Genetic engineering has enormous potential for crop improvement by rectifying single defects in elite crop varieties, and introducing novel traits into plant breeding programs. To realise this, it is important that transgenes are stably integrated and expressed in the primary transformants and in subsequent sexual generations. Moreover, the engineering process should not significantly alter the genetic integrity of the elite parent variety, otherwise expensive conventional breeding processes would be required to rectify the "collateral genetic damage' incurred.
Prior to this study, transgene inheritance and expression in sexual sugarcane progeny was undemonstrated. Also, field trials showed that most transgenic sugarcane lines regenerated from microprojectile-bombarded embryogenic callus were lower yielding than their 'parent' variety. It was considered plausible that these yield losses could have resulted from DNA sequence or DNA methylation changes as a consequence of tissue culture or gene-transfer. This thesis describes molecular and genetic analyses of transgenic and tissue-culture-derived sugarcane in order to demonstrate transgene inheritance and expression in sugarcane, and to investigate the source and molecular basis of yield loss in transgenic sugarcane.
Transgenic lines 78N146-7 and NCo310-D2 containing the selected marker (aphA) and luciferase reporter (luc) genes co-bombarded on separate plasmids, were crossed as females to non-transgenic sugarcane (Chapter 2). Pollen infertility of the transgenic parents precluded confirmation of paternal transgene inheritance. In both progenies, aphA and luc co-segregated by PCR and co-expressed at a ratio of 1:1. Southern analysis confirmed the inheritance of all transgene copies, and indicated that transgenes co-bombarded on separate plasmids tend to integrate into the sugarcane genome at linked sites interrupted by sugarcane DNA. In line 78N146-7, Southern analysis also detected non-expressed, truncated transgenes which segregated from the functional inserts without affecting the expression of the functional copies. Most importantly, transgene activity levels were similar in the progeny and their transgenic parent. Thus, transgenes can be introduced into sugarcane breeding programs with predictable Mendelian inheritance of the associated traits.
Efficient molecular marker technologies were used to characterise genomic changes in non-transgenic, tissue-culture-derived sugarcane clones of cvs. Q155 and Q117 (Chapter 3). A novel marker technology called amplified DNA methylation
polymorphism (AMP) was developed to detect DNA methylation changes at thousands of 5'-CCGG-3' sites dispersed throughout the genome. Surprisingly, all 24 tissue-culture clones assessed had widespread DNA methylation changes and unique AMP profiles. On average, 0.85% of 1,050 AMP markers were polymorphic between the tissue-culture clones and the parent variety, and both gain and loss of methylation was detected. Conventionally propagated plants showed DNA methylation polymorphisms at about half the level detected among tissue-culture clones. Randomly amplified DNA fingerprint (RAF) detected DNA sequence changes characterised by the loss of RAF fragments in 20% of the tissue-culture clones, indicative of probable DNA loss. These results indicate that collateral genetic damage could arise from metastable DNA methylation changes as well as stable DNA sequence changes.
RAF analysis detected DNA sequence alterations in all eight transgenic sugarcane lines assessed, all of which exhibited yield loss (Chapter 4). Between 0.4% and 2.4%o of RAF loci were lost in the transgenics. Segregation analysis showed that RAF loci lost in the transgenics were indicative of whole or partial chromosome loss. The extent of RAF polymorphism seemed to correlate with yield loss, but the correlation was not statistically significant. Co-dominant simple sequence repeat (SSR) markers were also lost in all transgenic lines, and the extent of SSR marker loss (0.8- 6.2%) was significantly correlated with yield loss in the transgenics (P = 0.017). AMP detected extensive DNA methylation changes in the same transgenic lines but the effect on yield loss could not be determined due to confounding effects of DNA loss.
Together, these results indicate that genomic changes arising during the tissue culture and gene-transfer stages of genetic engineering for sugarcane improvement are likely to result in a low frequency of commercially useful transgenic lines (low useful transformation frequency despite high absolute transformation efficiency). The DNA marker technologies described here provide ideal tools to identify tissue culture conditions that may minimise genomic change. Meanwhile, molecular marker analyses coupled with assays for desired transgene expression patterns, will be useful for the early selection of potentially elite genetically engineered sugarcane plants.