High-frequency regeneration and <i>Agrobacterium tumefaciens</i> -mediated transformation of broccoli ( <i>Brassica oleracea</i> var. <i>italica</i> )
Bibliographic record
Abstract
Summary An efficient protocol for high frequency regeneration and Agrobacterium tumefacies mediated transformation of Broccoli ( Brassica oleracea var. italica ) has been developed. Hypocotyl and leaf segment explants were transferred on to MS medium supplemented with different concentrations and combinations of IAA (0-0.57 μM) and Kinetin/BAP/Zeatin (8.9-22.2 μM). Hypocotyl explants showed the highest regeneration potential. Maximum number of shoots (16 per explant) were obtained on MS medium supplemented with 0.57 μM IAA and 22.2 μM BAP. The concentrations of extracellular calcium, nitrate and ammonium nitrogen in the medium had a marked effect on regeneration. The optimal media for maximum shoot proliferation (30 per explant) contained 3 mM CaCl 2 , 1900 mg l -1 KN0 3 , 825 mg l -1 (NH 4 ) 2 S0 4 , 22.2 μM BAP and 0.57 μM IAA. Rhizogenesis from isolated shoots was obtained on B5 medium supplemented with 2.85 μM IAA. The high frequency regeneration system served as an excellent tool to establish an efficient transformation method of broccoli. Hypocotyl explants were co-cultivated with the Agrobacterium tumefaciens strain LBA 4404, containing a binary vector with the neomycin phosphotransferase ( nptii ) gene and a β-glucuronidase ( gus ) gene, each under the control of a separate CaMV35S promoter. Primary selection of transformed explants was performed on high kanamycin (100 mg l -1 ) containing medium for 10 days and regeneration was achieved on the optimised regeneration medium containing lower concentration (50 mg l -1 ) of kanamycin. The frequency of transgenic plants was calculated on the basis of GUS activity detected by histochemical X-gluc test. The effect of pre-culture and growth of bacterial culture was studied. Explants precultured for 2-days prior to inoculation with A. tumefaciens resulted in improved transformation frequency. Bacterial cultures grown to the optical density of 2.0 resulted in the highest transformation rate (60%). Evidence for integration of nptii gene was obtained by the PCR. All transgenic plants grew normally. The present study demonstrates the improved high frequency regeneration and transformation of B. oleracea var. italica .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".