ABSTRACT
Three cDNAs (iAGPLI-1, iAGPLI-2, and iAGPLI-3) encoding an isoform of AGPase large subunit were isolated from a sweet potato cDNA library constructed from tuberous root tissue. iAGPLI-1 was 2,161 bp in length and contained an open reading frame of 517 amino acids with a calculated molecular mass of 57,689 Da. iAGPLI-2 and iAGPLI-3 were 1,804 and 1,524 bp in length, respectively, and contained partial open reading frames of 490 and 385 amino acids. Deduced amino acid sequence comparison analysis showed that iAGPLI-1 has sequence identity with iAGPLI-2 (97.9) and iAGPLI-3 (98.7%) while iAGPLI-2 and iAGPLI-3 have 96.8% sequence identity. iAGPLI-1 had the highest sequence identity of 77.8% with potato AGPase (sAGPL1). Steady-state levels of iAGPLI-1 transcripts were expressed predominantly in the stem, and moderately in the tuberous root, but not in either the roots or leaves. However, AGPase activity was present in all tissues. The expression level in the stem declined dramatically after a 12 h incubation in the dark to nearly 3% of the value under light, although the activity under a dark condition remained at half the levels in light. The activity levels were not correlated with the transcript levels. iAGPL transcripts in leaves were induced by sucrose feeding but not by glucose or fructose. Therefore, the expression of iAGPLI-1 is regulated in stem tissue preferentially and by sucrose. Southern blot analysis showed that the sweet potato genome contained several copies of iAGPLI gene probably due to polyploidy.
Subject(s)
DNA, Complementary/genetics , Nucleotidyltransferases/genetics , Solanaceae/genetics , Amino Acid Sequence , Blotting, Northern , Blotting, Southern , DNA, Complementary/chemistry , DNA, Plant/genetics , Gene Dosage , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Glucose-1-Phosphate Adenylyltransferase , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Nucleotidyltransferases/metabolism , Protein Subunits , RNA, Plant/genetics , RNA, Plant/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Solanaceae/enzymology , Tissue DistributionABSTRACT
The status of the transgene in tobacco plants transformed by Agrobacterium was analyzed with PCR. Twelve percent of the transgenic plants with the nptII gene showed different levels of transgene deletion, which was also found in transgenic watermelon (10-30%) and carrot (40-60%). It appeared that the percentage of transgenic plants carrying deleted transgenes depended on both the transgene and the plant. It is suggested that the transgene should be inserted between a right border and a selection marker to reduce the number of transgenic plants containing deleted transgenes after selection.
Subject(s)
Gene Deletion , Plants, Genetically Modified/genetics , Agrobacterium tumefaciens/genetics , DNA, Bacterial/analysis , DNA, Bacterial/genetics , DNA, Complementary/genetics , DNA, Plant/analysis , DNA, Plant/genetics , Glucose-1-Phosphate Adenylyltransferase , Nucleotidyltransferases/genetics , Plants, Toxic , Solanum tuberosum/enzymology , Solanum tuberosum/genetics , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F1 Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and α-naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl(-1) 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl(-1) 2,4-D and 1 mgl(-1) abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.
ABSTRACT
Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.
