In vitro conservation of citrus rootstocks using paclobutrazol and analysis of plant viability and genetic stability

The vulnerabilities to which citrus genetic resources are exposed under field conservation conditions make it important to ensure an in vitro backup. Thus, in this study, we aimed to establish a protocol for in vitro conservation of citrus rootstocks by analyzing the use of paclobutrazol to maintain a minimum growth and ensure the viability and genetic stability of conserved plants. In the first experiment, 1 cm-long apical microcuttings from plants of five rootstocks grown in vitro were placed in basal woody plant medium, supplemented with 0.2, 0.4, 0.6, 0.8, and 1.0 mg L-1 of paclobutrazol, and maintained in a conservation room at a temperature of 22 +/- 1 degrees C, photoperiod of 12 h, and photon flux density of 20 mu mol m(-2) s(-1) for 6 months. After this period, the plants from the medium selected for in vitro conservation of the rootstocks were acclimatized. For the second experiment, nucellar seedlings (confirmed by simple sequence repeat [SSR] markers) of 10 citrus rootstocks were multiplied and conserved in the previously defined medium. At the end of 12 months, the plants were subjected to three subculturing cycles to verify their viability. Molecular analyses were performed on plants from the third subculture using 21 SSR markers to detect somaclonal variants. Paclobutrazol was not efficient in maintaining reduced plant growth. Acclimatized plants grown in vitro exhibited 100% survival. SSR markers were efficient in characterizing the studied rootstocks. In general, the in vitro conservation conditions did not affect plant viability. The three subculturing cycles performed after conservation did not result in somaclonal variants. The conservation protocol described in this study can be used for the in vitro maintenance of the analyzed rootstocks and can be extended to other citrus genotypes.

Automated summary

The study aimed to establish a protocol for in vitro conservation of citrus rootstocks using paclobutrazol to ensure plant viability and genetic stability. Results showed that in vitro conservation did not affect plant viability, and molecular markers were efficient in characterizing the rootstocks. The protocol described in the study can be used for in vitro maintenance of the analyzed rootstocks and potentially other citrus genotypes.