Identification and characterization of spruce genes involved in somatic embryo development
Law, Derek Albert
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Somatic embryogenesis can provide researchers with an important tool to study the physiological and molecular mechanisms involved in embryo development. In spruce, few lines are able to produce fully developed embryos due to the presence of malformed meristems. Genes from two families known as KNOX (knotted-like homeobox) and ARGONAUTE (AGO) have previously been found to be involved in meristem development and maintenance. This work documents the discovery of a new member of the AGO family of proteins designated as PgAGO and the further study of a KNOX gene known as HBK2. The complete coding sequence of both PgAGO and HBK2 was obtained through screening of cDNA libraries generated from white spruce (Picea glauca) somatic embryos. RNA in-situ hybridization studies showed that PgAGO mRNAs accumulate preferentially within cells of the shoot and root apical meristems in developing spruce embryos. In addition, the expression of PgAGO was low in white spruce lines unable to produce embryos in culture. Norway spruce (Picea abies) embryogenic tissue was transformed via microprojectile bombardment with an antisense construct of PgAGO. Down-regulation of PgAGO altered proper development of the apical meristems and reduced embryo regeneration. RNA in-situ hybridization studies showed that HBK2 is specifically expressed in the sub-apical and cortical regions of developing embryos. Like PgAGO, HBK2 expression was diminished in white spruce lines unable to produce embryos in culture. Transformation experiments with antisense constructs of HBK2 completely arrested somatic embryo development. This study reveals the importance of a functional meristem during embryo development.