4.3 Ethephon releases the germination of dormant seeds of M. zehntneri
The treatments with phytoregulators have been used as a way to break dormancy in seeds of different Cactaceae species. The most important effects observed in the present study with M. zehntneri occurred when seeds were pre-treated with Ethephon (2-chloroethylphosphonic acid). The treatment using Ethephon promoted a germination percentage of 98% seeds in the culture medium free of other phytoregulators, compared to the 76% observed in the same culture medium without pre-treatment, and promoted the highest values of AGS and SGI.
Different commercial products containing Ethephon have been used as a direct substitute for the ethylene gas hormone, as it is easy to apply and effective in releasing ethylene in an alkaline solution (Zhang et al., 2010). In this way, Ethephon was the unique treatment promoting the germination of dormant seeds in M. zehntneri , but the knowledge about how this plant growth regulator affects cactus development is still limited. Among the few reports, ethylene is responsible for the closing, wilting, and pollination of flowers (Doorn 2002) and fruit ripening (Esparza et al. 2006) in some cactus species. However, the effects of ethylene on releasing seed dormancy in Cactaceae have not yet been reported.
Although little explored and used in Cactaceae, ethylene is considered a key hormone that regulates dormancy and seed germination, as well as the establishment of seedlings after germination in many plant species. This hormone is effective, at concentrations from 0.1 to 200 µL L-1, in releasing seeds from dormancy (Corbineaeu et al. 2014). In the present study, the pre-treatment with ethephon was effective to release the germination of M. zenhtineri seeds when applied by immersing seeds in a solution at 100 µL L-1for 24-h using the commercial product Ethrel®(Bayer®, Brasil), which contains 240 g L-1 Ethephon, thus, at a concentration of 24 µL L-1.
Ethylene acts in the release of seeds from dormancy, especially involving a complex interaction with other hormonal groups in seeds, such as ABA, Gibberellins, Nitrous Oxide (NO), and reactive oxygen species (ROS) (Arc et al. 2013). Different studies have demonstrated that, through the use of ethylene biosynthesis and action inhibitors, ET insensitive mutants, and the use of Ethylene biosynthesis precursors, such as 1-aminocyclopropane 1-carboxylic acid (ACC), ethylene is involved in overcoming the dormancy and promoting germination (Corbineau et al. 2014). In addition, this hormone is also capable of neutralizing the effects of ABA on seed dormancy.
Among the plant growth regulators used to study germination, one of the most used was Gibberellic Acid or GA3 (Mascot-Gómez et al. 2019). This plant growth regulator has shown divergent results in releasing dormant seeds of Cactaceae, in some cases even reducing the percentage of germinated seeds, as observed in Ferocactus species (Amador-Alférez et al. 2013). In the present study, seeds of M. zenhtneri did not respond to the presence of GA3 at 1.0 mg L-1 in the culture medium (72% germinated seeds) and did not differ from the control without phytoregulators (76% germinated seeds). These results differ from the in vitrogermination of M. sergipensis , in which the addition of GA3 at 2 mg L-1 for 6 hours increased the percentage of seeds germinated in this species. However, the maximum percentage of germinated seeds observed for this species and this treatment was 38% (Bravo Filho et al. 2019), much lower than observed in our study with M. zenhtnerii .
The main justification for the wide use of GA3 for overcoming seed dormancy is the fact that germination release in dormant seeds is associated with a balance between Abscisic Acid (ABA), which maintains seeds dormant, and some gibberellins such as GA3, which promotes seed germination (Yazaki and Kikuchi 2005; Rodríguez-Gacio et al. 2009; Yang et al. 2020). The external application of GA3 in seeds results in increased gibberellin contents in the embryos, which leads to dormancy overcoming and germination release in some species. Nevertheless, the limited positive results to increase germination by the use of GA3 in several cacti species may be related to the differential sensitivity of seed tissues to GA3 (Kucera et al., 2005). In addition, recent studies reported that the differential sensitivity of tissues to GA3 and ABA is also caused by the epigenetic control of the metabolism of GA3 and ABA (Sano and Marion-Poll, 2021; Smolikova et al. 2021).
Cytokinins and ethylene have been identified as hormonal groups that upregulate themselves and help to overcome seed dormancy (Zdarska et al. 2015). However, in the present study, the addition of BAP, a synthetic cytokinin, or the BAP + GA3 combination in the culture medium reduced the effects associated with germination promoted by the pre-treatment containing Ethephon. For example, in treatments containing this cytokinin in the culture medium, there were no differences in the percentage of germinated seeds pre-treated or not with Ethephon. The effects of cytokinins on the germination of Cactaceae seeds are practically non-existent, although positive effects of cytokinins both in increasing the germination percentage and GSI have been demonstrated in species from other plant families (Nikolić et al. 2006; Wang et al. 2011). In our study on Melocactus , BAP, and GA3reduced the GSP compared to the Ethephon pre-treatment. Cytokinins also have other positive effects, such as kinetin (KIN) and BAP that were successfully used to promote organogenesis in in vitro cladodes segments of Melocactus glauscecens , allowing the in vitroclonal multiplication of this species (Torres-Silva et al. 2018).
Other groups of phytoregulators, such as 3-indoleacetic acid (AIA), caused no effects on the germination of different Cactaceae species (Mascot-Gómez et al. 2019) or even inhibited the germination of these seeds (Amador-Alférez et al. 2013), such as the putrescine used for germination of Turbinicarpus lophophoroides and T. pseudopectinatus (Flores et al. 2007). Salicylic acid and Acetylsalicylic acid also considered phytoregulators, completely inhibited the germination of several cactus species (Mihalte et al. 2011).
Conclusions
Red light effectively contributed to the germination of M. zehntneri seeds, while blue light significantly decreased the percentage of germinated seeds. The exposure of seeds to darkness during germination, even for short periods, leads seeds to secondary dormancy. However, the greatest effect observed in this study on dormant seeds ofMelocactus was found with the use of ethephon, which induced 98% seed germination. The present study also can serve as a reference for breaking dormancy and promoting seed germination in Melocactus .In vitro cultivation can still serve as a tool for studying the germination and propagation of cacti species, aiming at their large-scale production or even conservation. The present study also pointed to a possible new mechanism that explains seed dormancy in Cactaceae seeds: the differential biosynthesis or sensitivity of cacti embryos to ABA.
Acknowledgements: The authors thank Willian Naves Duarte and Matheus Armelim Nogueira for their intellectual contributions to this manuscript.
Finnancial support: This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) (Process number 2021/01814-8) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ, Brazil) (Process number 311083/2018-8).
Conflict of interest declaration: Authors declares no conflict of interests