Criopreservação de gemas laterais e análises bioquímicas de Passiflora gibertii

Abstract

The Passiflora genus encompasses over 600 plant species worldwide, with high commercial potential for fruit production, ornamentation, and medicinal purposes. The diversity and intrinsic characteristics of Passiflora, such as seed dormancy and cross-fertilization, may require specific storage conditions and special care to maintain viability and genetic integrity over time, posing challenges in germplasm fields, a traditional conservation method. Cryopreservation is described as an increasingly established practice, offering an alternative for species conservation with advantages like space efficiency, low maintenance, and pathogen-free preservation. This study aimed to promote and assess the cryopreservation of axillary buds as explants from Passiflora gibertii plants in Murashige and Skoog (MS) standard culture medium supplemented with 6-benzylaminopurine (BAP) and sucrose. The cryopreservation technique employed was droplet vitrification. Beforehand, the explants underwent pre-treatment in a culture medium with a high sucrose concentration for 24 hours. Subsequently, they were subjected to Loading Solution (LS) for 30 minutes for dehydration. Aluminum strips were prepared at 0°C, and Vitrification Solution (VSL) drops were applied. The treatments were immersed in liquid nitrogen (LN) inside cryotubes for an hour. The re- warming process used Recovery Solution (RS) for 30 minutes. In the subsequent stage, explants were rehydrated in post-treatment medium with a high sucrose concentration for 24 hours. After this period, segments were transferred to a regeneration medium (MS + BAP) to assess survival rates. The treatment with the highest survival rate was 66.7%, achieved with a 45-minute exposure to VSL. Explant sections were made for qualitative anatomical evaluation. Surviving buds from the best treatment and dead buds were preserved, embedded, and analyzed under a microscope for comparison. Cells with ruptured membranes and cellular content leakage were prominently observed in dead buds, indicating that even with droplet vitrification, crystallization occurs in a significant portion of the explants. Biochemical analyses, covering various parameters such as sugars, antioxidant enzymes, and lipid peroxidation, were performed on cryopreserved explants compared to non-cryopreserved controls, both during cryopreservation and rooting, investigating physiology, antioxidants, and osmoregulation. In the cryopreservation process, exposure to the solution induced stress in the buds, reflected by a significant increase in malondialdehyde (MDA). High levels of MDA, combined with a reduction in protective antioxidants, indicated cell death. However, the application of VSL allowed survival and stimulated regeneration, evidenced by increased soluble sugars and hydrogen peroxide. Rooting differed between species, with Passiflora gibertii facing difficulties, while Passiflora alata demonstrated greater efficiency, supported by biochemical distinctions between rooted and non-rooted explants.