الفهرس 
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Abstract
The potato (Solnum tuberosum L.) is the world’s fourth most important crop in terms of food production . Potatoes are subjects to more than 40 viral diseases in addition to potato virus Y (PVY). PVY is considered one of the most damaging potato viruses causing significant yield depression reached to 80%.
The objectives of this study are
1- Isolate and identify PVY based on biological, chemical and molecular properties.
2- Elimination of PVY through meristem-tip culture, thermotherapy, chemotherapy, electrotherapy treatments.
3- Genetic stability of some potato genotypes to PVY by biochemical and molecular markers. As well as, production of PVY-free micro and macrotubers.
Tuber samples of naturally infected potato plants cvs. Diamond, Spounta and Loady rossette were collected from Qaluobia governorate during spring season. These samples were tested against PVY, PVX and PLRV using specific polyclonal antibodies (IgGPVY, IgGPVX and IgGPLRV) serologically by DAS-ELISA. It was found that some samples were infected with PVY, PVX and PLRV separatly, in addition to, other potato samples were found with mixed infections with (PVY+PVX), (PVX+PLRV), (PVY+PLRV) and (PVX+PVY+PLRV).
PVY was isolated biologically on ch. amaranticolor L., where as after 5-10 days, local lesions symptoms with morphological PVY characters of small chlorotic lesions were developed, also PVY particles were inoculated on healthy leaves of D. metel. L. The typical external symptoms of leave crinkle, mottling, sever mosaic, deformation and stunting produced after 20 to 30 days from inoculation.
PVY isolate was easily and rapid mechanically transmitted by infectious sap from infected potato cvs. Diamond, Spounta and Loady rossette to healthy potato, D. metel and Ch. amaranticolor. It was transmitted by peach aphid (Myzus persicae) via non presestant with the percentage 85% on potato plants and was transmitted by rubbing with 75% on D. metel and Ch. amaranticolor, but was transmitted with percent 100% through graft healthy tuber cv. Spounta with bud eye from infected tuber. The distinected symptoms of PVY isolate were appeared after 30 days post plantation under green house conditions.
In this study, host range of PVY isolate was tested on twenty three plant species and cultivars belong to six families that inoculated by rubbing infectious sap and the results were confirmed by DAS-ELISA. The results showed that PVY isolate was reacted with three categories with these plants. Two species out of twenty three tested plants belong to Chenopodiaceae, Ch. amaranticolor and Ch. moral were reacted with local infection which appeared chlorotic local lesions. Eleven plants belong to four families reacted systemically with PVY isolate. Ten plant species no reacted with PVY isolate.
Stability of PVY isolate in infectious crude sap was 65oC (TIP), 10-6 (DEP) and 36 hours (LIV).
PVY isolate was purified using polyethylene glycol (PEG) which gave PVY yield with 1.25 mg/100g leaf tissues and with UV spectra character of more than 1 (1.6).
The morphology of PVY isolate was demonstrated using transmission electron microscope (TEM) that revealed the long helical symmetry particles of PVY with 12x570 nm.
PVY isolate was identified serologically using specific polyclonal antibodies by DAS-ELISA.
The RT-PCR was used to detect of PVY isolate in infected potato plants using the CP gene oligonucleotides primers, transcribes of cDNA of PVY-RNA was transcribed in total RNA extracted from infected potato plants. The amplified DNA were in the expected size calculated (in related to DNA ladder) 530, 575 and 510 bp of Loady rossette, Spounta and Diamond respectively.
In this study, DAS-ELISA assay was used to detect PVY in potato plants and tubers cvs. Diamond, Spounta and Loady rossette which used to produce PVY- free plants as well as micropropagated plantlets. These tubers were divided into four groups, each group contain 10 tubers, first group was used for excised meristem-tip from sprouts; the second group treated with chemotherapy; group three treated with thermotherapy and the last one treated with electrotherapy of plantlets micropropagated in vitro, where the percentage of survival potato plantlets were increased in healthy meristem than infected one.
In chemotherapy treatment, the plantlets on MS medium treated with two antiviral compounds, virazole and 2-thiouracil were incorporated individually into MS medium with, 10, 20, 30, 40, 50 mg/L concentrations and the plantlets were incubated under convenient conditions for 30 days. It was found that, the percentage of survival of healthy and infected potato plantlets was decreased with concentration increasing of the antiviral compounds and the decreasing of survival percentage was increased in infected plantlets.
In thermotherapy, PVY infected potato plantlets were exposed to heat 30 and 35oC for 3 weeks. It was found that the survival percentage was decreased with increasing the temperature of each healthy and infected plantlets.
In Electrotherapy, each 5 sprouts of each potato tuber cvs. Diamond, Spounta and Loady rossette infected with PVY isolate were exposed to 5, 10 and 15 mA. for 5 and 10 min. It was found that the survival percentage was decreased with exposed to increasing mA and time. The results was determined according to DAS-ELISA using polyclonal antibodies poty virus specific.
Also in this study, in vitro production of microtubers PVY tested from shoot tips through the proliferation of plantlets resulted from thermotherapy and or chemotherapy treatments on MS medium containging 0.25, 0.5, 1.0 mg/L jasmonic acid and 5, 15, 25 mg/L coumarin individually. It was found that coumarin (25mg/L) gave highly significant increasing in number, size and weight of microtubers while jasmonic acid (0.25 and 0.5 mg/L) gave non-significant increasing compared with MS medium without growth substance.
SDS-PAGE technique was used to study the genetic stability of microtubers derived plantlets through determination of protein content and the specific activity of each of peroxidase and polyphenol oxidase in potato plantlets grown on MS medium containing jasmonic acid and coumarin individually related to BSA as standard protein. It was found that the coumarin induced high protein content 1.57, 1.75 and 1.70 mg/g fresh weight, than jasmonic acid 1.50, 1.35 and 1.60 mg/g fresh weight of potato plantlets cvs. Loady rossette, Spounta and Diamound respectively compared with untreated ones (1.28, 1.19 and 1.31 mg/g fresh weight).
Also, it was found that the total number of polymorphism polypeptides were 40 bands. Four monomorphic, common polypeptides of potato cultivars related to belonging Solanum tuberosum. Twenty three polymorphic, specific polypeptides of potato cultivars relate to jasmonic acid and coumarin treatment. Threetin unique bands, protein genetic markers reletad to chemo and thermo-therapeutics of infected potato plantlets.
On the other hand, it was observed that the jasmonic acid and coumarin due to increasing of the specific activity of each of peroxidase and polyphenol oxidase in all treated potato plantlets grown on MS medium related to protein content increasing.
Three ISSR primers were used to detect and identify the genetic stability or variation among tissue culture derived potato plants treated with jasmonic acid and coumarin. It was observed that the number of scored fragments were 14, 13 and 13 fragments per cvs. Diamond, Spounta and Loady rossette treated with jasmonic acid respectively with different moleculer weights ranged from 1575 to 105 bp, as well as 8,11 and 10 fragments per Diamond, Spounta and Loady rossette treated with coumarin respectively with different Mw ranged from 1321 to 125 bp. The genetic stability among microtubers of three potato cultivars treated with jasmonic acid and coumarin appeared identical DNA fragments (common amplified fragments MAF) with 23 (56%) and 17 (60%) respectively.
The genetic variability of microtubers for three potato cultivars treated with jasmonic acid and coumarin appeared the polymorphic amplified fragments (PAF) among potato cultivars were 8 with 20% and 9 with 30% as well as 9 and 3 unique fragments (genetic markers) with 22.5 and 10% respectively.
In the last part of this study, the produced microtubers from healthy and therapeutic PVY infected tubers which gave negative results by DAS-ELISA were used to produce potato minitubers (macrotubers) through growing under opening the mist in green house conditions. After two months from cultivation, the macrotubers were harvested, counted and size were determined. No significant differences of minitubers in number and size between healthy and therapeutic PVY infected tubers.