| dc.description.abstract | Located in the North of Spain, Basque Country has edaphoclimatic conditions that have allowedthe establishment and development of an important forestry sector at European level. In thissense, 54.8% of the territory is dedicated to natural and cultivated forests. The most cultivatedspecies in the Basque territory is Pinus radiata, whose first report of forest plantationestablishment dates back to 1897. From that year to the present, its cultivation has increased inthe Basque Country, reporting for the year 2022 the largest total area (25.9 %) and the highestforest productivity (80-85% of the annual cuts).Pinus radiata, has a natural distribution from the central coast of California to Mexico. Theversatility and quality of the wood, together with the rapid growth of the species, has allowed itsplantations to extended to countries such as Australia, Chile, New Zealand, South Africa andSpain. However, in recent years P. radiata in the Basque Country has been affected by severaldiseases caused mainly by fungi: Pine canker caused by Fusarium circinatum, Dothistromaneedle blight caused by Dothistroma septosporum and Dothistroma pini, and brown spot needleblight caused by Lecanosticta acicula. This last disease has affected 3,300 ha in Gipuzkoa,Basque Country. In addition, P. radiata plantations in the Basque Country have decreased from123,921 ha (2016) to 102,488 ha (2022), a decrease that coincides with a historical outbreak ofthe needle blight during the years 2018-2019. For all of the abovementioned reasons, andtogether with the increase in the world population that causes a strong demand for timber andnon-timber products, the forestry sector and HAZI (www.hazi.eus) proposed a list of candidatespecies to be used, compatible with the demand of the forestry sector, as well as socially andculturally accepted.Using the list of possible alternative species, the following were selected for this study:Cryptomeria japonica (Thunb. ex L.f.) D. Don, Pinus ponderosa (P. Lawson and C. Lawson),Sequoia sempervirens ( D.Don). Endl and apparently tolerant genotypes of P. radiata.However, these species display desirable characteristics for clonal propagation at maturity,when traditional vegetative propagation and rooting are unfeasible. Therefore, the developmentof alternative methods of propagation, such as in vitro propagation, for these species isnecessary. In vitro propagation through tissue culture could be performed mainly byorganogenesis or somatic embryogenesis; those techniques combined with traditional onescould be efficient for the vegetative propagation of the abovementioned alternative species. Inaddition, these methods allow to improve the quality, yield and health of commercially valuableforest species. In this context, organogenesis in species of the Pinaceae family is generallyrestricted to zygotic embryos, part of them or young seedlings as initial source of explant, andattempts to clone selected adult individuals have been feasible but have faced severaldifficulties. In this regard, in our laboratory at Neiker, several in vitro propagation protocolshave been successfully developed for Pinus pinaster Ait, Pinus sylvestris L., Pinus pinea L.,Pinus radiata or Pinus halepensis among others.Taking into account all this information, the general hypothesis of this work is that themodification of the physico-chemical environment in the micropropagation process caninfluence the regeneration efficiency of elite trees of forest species such as C. japonica, P.ponderosa and S. sempervirens to be considered as an alternative to P. radiata in the BasqueCountry. The second hypothesis to be corroborated is that adult P. radiata trees with apparenttolerance to pine needle blight can be used as a source of material for in vitro propagation.In this context, the objective of the first chapter of the thesis was to analyze the influence ofcollection dates and different concentrations of 6-benzyladenine (BA) in the shoot inductionphase, as well as the effect of different types of light on growth success of rooting. Also, theeffect of physico-chemical factors in the content of amino acids and carbohydrates in the shootsdeveloped in vitro was studied. Although reinvigorated shoots were obtained in both BA concentrations (22 or 44 ¿M), the highest BA concentration showed the best results in terms of shoot induction and number of shoots per explant when the explants were collected in the first week of February. In addition, in shoots developed in vitro a significantly higher differences were found in the different phases of the rooting process in the levels of fructose, glucose and sucrose and in the threonine and tyrosine contents.The objective in the second chapter was to optimize the micropropagation method of C. japonica using adult trees as a source of plant material. In a first experiment, the effect of different types of initial explants and three basal culture media on shoot induction was studied. In addition, two sucrose concentrations and two light treatments (LED versus fluorescent lights) were tested for rooting stage. In a second experiment, the effect of different growth regulators (6-benzylaminopurine, metatopolin and thidiazuron) on shoot induction and subsequent phases of the organogenesis process were analysed. As main results, the highest number of shoots was obtained when using basal explants longer than 1.5 cm of length grown in the QL culture medium (Quoirin and Lepoivre, 1977). The shoots produced a higher number of roots when grown under red LED lights. Finally, root induction was significantly higher in shoots previously induced with meta-topolin.The third chapter was focused on the analysis of the influence of different types of explants, basal culture media, cytokinins, auxins and light treatments on the in vitro propagation success in P. ponderosa. In this sense, whole zygotic embryos cultured in 44 ¿M BA performed better in terms of explant survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. Regarding the light treatments applied, a significantly higher percentage of rootable shoots was obtained under blue LED light. However, the percentage of acclimatization was higher in shoots previously cultivated under fluorescent light. Furthermore, anatomical studies using optical and scanning microscopy showed that light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs showed less stomata compared with needles from plantlets exposed to fluorescent light.The objective in the fourth chapter was to optimize the micropropagation of adult elite trees of S. sempervirens. To accomplish this objective, the effect of different cytokinins (6-benzylaminopurine, meta-Topolin and Kinetin), and four types of explants in shoot induction were studied. At rooting stage, the effect of two types of auxins was evaluated: 1-naphthalene acetic acid, indole-3-butyric acid and a mixture of both. The best induction rate was obtained when apical explants longer than 1.5 cm were induced with 4.4 ¿M BA. Regarding the rooting, shoots previously induced in 4.4 ¿M BA and rooted in a culture medium with 50 ¿M 1-naphthalene acetic acid showed the best result. However, acclimatization was better when explants had been cultivated with Kinetin and rooted in a culture medium with indole-3-butyric acid.Our study demonstrated that in vitro culture method can be used as an alternative for the propagation of elite individuals of C. japonica, P. ponderosa, P. radiata and S. sempervirens. Furthermore, the success of in vitro propagation in these species is influenced by genetic factors, collection dates, and physicochemical conditions of the culture environment. | es_ES |
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