| The oilseed Brassica species (B. napus, B. rapa or B. campestris, and B. juncea) are one of the most important vegetable oil and protein-rich meal crops in the world. Its cultivation has increased tremendously during the last decade and, by now, it is the second largest contributor to the world supply of vegetable oil. Oilseed Brassica is also very popular in China and it is the most important oil crop in China. This success is largely attributed to continuous and intensive breeding efforts. In the past years, the genetic improvement of B. napus has mainly been achieved by conventional plant breeding methods, especially F1 hybrid breeding. Recently, genetic engineering has opened a new platform to modify crops. The use of herbicide tolerant canola (B. napus) resulting from gene transfer accounts for nearly 50% of canola plantings both in Canada and Australia. Many possible agronomic and quality characters are expected to be genetically engineered into B. napus in the near future. An efficient tissue culture system is therefore thought to be crucial to the success of plant genetic engineering in Agrobacterium-based transformation protocols. Two primary factors have influenced the efficiency of Agrobacterium-based transformation, one being the infection frequency of Agrobacterium and the other, the regeneration frequency of the infected tissues. The main results observed are as follows:1. An efficient method for plant regeneration from cotyledon-petiole explants of Brassica napus was developed in the present study. The intact cotyledon-petiole was more regenerated than the half cotyledon-petiole. The maximum shoot regeneration frequency (96.7%) was obtained in the cotyledonary explant of 4~5d seedling age pre-cultured for 2d on the Murashige and Skoog (MS) medium supplemented with 2, 4-D1 mg/L, and then inoculated into the differentiation MS medium contained 3 mg/L 6-benzylaminopurine (BA), 0.15 mg/L 1-naphthaleneacetic acid (NAA) , 10 mg/L Ag2S2O3 , 30g/Lsugar and 7.5g/L agar (pH5.8). Using this method, the cotyledonary explants had swollen after 2 d culture and calli were observed from the cut end of cotyledon-petiole. The shoots were regenerated from some cotyledonary-petiole explant after cultured 7 days in differentiation MS medium. Some explant regenerated roots. The regeneration period from the explant to the seedling formation was shortened to about 25 days.2. Shoot regeneration ability from cotyledonary explant was strongly influenced by the genotype. The shoot regeneration frequency ranged from 10.0% for cv. R5 (B. rapa), to 83.6% for cv. Nl (B. napus). Two B. napus, one B. carinata and one B. juncea cultivar exhibited shoot regeneration frequency higher than 70.0%. In terms of the number of shoots produced per explant, B. rapa showedthe highest variation, ranging from 5.6 for cv. R3, to 1.3 for cv. R5. In general, B. juncea was relatively easy in terms of regeneration with the high shoot regeneration frequency (70.0%) and number of shoots per explant (3.89). B. rapa had the lowest frequency of regeneration from cotyledonary explants among the three basic diploid species, B. rapa, B. oleracea and B. nigra, and their synthetic amphidiploids, B. juncea, B. napus and B. carinata. B. oleracea and B. rapa which include the AA chromosome were more recalcitrant and their mean shoot regeneration frequencies reached only 22.2% and 24.8%, respectively. B. napus and B. carinata showed the relatively high mean shoot regeneration frequencies (47.9% and 48.9% respectively).3. In Brassica, while cotyledon explants showed high frequency of shoot regeneration success in genetic transformation using this explant is limited only to B. napus. The hypocotyl explants proved amenable for transformation. Protocols were developed for efficient shoot regeneration from hypocotyl explants of oilseed B. napus in this experiment. The maximum shoot regeneration frequency (96.7%) was obtained in the hypocotyl explant of 4~5d seedling age pre-cultured for 2d on the MS medium supplemented with 2, 4-D1.5 mg/L, and then... |
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