Harder, better, faster, stronger: Frost tolerance of Eucalyptus benthamii under cold acclimation.

Eucalypts are the most planted hardwood trees worldwide because of their very rapid growth, exceptional wood quality and adaptability. However, most commercial species and derived hybrids are sensitive to frost, which remains as the largest obstacle to their introduction in warm/temperate climates. As evergreen species, Eucalypts have developed the ability to tolerate frost events based on physiological and molecular responses triggered by previous exposure to cold temperatures, globally named cold acclimation. To characterize the acclimation process in two species with different tolerance to frost, E. grandis (Eg) and E. benthamii (Eb), seedlings were exposed for different times to low temperatures. Frost tolerance was estimated in leaves by an electrolyte leakage assay, and metabolome and morpho-physiological changes studied and correlated to the observed acclimation responses. Eb showed higher basal frost tolerance and an earlier and stronger acclimation response to cold temperatures than in the frost sensitive Eg. Eb was able to modify several morpho-physiological parameters, with a restriction in plant height, leaf area and leaf fresh weight during acclimation. Metabolome characterization allowed us to differentiate species and strengthen our understanding of their acclimation response dynamics. Interestingly, Eb displayed an early phase of sugar accumulation followed by a rise of different metabolites with possible roles as osmolytes and antioxidants, that correlated to frost tolerance and may explain Eb higher capacity to acclimate. This novel approach has helped us to point to the main metabolic processes underlying the cold tolerance acquisition process in two relevant Eucalyptus species.

Cold acclimation and freezing tolerance in three Eucalyptus species: A metabolomic and proteomic approach.

The ability of plants to cope with frost events relies on the physiological and molecular responses triggered by cold temperatures. This process, named acclimation, involves reprogramming gene expression in order to adjust metabolism. Planted Eucalyptus species are highly productive but most of them are frost sensitive. However, acclimation process varies among species and environmental conditions, promoting more or less frost damage in young plantations of frost-prone areas. To identify metabolites and proteins responsible for these differences, two acclimation regimes were imposed to seedling of Eucalyptus grandis Hill ex Maiden (Eg), Eucalyptus dunnii Maiden (Ed) and Eucalyptus benthamii Maiden Cambage (Eb), and leaves submitted to biochemical and molecular analyses. Further, seedlings were used for simulated frosts in order to test the acclimation status effect on frost tolerance. Eb showed higher frost tolerance than Ed and Eg under control and acclimation scenarios, possibly due to its higher accumulation of phenolics, anthocyanins and soluble sugars as well as lower levels of photosynthetic pigments and related proteins. Also, a rise in frost tolerance and in osmoprotectants and antioxidants was observed for all the species due to cold acclimation treatment. Interestingly, metabolic profiles differed among species, suggesting different mechanisms to endure frosts and, probably, different requirements for cold acclimation. Shotgun proteomics reinforced differences and commonalities and supported metabolome observations. An in depth understanding of these responses could help to safeguard planted forests productivity through breeding of tolerant genetic material.

Analysis of nutrient deficiencies affecting in vitro growth and development of Eucalyptus dunnii Maiden.

Although basal medium optimization is a key factor in the success of tissue culture, its mineral composition is frequently disregarded when optimizing in vitro propagation protocols. A previous work on Eucalyptus dunnii micropropagation suggests that excessive callus formation and leaf chlorosis are related to specific nutritional conditions of the basal media. Recently, a novel basal medium based on the mineral nutrient analysis of E. dunnii young stump shoots was developed and successfully tested in plant regeneration and micropropagation of E. dunnii, avoiding all these issues. Considering this basal medium as an ideal growth condition, a mild deprivation of each macro and micronutrient and of the total organic fraction was imposed to E. dunnii in vitro cultures for 30 d. As a result, K, Mg, Mn, Cl, Zn, Mo, Ni or Co deprivation quantitatively affected growth and development of axillary shoots. Moreover, leaf chlorosis and the development of organogenic callus under Fe deficiency, and leaf drop along with shoot tip necrosis under N deficiency were observed. These symptoms suggest that nutrient content in E. dunnii tissues needs to be above 420.3 mg kg-1 for Fe and 27.7 g kg-1 for N to avoid the symptoms of leaf chlorosis and shoot tip necrosis. Additionally, the main role of Mn in quantitative responses and the antagonism between ions, especially for Mg/K and Mg/Zn, were denoted by the multivariate analysis. Overall, these results make a relevant contribution to the optimization of in vitro propagation of E. dunnii and other hard-to-propagate related species.

TDZ pulsing evaluation on the in vitro morphogenesis of peach palm.

Peach palm (Bactris gasipaes Kunth.) cropping is an excellent alternative to native species exploitation; nevertheless, the problems with seed germination and conventional propagation justify the use of in vitro culturing. Aiming to asses TDZ pulsing effect on B. gasipaes morphogenesis, explants obtained from unarmed microplants were maintained in two treatments, half of them in MS free medium (without growth regulator) and the other half in MS with TDZ (0.36 µM). Both groups were transferred to growth regulator-free MS medium following 14 days of culture. After 84 days of culture, TDZ pulsing increased the growth and development of the shoots, restricted the growth and development of the roots, with no influence on adventitious bud induction or somatic embryogenesis. Furthermore, development of prickles, thickening of roots and chlorotic leaves were noted under TDZ pulsing. Leaf sheath histological analysis showed an epidermal origin and no vascularization of these prickles.