Ahmet Onay

In vitro micrografting of mature pistachio (Pistacia vera var. Siirt)

The success of various in vitro micrografting methods of shoot tips of pistachio (Pistacia vera L. var. Siirt) have been examined. Excised zygotic embryos that germinated in vitro were used as rootstocks. Current year shoot tips from mature trees of pistachio micrografted onto in vitro juvenile rootstocks, resulted in the restoration of shoot-bud proliferation. Variables tested include a size of microscion, grafting method, effects of culture medium and effects of time of the year at which shoot tips were used. The results indicate that the easiest and most successful method for grafting was slit micrografting. High levels of micrograft take were achieved with 2–4 mm (56.75%) and 4–6 mm (79.25%) long scions obtained from the regenerated shoot tips. The survival rate of the shoot tips was directly related to time of the year. The best growth of microscion was obtained with the in vitro forced shoot tips rather than with shoot tips excised from tree. Slow growth and lack of axillary shoot development on the micrografts was noticeable when the micrografts were cultured on hormone-free and germination medium. In vitro micrografted plantlets were successfully weaned and no problems were encountered with the establishment of micrografted plants in vivo.

Current status and conservation of Pistacia germplasm.

The genetic erosion of Pistacia germplasm has been highlighted in many reports. In order to emphasize this and to focus more attention on this subject, national and international (especially IPGRI and IFAR) institutions have initiated projects proposing to characterize, collect and conserve Pistacia germplasm. Therefore, this paper reviews recent research concerning conventional (in situ and ex situ) and unconventional biotechnological conservation strategies applied to the preservation of Pistacia germplasm. As regards conventional conservation, the majority of germplasm collections of Pistacia species are preserved on farms (in situ) and in seed and field genebanks (ex situ), as well as in the wild, where they are vulnerable to unexpected weather conditions and/or diseases. Hence, complementary successful unconventional in vitro methods (organogenesis, somatic embryogenesis and micrografting) and slow-growth storage conditions for medium-term preservation of Pistacia are presented together with the morphological and molecular studies carried out for the characterization of its species in this review. Moreover, special attention is additionally focused on cryopreservation (dehydration- and vitrification-based one-step freezing techniques) for the long-term preservation of Pistacia species. Possible basic principles concerning the establishment of a cryobank for the successful conservation of Pistacia germplasm are also discussed.

Micropropagation of mature male pistachio Pistacia vera L.

Summary Factors affecting the successful rapid proliferation and rooting of male pistachio (Pistacia vera L.) cv. Atlı, were studied. The most suitable type of cytokinin [6 benzyladenine (BA), kinetin (Kin), or thidiazuron (TDZ)], and the effect of eight different concentrations of BA (0.0675, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, or 8.0 mg l–1) were evaluated to optimise shoot proliferation. The auxins -naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), each at 2.0 mg l–1, or different concentrations (0.5, 1.0, 2.0, 4.0, or 8.0 mg l–1) of IBA, and the effect of explant size (1.0, 2.0, 3.0, or 4.0 cm) were assessed for root induction. The highest number of new microshoots per explant (5.64 ± 0.07) was obtained 4 weeks after culturing on Murashige and Skoog (MS) medium supplemented with 1.0 mg l–1 BA. Again, shoot length was highest in 1.0 mg l–1 BA, and decreased as the BA concentration increased. IBA was most effective in promoting root formation. The highest rooting frequency (73%) of microshoots was recorded for explants 4.0 cm in length, 4 weeks after culturing. In vitro-rooted plantlets were transferred to polyethylene pots filled with a 1:1:1 (v/v/v) mixture of soil, sand and peat.This treatment resulted in 90% survival of plantlets, which were acclimatised in a greenhouse.

Somatic embryogenesis of pistachio from female flowers

Summary Induction of somatic embryogenesis from the flower buds of pistachio (Pistacia vera L.) was studied with respect to growth regulators. Sixty-five percent of cultures formed embryogenic calli when placed on modified Murashige and Skoog medium containing 1 mg l–1 benzylaminopurine. In contrast, thidiazuron and α-naphthalene acetic acid reduced embryogenesis. Some of the somatic embryos had fused hypocotyls or multiple cotyledons. Abscisic acid was not necessary for maturation. Fewer somatic embryos germinated with 0.5 mg l–1 abscisic acid, but more converted into plantlets. Separate, morphologically normal, somatic embryos germinated on semi-solid germination medium, and developed into plantlets. Cytological analysis revealed a chromosome number of 2n = 2x = 30, similar to seedlings. The protocol described in this paper, for the induction of somatic embryos indirectly from female flowers of pistachio, represents a first step towards the production of clonal stocks.

Micropropagation of Pistachio

Pistachio (Pistacia vera L., family Anacardiaceae) is the only edible crop of 11 species in the genus Pistacia (Zohary 1952; Ozbek & Ayfer 1958; Crane 1984). The Pistachio tree is native to western Asia and Asia Minor, from Syria to the Caucasus and Afghanistan (Zohary 1952; Whitehouse 1957). Archaeological evidence in Turkey indicates that nuts were being used for food as early as 7,000 B.C. The tree was introduced into Europe at the beginning of the Christian era (Moldenke & Alma 1952). Subsequently its cultivation spread to other Mediterranean countries at approximately the beginning of the Christian era (Crane & Iwakiri 1981). The tree was first introduced into the United States in 1854 by Charles Mason, who distributed seed for experimental plantings in California (Lemaister 1959). Under favourable conditions the tree grows slowly to a height and spread of 5 to 9 metres, with one or several trunks. A strong apical dominance has also been reported to characterise developmental patterns of vegetative and floral Pistachio organs (Crane & Iwakiri 1985). Shoot extension begins late in March and terminates at the end of April to the middle of May (Crane 1984). Most of the axillary buds differentiate inflorescence buds during April and grown to their ultimate size for the season by late June (Takeda et al. 1979).

Novel Methods in Micropropagation of Pistachio

The focus of this paper is to describe the novel methods developed for the different stages of micropropagation: installation of mature apical shoot tips and elimination of browning exudates; forcing hardwood shoots from the lignified stem sections; forcing axenic leaves; initiation of embryogenic masses (EMS); encapsulation of somatic embryos and cryopreservation of axillary buds for storage, and the facilitation of rooting. These developed methods are not being used by commercial micropropagation laboratories yet. Exudation of phenolics is inhibited when the disinfested and rinsed explants are cut at the base of the shoot tips and washed twice for 1 h by shaking them in sterile distilled water on a shaker at 100 rpm. For shoot initiation, 15–20 cm long terminal stem sections are cut into 3–4 cm sections and placed in pots filled with peat, perlite and soil. Two or three weeks later, the developed softwood shoots are excised; or freshly harvested three to nine apical tips of 1–2 cm are disinfested and used as explants. Leaves excised from axenic shoot cultures were also used to induce organogenesis on a Murashige and Skoog (MS) medium with Gamborg vitamins supplemented with combinations of different concentrations of 6-benzylaminopurine (BA) and indole-3-asetic acid (IAA). Calcium alginate gel was used to encapsulate somatic embryos to produce synthetic seeds. The encapsulated somatic embryos can be stored under refrigerated conditions for short-term conservation. For long-term conservation, some axillary buds can also be stored by using vitrification and one-step freezing technique; however, other cryogenic techniques should also be tested. With these improved stages, application of Pistachio vera L. micropropagation in commercial clonal orchards will be feasible as an alternative to traditional propagation in the near future.

Pistachio (Pistacia vera L.)

The pistachio tree (Pistacia vera L.), a deciduous, dioecious, and wind-pollinated tree species, is a member of the Anacardiaceae, a family that also includes cashew, mango, poison ivy, poison oak, pepper tree, and sumac. The genus Pistacia includes at least 11 species (Zohary, 1952); other authors recognise as many as 15 (Whitehouse, 1957). P. vera L. is the only economic importance for bearing edible fruits and serving as rootstocks. P. vera L. is an Irano-Turanian species, the main range of which covers the middle Asian republics of Uzbekistan, Tadzhikistan, Kirgiziya and southern most parts of Turkmenia and Kazakhstan. In the north, P. vera extend to about latitude 43 N. to the Karatau, Kirgizskiy and Talasskiy Alatau mountain ranges, while in the south and south-western Afghanistan, in the Paropamisus mountain. in the Herat province it reaches a latitude of 35 N. It grows most abundantly and on the most extensive areas in Tadzhikistan, where it occupies about 115.000 ha; in the whole of central Asia natural thickets cover about 300.00 ha (Browicz 1988).

Determination of the Fatty Acid Composition of the Fruits and Different Organs of Lentisk (Pistacia lentiscus L.)

Abstract This paper reports the fatty acid composition of the oil extracts from seeds and in vivo and in vitro grown organs of Pistacia lentiscus L. were determined by using gas chromatography. The main fatty acids were linoleic (LA), palmitic (PALM), oleic (OLA) and linolenic (ALA) acids in the fruits, resins and in both in vivo and in vitro grown root, leaf and stem sections of male or female tree. The major fatty acids were represented by polyunsaturated fatty acids (PUFA) accounting for 56.94 %, 64.44 % and 55.57 % in root, leaf and stem part of male tree grown in vivo, respectively. The prominent class of fatty acid composition of different male organs of P. lentiscus L. regenerated in vitro was represented by PUFA accounting for 63.24 %. The monounsaturated fatty acid (MUFA), OLA and PUFA, LA were determined in the oils of the two genotypes studied.

AN EFFECTIVE PROTOCOL FOR IN VITRO GERMINATION AND SEEDLING DEVELOPMENT OF LENTISK (Pistacia lentiscus L.)

Different nutrient media (MS [Murashige and Skoog 1962]; QL [Quoirin and Lepoivre 1977] and WPM [Lloyd and McCown 1980]); plant growth regulators BA (benzil adenin), GA3 (gibberellic acid), IBA (indole-3-butyric-acid), NAA (naftalen acedic acid); and sucrose concentrations were studied to determine the in vitro culture effects on healthier and faster seedling development from mature lentisk (Pistacia lentiscus L.) seeds. After 28 days of culture, the percentage of germinated seeds was the highest (70%) in the full-strength MS medium. The cytokinin BA was superior to other tested treatments in terms of its ability to promote germination of lentisk seeds. When tested at different concentrations, sucrose gave the best results obtained at concentrations of 1–4%, whereas high concentrations (6 and 8%) mainly decreased germination rate and there was no a regular pattern for elongation of the aerial parts of plants. With this described protocol, on average 76.67% seeds germinated 4 weeks after culture. Developed seedlings were satisfactorily acclimatized in sterilized peat, soil and perlite containing compost, with high percentage survival viability was obtained 9 months after transfer to in vivo conditions (93.33%). The results obtained showed that the enriched full-strength MS medium supplemented with 1 mg L BA and 3% sucrose induced homogeneous and healthy seedling development in a period of 4 to 8 weeks of culture.

Biotechnological Approaches for Conservation of the Genus Pistacia

Climate change with the combination of increased population growth, unplanned urbanization, habitat loss and degradation, pollution and diseases, overexploitation of valuable species are the major causes of the loss of plant biodiversity. Moreover, since environment and environmental factors will change faster than the most of the plant’s adaptation, conservation strategies become critically essential for preservation of plant species. In this chapter, we described the current status of biotechnological conservation approaches for Pistacia species. Since in vitro conservation and most of cryopreservation methods rely on tissue culture-based methods, we described here micropropagation, micrografting, and somatic embryogenesis procedures in addition to in vitro conservation and cryopreservation in Pistacia species. Because there is no one ‘universal protocol,’ biotechnological conservation studies for Pistacia are still limited with a few species of the genus as described in this chapter. Development and optimization of appropriate techniques especially for long-term preservation of different Pistacia species can lead to establish cryobanks to conserve germplasm. Furthermore, the developed technologies will provide to prevent possible genetic erosion of Pistacia species.