Cryopreservation of mature zygotic embryos, shoot bud regeneration, and feld establishment of Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis in vitro‑derived plants

AYALA, L; LUNA C; ELSA A. BRUGNOLI; ESPASANDIN F.; DUARTE, MJ; GONZALEZ A.M; GAUCHAT, ME; MONCALEÁN GUILLÉN, P; SANSBERRO P

TREES-STRUCTURE AND FUNCTION

SPRINGER

Lugar: Berlin; Año: 2023 vol. 37 p. 417 - 443

0931-1890

The developed protocol for organogenesis, in vitro plantlet production, and cryopreservation opens the possibility for mass propagation of hybrid pine (Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis).Abstract The low seed production of the interspecifc hybrid Pinus elliottii var. elliottii x Pinus caribaea var. hondurensis restricts its commercial expansion, making it necessary to ensure efcient cryopreservation and a propagation protocol with no genetic variability. Mature zygotic embryos (MZEs) were cultured in Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and thidiazuron (TDZ). After 45 days in culture, the highest rate of regeneration (86.7±8.8%) and the maximum number of diferentiated buds per responsive explant (15.5±2.8) were achieved from explants cultivated on MS with BA and TDZ (0.5 μM each). Cryopreservation of zygotic embryos using a simple desiccation step and a direct immersion into liquid nitrogen did not afect regeneration and would enhance embryo storage duration. Half-strength MS enriched with sucrose (0.09 M) and gelled with gellan gum (4 g L− 1) under forced ventilation culture was used for shoot elongation. Subsequently, 73±6.7% of shoots produced roots after pretreatment with 1.25 mM indole-3-butyric acid solution for 5 min and culture on quarter-strength MS with sucrose (0.045 M). The regenerated plantlets were successfully transferred to ex vitro conditions. The procedure took 160 days and comprised the adventitious bud formation from cryopreserved MZEs, shoot elongation, rooting, and plantlet acclimation. Considering that water defcit is the major strain during forest establishment, a controlled experiment was carried out to determine the competence of plantlets to overcome this stress. Next, feld studies assessed the survival rate and growth of 16-month-old plants. Our results indicated that the feld performance of tissue-culture-derived plants is similar to seedlings and rooted cutting plants. Additionally, inter-simple sequence repeat marker analysis revealed the genetic uniformity among the in vitro-raised plants, demonstrating the reliability and validity of the procedure. Thus, the developed regeneration and cryopreservation protocol for mature zygotic embryo explants is a valuable alternative for breeding programs and commercial P. elliottii x P. caribaea propagation.