MOLECULAR ASPECTS OF DIRECT SOMATIC EMBRYOGENESIS IN ALFALFA (MEDICAGO)

Somatic embryogenesis follows the developmental pattern of the zygote in vitro so it provides a model for early plant development which is more accessible for experimental manipulations. Most of the fundamental research in this area has exploited indirect embryogenic systems which require cells to pass through a stage of dedifferentiation before they became committed to produce somatic embryos. In contrast, direct somatic embryogenesis proceeds from cells within organised tissues, such as leaves, which are termed Pre-Embryogenic Determined cells (PEDCs). Such cells can be directed to produce embryos by external stimuli (wounding and auxins) without cell dedifferentiation. In this system, embryogenic competence and the initial embryogenic cell divisions can be linked to expression of the genes (such as cyclin-dependent kinases and cyclins) which determine and regulate cell division competence and cell division, respectively. A system previously established for studying direct somatic embryogenesis from differentiated leaf cells of Medicago falcata was used for this investigation. A RT-PCR strategy has been used for cloning two A-like cyclin genes termed cycMfl and cycMf2 and a cdc2 cDNA fragment from alfalfa. The full cDNA and genomic sequence of cycMfl was determined by the RACE procedure. Several cycMfl 5' end cDNA sequences, representing different allelic variants in the tetrapioid alfalfa genome, were also isolated. The expression of the cycMfl, cycMf2 and cdc2 genes was characterised by Northern, whole-mount and in situ hybridization analysis during alfalfa plant development and/or during the early stages of direct somatic embryo induction. cycMfl together with cdc2 was expressed in non-dividing but embryogenically competent leaf cells whereas cycMf2 was detected only after the initiation of embryogenic cell divisions. Wounding and phytohormone treatment of the leaf explants did not significantly change the level of cdc2 RNA during the induction period. The expression of cycMft, however, was transcriptionally activated upon induction treatment but no cell- or tissue specific localization of the transcripts in the alfalfa leaves was detected. Thus, the presence of cycMf1/cdc2 inactive complexes in embryogenic leaf cells may determine a high degree of cell division competence which could be an important feature of embryogenic competence. However cycMft expression is certainly not the sole factor in determining the embryogenic pathway.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

1996

Author dc:creator

• Russinova, Eugenia Tzvetanova

Licence

• https://dora.dmu.ac.uk/bitstreams/7960a764-0449-41b8-a2c4-bc432e6fdcef/download