In Vitro Plant Regeneration in Conifers: The Role of WOX and KNOX Gene Families

[EN] Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the WOX and KNOX homeobox gene families, whose function has been widely described in Arabidopsis thaliana. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of WOXX, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of Pinus pinaster.SIThis research was funded by “Fondo Europeo de Desarrollo Regional” (FEDER)/“Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación” (RTA2017-00063-C04-04)

An improved micropropagation protocol for stone pine (Pinus pinea L.)

[EN] A protocol for micropropagation from isolated cotyledons from Pinus pinea L. has been developed. Major improvements are the increase in rooting rates and the description for the first time of a successful continuous multiplication procedure. For bud induction, isolated cotyledons were cultured on 1/2 LP with 44.4 μM BA during 4 days. Shoot development was obtained by transfer to 1/2 LP hormone-free medium with activated charcoal. Since an additional 20 weeks were required for shoot elongation and rooting, at least 70 plantlets can be expected per seed after 29 weeks based on a 70% success rate. Besides, shoots can be micropropagated successively by subculturing on 1/2 LPC.[FR] Amélioration du protocole demultiplication par micropropagation du pin pignon (Pinus pinea L.). Nous avons développé un protocole de multiplication par micropropagation de Pinus pinea L. à partir de cotylédons isolés. Les principaux progrès réalisés concernent l’augmentation du taux d’enracinement et la description pour la première fois d’une méthode de multiplication en continu. Pour l’induction des bourgeons, des cotylédons isolés ont été cultivés dans le milieu 1/2 LP avec 44.4 μM BA pendant 4 jours. Le développement des pousses a été obtenu en transférant les bourgeons dans un milieu 1/2 LP sans hormone avec du charbon actif. Compte tenu des délais nécessaires (20 semaines) pour obtenir l’élongation des pousses et l’enracinement, au moins 70 plantules par graine peuvent être obtenues au bout de 29 semaines, sur base d’un taux d’enracinement de 70 %. De plus, les pousses peuvent être multipliées avec succès par micropropagation par sous-culture dans le milieu 1/2 LPC.SIPablo Alonso and this work were supported by Ministerio de Ciencia y Tecnología de España (MCT-02-AGL-00867)

EuroPineDB: a high-coverage web database for maritime pine transcriptome

Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases

Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth

In Vitro Plant Regeneration in Conifers: The Role of WOX and KNOX Gene Families

Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the WOX and KNOX homeobox gene families, whose function has been widely described in Arabidopsis thaliana. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of WOXX, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of Pinus pinaster

An improved micropropagation protocol for stone pine (Pinus pinea L.)

A protocol for micropropagation from isolated cotyledons from Pinus pinea L. has been developed. Major improvements are the increase in rooting rates and the description for the first time of a successful continuous multiplication procedure. For bud induction, isolated cotyledons were cultured on $1/2$ LP with 44.4 $\mu$M BA during 4 days. Shoot development was obtained by transfer to $1/2$ LP hormone-free medium with activated charcoal. Since an additional 20 weeks were required for shoot elongation and rooting, at least 70 plantlets can be expected per seed after 29 weeks based on a 70% success rate. Besides, shoots can be micropropagated successively by subculturing on $1/2$ LPC.Amélioration du protocole de multiplication par micropropagation du pin pignon (Pinus pinea L.). Nous avons développé un protocole de multiplication par micropropagation de Pinus pinea L. à partir de cotylédons isolés. Les principaux progrès réalisés concernent l'augmentation du taux d'enracinement et la description pour la première fois d'une méthode de multiplication en continu. Pour l'induction des bourgeons, des cotylédons isolés ont été cultivés dans le milieu $1/2$ LP avec 44.4 $\mu$M BA pendant 4 jours. Le développement des pousses a été obtenu en transférant les bourgeons dans un milieu $1/2$ LP sans hormone avec du charbon actif. Compte tenu des délais nécessaires (20 semaines) pour obtenir l'élongation des pousses et l'enracinement, au moins 70 plantules par graine peuvent être obtenues au bout de 29 semaines, sur base d'un taux d'enracinement de 70 %. De plus, les pousses peuvent être multipliées avec succès par micropropagation par sous-culture dans le milieu $1/2$ LPC

Micrografting of mature stone pine (Pinus pinea L.) trees

This paper describes an in vitro micrografting method for selected mature Pinus pinea L. trees. Needle fascicles of five selected clones were micrografted onto hypocotyls of two weeks-old germinated isolated embryos. Fascicle meristems outgrowth was recorded after one month of culture and the performance of the clones assayed was evaluated. Clones behave statistically different in establishment and development rates. Overall success of our protocol reached 43% of the graftings made.Micro-greffage de clones sélectionnés de Pinus pinea L. Ce rapport décrit une méthode pour le greffage in vitro de Pinus pinea L. Des brachyblastes de cinq clones ont été greffés sur embryons isolés germés, âgés de deux semaines. Le développement des méristèmes fasciculaires a été quantifié pour chaque clone après un mois. Les pourcentages d’établissement et de développement sont statistiquement différents pour les cinq clones. Le taux de succès du greffage réalisé par notre méthodologie s’élève à 43 %

Improving plantlet yield in <i>Pinus pinaster</i> somatic embryogenesis

<div><p></p><p>Somatic embryogenesis is expected to play a significant role in the future forest tree improvement programmes. The main bottleneck of this technique is still the progression from immature embryogenic cultures to mature cotyledonary embryos able to develop properly into well-growing plants. In this work, we present an improved protocol focused on increasing the maturation and conversion rate of <i>Pinus pinaster</i> Ait. embryogenic cultures. Results showed that the optimisation of the nutrient composition in the maturation medium increased the number of mature embryos by 25% (187.8 embryos per gram of fresh mass in average compared to 144.5 embryos in regular medium). It was also shown that 12-month cryostorage did not reduce viability or embryogenic ability of maritime pine cultures. A further increase in the yield of the protocol could be obtained by using benzyladenine in the conversion medium, promoting the bud-break of axillary buds that yielded 5.7 shoots in average per somatic embryo. Rooting of axillary shoots reached 84.3%. This methodology offers an alternative to overcome some problems associated with low somatic embryo production since the plantlet yield could be increased fivefolds.</p></div

Ordas (02-1291).vp

Summary As part of a study aimed at understanding the physiological and molecular mechanisms involved in adventitious shoot bud formation in pine cotyledons, we conducted a transcriptome analysis to identify early-induced genes during the first phases of adventitious caulogenesis in Pinus pinea L. cotyledons cultured in the presence of benzyladenine. A subtractive cDNA library with more than 700 clones was constructed. Of these clones, 393 were sequenced, analyzed and grouped according to their putative function. Quantitative real-time PCR analysis was performed to confirm the differential expression of 30 candidate genes. Results are contrasted with available data for other species