Tissue culture and agrobacterium-mediated gene transfer in citrus

Tissue culture cloning can aid in molecular genetic engineering of pummelo and tangerine. Immature seeds, with their micropylar end removed, of 'Thong Dee' pummelo were cultured on modified Murashige and Tucker (1969) (MT) media with combinations of 30 and 50 mg/l sucrose, 0 and 500 mg/l malt extract (ME), 0 and 20 mg/l BA and 0 and 0.1 mg/l 2,4-D. Both compact callus and friable callus were formed on all media. Immature seeds, with their mycropylar end removed, of 'Thong Dee' pummelo were cultured on modified MT media with 50 g/l sucrose, 500 mg/l ME and combination of 0, 5, 10, 15, 20 and 25 mg/l BA and 0 and 0.1 mg/l 2,4-D. The highest percentage of compact callus formation (34.6 percent) and cotyledon like bodies (clbs) (7 percent) were observed on the medium containing 5 mg/l BA. The highest percentage of friable callus formation (11.6 percent) were observed on the medium containing 25 mg/l BA. When both types of callus were cultured on the media containing 0.5 and 5.0 mg/l BA in combination with 0 and 0.1 mg/l 2,4-D or 0 and 0.1 mg/l NAA, clbs were developed only from friable calli on the medium with 5 mg/l BA and 0.1 mg/l 2,4-D. Two types of callus and clbs derived from friable calli and immature seeds were cultured on the medium containing 5 mg/l BA with 0 and 0.1 mg/l 2,4-D and 0, 1, 2 and 4 mg/l GA. The highest number of shoot were obtained from clbs derived from either friable calli or immature seeds on the medium with 5 mg/l BA and 4 mg/l GA. Immature seeds, with their micropylar end removed, of tangerine were culture on modified MT media with 50 g/l sucrose, 500 mg/l ME and combinations of 5 mg/l BA and 0, 0.01, 0.05 and 0.1 mg/l 2,4-D. The highest frequency of compact callus formation (5.7 percent) were obtained on the media with 5 mg/l BA and 0.1 mg/l 2,4-D. Whilst the highest frequency of friable callus formation (8.3 percent) were obtained on the media with 5 mg/l BA and 0.05 mg/l 2,4-D. When calli were cultured on the media containing 0.1, 0.5 and 1.0 mg/l BA, 2 g/l activated charcoal was only the best for shoot formation on compact callus. Embryoid formation was founded on the medium containing 18.0 g/l galactose and 18.0 g/l sorbitol. Embryogenic callus of tangerine were co-cultivated with 1/2 dilution of Agrobacterium tumefaciens suspension with 200 micro M acetoslyringone for 2 days. Based on GUS assays, acetosyringone could enhance transformation frequency. The expression of GUS gene in 3 areas on tangerine callus cells. Embryoid were formed when placed the transformed calli on a medium containing 18.0 g/l galactose, 18.0 g/l sorbitol and 200 mg/l kanamycin.