Olive Medium Research Articles

Micropropagation and cryopreservation of the rare endemic Colchicum figlalii germplasm

BACKGROUND: The natural population of Colchicum figlalii (Varol) Parolly & Eren grows in a narrow area of serpentine rock clearings at an altitude of 1900-2100 m in Southwestern Anatolia (Sandras Mountain, Mugla, Turkey). The species is regarded as endangered according to the IUCN Red List Categories. OBJECTIVE: To develop an optimum procedure for in vitro propagation and cryopreservation of germplasm of this rare endemic. MATERIALS AND METHODS: A total of 281 bulbs were used as in vitro culture starting material and after surface sterilization, clean material was obtained from 157 of them. Woody Plant Medium (WPM), Olive Medium (OM), and Murashige and Skoog medium (MS) were used for in vitro culture establishment. RESULTS: The maximum regeneration rate (˜67.3%) was obtained after four weeks of incubation on OM. The calli were successfully induced by using OM supplemented with 10.7 μM NAA from leaves of in vitro grown C. figlalii bulbs. A PVS2-vitrification procedure was used for cryopreservation of C. figlalii callus tissue. After cryo-storage, the best result for regeneration (66.7%) was obtained from calli treated with PVS2 for 75 min before plunging into liquid nitrogen. All rooted seedlings derived from cryopreserved calli were successfully acclimatized to greenhouse conditions. CONCLUSION: This study is an effective reference for future long-term conservation of similar species that are difficult to cryopreserve.

Delineation of the impacts of varying 6-benzylaminopurine concentrations on physiological, biochemical and genetic traits of different olive cultivars under in vitro conditions.

Abstract. The plant growth regulator 6-benzylaminopurine (BAP) is an important component of plant nutrient medium with tendency to accelerate physiological, biochemical and molecular processes in woody plants such as olive. To date, limited knowledge is available on the role of BAP in mediating physiological, biochemical and genetic activities in olives under in vitro conditions. To cover this research gap, the current study was conducted with the objective of studying the role of BAP in regulating physiological traits (chlorophyll, CO2 assimilation), antioxidant enzymes (superoxide dismutase, catalase and peroxidase), metabolic contents (starch, sucrose and flavonoids) and gene expression (OeRbcl, OePOD10, OeSOD10, OeCAT7, OeSS4, OeSuSY7, OeF3GT and OeChlH) under varying concentrations (0, 0.5, 1.5 and 2.5mg L-1) within the provided in vitro conditions. The explants obtained from different olive cultivars ('Leccino', 'Gemlik', 'Moraiolo', 'Arbosana') were cultured on olive medium (OM) provided with different BAP concentrations using a two-factorial design, and data were analysed statistically. All traits increased significantly under in vitro conditions due to increasing concentrations of BAP; however, this increase was more dramatic at 2.5mg L-1 and the least dramatic at 0.5mg L-1. Moreover, correlation, principal component analysis and heatmap cluster analysis confirmed significant changes in the paired association and expression of traits with changing BAP concentration and type of olive cultivars. Likewise, the expression of all genes varied due to changes in BAP concentration in all cultivars, corresponding to variations in physiological and biochemical traits. Moreover, the spectrographs generated via scanning electron microscopy further indicated the variations in the distribution of elements in olive leaf samples due to varying BAP concentrations. Although all cultivars showed a significant response to in vitro varying concentrations of BAP, the response of Arbosana was statistically more significant. In conclusion, the current study proved the dynamic impact of the varying BAP concentrations on regulating the physiological, biochemical, and molecular attributes of olive cultivars.

Micropropagation of autochthonous olive varieties from Türkiye

Olive (Olea europaea L.) is one of the oldest cultivated fruit species in the world. Fruits and oils of autocthonous olive varieties (native Turkish olive varieties) with unique sensory properties (taste, smell and aroma) gain importance recently. Particularly olive oil companies looking for varieties that have distinct taste, smell and aroma in their oil. Propagation of olive varieties by cuttings and grafting is very difficult and expensive, therefore it is important to find solution for easy and mass propagation. Micropropagation is particularly beneficial to propagate plants that are difficult to reproduce conventionally or to ensure virus-free plants or plants with particular qualities. In this study, in vitro micropropagation success of two autocthonous olive varieties (‘Mavi’ and ‘Guleki’) grown in the origin center of olive, Southeast Anatolia, Türkiye was investigated. Both varieties, have distinct smell, taste and aroma and are difficult to root by cuttings. The effects of three different medium OM (Olive Medium), WPM (Woody Plant Medium) and DKW (Driver-Kuniyuki Walnut Medium) and two different growth regulators BAP (6-Benzylaminopurine) and Zeatin on shoot induction (proliferation) in the in vitro micropropagation of the olive varieties were examined. Obtained shoots were later subjected to in vitro rooting and acclimatization. The highest proliferation efficiency and shoot length for both varieties were obtained with the use of 1 mg Zeatin+0.1 mg GAɜ hormone combinations on OM medium. ‘Mavi’ variety formed more roots compared to ‘Guleki’ (3.33 vs. 2.75 per shoots), and gave the highest rooting rate of 74.33% with 2 mg IBA (Indole Butyric Acid) treatment on ½ OM medium. In terms of rooting rate, Guleki gave the highest rooting (100%) on medium containing ½ OM, 0.2 mg GA3 and 4 mg IBA. 60% and 75% of the micropropagated plants of ‘Mavi’ and ‘Guleki’ varieties adapted well to the external conditions.

In vitro regeneration from adult node explants of Juniperus oxycedrus

In the present study the in vitro propagation from adult native plants of Juniperus oxycedrus was investigated. The effect of seasonal explant collection (spring, summer, winter), in varying media and cytokinin type were examined. The decontamination process of explants taken in the summer was more efficient than that of the other seasons (57.3% of the explants survived). In the spring a low performance in shoot formation took place both on hormone free Murashige and Skoog (MS) media and Rugini Olive Medium (OM) or each containing 6-benzyladenine (BA) at 1.0 mg L-1 by which shoots did not elongate more than 0.5 cm. In the summer OM was used, and a higher in vitro performance was observed on medium containing 0.1 mg L-1 6-(γ,γ-Dimethylallylamino)purine (2iP); the shoot formation percentage was 72.0%, and respectively 3.1 shoots, 1.4 cm in length, were formed. In the winter, the shoot formation percentage was 92.0-100.0%, and respectively 3.9 shoots of 0.4 cm length were formed on medium containing 1.0 mg L-1 2iP. The multiplication phase on cytokinin-containing media led to a satisfactory proliferation rate in terms of shoot formation percentage and shoot number. The higher shoot number (3.3) was recorded on media containing 2iP at 1.0 mg L-1, while the shoot length was longer (2.0) cm, on hormone-free media. Initiation of roots was not observed during the rooting phase on hormone-free half-strength OM, or supplemented with 1.0 and 2.0 mg L-1 IBA. Spontaneous rooting at a low percentage (10.0%) was observed on OM media supplemented with 0.1 mg L-1 2iP and successful acclimatisation took place on a 1:1 (v/v) ratio of peat to perlite substrate.

The First Evidence of the Beneficial Effects of Se-Supplementation on In Vitro Cultivated Olive Tree Explants.

Selenium is an essential micronutrient that provides important benefits to plants and humans. At proper concentrations, selenium increases plant growth, pollen vitality, the shelf life of fresh products, and seems to improve stress resistance; these effects can certainly be attributed to its direct and indirect antioxidant capacity. For these reasons, in the present work, the effects of selenium at different dosages on in vitro cultivated olive explants were investigated to observe possible positive effects (in terms of growth and vigor) on the proliferation phase. The work was carried out on four different olive cultivars: “San Felice”, “Canino”, “Frantoio”, and “Moraiolo”. The explants were cultured in aseptic conditions on olive medium (OM), with the addition of 4 mg·L−1 of zeatin, 30 g·L−1 of sucrose, and 7 g·L−1 of agar. The experimental scheme included a comparison between explants grown with five different concentrations of Na2SeO4 (0, 10, 20, 40, and 80 mg L−1) added to the medium during three successive subcultures. Interesting information has emerged from the results and all varieties responded to different concentrations of Selenium. The optimal Se dosages varied for each cultivar, but in general, Se concentration between 10 and 40 mg L−1 increased fresh and dry weight of the explants and shoot lengths. Se treatment induced in all cultivars and for all dosages used an increase in total Se content in proliferated explants. Furthermore, as the subcultures proceeded, the ability of the explants to absorb Se did not diminish. The Se content ranged from 8.55 to 114.21 µg kg−1 plant DW in ‘Frantoio’, from 9.83 to 94.85 µg kg−1 plant DW in ‘Moraiolo’, from 19.84 to 114.21 µg kg−1 plant DW in ‘Canino’, and from 20.97 to 95.54 µg kg−1 plant DW in ‘San Felice’. In general, the effect of selenium tends to decrease with the progress of subcultures and this suggests a sort of “adaptation” effect of the explants to its presence. The present study highlights for the first time the possibility of using in vitro cultures as biotechnological support to study supplementation with selenium and its effects on in vitro olive plant growth.

Effect of Silver Nanoparticles, Medium Composition and Growth Regulators on in vitro propagation of Picual Olive Cultivar

Micro-propagation of olive provides the possibility of producing clones of native plants with appropriate attributes and mass production in short period of time, compared to conventional methods. Micropropagation of olive requires optimization of protocols and adaptation to specific cultivars requirements. This experiment was carried out during 2020 and 2021 seasons in tissue culture laboratory of Pomology Department, Faculty of Agriculture, Cairo University in order to evaluate the potential effects of nano silver at (0, 5, 10 and 20 mg L-1) to improve sprouting percentage and shoot growth of Picual olive cv. The effect of nutrient media composition (MS modified, OM and OM modified) and cytokinins (BAP and Zeatin) on multiplication rate and shoot growth were examined. IBA and NAA (2 and 4 mg L) was examined during rooting stage. The obtained results cleared that Silver Nanoparticles (AgNPs) at 5 mg L-1 recorded the highest percentage of bud sprouting, shoot length, number of shoot/explant and number of leaves/ shoot. Olive medium (OM) recorded the highest shoot length, number of shoot/explant and number of leaves/ shoot compared with OM modified and MS modified media. Also, Zeatin at 4 mg L-1 recorded the highest shoot growth parameters during in vitro multiplication stage. During in vitro rooting stage, both the type and concentration of auxins have a significant influence on number of roots, root length and rooting % of Picual olive cultivar. In conclusion, it is recommended that very low concentrations of nano-silver particles be used in plants tissue culture on olive media (OM) supplemented with Zeatin at 4mgL-1.

Peroxidase Enzyme Fractions as Markers of Somatic Embryogenesis Capacities in Olive (Olea europaea L.).

As part of the search for biochemical markers of somatic embryogenesis in tissue cultures of olive (Olea europaea L.), peroxidases (POXs) in both the soluble and ionically wall-bound fractions were studied in two reputed olive cultivars (cvs.): “Picholine Marocaine” and “Dahbia”. In order to carry out embryogenesis induction, proximal cotyledons were cultured in modified olive medium (OMc) supplemented with 25 μM indole-3-butylic acid (IBA) and 2.5 μM 2-isopentenyladenine (2iP), while distal leaf fragments (somatic explants) were cultured in OMc supplemented with 4.56 µM zeatin riboside (ZR) and 10.25 µM 1-naphthaleneacetic acid (NAA). Regarding embryogenic potentials, the zygotic explants (cv. Picholine Marocaine: 43.39%; cv. Dahbia: 53.41%) were more regenerative than the somatic explants (cv. Picholine Marocaine: 13.05%; cv. Dahbia: 19.51%). The enzyme assay showed a higher POX activity in embryogenic calluses (ECs) than in nonembryogenic calluses (NECs) for the zygotic explants in both studied cultivars. When expressed as units per milligram of proteins (U mg−1 proteins), the highest total POXs activities (soluble POXs + ionically wall-bound POXs) were found in the ECs derived from the zygotic explants; for cv. Dahbia, 65% of the enzyme activities came from the ionically wall-bound fractions. Polyacrylamide gel electrophoresis showed that the ECs of the highly active cv. Dahbia were characterized by highly active isoperoxidases that were revealed in four migration zones, particularly a doublet in the A4 zone (Rf 0.70–0.73) present in the ionically wall-bound POXs. The fast-moving anodic POXs of the ionically wall-bound fractions could be adopted as an early electrophoretic test to determine the embryogenesis capacities in olive tissue culture materials. As biochemical markers, the POX enzyme and its profile in fractions, i.e., as soluble POXs and ionically wall-bound POXs, can offer a valuable tool for improving the tissue culture of olive via somatic embryogenesis.

Bazı Turunçgil Melezlerinin in vitro Koşullarda Mikroçoğaltım ve Köklenme Performanslarının Araştırılması

Turunçgiller yetiştiriciliği yapılan en önemli ve yüksek tür çeşitliliğine sahip meyve gruplarından biridir. Taze olarak tüketilmesi ve farklı endüstrilerde ham madde olarak kullanılması turunçgillerin önemini artırmaktadır. Bu nedenle turunçgillerde yeni çeşit ve anaç geliştirme çalışmaları oldukça önemlidir. Melezleme çalışmaları sonucunda elde edilen değerli bitkilerin çoğaltılması için bitki doku kültürü çalışmaları güçlü bir alternatiftir. Bu çalışmada, Sunki mandarin X Rubidoux üç yapraklı melezlerinin in vitro koşullarda mikroçoğaltımında genotip ve farklı besin ortamlarının (Murashige and Skoog Medium -MS, Woody Plant Medium -WPM, Rugini Olive Medium -ROM) etkisi incelenmiştir. Ayrıca bitkilerin köklendirilmesi üzerine genotip ve farklı bitki büyüme düzenleyicilerin (indol bütirik asit - IBA ve naftalen asetik asit - NAA) etkisi araştırılmıştır. Elde edilen köklü bitkiler dış koşullara alıştırılmıştır. Çalışmada kullanılan genotiplerden mikroçoğaltım aşamasında en başarılı genotipler SR-60 ve SR-75 olurken, köklenme aşamasında en başarılı genotipler SR-41, SR-47 ve SR-60 olmuştur. Besin ortamlarının mikroçoğaltım üzerine etkisi istatistiksel olarak önemsiz bulunmuştur (P<0.05). Köklenme denemelerinde bitki boyu ve kök uzunluğu için en başarılı bitki büyüme düzenleyici IBA, kök sayısı için en başarılı bitki büyüme düzenleyici ise NAA olarak belirlenmiştir.

Oily in situ gels as an alternative floating platform for ketoconazole release

Sodium alginate, calcium carbonate, and guar gum were mixed with oils such as olive oil (OO), sesame oil (SO), and medium chain triglyceride (MCT). The oily formulations were found to simplify the preparation of in situ floating gel. This was the aim of this study using ketoconazole (keto) as a model drug. The investigations for the floating property were established by In vitro gelling capacity study and In vitro floating study. Additionally, in vitro release study was applied to find the best formulations to delay the release of keto. Then, selected formulations were studied by FTIR and SEM. Lastly, in vivo gelation was performed to examine the gelation in the rat’s stomach. The results showed all formulations were floating after successful gelation as the least amount of sodium alginate to gel oils was 20% w/w. The gels in SO and OO were better than MCT in delaying keto release, and 30% w/w sodium alginate in SO was the best to delay the release of keto within 8 hours of the release study. Selected gels showed interactions between the keto molecules and the molecules of the gel contents by FTIR study, and SEM showed a difference in the internal structure of selected formulations. Lastly, the 30% w/w sodium alginate in SO proved to gel and remain in the rat's stomach in the following periods: 30 min, 1 hour, 2 hours, and after 8 hours. Oily suspension formulations showed floating properties in the stomach and slowed the release of keto and specifically 30% w/w of sodium alginate in SO.

Micropropagation and prevention of hyperhydricity in olive (Olea europaea L.) cultivar ‘Gemlik’

Olive (Olea europaea L.) is an economically important crop because of its fruit and oil. Successful olive micropropagation is highly dependence on cultivar, shoot proliferation rate, which is generally low, the rooting of micropropagated olive plantlets is difficult, and the rate of post-transplanting losses is high. In addition, hyperhydricity, a common problem in vitro culture was found to be prevalent. The aim of this study was to establish a micropropagation system for the Turkish O. europaea L. cv. Gemlik. Initially, five different basal media were tested to determine appropriate medium for establishment of in vitro culture and Woody Plant Medium (WPM) was the most efficient. Nodal explants were cultured on WPM containing different plant growth regulators (PGRs) for shoot regeneration. Maximum regeneration frequency and number of shoots per explant were achieved from nodal explants cultured on WPM supplemented with 4.0 mg/L 6-benzyladenine (BA). However, all cultures showed high hyperhydricity and an experimentation was also conducted to resolve the hyperhydricity problem. Hyperhydricity was prevented by changing the gelling agent to Agar-Agar. The shoots regenerated from nodal explants and still attached to initial woody nodal explant were transferred to four different medium formulations each containing 2.0 mg/L zeatin (ZEA) for shoot elongation. Modified Olive Medium (MOM2: OM with three times the concentrations of KNO3) fortified with 2.0 mg/L ZEA was found to be the best for shoot elongation. The elongated shoots were rooted on Olive medium (OM) containing 160 mg/L Putrescine, 1.5 mg/L naphthaleneacetic acid (NAA), 30 g/L mannitol and solidified with 0.65% (w/v) Agar-Agar. Finally, all plantlets were successfully acclimatized in a climate chamber and the plants were transferred to greenhouse conditions.

Establishment of efficient adventitious shoots induction system and ex vitro rooting in Vaccinium corymbosum (Ericaceae)

Background: The extension of the blueberry industry in China is restricted by the low performance of proliferation coefficient, transplanting survival rate and the long cycle production.Hypothesis: We hypothesized the suitable medium with optimal concentration and type combination would improve the survival rate of Vaccinium corymbosum. Species studied: Vaccinium corymbosum L. cultivar ‘O’Neal.Study site and years of study: Our study was conducted in Yunnan Breeding and Cultivation Research and Development Center of Endangered and Daodi Chinese medicinal materials, Yunnan University of Chinese Medicine since November 2015.Methods: Efficient protocol of plant regeneration about Southern highbush blueberry (cultivar ‘O’Neal) was established using annual shoots via single factor and orthogonal experiments.Results: Olive medium with 2.0 mg·L-1 zeatin was found to be most suitable for initiation culture. The highest callus induction and bud formation rate were determined with 93.67 % and 95.57 %, respectively. Furthermore, as the germination of axillary buds, numerous shoots were formed with the proliferation coefficient (> 60). Well-developed shoots were obtained using rejuvenation culture on half-strength Olive medium supplemented with combination of 2.0 mg·L-1 indole-3-butyric acid, 1.0 mg·L-1 naphthaleneacetic acid, 0.01 mg·L-1 chlormequat chloride and 0.5 g·L-1 activated charcoal. The rooting rate could reach to 100 % using 2 cm length of well-developed shoots transferred into the 5:1 sterilized peat:perlite, which was enhanced from 27.61 up to 95 % after transplanting to the field.Conclusions: An efficient system for plant regeneration from bud induction to transplanting to the field was established to maintain the characteristics of southern highbush blueberry varieties.

Does dikegulac affect in vitro shoot proliferation and hyperhydricity incidence in olive explants?

Dikegulac was tested as a lateral shoot-inducing agent on micropropagated olive (Olea europaea ‘Chondrolia Chalkidikis’) shoots. Rugini olive medium was supplemented with dikegulac at 0, 16.9, 33.8, 66.7, 100.5 or 133.4 μΜ. Dikegulac was not phytotoxic and the explants treated with 100.5 μΜ had higher number, length, and weight of new shoots. Hyperhydricity (or vitrification) symptoms were diminished by increasing dikegulac concentration in the medium dose (66.7–133.4 μΜ). Also, dikegulac stimulated the production of large amounts of callus at the base of olive explants.

Neem Oil Used as a “Complex Mixture” to Improve In Vitro Shoot Proliferation in Olive

Shoots of the olive cultivar Moraiolo were previously cultured in aseptic conditions on Olive Medium (OM), with the addition of 4 mg·L−1 of zeatin, 30 g·L−1 of sucrose, and 7 g·L−1 of agar. Then, 1-cm long uninodal explants with two leaves and two axillary buds were excised from the proliferated masses and placed on the same proliferation medium enriched with four concentrations of neem oil (0—control, 0.1, 0.5, and 1.0 mL·L−1), added before sterilization. The addition of 0.1 mL·L−1 of neem oil to the medium gave an improvement in shoot regeneration. More vigorous shoots (longer proliferated shoots) were obtained along with a higher number of nodes (multiplication rate). Overall, there was a significant increase in the total fresh and dry proliferated weights. To our knowledge, this is the first report showing a strong and beneficial effect of neem oil, used as a “complex mixture,” on in vitro plant regeneration.

A New Approach for Olive (Arbequina cv.) Micropropagation: Effect of Dikegulac, Light and Carbon Source

Micro-propagation of olive provides the possibility of producing clones of native plants with appropriate attributes and mass reproduction in short period of time, compared to conventional methods. Apical dominance in olive explants limits the growth of lateral branches in vitro. The effect of Woody Plant Medium (WPM), Olive Medium (OM) and Murashig and Skoog (MS) with carbohydrate source (mannitol and sucrose), was investigated for in vitro shoot proliferation of olive. Different concentrations of sodium dikegulac (0.0, 2.5, 5.0, 7.5 and 10 mg L-1) along with 3 mg L-1 zeatin and 0.5 mg L-1 BAP and two light qualities (red and white) were used to overcome apical dominance. Effects of different IBA concentrations on the in vitro root induction were also investigated. More than 74% of explants rooted when IBA was used at 2 mg L-1. OM was the most effective medium, resulting in better and morphologically superior microshoots. Mannitol showed a positive effect on shoot proliferation. Using sodium dikegulac at 5 mg L-1 under white light significantly stimulated axillary bud growth. In the corresponding concentrations of sodium dikegulac, red light had a significant effect on longitudinal growth compared to the white light.

In vitro propagation of Picconia azorica (Tutin) Knobl. (Oleaceae) an Azorean endangered endemic plant species

Picconia azorica (Tutin) Knobl. (Oleaceae) is a woody plant endemic from the Azores. It is considered as a part of a true relict taxon, reflecting its phylogeny, genetics and geography. Given the fragile status of this species, effective management and conservation are necessary for its survival and repopulation, initially for ecological reasons, but ultimately for forestry purposes as a valuable source of timber. Micropropagation is a useful tool for conservation programs, and in vitro culture techniques have, therefore, been developed for P. azorica by testing different combinations of media and growth regulators at each micropropagation stage. Adult plants from the field were successfully established in vitro on Rugini olive medium supplemented with 24.6 µM 2-isopentenyl adenine (2iP), resulting in 91.2 % sprouting of the nodal explants that survived the disinfection stage, although similar results were achieved using 18.2 µM zeatin. At the multiplication stage, the best shoot quality and the best compromise between shoot length and new axillary shoot development were achieved using the modified macronutrients Driver and Kuniyuki walnut medium supplemented with 24.6 µM 2iP and 0.6 µM indole-3 acetic acid. Rooting was most successful with half-strength macronutrients combined with 10 µM indole-3 butyric acid. Using this micropropagation protocol, a wide range of accessions were successfully established in vitro and are now being propagated on a larger scale for ecosystem restoration.

Physiological Response of In Vitro Cultured MAGNOLIASP. to Nutrient Medium Composition

Abstract The objective of this study was to assess the regeneration response of in vitro cultured Magnolia × soulangeana ‘Alexandrina’ and Magnolia liliiflora ‘Nigra’ to nutrient medium composition. In the primary culture (initiated from dormant axillary buds) combinations of Murashige and Skoog (MS) basal salts with 6-benzylaminopurine and α-naphthaleneacetic acid were tested. The primary explants of cv. ‘Alexandrina’ expressed higher regeneration rate than cv. ‘Nigra’. For both species, the regen eration was most strongly potentiated at addition of 0.25 mg dm−3 of the cytokinin alone. The auxin exerted undesir–able effects. Several basal salts media were applied in proliferation stage and their physiological effects were evaluated in reference to traditionally used MS. At culturing on Chée & Pool C2d Vitis Medium (VM) that is for the first time introduced to magnolia and on MS, M. liliiflora formed more but less elongated shoots than M. soulangeana. However, on VM, substantial increase (25-30%) of the number of axillary shoots and leaves, shoot length and fresh and dry weights over MS was established for both species. This suggested VM as promising composition of nutrients in multiplication stage. Microshoots obtained on MS, VM, Rugini Olive Medium and DKW Juglans Medium were successfully rooted in vitro and subsequently established ex vitro. The findings expand the information on magnolia response to culture conditions and contribute to elaboration of innovative elements of protocols for establishing tissue cultures with high regeneration capacity.

Trocas gasosas influenciam na morfogênese in vitro de duas cultivares de oliveira (Olea europaea L.)

Os objetivos deste trabalho foram estabelecer in vitro as cultivares de oliveira 'Arbequina' e 'Maria da Fé' e avaliar a influência das tampas com membranas permeáveis a gases na morfogênese in vitro dessas cultivares. Inocularam-se segmentos nodais com gemas previamente descontaminadas pelo protocolo aqui desenvolvido. Utilizaram-se o delineamento inteiramente casualizado (DIC) em esquema fatorial 2³, duas cultivares; dois meios de cultura OM (Olive medium) (OM + 20 µM de zeatina [1]; e OM + 20 µM de zeatina + 10 µM de GA3 [2]); dois tipos de vedação (tampa rígida sem orifício e com membrana porosa) com cinco repetições/ tratamento; e a unidade experimental constituída por quatro tubos de ensaio. Avaliaram-se: a porcentagem de contaminação total; a porcentagem de contaminação fúngica e bacteriana; o número de gemas intumescidas; o número de brotos; e a porcentagem de oxidação. Aos 30 dias de cultivo, constatou-se a contaminação de 15% e 8,8% dos explantes de 'Arbequina' e 'Maria da Fé', respectivamente. Em 'Arbequina', 33,3% e 66,7% ocorreram por contaminação fúngica e bacteriana, respectivamente. Em 'Maria da Fé', 28,6% e 71,4% decorreram de contaminação fúngica e bacteriana, respectivamente. O número de gemas foi superior (p<0,05) em 'Arbequina', comparativamente à 'Maria da Fé', quando se utilizou tampa com membrana porosa para vedar os frascos. Em tampa rígida não houve diferença entre cultivares. O número de brotos no meio 1 foi superior estatisticamente (p<0,05) ao no meio 2. Não houve diferença estatística em porcentagem de oxidação. Sugere-se a utilização do protocolo de desinfestação aqui desenvolvido, como também do meio 1 e tampas com membranas porosas, pois isso favorecerá o desenvolvimento das gemas e a posterior formação de plantas.

THE EFFECT OF EXPLANT TYPE AND NUTRIENT MEDIUM ON THE IN VITRO PROLIFERATION OF OLIVE (OLEA EUROPAEA L. 'CHONDROLIA CHALKIDIKIS')

The effect of explant type and nutrient medium on in vitro proliferation of olive (Olea europaea L. ‘Chondrolia Chalkidikis’) was studied. The explants, taken from shoots of olive plants grown in a greenhouse, were divided into three groups: young shoot tips, uni- and bi-nodal explants. Furthermore, half of the explants maintained part of their leaves while the rest were defoliated. The explants were established in the OM nutrient medium containing BA (1.0 mg L-1) and IBA (0.5 mg L-1). After a 4-week period, it was observed that the type of explant affected significantly the in vitro proliferation of olive. Specifically, the bi-nodal explants without leaves had the highest proliferation rate and developed the greatest number and the longest new shoots. The young olive shoot tips died as a result of the disinfection process conducted before the establishment of the in vitro culture. Afterwards, three types of nutrient media were used for the in vitro multiplication of olive: the Olive Medium (OM), the Murashige and Skoog (MS) and the Woody Plant Medium (WPM). The concentration of the macronutrients of the above media was the normal or reduced to half or doubled (OM, ½OM, 2OM, MS, ½MS, 2MS, WPM, ½WPM, 2WPM). In all treatments BA (1.0 mg L-1) and IBA (0.5 mg L-1) were included. The experiment lasted 4 weeks. Not only the nutrient medium but also the macronutrients’ concentration affected the in vitro proliferation of olive. The highest proliferation rate was observed when the full strength OM nutrient medium was used, while the MS medium presented the smallest rate, independently of the salt concentration. The greatest number of new shoots was developed in the 2WPM medium, while the longest ones in the full strength OM medium. Finally, hyperhydricity was observed in all the treatments but it was more severe in the cases of the explants cultured in the MS culture medium.

Embryogenic competence of calli and embryos regeneration from various explants of Dahbia cv, a Moroccan olive tree (Olea europaea L.)

The olive (Olea europaea L.) is an economically important species in the Mediterranean basin. Studies on the improvement of the olive using biotechnological techniques as somatic embryogenesis were always hampered by the recalcitrant aspect of its explants to regeneration in vitro. Thus, we carried out this investigation in order to test the embryogenic capacity of different types of explants of the Moroccan olive cultivar Dahbia: ovaries and stamens (taken from unpollinated flowers), radicles and cotyledons (excised from zygotic embryos) and leaves andpetioles (taken from micropropagated plantlets which were maintained in vitro for fouryears) were cultured for three weeks on an induction medium (OMi) consisting of abasal olive medium (OM), supplemented with 25 µM indole-3-butyric acid (IBA) and 2.5 µM 2-isopentenyladenine (2iP), transferred to OM medium devoid of growth regulators for four weeks, and then to an olive cyclic embryogenesis medium (ECO)supplemented with 0.25 µM (IBA), 0.44 µM 6-benzylaminopurine (BAP) and 0.5 µM 2iP. All the obtained calli were morphologically identical on ECO supplemented with growth regulators; they were compact and brownish and exhibited nodules on their periphery. The histological observation showed cells with embryogeniccharacteristics. Although, acquisition of embryogenic competence was shown on all the calli, the expression of embryogenic cells into somatic embryos were observed on the sole zygotic origin (radicles and cotyledons), indicating that, within the same cultivar and the same culture conditions, the expression capacity varies according to the explants nature. Key words: Somatic embryogenesis, embryogenic competence, Olea europaea L., Dahbia cv.

Micropropagation of Juniperus navicularis, an endemic and rare species from Portugal SW coast

Juniperus navicularis Gand (= J. oxycedrus L. subsp. transtagana Franco) is an endemic dioecious shrub from the Plio-Plistocene transition sands of the west Portuguese coast line. It grows in sole or mixed stands along with Pinus pinea and P. pinaster and, more rarely, with Quercus lusitanica. In order to reduce fire incidence in these Mediterranean mixed stands, forest management practices of mechanical or chemical treatments, have drastically reduced J. navicularis populations. In addition, the fast expansion of touristic areas along with a low seed germination rate further endangers this species. In this study we developed a protocol for micropropagation of adult J. navicularis. Microcuttings were obtained from lateral and apical twigs of both female and male mature plants, and used as explants. Microcuttings with axillary buds were grown on different media and plant growth regulators combinations. Developmental parameters were evaluated to define the best medium and plant growth regulator concentration for shoot bud induction during the multiplication cycles. Olive Medium and Gupta and Durzan medium, both supplemented with 0.45 μM 6-benzyl-amino-purine, were the most favourable of all combinations tested achieving more than 3 new shoots per explant. The highest rooting (60%) was obtained in microshoots cultured in Olive Medium when supplemented with indole-3-butyric acid at 12.3 μM. With this multiplication protocol, it was possible to obtain more than 500 shoots from the initial 20, after three multiplication cycles in 3 months.