Somatic embryogenesis receptor-like kinase (SERK) : aplicabilidade como marcador embriogênico em algodoeiro

Abstract

The release of transgenic cultivars of recalcitrant species such as cotton can be delayed due to the difficulty in regenerating the transformed tissue. Existing regeneration protocols are limited to a few responsive varieties, such as Coker, commonly used as recipients during genetic transformation. One of the factors that may be related to recalcitrance is the inefficiency in the activation of different genes during in vitro culture that may be associated with the acquisition of embryogenic competence. Among the genes mentioned in the literature, the Somatic Embryogenesis Receptor-Like Kinase (SERK) is shown to be responsible for the transition from the somatic cell state to the embryogenic in different plant species. The potential of this gene as a marker of somatic embryogenesis has been investigated. In this work, the relationship of the levels of GhSERK1 expression with the acquisition of embryogenic competence in cotton genotypes was analyzed. For this, a short sequence (186 bp) of this gene was selected from the conserved region between cotton and five other dicotyledons (Theobroma cacao, Citrus sinensis, Vitis vinifera, Glycine max and Phaseolus vulgaris) in order to allow the probe can be applied in the selection of other cultures. Initially, the RT-qPCR gene expression tests were performed in six genotypes with known embryogenic capacity (embryogenic - Coker 312, BRS Rubi and BRS Seridó and non-embryogenic - BRS 201, CNPA Precoce 1 and BRS Topázio), from RNA extracted from meristematic zygotic tissues. The aforementioned genotypes were induced to somatic embryogenesis in medium supplemented with naphthaleneacetic acid and kinetin. Corroborating with the result obtained via RT-qPCR, somatic embryos were formed only in Coker 312, BRS Seridó and BRS Rubi. In the latter, the percentage of fertile plants obtained was higher (50%) than Coker 312 (41.6%). The selective applicability of GhSERK1 was validated in four genotypes top lines (CNPA 286, CNPA BA 139, CNPA BA 1366 and CNPA BA 2247) with unknown in vitro behavior. Simultaneously, we performed induction tests on somatic embryogenesis to verify the expression results. In the genotypes in which the relative expression was higher (Coker 312 and CNPA BA 139), somatic embryos were formed. A nonradioactive probe was then grown from the 186 bp fragment and attested for its efficiency via Southern Blot in the top line genotypes, robust spots were observed in Coker 312 and CNPA BA 139. Based on these results, the latter was selected as the recipient genotype in future transformation studies. In addition, the sequence used allowed to identify genotypes responsive to somatic embryogenesis from zygotic tissues, optimizing time and costs. This work provides collaborations for the understanding of factors that may be involved in the acquisition of embryogenic competence generating perspectives of new studies to strengthen the programs of genetic improvement of the culture.