Investigation of flowing time, flower structure, microspore density, microspore vitality and microspore-derived embryo (MDE) formation rate according to the light quality treatment on broccoli donor plant was accomplished. The material was 08-8-3 line yielding high MDE production rate having 4.0 ± 0.5 mm flower bud length. The donor plant was cultivated with light quality treatment of red LED light, red+blue+white LED light and fluorescent light. The light intensity was 50 μ molm-2s-1 and photoperiod was 16/8 hours (light/dark). The flowering time was fastest at red LED light treatment compared to the other light treatment condition. 100.0, 36.4 and 18.2% of flower bud with longer stigma length than floral leaf which reported high MDE production rate were found under red LED light, Red+Blue+White LED light and fluorescent lights respectively. The microspore density and MDE production rate per single flower bud was highest at Red LED light. Suitable flower bud and high MDE production rate could be achieved in a short period if using LED light to broccoli donor plant cultivation. The above result is thought to be very useful for the development of a new cultivar of broccoli and other many crops including Brassica using haploid breeding technology. This journal was supported by the National Research Foundation

Heat shock pretreatment, dark culture period and washing medium could have marked effects on microspore embryogenesis. A heat shock pretreatment of microspores at 32.5°C for 48 hours gave high production rate of microspore-derived embryo (MDE) when compare to shorter and longer period. The yield of MDE increased significantly when microspore cultured for 15 days at 25℃ in dark condition followed by heat shock pretreatment. MDE were browned and lost vitality when dark treatment period extended longer than 15 days. This is caused by an insufficient oxygen and light for growing embryo which already formed during dark treatment period. The vitality of a microspore isolated from flower bud stored at 4℃ become decreased at the very first day and the vitality of microspore stored at 4℃ in the form of flower bud itself become decreased from the 5th day after storage. This shows the possibility of getting a certain period of storage for a suitable flower bud in MDE formation. The yield of MDE was most effective when isolated microspore was had with MS medium compared to B-5 and NLN medium and also showed most effective result with sucrose 130 g􀂂L-1 in additional sucrose concentration. The above result is thought to be very useful for the development of a new cultivar of radish and other many crops including Brassica using haploid breeding technology.

Transient expression profiles for several chimeric β-glucuronidase (GUS) gene constructs were determined in microspore-derived embryos of wheat following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by six different promoters [Cauliflower Mosaic Virus 35S (CaMV35S), Nopaline synthase (NOS), Mannopine synthase (MAS), Chlorella Mosaic Virus Adenin methyltransferase (AMT), maize Ubiquitin 1 (UBI1), and enhanced 35S (E35S)]. The total numbers of GUS blue spot were determined manually under a dissecting microscope after histochemical staining for GUS. Results suggest that the E35S promoter is the most active and UBI1 promoter is the second active in embryos or embryogenic calli derived from wheat microspore. In addition, by flurometric assay on GUS, the E35S promoter was the best. Therefore, both UBI1 and E35S promoter are suitable for constitutive expression of the gene of interest in microspore-derived embryos of wheat. This information describing promoter functionality in wheat will be important when designing gene constructs for traits modification and when choosing appropriate cultivars for improvement through gene transfer experiments.

The effect of osmotic condition on β-glucuronidase (GUS) transient expression was evaluated in microspore-derived embryos of wheat. Microspore explants were treated on medium containing various mannitol concentrations prior to and post bombardment with plasmid DNA pAHC25 containing uidA gene controlled by maize ubiquitin 1 (UBI1) promoter. GUS expression in the bombarded explants was examined by histochemical and fluorometric assays. Increased GUS expression was observed with mannitol treatment when compared to untreated explants. The histochemical study showed that the number of blue (GUS) foci were the highest in the bombarded explants treated with 0.6 M mannitol medium. The fluorometric assay of bombarded explants also proved 3.5-fold increase in GUS activity with 0.6 M mannitol treatment when compared to without mannitol treatment. These results indicate that 0.6 M mannitol is beneficial for improving transformation efficiency of wheat microspore-derived embryos or embryogenic calli through biolistic transformation.