4× Philippine cebu 1’, had higher pollen fertility and germinability than the diploid counterparts, i.e., in the diploid strains, pollen fertility ranged from 0.3 to 6.2% and the germination rate from 0.0 to 0.1%, while in the tetraploid strains, pollen fertility ranged from 27.4 to 74.2% and the germination rate from 4.8 to 12.9%.     

In vitro stable regeneration of onion and garlic from suspension culture and chromosomal instability in solid callus culture

An efficient regeneration system from bulb-derived callus tissues in suspension of onion (Allium cepa L.) and garlic (A. sativum L.) was established. Callus culture was induced in Gelrite^®-solidified Murashige and Skoog's (MS) modified basal medium with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.93 μM kinetin supplements. After 4 weeks of induction, the callus tissue was partly transferred to liquid MS media containing different levels of α-naphthaleneacetic acid (NAA) and kinetin for plant regeneration and the rest was maintained in the same medium for chromosome analysis and nuclear DNA quantification following in situ microspectrophotometry. The cultures in suspension, maintained in agitated condition for 8 weeks, showed a high frequency of rapidly regenerated plants after transferring to Gelrite^®-solidified one half strength of MS basal medium. Chromosome analysis of the regenerated plants, transferred to the field with 90% survival rate, revealed stable chromosome number (2n = 16) in both species. On the other hand, callus tissues maintained in solid induction medium for long period showed abnormality in chromosome behavior leading to the formation of both hypo- and hyper-diploid cells along with the diploid cells. The frequency of aneuploid cells (2.2–48.9%) increased with callus age in both species with high and statistically significant number of hyperdiploid cells. The role of endoreduplication as well as non-disjunction of chromosomes resulting in instability in chromosome number has been suggested. This was also supported by the nuclear DNA value in successive passages with statistically significant increase.     

Direct shoot organogenesis on shoot apex from seedling explants of Capsicum annuum L.

In vitro direct multiple shoot formation from seedling explants of Indian high pungent varieties of Capsicum annuum cv. Arka Abhir (AA) and Arka Lohit (AL) was successfully obtained. We were able to induce regeneration potency in these varieties by inverting the explant. Aseptically grown seedling explants with decapitated roots, apical meristem and cotyledonary leaves were inoculated in an inverted position in bud induction medium comprising of Murashige and Skoog's basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid (MES) buffer along with 26.63 μM benzyl adenine (BA), 2.28 μM indole-3-acetic acid (IAA) and 10 μM silver nitrate. Profuse shoot bud induction with 20–25 shoot buds per explant was obtained. Supplementation of phloroglucinol in the bud induction medium resulted in 17 and 18% enhancement in bud induction response in Arka Abhir and Arka Lohit variety, respectively on the inverted hypocotyls. Auxin transport inhibitor tri-iodo benzoic acid (TIBA) in the bud induction medium resulted in induction of buds in a shorter period of 40–45 days when compared to bud induction (BI) medium which takes 55–65 days for bud induction. These buds were transferred to MS medium containing 2.8 μM gibberellic acid and 10 μM silver nitrate resulting in elongation of shoot buds. Transfer of shoots to MS basal medium induced rooting to produce plantlets. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. annuum tissues.     

In vitro response of pistachio nodal explants to silver nitrate

The effect of silver nitrate on shoot differentiation and shoot growth was examined in order to improve the regeneration efficiency of pistachio (Pistacia vera L. cv. Kirmizi) in vitro. Nodal explants of in vitro-grown seedlings were used to test various concentrations and combinations of 6-benzyladenine (BA), kinetin (KIN), gibberellic acid (GA₃) and silver nitrate (AgNO₃). Addition of AgNO₃ up to 48.0 μM to the culture medium improved the regeneration frequency and shoot growth, and reduced basal callus formation in all regenerated explants. The highest regeneration frequency (100%) was recorded on Murashige and Skoog (MS) medium containing 9.0 μM BA, 0.2 μM GA₃ and 24.0 or 48.0 μM AgNO₃ in combination. The best proliferation response in terms of both shoot formation and low callus production was obtained in the medium containing a combination of 9.0 μM BA, 0.2 μM GA₃ and 12.0 μM AgNO₃. Regenerated shoots, coming from three cycles of subculturing in proliferation media, were rooted in half-strength MS medium containing 12.0 μM indole-3-butyric acid (IBA). Well rooted plantlets were acclimatized and eventually established in peat and perlite. The development and optimization of an effective micropropagation protocol that is presented in this paper can give an important contribution to improve the quality of pistachio plants and, as a consequence, of orchard production in Middle East countries.     

Factors affecting tissue culture of Damask rose (Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N⁶-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.     

Culture method and photosynthetic photon flux affect photosynthesis,growth and survival of Limonium ‘Misty Blue’ in vitro

Microcuttings (shoots each with two leaves) of Limonium ‘Misty Blue’ were cultivated in vitro for 28 days under photoautotrophic (sucrose-free culture medium; CO₂ and photosynthetic photon flux (PPF) enriched conditions), photomixotrophic (medium with 30 g l⁻¹ sucrose; CO₂ and PPF enriched conditions) and heterotrophic (medium with 30 g l⁻¹ sucrose; CO₂ non-enriched conditions) methods. Several growth variables were measured during and at the end of cultivation: shoot fresh and dry weight, percentage of shoot dry matter, root fresh weight, number of leaves, leaf area, chlorophyll and sugar content of leaves, stomatal density and size, net photosynthetic rate (NPR) and percent survival of plantlets ex vitro. Plantlets grown in photoautotrophic and photomixotrophic methods had more leaves, high chlorophyll and sugar contents, high NPR, and showed high percent survival. However, these plantlets possessed less number of stomata per square millimeter. In contrast, the plantlets grown by the heterotrophic method showed decreased values of these growth variables except for the number of stomata per square millimeter. These results indicate that CO₂ enrichment for plantlets in vitro at a relatively high PPF would promote photosynthesis and hence growth of chlorophyllous explants/plantlets in vitro. The resulting plantlets were acclimatized better and sooner on ex vitro transplantation.     

Somatic embryogenesis from floral tissues of feijoa (Feijoa sellowiana Berg)

The objectives of the present work were to study the embryogenic competence of floral tissues of Feijoa sellowiana and to investigate the influence of plant growth regulators on somatic embryo induction and development in order to establish a somatic embryogenesis protocol starting from somatic tissues. Petals, stamens and ovaries of floral buds were cultivated onto LPm basal medium supplemented with different levels of 2,4-D, Picloram, 2-iP, Kin and BAP. The highest embryogenic callus induction was obtained with Picloram (10 μM) and Kin (1 μM). Rates of embryogenic calluses induction in stamens and petals were significantly affected by PGRs. Embryogenic calluses were transferred to the same medium, supplemented with gradually reduced levels of PGRs-free medium. After 60 days in suspension cultures with 2,4-D (1 μM) and 2-iP (1 μM) calluses were transferred to PGR-free medium. After 30 days it was observed the development of globular somatic embryos on the surface of 18% of friable calluses previously induced with Picloram (10 μM) and Kin (1 μM). Only embryogenic calluses derived from stamens gave rise to this morphogenetic pattern.Torpedo and cotyledonary somatic embryos transferred to PGR-free culture medium were converted to complete plantlets. This is the first report of somatic embryogenesis in this species starting from somatic tissues.     

Daylength and photon flux density influence the growth regulator effects on morphogenesis in epicotyl segments of Troyer citrange

The optimal growth regulator addenda for adventitious shoot regeneration in epicotyl cuttings of Troyer citrange (Citrus sinensis×Poncirus trifoliata) varied with the conditions of illumination. The response to illumination and to growth regulators differed for the direct and the indirect (through callusing) pathways of regeneration. Shoot formation through the direct organogenic pathway decreased as the concentration of benzyladenine (BA) in the medium was increased in the range 2.2–22 μM, when the explants were incubated in the dark or under an 8 h daylength. For explants incubated under a 16 h daylength, the number of shoots formed increased with BA concentration. Optimal conditions of incubation for shoot formation through the direct pathway were either an 8 h daylength with a photon flux density of 74 μmol m⁻² s⁻¹ in the presence of 2.2 μM BA, or a 16 h daylength with a photon flux density of 37 μmol m⁻² s⁻¹ in the presence of 22 μM BA. Irrespective of the conditions of incubation, shoot formation through the indirect organogenic pathway was suppressed by the addition of 22 μM BA to the medium. Optimal conditions for shoot formation through this pathway were incubation under an 8 h daylength at a photon flux density of 74 μmol m⁻² s⁻¹ in the presence of 2.2 μM BA. At the optimal conditions indicated, the addition of the synthetic auxin naphtaleneacetic acid (0.54 μM) reduced shoot formation. Irrespective of the pathway of regeneration, the number of shoots formed decreased markedly with the distance of the cutting from the cotyledonary node.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

The rootstock effects on plant adaptability,production, fruit quality,and nutrition in the peach (cv. ‘Suncrest’)

In clayey and calcareous soils without a stable irrigation and fertilization system, the type of rootstock can particularly affect both the vegetative and productive properties of ‘Suncrest’ peach (Prunus persica L. Batsch) plants, and the qualitative and nutritional attributes of their fruit. The GF677 rootstock (P. persica × Prunus amygdalus) promoted the highest vegetative development, followed by Julior (Prunus insistitia). The ‘Suncrest’ on Ishtara [(Prunus cerasifera × P. persica) × (P. cerasifera × Prunus salicina)] and Barrier1 (P. persica × Prunus davidiana) had lower, but similar, plant vigour, but the latter rootstock differed in its higher production of pruned wood. The lowest adaptability to these cultivation conditions was observed for ‘Suncrest’ grafted onto Citation (P. persica × P. salicina), which showed the lowest plant development and production. For the plant yield, the ‘Suncrest’ grafted onto GF677, Julior, Ishtara, and Barrier1 were all similar. The fruit yield and both the canopy volume and pruned wood of ‘Suncrest’ grafted onto Ishtara showed a particular relationship, giving the best indices of yield efficiency and plant physiological equilibrium. Moreover, the same rootstock promoted the largest fruit size, while the smallest fruit were found on GF677 rootstock. The fruit from the Citation ‘Suncrest’ ripened 3 days early, while those from Barrier1 had a late fruit ripening. Firmness, soluble solids and the soluble solids to total acidity ratio were only affected slightly by the different rootstocks, while the total acidity of the fruit varied significantly according to rootstock; the fruit from ‘Suncrest’ grafted onto Barrier1, Julior, and Citation had the highest total acidities. The rootstock effects on the nutritional attributes of the fruit were relevant. The ‘Suncrest’ on Julior and GF677, followed by Ishtara, produced fruit with the greatest antioxidant activities and total phenolic contents. The ‘Suncrest’ on Citation and, especially, Barrier1 had reduced nutritional values of the fruit. The variations in antioxidant activities and total phenolic contents showed a positive correlation.     

Seasonal changes in CO2 assimilation in leaves of five major Greek olive cultivars

Leaf CO₂ assimilation rate, stomatal conductance (g_s), internal CO₂ concentration (C_i), chlorophyll (a + b) content, specific leaf weight (SLW) and stomatal density were measured during the season, under field conditions, for five major Greek olive cultivars, ‘Koroneiki’, ‘Megaritiki’, ‘Konservolia’, ‘Lianolia Kerkiras’, and ‘Kalamon’, with different morphological and agronomic characteristics and diverse genetic background. Measurements were taken from current-season and 1-year-old leaves, and from fruiting and vegetative shoots, throughout the season, from March to November in years 2001 and 2002. CO₂ assimilation rates showed a substantial seasonal variation, similar in all cultivars, with higher values during spring and autumn and lower values during summer and late autumn. Stomatal conductance (g_s) followed similar trends to leaf CO₂ assimilation rates, increasing from March to July, following by a decrease during August and increasing again in autumn. ‘Koroneiki’ had the highest leaf CO₂ assimilation rate and g_s values (21 μmol m⁻² s⁻¹ and 0.45 mol m⁻² s⁻¹, respectively) while ‘Lianolia Kerkiras’ and ‘Kalamon’ showed the lowest leaf CO₂ assimilation rate and g_s values (13–14 μmol m⁻² s⁻¹ and 0.22 mol m⁻² s⁻¹, respectively). One-year-old leaves had significantly higher leaf CO₂ assimilation rate than current-season leaves from April to June, for all cultivars. From August and then, leaf CO₂ assimilation rate in current-season leaves was higher than in 1-year-old leaves. There were no significant differences in leaf CO₂ assimilation rate between fruiting and vegetative shoots. Total chlorophyll (a + b) content increased with leaf age in current-season leaves. In 1-year-old leaves chlorophyll content increased in spring, then started to decrease and increased slightly again late in the season. Chlorophyll content was higher in 1-year-old leaves than in current-season leaves throughout the season. Total specific leaf weight (SLW) increased throughout the season for all cultivars. Stomatal density in lower leaf surface was lowest for ‘Koroneiki’ (399 mm⁻²) and highest for ‘Megaritiki’ (550 mm⁻²). Our results showed differences in leaf CO₂ assimilation rate among the five different olive cultivars, with a diverse genetic background, ranging from 12 to 21 μmol m⁻² s⁻¹. From the five cultivars examined, ‘Koroneiki’, a drought resistant cultivar, performed better and was able to maintain higher leaf CO₂ assimilation rate, even under high air vapor pressure deficit. All cultivars had a pronounced seasonal variation in leaf CO₂ assimilation rate, affected by date of the year, depending on ambient conditions. The high temperatures and high air vapor pressure deficit occurring during summer caused a reduction in leaf CO₂ assimilation rate in all cultivars. Leaf CO₂ assimilation rate was also affected by leaf age for all cultivars, with old leaves having significantly higher leaf CO₂ assimilation rate than young leaves early in the season.     

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ Kn, which contained only half concentration of NH₄NO₃. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm²) was transferred to solid media (MS + 0.2 mg l⁻¹ 2,4-D + 5.0 mg l⁻¹ BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.     

Microsporogenesis and anther culture in carob tree (Ceratonia siliqua L.)

An in vivo study was made on male flowers of carob tree (Ceratonia siliqua L.), in order to establish a correlation between the flower and anther development, and microsporogenesis. In addition studies were conducted to find which phase is more appropriate for anther culture and haploid production. During the development of male flowers, six stages were identified. The male gametophytic cycle begins when flowers are in developmental phase 0, with the formation of the epidermis, endothecium, primary sporogeneous tissue, primary parietal cells and pollen mother cells. During developmental phase I we observed the formation of pollen mother cells, the microspore tetrads, and uni- and binucleate pollen grains. At developmental phase II, uni- and binucleate microspores, and completely formed pollen grains were observed. In developmental phase III we could observe mature pollen grains ready to be released from the anthers as single binucleate pollen grains. Anthers from flowers at developmental phases I and II, with microspores at late uninucleate to early binucleate stage, were cultured in semi-solid Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) combined with one of the citokinins: N⁶-benzyladenine (BA), kinetin (Kin), zeatin (Zea) and thidiazuron (TDZ). To obtain embryogenic calli anthers should be collected from flowers in developmental phase I. High frequencies of callogenesis were obtained, and the best medium for calli induction was MS supplemented with 0.5 mg l⁻¹ 2,4-D + 4 mg l⁻¹ TDZ. The frequency of haploid cells was found to be 17.2%.     

Factors controlling high efficiency adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida L.)

Studies were conducted to improve adventitious bud regeneration in roses (Rosa hybrida L.), specifically to extend the protocol to different genotypes and to initiate production of multiple shoots per explant. The best results were obtained by using a two-stage procedure where excised leaflets were incubated on Murashige and Skoog (MS) (1962) induction medium with 6.8 μM TDZ plus 0.49 μM IBA in the dark for 7 days and subsequently transferred to an MS-based regeneration medium with 2.22 μM BA plus 0.049 μM IBA exposed to a PPFD of 15 μmol m⁻² s⁻¹ PAR. Bud formation capacity was also significantly affected by the genotype and the environment, such as the use of bottom cooling creating a lower RH in the vessel. The addition of silver nitrate to the induction medium also significantly improved the percentage of regeneration in three genotypes tested. Regenerated shoots failed to elongate when transferred to MS proliferation medium containing 0.5 mg l⁻¹ BA, however maximum bud development and elongation were achieved when kinetin in the range 1–2 mg l⁻¹ was used. Elongated shoots were excised and rooted best on zero growth regulator half-strength MS modified medium. Rooted plantlets were acclimatized under greenhouse conditions for evaluation of somaclonal variation.