Expression of two promoter-GUS fusion genes in Brassica oleracea var. italica: A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Lincoln University

Abstract

Two chimeric gene constructs, consisting of the β-glucuronidase (gus) gene under the control pf either the asparagus (Asparagus officinalis) asparagine synthetase (AS) promoter (AS/G), or the Antirrhinum majus myb305 promoter (MYB/G), were introduced into broccoli (Brassica oleracea var. italica), so that expression patterns of these promoters could be studied by GUS histochemical assays throughout the broccoli life-cycle and during post­harvest.

Multiple regeneration and transformation experiments were conducted in an attempt to develop a high efficiency Agrobacterium tumefaciens-mediated transformation system. Regeneration experiments were conducted for three explant sources (cotyledons, light­ germinated hypocotyls and dark-germinated hypocotyls); three broccoli cultivars (Shogun (Sh), Marathon (Mt) and Green Belt (Gt)); and for three concentrations of N⁶ benzylaminopurine (BA) (1.0, 2.0 and 5.0 mg/L). The treatment interactions which significantly affected variation (p :'.5: 0.05) changed over the time course of the experiment. The culture conditions that produced the best results after 28 days of tissue culture were applied to subsequent transformation experiments. At this point the treatment interactions showing significant variation were Cultivar X Explant and [BA] X Cultivar. Sh and Mt dark­germinated hypocotyls produced a significantly higher percentage of explants regenerating shoots than other explant types (81.7% and 62.8% respectively, light-germinated 26.9% and 15.3%, cotyledons 56.5% and 13.9%). Dark-germinated hypocotyls on 2 mg/L BA regenerated significantly more shoots (63.9%) than dark grown hypocotyls on 1 mg/L BA (50% ). After 44 days of tissue culture, [BA] no longer had a significant effect on regeneration. However, explant and cultivar treatments still exerted a significant effect on the percentage of explants regenerating shoots.

Transformation experiments were conducted to test the effect of a number of factors on transformation. These used A. tumefaciens strain LBA4404, two explant sources (light germinated hypocotyls, dark-germinated hypocotyls), the three broccoli cultivars used previously, two acetosyringone levels (0.0 µMIL and 5.0 µMIL), two co-cultivation dish sealants (Gladwrap and Micropore tape (3M)) and co-cultivation conditions of light vs dark. The transformation efficiency in these experiments was too low for statisical analysis, but statisical analysis was conducted on data for explant mortality and shoot regeneration. The percentage of explant mortality was shown to be affected by cultivar (Experiments 9 and 12, Appendix 7) (Sh 37 .5%, 40.6%, Mt 60.1 %, 60.6%, Gt 60.1 %, 68.1 % ) and sealant (Micropore 37.9%, Gladwrap 67.2%).

Thirty-five AS/G transformants from five plant cultivars and thirty-eight MYB/G transformants from two plant cultivars were obtained. Plants from sixteen lines of each were transferred to a containment greenhouse. Transformation was confirmed using growth on selective levels of kanamycin as well as by molecular analyses. Molecular analyses involved PCR and Southern hybridisation to confirm the presence of the GUS reporter gene and the npt II selectable marker gene.

GUS expression was observed in both AS/G and MYB/G derived transformants using the X-Glu (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) histochemical assay. The AS promoter was sufficient to drive high levels of transgene expression. A post-harvest increase in GUS expression was found in the leaves of transformants (1-4 months in the greenhouse), 24 hours after excision. These same plants were shown to be relatively insensitive to light regulation. Expression was shown not to be dependent on post-harvest (time-24 h), and evidence for sugar dependent down-regulation and nitrogen dependent up-regulation of asparagus AS promoter activity was obtained.

The myb305 promoter was also sufficient to drive gus transgene expression in broccoli. GUS expression was observed in the sepals of mature (open) flowers, in stems at leaf axils, at the base or heal of leaf petioles when tom from the stem, in the vascular tissue of leaf petioles, at the base of cauline leaves when torn from the stem and in the leaf of one MYB/G line.