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Pinus Canariensis Plant Regeneration Through Somatic Embryogenesis

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Pinus Canariensis Plant Regeneration Through Somatic Embryogenesis Forest Systems 29 (1), eSC05, 6 pages (2020) eISSN: 2171-9845 https://doi.org/10.5424/fs/2020291-16136 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) SHORT COMMUNICATION OPEN ACCESS Pinus canariensis plant regeneration through somatic embryogenesis Castander-Olarieta Ander (Castander-Olarieta, A)1, Moncaleán Paloma (Moncaleán, P)1, Montalbán Itziar A (Montalbán, IA)1* 1 Department of Forestry Science, NEIKER-Tecnalia, Arkaute, Spain Abstract Aim of the study: To develop an efficient method to regenerate plants through somatic embryogenesis of an ecologically relevant tree species such as Pinus canariensis. Area of study: The study was conducted in the research laboratories of Neiker-Tecnalia (Arkaute, Spain). Material and methods: Green cones of Pinus canariensis from two collection dates were processed and the resulting immature zygotic embryos were cultured on three basal media. The initiated embryogenic tissues were proliferated testing two subculture frequencies, and the obtained embryogenic cell lines were subjected to maturation. Germination of the produced somatic embryos was conducted and acclimatization was carried out in a greenhouse under controlled conditions. Main results: Actively proliferating embryogenic cell lines were obtained and well-formed somatic embryos that successfully germinated were acclimatized in the greenhouse showing a proper growth. Research highlights: This is the first report on Pinus canariensis somatic embryogenesis, opening the way for a powerful bio­ technological tool for both research purposes and massive vegetative propagation of this species. Key words: acclimatization; Canary Island pine; micropropagation; embryogenic tissue; somatic embryo. Abbreviations used: embryogenic tissue (ET); established cell line (ECL); somatic embryogenesis (SE); somatic embryos (Se’s). authors’ contributions: PM, IM and ACO conceived and planned the experiments. ACO performed the experiments. ACO wrote the manuscript and all authors provided critical feedback and helped shape the research, analyses and manuscript. Citation: Castander-Olarieta, A., Moncaleán, P., Montalbán, I.A. (2020). Pinus canariensis plant regeneration through somatic embryogenesis. Forest Systems, Volume 29, Issue 1, eSC05. https://doi.org/10.5424/fs/2020291-16136. Received: 02 Dec 2019 Accepted: 25 Mar 2020 Copyright © 2020 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Funding Agencies/Institutions Project/Grant MINECO (Spanish Government) AGL2016-76143-C4-3R CYTED P117RT0522 DECO (Basque government) Ayudas de formación a jóvenes investigadores y tecnólogos Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial rela­ tionships that could be construed as a potential conflict of interest. Correspondence should be addressed to Itziar A. Montalbán: [email protected] Introduction ing trend derived from climate change, coupled with reduced precipitation, is expected to constraint tree The Canary Island pine (Pinus canariensis Chr. Sm. growth and tree ring width (Brito et al., 2016). Further­ Ex DC) is a subtropical species endemic to the western more, more frequent intense wildfires have been dem­ Canary Islands, growing across contrasting habitats, onstrated to cause long-term changes in the composi­ from xeric conditions to areas with > 1200 mm of an­ tion of soil and negatively affect the vitality of adult nual rain, and from the sea level up to 2400 m (López trees and the establishment of new plantlet (Durán et De Heredia et al., 2014). Despite the well-known great al., 2010; Otto et al., 2010). adaptability of this species to drought conditions and Ecologically it is a relevant species because it colo­ resistance to forest fires, exemplified by xeromorphic nizes volcanic soils where no other tree species can needles, serotinous cones and deep tap roots, the warm­ compete, thus maintaining soil stability on the higher 2 Castander-Olarieta Ander, Moncaleán Paloma, Montalbán Itziar A slopes of the islands. Besides, P. canariensis is an im­ comprising a total number of 240 initial explants. All the portant timber-producing species in the Canary Islands, megagametophytes were kept at 23ºC in darkness and employed in cabinetmaking and outdoor carpentry after 12 weeks on the initiation medium, initiation rates thanks to its rot-proof nature. Growth is rapid and the were calculated. These incubation conditions were main­ heart-wood is of extremely high quality, which pro­ tained during proliferation and maturation. moted its introduction as a forest and ornamental spe­ Proliferation of ET was carried out using EDM pro­ cies to several continents, becoming a successful forest liferation medium, increasing the gellan gum concentra­ tree in South Africa (Martinez Pulido et al., 1990). tion to 4.5 gL-1. In this case, small parts of about 0.5 Therefore, the development of a rapid clonal propaga­ cm in diameter from the generated embryonal masses tion method would be of considerable value. were divided in different Petri dishes (>3); one part Successful micropropagation and regeneration of was subcultured every two weeks while the other was plantlet using organogenic techniques from cotyledon­ subcultured monthly. Maturation was carried out using ary explants have already been achieved (Martinez EDM basal medium supplemented with 60 µM abscisic Pulido et al., 1990; 1992; 1994). However, as far as we acid and 9 gL-1 gellan gum following Montalbán et al., know, there are no studies of somatic embryogenesis (2010); 90 mg of ET inoculum per Petri dish, two es­ (SE) in this species. SE is the most efficient biotech­ tablished cell lines (ECL) and five replicates per ECL nological approach for conifer clonal propagation and were employed. After 14 weeks, the maturation success it can be combined with cryopreservation, becoming was evaluated and the number of mature somatic em­ this technique a useful tool to increase the availability bryos (Se’s) per gram of ET was calculated. of elite plant material. Furthermore, recent research has Germination and acclimatization in the greenhouse demonstrated that the culture conditions during SE can followed the procedure described by Montalbán & determine both the success of the process (García- Moncaleán (2018). The Se´s were germinated on Petri Mendiguren et al., 2016; Pereira et al., 2016; Castand­ dishes with half-strength macronutrients LP medium er-Olarieta et al., 2019) and the behaviour of the gener­ (1/2 LP, Quoirin & Lepoivre 1977 modified by Aitken- ated plants ex vitro (García-Mendiguren et al., 2017). Christie et al., 1988) supplemented with 2 gL-1 acti­ Considering the abovementioned information, the vated charcoal and 9 gL-1 gellan gum (Difco® Agar aim of this work was to evaluate the feasibility of SE granulated). After nine weeks, germination rates were in P. canariensis using immature zygotic embryos as evaluated and successfully germinated seedlings were initial explants. subcultured to glass jars with medium of the same composition. After another nine weeks, the somatic plants were transferred to individual pots containing Materials and methods peat: vermiculite (8:2, v/v) and acclimatized in a green­ house under controlled conditions (T = 23 ± 3ºC and One-year-old green female cones of P. canariensis were RH = 70 ± 5%) and regular watering. collected in July and September 2018 from open-pollinat­ Prior to acclimatization, the plants that had not de­ ed trees in Orio (Gipuzkoa, Spain; latitude: 43º29’63’’N, veloped a proper root system were transferred to a longitude: 2º08’54’’W, elevation: 296m). Immature O118/80+OD118 microbox (SacO2) containing megagametophytes were extracted and sterilized follow­ perlite:peat (7:3, v/v) moistened with 1/2 LP liquid ing Montalbán et al., (2014) and the developmental stage medium supplemented with 1 μM 1-naphthalenacetic of the zygotic embryos was determined using a Leica acid and 0.5 μM indol-3-butyric acid. Seven weeks DMS 1000 microscope (Montalbán et al., 2012). later they were carefully removed from the microbox, In order to induce the initiation of embryogenic tissue the rooting rate was evaluated, and all plants were ac­ (ET), three media were tested: EDM initiation medium climatized in the greenhouse as previously described. (Walter et al., 2005), DCR initiation medium (Gupta & The results of all the experiments were analysed by Durzan 1985) and a modified MCM medium (Bornman ANOVA. However, for both the number of initiations 1983). EDM initiation medium was supplemented with and Se’s, the analysis of variance did not fulfil the 4.5 μM 2,4-dichlorophenoxyacetic acid and 2.7 μM normality hypothesis, and thus, a Kruskal-Wallis test 6-benzylaminopurine, while the other two media were was performed. supplemented with 9 μM 2,4-dichlorophenoxyacetic acid and 2.7 μM kinetin. All media were solidified with 3.5 gL-1 gellan gum (Gelrite®) and, after autoclaving, EDM Results and discussion amino acid mixture was added (Walter et al., 2005). Eight megagametophytes per Petri dish and five Petri dishes The microscopic analysis of the zygotic embryo per initiation medium and collection time were cultured, along the different collection times revealed the pres­ Forest Systems April 2020 • Volume 29 • Issue 1 • eSC05 Somatic embryogenesis in Pinus canariensis 3 ence of several developmental stages. All the megaga­ ing that ECLs subcultured monthly showed a more metophytes
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