Testing Explant Sources, Culture Media, and Light Conditions for the Improvement of Organogenesis in Pinus ponderosa (P. Lawson and C. Lawson).

Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15-30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µΜ 6-benzyladenine showed the best results in terms of explants 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. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m-2 s-1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m-2 s-1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets.

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL-1 of L-glutamine, 1,050 mgL-1 of L-asparagine, 350 mgL-1 of L-arginine, and 35 mgL-1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.