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Digitized by Kansas Correctional Industries

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True potato (Solanum tuberosum) seed (TPS) is used for preservation of variable genetic lines of wild and cultivated potatoes (Hawkes et al., 2000) and for propagation of food crops in some developing countries. TPS has advantages over seed potato tubers in storage and transportation and favors lower virus infection levels in fields. However, TPS has thermodormancy and will not readily germinate at 25°C and above (D'Antonio and McHale, 1988; Pallais, 1995a, b; Alvarado et al., 2000). TPS can be extremely unreliable when planted directly in fields due to poor emergence related to diseases and soil crusting. Germination tests were conducted with two lots of TPS derived from cvs. EB-8109 and All Blue, respectively, to study dormancy mechanisms. Seeds were germinated under four temperature regimes (10°C, 15°C, 20°C and 25°C). The two lots showed distinctly different germination characteristics. EB-8109 seeds showed only thermodormancy whereas All Blue seeds showed very deep dormancy. A carotenoid synthesis inhibitor, fluridone, which blocks abscisic acid (ABA) synthesis, effectively broke thermodormancy in EB-8109 TPS but did not break primary dormancy in All Blue seeds. Additional treatments, including pre-chilling and hormonal regimes, also failed to break All Blue deep dormancy. When the micropylar region of the endosperm (endosperm cap) was removed from seeds of both seed lots, radicle elongation was observed, suggesting that mechanical resistance from the endosperm cap restrains radicle protrusion, and that weakening of the endosperm cap is requisite for TPS germination. Endo-β-mannanase expression was measured to help characterize mechanism underlying the weakening of endosperm cap tissues. This enzyme is thought to permit radicle protrusion by degrading cell walls thereby weakening the tissues of the endosperm cap (Groot et al., 1988). The coding region of germination-specific mannanase was isolated from the potato genome by use of polymerase chain reaction (PCR) with primers specifically designed for the tomato germination-specific mannanase gene (LeMAN2, Nonogaki et al., 2000). The cDNA of the TPS mannanase was identical to that of LeMAN2. The expression of mannanase mRNA was detected in the endosperm cap of germinating TPS after 72 h of imbibition at 15°C, while no expression was detected at 25°C (thermodormant condition). Fluridone induced mannanase expression in the micropylar region of the endospem at 25°C. Thus, there was a correlation between induction of mannanase and dormancy breakage. A major increase in TPS post-germinative endo-β-mannanase activity was detected by use of gel diffusion assay. Two isoforms of mannanases were detected in the protein extracts of germinated TPS by activity staining of native polyacrylamide gel electrophoresis. The post-germinative mannanase was detected in the whole endosperm of germinated TPS by using tissue printing with the LeMAN1 (Bewley et al., 1997) RNA probe. These results suggest that, as with tomato, TPS also expresses post-germinative mannanase activity. The promoter region of a new tomato mannanase was isolated during this research. This promoter was shown to be involved in anther-specific expression of mannanase. / Graduation date: 2003

In vitro culture techniques are widely used in potato for rapid production of specific pathogen tested plantlets and microtubers, which are essential for seed certification programs. The limited calcium (Ca) uptake in vitro promotes physiological disorders in many micropropagated species including potato. Factors that may affect Ca uptake by potato plantlets, including cations, osmotic potential, and boron (B) were examined. The possibility of improving microtuber yield by low pH shock or successive harvests, which stimulated greenhouse potato production, were also investigated. Ca uptake by cvs. Norland and Bintje potato plantlets grown on modified Murashige and Skoog (MS, 1962) media was enhanced by diluting the total macroelements in the nutrient medium, except Ca. Increasing Ca:specific cation ratios, by lowering either NH 4, Mg or K, had little effect on Ca uptake by Norland plantlets. At equivalent Ca:total cation, Ca uptake was promoted further when the total macroelement concentration was reduced by half than when the Ca level was doubled (from 3 to 6 mM). This suggested that the increase in the osmotic potential of the medium had a great impact on Ca uptake, probably by affecting the root pressure. B contamination from impurities in the nutrient chemical components always occurred. The addition of the recommended H3BO 3 level of 0.1 mM (1.08 ppm) or more (0.3 mM) to the medium decreased Ca content of leaves and shoots in Norland but not Bintje, while addition of only 25% of this level was sufficient to provide plantlets with adequate B tissue concentration without compromising normal growth. Macroelement dilution along with Ca addition, and lower B levels than commonly used in MS (0.1 mM) are recommended for optimal Ca uptake in potato and possibly other plant species. Microtuber yield was not improved by a low pH shock. Adjusting microtuberization medium pH to less than 5.7 (3.5, 4.3 or 5.2), prior to autoclaving, reduced microtuber number and/or weight

A protocol is presented for the rapid (28 days) induction of microtubers on micropropagated layered potato plantlets of 'Kennebec', 'Russet Burbank' and 'Superior' in medium devoid of growth regulators. With this method the addition of coumarin, 6-(2-chloroethyl)-trimethylammonium chloride and 6-benzylamino-purine to the microtuberization medium either had no effect or significantly reduced microtuber weight per plantlet. Increasing the incubation period from 28 to 56 days significantly increased the weight of microtubers per plantlet and the proportion of microtubers heavier than 1 gram. Increasing the volume of microtuberization medium from 50 to 100 ml significantly increased the number of microtubers per plantlet. Microtuber dormancy periods were cultivar-specific and microtubers $ le$250 mg had longer dormancy periods as compared to microtubers $>$250 mg. A positive correlation was established between endogenous abscisic acid levels and microtuber dormancy periods. Microtubers $ le$250 mg had lower specific gravity, fewer eyes and produce fewer sprouts than microtubers $>$250 mg. Microtuber-derived plants were generally single-stemmed. Severe physiological ageing treatment ($>$2500 degree-days) had no effect on microtuber sprout development, stem number, tuber number and only minimally influenced tuber weight of microtuber-derived plants. Decreasing field in-row planting density from 30 to 10 cm reduced tuber weights and numbers per plant but increased them on a per hectare basis. Economic analysis indicated that optimum planting density varied depending on plantlet cost. The optimum planting density was 10 cm if the cost of the plantlet was $0.10 or less, 20 if plantlet cost were from $0.10 and $0.20 and 30 cm for plantlet cost greater than $0.20. A potato seed tuber certification program adapted to the needs and constraints of Egypt is presented.

Various aspects of Ca2+ uptake into greenhouse-grown potato plants and micropropagated potato plantlets were examined, including the movement of Ca2+ into potato plants and tubers, identification of Ca-packing genotypes and assessment of the effect of improved medium Ca2+ level on different parameters such as plantlet growth, microtuber induction, yield and tissue Ca concentration. The effect of exogenous vitamin Ds and UV-elicited endogenous vitamin D synthesis on regulation of 45Ca 2+ uptake into plantlet shoots was also examined. Safranin dye was not as suitable as the tracer 45Ca2+ for monitoring translocation of Ca2+ into tubers. Uptake of Ca2+ into tubers occurred indirectly through the main basal roots and stolon roots and directly through tuber periderm. Liquid scintillation counting and flame atomic absorption spectrophotometry were used to screen six potato cultivars and two wild species for ability to take up Ca2+ from treatment solutions containing high or low Ca2+ levels. In vitro potato micropropagation, microtuberization, and tissue calcium concentration were compared for six cultivars when Murashige-Skoog basal medium Ca2+ level was increased from 3, to 5 or 15 mM. All aspects of growth including shoot dry weight, early microtuber induction, microtuber yield and tissue Ca concentration were improved when medium Ca2+ level was 15 mM. Cultivar Bintje was the most efficient genotype at accumulating Ca 2+ from treatment solutions or growth media containing high or low Ca2+ levels. Vitamin Ds improved 45Ca 2+ uptake into shoots of micropropagated potato plantlets and D 3 was more efficient in this regard than D2 or a combination of D3 and D2. Plantlets had increased 45Ca 2+ uptake when exposed to UV irradiation compared with the untreated control plantlets. Plantlets given a 24 hour interval in the dark after UV exposure had even greater 45Ca2+ uptake, suggesting that vitamin D, metabolites, specifically calcitriol (1, 25(OH)2-v