Broccoli is a crop of commercial importance, in the UK alone 7,086 hectares of broccoli was planted in 2007 (DEFRA). Consumers include broccoli in their diets as it is a vegetable with many health promoting properties (Leja et al, 2001). Broccoli is known as a ‘functional food’ as it contains high levels of vitamins, minerals, glucosinolates and flavonoids (Vallejo et al, 2003; Heimler et al, 2006). These phytochemicals are known to have a protective role against cancer and cardiovascular disease (Bengtsson et al, 2005; Vallejo et al, 2004). Broccoli heads are composed of immature florets arrange in whorls on a fleshy stem (Buchanan-Wollaston et al, 2001; Nishikawa et al, 2005). Each floret contains hundreds of immature flower buds; the sepals that protect the flowers are rich in chlorophyll, which gives broccoli its characteristic green appearance (Buchanan-Wollaston et al, 2001; Nishikawa et al, 2005). However, during storage the sepals become susceptible to rapid yellowing (Buchanan-Wollaston et al, 2001: Nishikawa et al, 2005).
In broccoli, senescence in induced prematurely as a result of harvesting and post-harvest stresses (Coupe et al, 2003; Gapper et al, 2005). This initiates rapid deterioration and quality loss such as senescing of buds, opening of buds to expose the petals, softening of tissue and wilting, all of which causes broccoli to become unmarketable (Wurr et al, 2002; Serrano et al, 2006). Post-harvest senescence is also observed in other perishable crops such as asparagus, lettuce and cauliflower (King & O’Donoghue, 1995; Downs et al, 1997). As with broccoli, these crops are all harvested before reaching maturity and are still undergoing rapid pre-harvest growth (King & O’Donoghue, 1995; Downs et al, 1997). Deterioration in young plant organs such as those described above is associated with the inability to maintain metabolic homeostasis (King & O’Donoghue, 1995), as losses due to respiration and transpiration can no longer be replaced (Brosnan & Sun, 2001). Decapitation of the crop removes the supply of energy, water, nutrients and hormones and therefore triggers the onset of post-harvest senescence (Brosnan & Sun, 2001). Other factors that promote post-harvest senescence are respiration rate, storage temperature and sensitivity to ethylene (Brash et al, 1994; Albanese et al, 2007). As broccoli is harvested when it is immature it has a high respiration rate, which has been shown to increase with temperature (Brash et al, 1995). Respiration rate and storage temperature are limiting factors of shelf life, as broccoli heads stored at 10oC have a respiration rate of 38-43 ml CO2/kg.hr rising to 80-90 ml CO2/kg.hr when stored at 15oC (UC Davis). Broccoli stored at 10oC has a shelf life of ~5 days. However, most retailers issue shelf life guidelines of only 4 days from delivery to consumption (Wurr et al, 2002). The presence of ethylene is also a limiting factor of shelf life. Endogenous ethylene has a detrimental effect on broccoli as it is a regulator of chlorophyll loss (Gong & Mattheis, 2002; Suzuki et al, 2005). Ethylene stimulates chlorophyllase activity, an enzyme in the chlorophyll breakdown pathway, which dephytylates chlorophyll to produce chloropyllide (Hortensteiner, 2006; Gong & Mattheis, 2002).
Post-harvest senescence in broccoli is also associated with a decline in metabolites. Unlike humans, plants have the ability to synthesize L-ascorbic acid (vitamin C), which is crucial for the modulation of plant development through hormone signalling (Valpuesta & Botella, 2004). Humans however, have to acquire vitamin C through consumption of fruits and vegetables (Leskova et al, 2006; Kurilich et al, 1999). Vitamin C is an essential part of the human diet and has many physiological roles within the body; it acts as a co-factor to enzymes involved in the synthesis of collagen, carnitine and neurotransmitters (Naidu, 2003). Vitamin C is also with associated with general health and well being, it has been shown to have a role in the prevention and relief of common colds and is essential for wound healing and repair (Naidu, 2003). The benefits of vitamin C also prevent diseases such as cancer and atherosclerosis (Nadiu, 2003). The recommended daily allowance for vitamin C is 60 mg per day for adults (FSA). A 100g serving of raw broccoli provides 89.2 mg of Vitamin C with 100g of cooked broccoli (boiled) providing 64.9 mg of vitamin C (USDA nutritional database). However, it is unclear how stable vitamin C content is once broccoli has been harvested and when the optimal time (days) after harvest is to consume broccoli to benefit most from the active phytochemicals it contains.
Post harvest quality loss, such as bud yellowing, the loss of turgor causing and the decline in nutrients, are the main concerns for broccoli growers, retailers and consumers. Kader (2002) states that a high percentage of harvested fruit and vegetables are never consumed, estimating post harvest losses of between 5% and 25% for fruit and vegetables. To reduce post harvest losses in broccoli, shelf life needs to be extended, and a logical approach to do this is to utilise the natural variation displayed within broccoli cultivars. Evidence suggests that shelf life and vitamin C content are under genetic control as these traits vary with genotype. A study by Buchanan-Wollaston et al, (2003) investigated the shelf life length of doubled haploid lines derived from the commercial variety Marathon. Although the doubled haploid lines used in the study shared the same genetic makeup, the shelf life of lines varied, suggesting that different combinations of genes affect shelf life length.
By utilising natural variation seen within the mapping population, improved shelf life and vitamin C content can be selected for and bred into existing commercial cultivars. The use of doubled haploid (DH) lines to study agronomic traits such as yellowing and vitamin C content has many advantages. Populations of DH lines are homozygous and ‘genetically fixed’ which allows the performance of DH lines to be evaluated in replicated field trials indefinitely (Pink et al, 2008). This allows the collection of vast amounts of phenotypic and quantitative data. Doubled haploid lines are also useful in the construction of genetic linkage maps, as they can easily be scored, as only one parental genotype will segregate at each locus (Snape and Simpson, 2004). Many published B.oleracea maps use DH lines such as the integrated B.oleracea map produced by Sebastian et al, (2000). The integrated map consists of marker information from the AG population and the NG population. The AG population was produced from a cross between a DH line A12DHd of B.alboglabra and a DH line GDDH33, derived from the F1 broccoli cv. Green Duke, where as the NG population was produced from a cross between DH lines derived from the cauliflower cv. Nedcha (CA25) and a DH line derived from the Brussel sprout cv, Grower (AC498).
Another advantage of DH lines is the identification of QTLs associated with quantitative traits. In this study this has the potential to identify senescence associated genes (SAGs) and vitamin C biosythesis (VTC) genes found in QTL regions (Buchanan-Wollaston et al, 2003). Chen et al, (2008) reports on the identification of 42 SAGs within the senescence community that are associated with broccoli florets, these included ethylene related genes, metabolic related genes and genes with other functions, but the location of these genes in the genome is unknown. In Arabidopsis thaliana 4 VTC gene have been identified and mapped, providing the potential for gene orthologs to be identified in broccoli.
The main aim of this project is to exploit natural variation in various aspects of broccoli shelf life to identify genetic loci and genes that improve post harvest quality. A new broccoli x broccoli linkage map is being developed which will be used to identify QTL relating to shelf life, vitamin C content and vitamin C stability. Eventual identification of the genes controlling these traits is the long term aim.
- To develop a new broccoli x broccoli framework linkage map
- To characterise the shelf life phenotypes of the DH mapping population.
- To determine the vitamin C content of DH lines at harvest and throughout storage.
- To map QTLs for shelf life, vitamin C content at harvest and vitamin C stability during storage.
- To identify and map vitamin C biosynthesis gene orthologs from Arabidopsis thaliana in broccoli.