In vitro evaluation of endochitinase transgenic royal gala apple lines for resistance to apple scab (venturia inaequalis (cke.) wint)
Endokitinaz transgenik royal gala elma hatlarının elma kara leke (venturıa ınaequalıs (cke.) wınt) hastalığına karşı dayanıklılığının ın vıtro değerlendirilmesi
- Tez No: 711445
- Danışmanlar: PROF. DR. HERBERT S. ALDWINCKLE
- Tez Türü: Yüksek Lisans
- Konular: Ziraat, Agriculture
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 1997
- Dil: İngilizce
- Üniversite: Cornell University
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Bitki Koruma Ana Bilim Dalı
- Bilim Dalı: Bitki Koruma Bilim Dalı
- Sayfa Sayısı: 59
Özet
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Özet (Çeviri)
The purpose of this research was to characterize putative endochitinase-transgenic Royal Gala apple lines and to evaluate their resistance to Venturia inaequalis. Seven primers, specific for the apple genome, were used in Randomly Amplified Polymorphic DNA (RAPD) analysis of the putative endochitinase-transgenic lines, and a putative marker-gene transgenic Royal Gala and non-transformed Royal Gala as controls. The amplified band patterns of most of the putative endochitinase-transgenic lines were similar to the band pattern of non-transformed Royal Gala, indicating that the putative lines were in fact derived from Royal Gala. All putative endochitinase-transgenic lines amplified at least one primer, and gave similar band profiles as Royal Gala. I concluded that all putative endochitinase-transgenic Royal Gala apple lines were derived from Royal Gala. The transgenic Royal Gala lines were assayed for expression of endochitinase using a multi-well plate fluorescent enzyme assay. Purified endochitinase from Trichoderma harzianum was diluted to provide a series of enzyme concentrations with which the activity of endochitinasetransgenic, marker-gene transgenic and non-transgenic lines were compared, based on fluorescence in UV light of methylumbelliferone produced by endochitinase from the substrate, 4-methylumbelliferyl B-d-N, N', N" triacetylchitotrioside. Four endochitinase-transgenic lines gave high fluorescence, indicating much endochitinase activity. Two lines gave low fluorescence indicating low endochitinase activity, and the fluorescence of two was very low and visually indistinguishable from nontransformed Royal Gala and marker-gene transgenic Royal Gala. An in vitro assay was used for the evaluation of endochitinasetransgenic Royal Gala lines for resistance to V. inaequalis. In two experiments, leaves from micropropagated shoots and in one experiment, leaves from in vitro rooted plants of endochitinase-transgenic lines, a marker gene transgenic line, non-transformed Royal Gala and the other cultivars of known susceptibility to scab were inoculated in vitro. Excised leaves were sprayed with a mixed conidial suspension of five races of V. inaequalis and incubated at 19°C, 16 hr photoperiod of 40 µE m-2s-1 light, in moist Petri plates. Following inoculation, leaves were observed at intervals with a stereomicroscope and disease severity ratings (DSR) were recorded after four weeks later, conidia were counted after removal from leaves by shaking in water. The several lines were ranked by the two ratings, but the variance in disease severity was lower than in conidial numbers. As expected from field observations of disease, DSR ranked Mclntosh as the most susceptible cultivar and Liberty as the most resistant cultivar, of the cultivars Golden Delicious, Liberty, Mclntosh and Royal Gala. Golden Delicious and Royal Gala were intermediate in DSR between Mclntosh and Liberty. The reaction of marker-gene transgenic Royal Gala was almost the same as non-transformed Royal Gala. Only a few no and low chitinase expressing lines of Royal Gala were inoculated: these appeared rather more resistant, based on DSR, than nontransformed Royal Gala. Three of four high chitinase-expressing lines of Royal Gala had significantly lower DSRs than that of non-transformed Royal Gala. Many of these lower DSRs were significantly different from the Royal Gala DSR. Although these data are preliminary, and should be confirmed by testing whole plants of the transgenic lines for scab resistance, my in vitro inoculation test appear useful for identifying resistant transgenic apple lines.
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