CELL DIVISION AND EMBRYOGENIC COMPETENCE IN ALFALFA {Medicago falcata L.)

This investigation exploited a procedure for the production of somatic embryos directly from alfalfa leaf explants. Chopped leaf fragments were incubated in a liquid medium containing 2,4-D. The organisation of the swollen leaf tissue was disrupted and after 15 days the globular clusters of small dividing cells (pro-embryos) which had formed within the leaf tissue were released into the medium. After transfer to a development medium, the pro-embryos proceeded through the classical heart and torpedo stages to the regeneration of whole plants. The role of cell division regulation during this process was investigated. After 1-2 days, the incorporation of 5-bromodeoxyuridine was observed only in cells flanking the vascular elements of the leaf but by three days virtually all of the leaf cells were active in DNA synthesis. After 10-15 days, DNA synthetic activity was concentrated in the globular pro-embryos, but was still observed in non-embryogenic cells. The expression of cell cycle regulatory genes in the induced alfalfa leaf explants was studied by in situ hybridisation and transformation with promoter:guzA constructs. Direct and indirect transformation and regeneration systems have been developed. Transgenic plantlets were produced within 10-14 weeks in the direct (MSH) system and 12-16 weeks in the indirect (Bsh) system. The MSH system appears to be the fastest transformation system reported for Leguminous species to date. The successful transformation of embryogenic lines 47/1-150 and 47/1-5 has facilitated the investigation of the regulation of cell division during direct somatic embryogenesis. A comparison of the expression from Arabidopsis cdc2a, cyc1a and cyc3a promoters showed that in all three cases there was a marked increase in expression two days after induction. Histochemical staining for GUS activity and in situ hybridisation analysis showed the same pattern as for DNA synthesis; initial activation in the vascular tissue prior to expression throughout the entire leaf tissue. However, by ten days after induction, cyc1 expression was restricted to the globular pro-embryo clusters within the leaf, whilst cdc2a and cyc3a expression remained detectable within the surrounding leaf tissue. The expression of all three genes and subsequent pro-embryo formation was induced by 2,4-D treatment alone. Neither kinetin nor wounding was able to induce cdc2a nor cyclin gene expression in the absence of 2,4-D, and somatic embryogenesis did not occur under these conditions. 2,4-D treatment for as little as 1 hour was sufficient to induce cell cycle gene expression, but roots, rather than somatic embryos, were formed after leaf explants were transferred to development medium. The patterns of cell growth and division and cell cycle gene expression during the induction phase were similar after one hour treatment or during continuous exposure to 2,4-D. It is concluded that the activation of cell division is a necessary component of embryogenesis competence, but somatic embryogenesis requires the continued presence of 2,4-D. Preliminary studies of the expression of genes isolated by subtractive hybridisation were made by dot blot array hybridisation. The expression patterns of a large number of different clones have been identified, some may prove to be useful molecular markers for specific stages of embryogenesis. The most abundant mRNAs were those encoding ribosomal proteins. Most of the sequences could be detected on the third day after induction and they generally reached a peak of expression after 5-10 days. This catalogue of gene expression provides an entry point for a comprehensive analysis of gene regulation during early embryogenesis.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2000

Author dc:creator

• SHAO, CUI-YING

Licence

• https://dora.dmu.ac.uk/bitstreams/edee86e8-46cb-4b6e-b417-1fad81bc61a5/download