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.     

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.     

Optimization of somatic embryogenesis in suspension cultures of horsegram [Macrotyloma uniflorum (Lam.) Verdc.]—A hardy grain legume

Cell suspension cultures were established from immature cotyledon derived calli from drought tolerant legume horsegram [Macrotyloma uniflorum (Lam.) Verdc.]. Embryogenic callus could be originated from cut slices of the immature cotyledons on MS solid medium [Murashige, T. Skoog, K., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497] augmented with 1.0 μM zeatin and 4.5 μM NAA. Numerous somatic embryoids (26.4%) appeared on MS liquid basal nutrient medium with 5.6 μM NAA and with absence of zeatin during 3 weeks culture. Sustained cell division resulted in the formation of cell aggregates, and then progressed to globular, heart and further if they differentiate properly to torpedo and cotyledonary stages within 5 weeks. Transfer of individual embryos on to a fresh MS basal medium with no plant growth regulators was able to achieve complete maturation. Only a relatively few number of embryos developed into root/shoot when transferred to 0.9 μM GA₃, 15 g/l⁻¹ sucrose and 2.4 g/l⁻¹ gelrite containing medium. Substitution of sucrose associated with the use of l-glutamine gave, in the range of concentrations tested, the strongest enhancement of the embryo growth and development. About 5% of somatic embryos were converted into true-to-type fertile plants.     

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.     

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.     

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.     

Protocol for rapid propagation,and to overcome delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L.

Single medium based efficient protocol for rapid propagation, and to overcome the delayed rhizome formation in field established in vitro derived plantlets of Kaempferia galanga L. through in vitro rhizome induction was achieved. MS medium with combination of 8.87 μM N⁶-benzyladenine (BA), and 2.46 μM indole-3-butyric acid (IBA) induced a mean of 6.2 shoots per explant. Addition of 11.7 μM silver nitrate to 8.87 μM BA and 2.46 μM IBA supplemented medium facilitated the highest number of shoots (mean of 8.3 shoots) as well as roots within 60 days. Subculture of isolated shoots on medium with the same concentration of BA, IBA and silver nitrate increased the number to a mean of 12.1 shoots. Silver nitrate enriched medium developed rhizome at the base of shoots. Increase of sucrose concentration (6–8%) in medium with BA, IBA and silver nitrate favoured the best rhizome development. Ninety five per cent of the plantlets survived in field conditions. The plantlets established in field without in vitro developed rhizome (from medium with BA and IBA) did not form rhizome even at 7 months after transplantation. Instead, they developed tuberous roots only. The plantlets with in vitro developed rhizome (on medium having BA, IBA, silver nitrate, and 6–8% sucrose), and that established from conventional way (through splitting of old rhizome) showed no difference in growth of the rhizome. The present study emphasizes the efficacy of silver nitrate and sucrose to develop rhizome in vitro, which enabled to overcome the delayed development of rhizome, and reduced yield of plantlets established in field without in vitro developed rhizome.     

Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine

A brief culture of mulberry leaves for 8–10 days on MS medium with 18.17 μM TDZ followed by transfer to 8.88 μM BAP supplemented medium triggered high frequency shoot organogenesis (77.6–89.2%) and favoured shoot elongation in Morus spp. Shoot proliferation was highest in the presence of 2.22 μM BAP with induction of 9.4–10.6 shoots per culture. High frequency of root induction (76.0–86.6%) was observed on medium supplemented with 0.49 μM IBA whereas increase in the level of IBA (4.92 μM) resulted in induction of roots along with development of callus from the base of the shoots. The regenerated plants established in soil at higher frequency in rainy season compared to winter and summer.     

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.     

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.     

An investigation on the effect of different plant growth regulating compounds in in vitro shoot tip and node culture of lemon seedlings

The possible application of some less commonly used in vitro growth regulating compounds is outlined. A number of treatments were applied to determine the best way of inducing in vitro shoot proliferation and rooting on a modified Driver–Kuniyuki [HortScience 19 (1984) 507] basal medium of lemon (Citrus limon (L.) Burm, f. cv. Interdonato) seedlings. 6-Benzyladenine (BA) alone (1, 2 and 4 mg l⁻¹) and in combination with either orange juice (10%, v/v), silver nitrate (3 mg l⁻¹), gibberellic acid (GA₃) (0.1 mg l⁻¹ at the establishment stage and 0.5 mg l⁻¹ at all combinations during the proliferation stage) or abscisic acid (ABA) (0.2 mg l⁻¹ only at the establishment stage) were used to stimulate shoot formation during the establishment and the proliferation stage. The combination of BA with ABA gave a high rate of shoot formation, while GA₃ and silver nitrate enhanced shoot elongation. When these shoots were transferred to the rooting stage, the effect of application of two different auxins (indole-3-butyric acid (IBA) and α-napthaleneacetic acid) was examined, as well as different methods of application (auxin added to the basal medium and auxin application by dipping the base of the explant in auxin solution). Dipping the base of the explants in a 50% ethanol solution of IBA at 1000 mg l⁻¹ for 5 s resulted in 80% rooting with subsequent 90% survival of these explants, during acclimatization under mist.     

In vitro plantlet regeneration of Olea europaea ssp. maderensis

Different media were assayed for Olea europaea L. ssp. maderensis Lowe micropropagation. Shoot elongation and propagation was more efficient on DKW medium supplemented with 4.4 μM BA and 0.4 μM IBA. Higher branching was obtained on OM medium supplemented with 18.2 μM zeatin. Rooting was achieved at higher rates on half strength DKW medium supplemented with 20.7 μM IBA. Plants were acclimated to greenhouse and up to now no morphological changes were observed among the regenerated in vitro plants.     

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.     

Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia (Christm.) Swing

Multiple shoots were produced from node explants of lime (Citrus aurantifolia (Christm.) Swing) on MS medium supplemented with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (kinetin) and α-naphthaleneacetic acid (NAA). The highest number of shoots, nine shoots per node, were produced on a medium containing 2 mg l⁻¹ BAP (8.8 μM), 1 mg l⁻¹ kinetin (4.6 μM) and 1 mg l⁻¹ NAA (5.4 μM). Depending on the concentration of BAP and kinetin, NAA either inhibited, stimulated or did not affect shoot multiplication, which also depended on the cytokinin level. Maximum shoot length was obtained from treatments containing 0.5 mg l⁻¹ BAP (2.2 μM) combined with 1 mg l⁻¹ kinetin (4.6 μM) and 0.5 mg l⁻¹ NAA (2.7 μM). The largest leaves of resultant shoots were produced on a medium containing 0.5 mg l⁻¹ each of kinetin (2.3 μM) and NAA (2.7 μM). Transferring in vitro shoots to rooting media containing indole-3-butyric acid (IBA) and NAA produced complete plantlets. The highest rooting percentage was obtained on a medium containing either 1 mg l⁻¹ NAA (5.4 μM) alone or 0.5 mg l⁻¹ NAA (2.7 μM) combined with 2 mg l⁻¹ IBA (9.6 μM), whereas the highest number of roots were produced on a treatment containing both 2 mg l⁻¹ NAA (10.8 μM) and 2 mg l⁻¹ IBA (9.6 μM). Roots elongated most on treatments containing 0.5 mg l⁻¹ of either NAA (2.7 μM) or IBA (2.4 μM). Shoot growth associated with the rooting phase was the highest in response to 2 mg l⁻¹ IBA (9.6 μM) or 0.5 mg l⁻¹ NAA (2.7 μM). Plantlets that survived acclimatization, 82%, exhibited normal growth in soil under greenhouse conditions.     

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.     

Net CO2 exchange rate of in vitro plum cultures during growth evolution at different photosynthetic photon flux density

CO₂ concentration was monitored during three 15-day subculturing cycles in vessels containing actively proliferating plum cultures of Prunus cerasifera, clone Mr.S. 2/5. The effects of two photosynthetic photon flux density regimes: 50 ± 5 μmol m⁻² s⁻¹ and 210 ± 5 μmol m⁻² s⁻¹ were compared. Three distinct phases in the CO₂ trend were distinguished during each culturing cycle of both light treatments. In the first, occurring at the beginning of the culture cycle, the amount of CO₂ emitted by the cultures during dark periods was greater than that assimilated during the light periods. In the second phase, the opposite trend was detected, while in the third, the range of CO₂ day–night fluctuations increased or remained stable according to the number of explants per vessel. The treatment with 210 ± 5 μmol m⁻² s⁻¹ did not modify the CO₂ phase trend but induced more pronounced fluctuations in day–night CO₂ concentration. Under this light treatment, cultures reached CO₂ compensation point for a period as long as 48% of the total number of light hours, while under 50 ± 5 μmol m⁻² s⁻¹, it was only 8%. The different range in CO₂ day–night fluctuations monitored throughout a subculturing cycle, appeared to be mainly induced by changes in culture growth dynamics.     

Shoot induction and plant regeneration from receptacle tissues of Lilium longiflorum

An efficient system has been developed for the in vitro plant regeneration of Lilium longiflorum Thunb. by culturing receptacle sections from flower buds. The sections were cultured on one-half MS medium plus 30 g l⁻¹ sucrose, 8 g l⁻¹ agar, 5.4 μM NAA or 4.9 μM IBA plus 2.2 μM BAP. A section size of 3–4 mm was found to be optimal. After 60 days an average of 41 shoots were formed per explant. More vigorous shoots were obtained by subculturing on hormone-free medium with 20 g l⁻¹ sucrose. Rooting occurred on one-half MS medium with 1.1 μM NAA. Rooted plants were hardened-off in a greenhouse for two months, and normal flowering plants were produced.     

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.     

Rapid in vitro multiplication and conservation of Garcinia indica: A tropical medicinal tree species

A simple and efficient method has been developed for rapid regeneration of plantlets via adventitious bud differentiation on mature seeds of Garcinia indica (Thouars) Choisy, a medicinally important facultative apomictic tropical tree species. High frequency direct shoot proliferation was induced in seed segments cultured on Murashige and Skoog's medium supplemented with cytokinins (BAP, kinetin and TDZ) alone and in combination with auxin (NAA). Amongst the various combinations used, BAP proved to be the most effective. Multiple shoots formed within 4–5 weeks of culture. The shoot forming capacity of the seeds was influenced by the BAP concentration tested (5–50 μM) and optimal response was observed at different concentrations (12.5–50 μM) in different genotypes investigated. Significant differences were recorded in terms of percent response (27.78–100%) as well as average number of shoots per explant (3.49–57.67) among the four genotypes investigated. Elongation of the induced shoots was achieved on MS basal medium containing 0.2% activated charcoal. The induction medium had a profound effect on rate of bud elongation with shoots induced on lower concentrations of BAP showing as much as four-fold elongation within 4 weeks. Proliferating shoot cultures were established by repeatedly subculturing the shoot nodes on MS medium supplemented with 5 μM BAP. Maximum rooting (91.66%) occurred in shoots cultured on half-strength MS medium supplemented with 10 μM IBA. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 15–17 weeks. For in vitro conservation, the shoot cultures were maintained on medium supplemented with 0.5 μM BAP and the subculture duration could be enhanced up to maximum of 11 months.     

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.