Growth, anatomy and histochemistry of fast growing species under in vitro conservation through mineral oil and low-temperature combination

We have developed an efficient method for the in vitro storage of shoots of fast growing species through mineral oil and low-temperature combination. In a first experiment, nodal segments of Pfaffia glomerata and Lippia filifolia, two fast growing species in vitro, were initially cultured for up to 120 days in MS medium without cover or completely covered with distilled water or mineral oil. In a second experiment, shoots were storage for 360 days in different volumes of mineral oil (5, 10, 15 mL) and at different storage temperatures (15, 20, 25 °C). During these experiments, we evaluated monthly the survival (%) and height (cm) of the cultures. Finally, we carried out anatomical and histochemical analyses on the cultures from the best conservation treatment. At the end of the first experiment, the highest percentages of survival and the lowest growth rates were obtained when the propagules of both species were conserved immersed in mineral oil. According to results of the second experiment, P. glomerata can be maintained for up to 360 days with 100% survival when in 5 mL of mineral oil, as long as this process is performed at a temperature of 15 °C. Under these same conditions, the propagules of L. filifolia have survival rates above 50% after 360 days. Anatomically, some adaptations were observed to the low availability of oxygen imposed by the use of mineral oil. Plants conserved partially or completely submerged in mineral oil developed palisade parenchyma cells in the mesophyll of their leaves. Moreover, the development of stem vascular bundles was mitigated. Morphoanatomical abnormalities were not identified. Histochemically, neutral polysaccharides were present as reserve material in the leaves or stem of these propagules. This protocol can assist banks, collections or laboratories that maintain germplasm and are all or part of fast-growing species, avoiding the frequent need to manipulate the cultures. The in vitro conservation through mineral oil and low-temperature combination can aid those banks, collections or laboratories that keeps germplasm and are constituted, in whole or in part, of fast-growing species.