P. Moncaleán

In vitro response of Actinidia deliciosa explants to different BA incubation periods

Actinidia deliciosa apical shoots were cultured in MS liquid medium with cellulose plugs as support for the explants. Different BA (4.4 μM) incubation periods were tested in order to improve the effectiveness of the micropropagation system by reducing the cytokinin incubation period. At the end of 3 successive subcultures, the explants were analysed and a number of parameters (number, weight and length of shoots, presence and weight of callus, multiplication index, etc.) were measured. Different BA incubation periods have a long-term effect since the best results at the end of multiplication stage were not followed by better growth at the end of the acclimatised period studied. The highest quality plants were those obtained from culturing in the presence of BA for 1 day. Our results show that BA not only has an important effect on the different phases of micropropagation, but will also regulate the future development of the regenerants.

Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees.

In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the trees organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work, we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin (CK) types [N⁶-benzyladenine (BA), meta-topolin (mT) and trans-zeatin] and concentrations (25 and 50 µM), and studied the effect of culture conditions on endogenous CK and indole-3-acetic acid (IAA) levels at different stages of the organogenic process. To this end, the levels of 43 CKs and IAA were determined in P. radiata buds before and during the reinvigoration process. When BA or mT was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid CK content. We also report for the first time the presence of O-glucosides in non-treated P. radiata explants from the field and remark the importance of O-glucosides as storage forms.

Cytokinins and Mineral Nutrition in Actinidia deliciosa (Kiwi) Shoots Cultured In Vitro

Summary Benzyladenine (BA) uptake and metabolism, zeatin-type cytokinins and residual macronutrients in the culture medium were measured after 0, 0.5, 1, 2, 8 and 16 hours of culture in Actinidia deliciosa explants cultured in liquid medium using cellulose plugs as support of the explants. At the end of the culture period, low percentages of hyperhydric shoots were found. During the first 30 min of culture the amount of BA in the culture medium dramatically decreased. 8-[ 14 C] BA was rapidly metabolized by rootless shoots of kiwifruit cultured in liquid medium and it could be the reason of the low percentage of hyperhydric shoots. The levels of zeatin-type cytokinins remained constant during the first 16 h of culture, which could suggest that BA acts «per se» and not through endogenous cytokinins in the development of kiwifruit explants. Ammonium and phosphate were mainly uptaken during the first hours of culture and these ions were the most consumed at the end of the culture period (35 days). MS medium could be too rich for the culture of kiwifruit in liquid medium except for phosphate which concentration should be even increased.

Solute accumulation and elastic modulus changes in six radiata pine breeds exposed to drought

Drought is one of the main abiotic factors that determine forest species growth, survival and productivity. For this reason, knowledge of plant drought response and the identification of physiological traits involved in stress tolerance will be of interest to breeding programs. In this work, several Pinus radiata D. Don breeds from different geographical origins were evaluated along a water stress period (4 weeks) and subsequent rewatering (1 week), showing different responses among them. Leaf water potential (Ψ(leaf)) and osmotic potential decreases were accompanied by a variation in the total relative water content (RWC, %). The most tolerant breeds presented the lowest leaf water potential and RWC at turgor loss point, and showed the lowest elastic modulus (ε) values. A high ε value was a characteristic of a less-drought-tolerant plant and was related to membrane alterations (high electrolyte leakage percentages) that could favor cell water loss. Of the group of solutes that contributed to osmotic adjustment, soluble carbohydrates were the most abundant, although stressed plants also increased their content of free amino acids [mainly proline (Pro) and glutamic acid (Glu), and γ-aminobutyric acid (GABA)] and free polyamines. In addition, the most sensitive breeds had a higher GABA/Glu ratio. After rewatering, Pro and GABA were higher in rehydrated plants than in controls.

Enhancing initiation and proliferation in radiata pine (Pinus radiata D. Don) somatic embryogenesis through seed family screening, zygotic embryo staging and media adjustments

In Pinus spp., initiation of somatic embryogenesis (SE) is influenced by the developmental stage of immature embryos, the genotype of the parent trees and the formulation of tissue culture medium. Optimizing all these factors can lead to improved initiation and proliferation response; however, few studies have focused on improving these stages. For this reason, the objectives of this research were to determine the best immature zygotic embryo developmental stage for initiation and to test the effect of different sources of organic nitrogen in the initiation and proliferation steps in Pinus radiata SE. We have determined and verified the optimum zygotic embryo developmental stages 2–4 for embryogenic tissue (ET) initiation and proliferation and identified the most responsive seed families in two consecutive years. Besides EDM (Walter et al. 1998), medium with high gellan gum content during ET proliferation maintained the embryogenic tissue in a better micro-morphological arrangement for a longer time.

Immunolocalization of IAA and ABA in roots and needles of radiata pine (Pinus radiata) during drought and rewatering.

Anatomical, physiological and phytohormonal changes involved in drought tolerance were examined in different Pinus radiata D. Don breeds subjected to soil drying and rewatering. Breeds with the smallest stomatal chamber size had the lowest transpiration rate and the highest intrinsic water-use efficiency. Xylem cell size was positively correlated with leaf hydraulic conductance and needle indole-3-acetic acid (IAA) concentrations, whereas transpiration rate was negatively correlated with needle abscisic acid (ABA) levels. Since these two phytohormones seem important in regulating the P. radiata drought response, they were simultaneously immunolocalized in roots and needles of the most tolerant breed (P. radiata var. radiata × var. cedrosensis) during two sequential drought cycles and after rewatering. During drought, IAA was unequally distributed into the pointed area of the needle cross-section and mainly located in mesophyll and vascular tissue cells of needles, possibly inducing needle epinasty, whereas ABA was principally located in guard cells, presumably to elicit stomata closure. In the roots, at the end of the first drought cycle, while strong IAA accumulation was observed in the cortex, ABA levels decreased probably due to translocation to the leaves. Rewatering modified the distribution of both IAA and ABA in the needles, causing an accumulation principally in vascular tissue, with residual concentrations in mesophyll, likely favouring the acclimatization of the plants for further drought cycles. Contrarily, in the roots IAA and ABA were located in the exodermis, a natural barrier that regulates the phytohormone translocation to other plant tissues and hormone losses to the soil solution after rewatering. These results confirm that immunolocalization is an efficient tool to understand the translocation of IAA and ABA in plants subjected to different water stress situations, and clarify their role in regulating physiological responses such as stomata closure and epinasty in needles and root development.

Plant growth regulators as putative physiological markers of developmental stage in Prunus persica

Prunus persica plants of different ages and statesof maturation were analysed to compare their phytohormonal status. Endogenouslevels of indole-3-acetic acid (IAA), abscisic acid (ABA), and severalcytokinins (Cks): zeatin (Z), zeatin riboside ([9R]Z), dihydrozeatin ((diH)Z),dihydrozeatin riboside ((diH)Z[9R]), isopentenyl adenine (iP) and isopentenyladenosine ([9R]iP), were measured in order to determine their possible use asphysiological indices of phase change and maturation. A decrease in Ck levels(Z, [9R]Z, (diH)Z, (diH)[9R]Z, and iP, [9RiP) was found from the embryonic tojuvenile stage as well as a decrease in the ratio of iP-type (iP and[9R]iP)/Z-type Cks paralleling the increase in tree age. ABA levels increasedduring maturation in Prunus persica and the ratio ofCks/IAA decreased with tree age. From our results, we propose that the balancesof Cks/IAA and iP-type/Z-type Cks are good indices of different developmentalstates in Prunus persica.

Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest

Pinus halepensis Mill. is a common forest species in the Mediterranean area and it is important for environmental conservation. This study established a method of regenerating Pinus halepensis Mill. through somatic embryogenesis. The effect of culture medium (mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was analysed during the first steps of embryogenesis process. This study showed a marked effect of the culture medium tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture medium and the proliferation stage was significantly affected by the combination of plant growth regulators. At the end of the maturation phase the presence of activated charcoal was also evaluated. Finally, maturation of embryogenic tissue was affected by the nitrogen source in the culture medium and these results were different for high and low mature embryo producing cell lines. To the best of our knowledge, this is the first report on Aleppo pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.

Improved micropropagation protocol for maritime pine using zygotic embryos

Abstract Pinus pinaster Ait. is a conifer widely distributed through Spain. It is considered one of the major forest species because of wood and resin production. This fact explains the efforts of companies for capturing and enhancing the genetic gain in breeding programs. Micropropagation is a useful strategy to obtain identical plants from selected material, but nowadays, organogenesis protocols are the unique techniques to obtain clonal plants from a high number of elite genotypes. In this work, an improved micropropagation protocol for maritime pine using zygotic embryos as initial explant was developed. To optimize the protocol, the study of culture medium and exogenously applied cytokinins was needed. Embryos cultured on 1/2 DCR as basal medium exhibited high organogenic responses during induction, elongation, and rooting periods. Explants cultured on medium with 2.2 µM meta-topolin showed the highest values in all parameters analyzed along the different micropropagation phases, from induction to acclimatization. These explants showed a survival percentage of 90%, embryos forming shoots of 96.7%, and bud forming capacity and shoot elongation capacity of 4.38 and 3.39, respectively. Moreover, 70% of rooted shoots were obtained. All rooted plants survived in the acclimatizing stage after two months under ex vitro conditions. Use of 1/2 DCR as basal medium may provide an efficient and simplified in vitro protocol from whole zygotic embryos, ensuring the large-scale production of Pinus pinaster superior genotypes.

Metabolites and hormones are involved in the intraspecific variability of drought hardening in radiata pine.

Studies of metabolic and physiological bases of plant tolerance and hardening against drought are essential to improve genetic breeding programs, especially in productive species such as Pinus radiata. The exposure to different drought cycles is a highly effective tool that improves plant conditioning, but limited information is available about the mechanisms that modulate this process. To clarify this issue, six P. radiata breeds with well-known differences in drought tolerance were analyzed after two consecutive drought cycles. Survival rate, concentration of several metabolites such as free soluble amino acids and polyamines, and main plant hormones varied between them after drought hardening, while relative growth ratio and water potential at both predawn and dawn did not. Hardening induced a strong increase in total soluble amino acids in all breeds, accumulating mainly those implicated in the glutamate metabolism (GM), especially L-proline, in the most tolerant breeds. Other amino acids from GM such as γ-aminobutyric acid (GABA) and L-arginine (Arg) were also strongly increased. GABA pathway could improve the response against drought, whereas Arg acts as precursor for the synthesis of spermidine. This polyamine showed a positive relationship with the survival capacity, probably due to its role as antioxidant under stress conditions. Finally, drought hardening also induced changes in phytohormone content, showing each breed a different profile. Although all of them accumulated indole-3-acetic acid and jasmonic acid and reduced zeatin content in needles, significant differences were observed regarding abscisic acid, salicylic acid and mainly zeatin riboside. These results confirm that hardening is not only species-dependent but also an intraspecific processes controlled through metabolite changes.