Micropropagation of a recalcitrant pine (Pinus pinea L.): An overview of the effects of ectomycorrhizal inoculation

Stone pine (Pinus pinea L.) is an economically important forest species in some regions of Iberian Peninsula. Portugal and Spain have nearly 500,000 ha of stone pine stands, representing 85% of worldwide distribution. The main use of this species is for the production of seeds (pinion) for food industry. In addition to its enormous profitability as a producer of seeds, it has beneficial impact on soil protection, dunes fixation and is a pioneer species particularly for cork and holm oaks degraded ecosystems. Stone pine plantations are today a major source of income for forestry holdings. Investments have targeted breeding, reforestation, forest management and harvesting. The maternal inheritance of desirable characteristics such as cone weight, number of seeds per cone and seed length is considerably high in this species thus encouraging the selection of seeds from “plus” trees. The selected trees have been propagated by grafting and micropropagation. However, grafting generates high variability due to scion-rootstock interaction that varies production levels. The production of clonal plants from selected seeds by micropropagation techniques has advanced very slowly due to the recalcitrance of this species in tissue culture and particularly to adventitious rooting of microshoots. Due to the tremendous importance of developing a reproducible tissue culture method for clonal propagation, a study has been carried out for over a decade to enhance rooting and acclimation. During this period of time, continuous increments in the multiplication rate and rooting frequency were achieved by introducing variations in culture media composition and conditions. Auxins, carbohydrates, light quality and duration, temperature at different concentrations and levels as well as compounds such as coumarin; salicylic acid, polyamines, etc. were tested for induction and expression phases of adventitious rooting. Despite these efforts, microshoots regenerated through organogenesis from mature embryo cotyledons failed to root or to have sustained root growth. At this point, an in vitro co-culture technique of stone pine microshoots with ectomycorrhizal-fungi was introduced to overcome the adventitious root growth cessation in vitro and improve root development during acclimation phase. An overview of the results showing the positive effect of fungal inoculation in promoting root growth in vitro and on plantlet survival during acclimation will be presented. Preliminary results of biochemical signals between Pinus pinea/Pisolithus arhizus during early steps of in vitro culture detected by liquid chromatography-mass spectrometry that might be responsible for the positive effect on root growth will be also presented

Root and shoot growth of Pinus strobus x P. wallichiana somatic plants is influenced by the nitrogen composition of the germination medium

Somatic embryogenesis (SE) in conifers has proven to be an useful biotechnological tool for species conservation and mass propagation, but to make it commercially viable, adaptive research is required to convert laboratory protocols into plantation reality. Hybrid white pines are of interest to forest industry in North America due to the potential resistance to white pine blister rust (Cronartium ribicola). Somatic embryos of many pine species are typically germinated on the same nutrient medium as used for earlier stages of SE. For most of the genotypes of white and hybrid white pine, the time required for the plantlets to grow a 2 cm root takes approximately five to six months at which time the plantlets can be potted and transferred to a greenhouse. The survival of plants is however sketchy and not always reliable. In order to accelerate and improve the root growth and survival of the plants in a greenhouse a study was initiated on the manipulation of the nitrogen composition of the germination medium. We were interested in learning whether the pine somatic seedlings had a preference for organic or inorganic forms of N during the germination step and root growth. Experiments have been conducted to determine the uptake and content of 15N in those roots by submerging them in a hydroponic solution for two hours. The somatic seedlings’ root growth was dramatically affected by the N composition of the medium as well as it did affect the uptake of 15N. To follow up the nitrogen metabolism in somatic seedlings, we have performed microarray and qPCR analysis for nitrogen or nitrogen-related metabolism genes. The results of the analysis will be discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Mycorrhiza-like structures in rooted microshoots of Pinus pinea L.

Pinus pinea L. (stone pine) is one of the major plantation species in Iberian Peninsula, being Portugal the largest edible seed producer in the world. The induction and improvement of in vitro rhizogenesis of microshoots of Pinus pinea was developed in our laboratory using a co-culture system with ECM fungi. In the acclimation phase in mixed substrates, or in rhizotrons, anatomical and morphological studies were done to observe the evolution of the root system in microshoots from the co-culture system vs. control plants. Extensive dichotomous and coralloid branching of lateral roots occurred spontaneously in inoculated and control plants as well. Moreover, similar branching occurred in liquid culture of excised seedling roots without the presence of ECM fungi. The striking similarity of these organs with pine ectomycorrhizas prompted their anatomical analysis; however the presence of Hartig net was not confirmed. These results suggested that the development of ECM-like structures might have occurred spontaneously.info:eu-repo/semantics/publishedVersio

O-coumaric acid ester, a potential early signaling molecule in Pinus pinea and Pisolithus arhizus symbiosis established in vitro

During ectomycorrhizal (ECM) establishment, biochemical signals lead to the development of complex structures in both the plant and the fungus that ultimately result in the formation of an ectomycorrhiza. The cross-talk between partners begins before physical contact. Our objective was to investigate the chemical nature of the signals during the first stages of in vitro mycorrhization of Pinus pinea with Pisolithus arhizus. For this purpose a double-phase solid liquid medium was expressly developed for the co-culture in order to simplify the extraction and further molecules analysis. O-coumaric acid ester was identified using HPLC UV and LC DAD MS on the second day of co-culture and its presence was detected for up to 10 days. These results contribute to the characterization of biochemical signals during pre-colonization involving conifer species and an ECM fungus, and demonstrate the suitability of the double-phase medium developed for the growth of both organisms and for the analysis of released chemical mediators.info:eu-repo/semantics/publishedVersio

Pisolithus arhizus (Scop.) Rauschert improves growth of adventitious roots and acclimatization of in vitro regenerated plantlets of Pinus pinea L.

Stone pine (Pinus pinea L.) is an economically important forest tree in the Mediterranean region and has been the target of breeding and selection through micropropagation mainly for its ecological and ornamental aspects. A crucial step in micropropagation is adventitious rooting of microshoots, which often is highly inefficient in most conifer species including stone pine. Hence, we conducted in vitro co-culture of Pinus pinea microshoots with the ectomycorrhizal fungus Pisolithus arhizus (isolated from natural stands) in order to promote adventitious root growth and plant survival during acclimatization. Significant differences were found in the number of branches, in the number of roots plus branches, in total length of roots, in total length of roots plus branches, in average root length and in the length of the longest root in inoculated plants during in vitro rooting compared with non-inoculated plants. The roots of inoculated plants also grew better in vermiculite and during acclimatization in a mixed substrate compared with roots of control plants resulting in the development of vigorous root system. Overall, mycorrhizal inoculation increased the survival rate of the regenerated pine

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISICACID3 (ABI3) and its Zeamays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers

Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis

The involvement of two R2R3-MYB genes from Pinus taeda L., PtMYB1 and PtMYB8, in phenylpropanoid metabolism and secondary cell wall biogenesis was investigated in planta. These pine MYBs were constitutively overexpressed (OE) in Picea glauca (Moench) Voss, used as a heterologous conifer expression system. Morphological, histological, chemical (lignin and soluble phenols), and transcriptional analyses, i.e. microarray and reverse transcription quantitative PCR (RT-qPCR) were used for extensive phenotyping of MYB-overexpressing spruce plantlets. Upon germination of somatic embryos, root growth was reduced in both transgenics. Enhanced lignin deposition was also a common feature but ectopic secondary cell wall deposition was more strongly associated with PtMYB8-OE. Microarray and RT-qPCR data showed that overexpression of each MYB led to an overlapping up-regulation of many genes encoding phenylpropanoid enzymes involved in lignin monomer synthesis, while misregulation of several cell wall-related genes and other MYB transcription factors was specifically associated with PtMYB8-OE. Together, the results suggest that MYB1 and MYB8 may be part of a conserved transcriptional network involved in secondary cell wall deposition in conifers