M.Sc. / Mnandi and Astrid are two commercially important potato cultivars in South Africa. These two cultivars are closely related and are morphological virtually identical. It is, however, necessary to be able to distinguish between these two cultivars, because each of these cultivars has certain desirable characteristics. It was decided to use DNA markers, since DNA markers are not influenced by the environment and the polymerase chain reaction (PCR) based DNA markers are relatively easy, cheap and fast. It was decided to develop a sequenced characterized amplified region (SCAR) due to the problems with the reproducibility of random amplified polymorphic DNA (RAPDs). SCARs are derived from RAPD fragments by using the sequence of a RAPD derived fragment to design a set of new longer primers (usually 20-24mer) which are less sensitive to PCR conditions. Ten commercial potato cultivars (Astrid, Mnandi. BP,, Buffelspoort, VanderPlank, Up-to-Date, Hoevelder, Hertha, Pimpernel and Agria) were used in this study. Commercially available RAPD primers (102) were evaluated to seek a polymorphism unique to either Mnandi or Astrid. Thirtyseven polymorphisms between Astrid and Mnandi were identified but only three were unique. The polymorphism obtained with OPH-15 was however, not reproducible. The polymorphisms obtained with UBC 509 and 582, corresponding to the presence in Mnandi of a 300 and 900 by fragment respectively, were reproducible. These two fragments, UBC 509 3" and UBC 582900, were cloned into the pMosBlue TA cloning vector and sequenced. The identity if the inserts in the recombinant plasmids were verified with PCR and Southern blotting. The sequences were used to develop two sets of SCAR primers, SCAR UBC 509 3" and SCAR UBC 582900 . The two SCAR primer pairs were then used in PCR reactions. The SCAR UBC 509 300 primer pair amplified a fragment of 230 by in both Astrid and Mnandi and a fragment of 260 by in Mnandi. The polymorphism is thus retained and SCAR UBC 509 3" can be used to distinguish between Astrid and Mnandi. The SCAR UBC 582' primer pair amplify a fragment of 500 by in both Astrid and Mnandi as well as some other longer fragments. It was not possible to regain a polymorphism by either elevating the annealing temperature or by digesting the amplification products with restriction enzymes. SCAR UBC 582' could thus not be used to distinguish between Astrid and Mnandi.

The effects of seed size on sprout growth of potato seed pieces were investigated in controlled temperature conditions. The objectives were to quantify the effects of seed size, plant growth regulators (gibberellin and paclobutrasol) and calcium availability on sprout length, sprout and root dry mass, as well as the fraction of seed reserves utilised for sprout development. The interactive effect between seed size and temperature was also investigated Sprout length did not differ among seed pieces ranging from 1 to 8g in mass. For larger seed pieces sprout length, dry mass and dry mass per unit length, and the root dry mass increased with increasing seed size, reaching a maximum at a seed mass of 24g after 40 days. The smaller the initial seed piece the larger was the fraction of assimilates utilised for sprout growth. Sprout length and dry mass as well as assimilate mobilisation for growth, were promoted by dipping seed pieces in gibberellin solutions, with the greatest effect obtained with 30 ppm GA, the highest concentration used in the experiment. Paclobutrasol showed inhibiting effects on all growth parameters except for sprout thickness. The root dry mass did not differ among treatments. Sprout growth was enhanced by calcium availability, with the greatest effect obtained with exogenous supply of calcium at 10 mmol/I as CaS04. A similar effect was noticed when complete nutrient solution was supplied. CaC03 was less effective than CaS04. Calcium availability to sprout tips affected sprout growth only when the roots were deprived of nutrients. Sprout length of the two seed pieces (3 and 5g in mass) was similarly affected by temperature over time, with the sprouts at the low temperature initially shorter, but reaching the same lengths as those at the high temperature after 30 and 40 days. Sprout dry mass and thickness of 3 and 5g seed pieces were similarly affected by temperature, with higher dry mass and greater thickening at 16°C than at 26°C. Exposure to low temperature (16°C) was associated with the transfer of a large fraction of seed reserves to the sprouts. / Dissertation (MSc (Agric): Agronomy)--University of Pretoria, 2006. / Plant Production and Soil Science / unrestricted

Complementary DNA (cDNA) clones representing approximately 5800 nucleotides of potato leafroll virus (PLRV) genomic RNA were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) that could encode a 23 kDa protein was identified and further characterized. Comparison of the deduced amino acid sequence with the coat protein amino acid sequence of the PAV strain of barley yellow dwarf luteovirus (BYDV-PAV) showed significant similarity. This observation together with its size and internal location within the genome suggested that this gene encoded the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including a 17 kDa ORF within the ORF encoding the 23 kDa coat protein, and termination of the latter with an amber codon which is immediately followed by a large ORF in the same reading frame. Three PLRV coat protein gene sequences were used to transform tobacco and the potato cultivars 'Desiree' and 'Russet Burbank' via Agrobacterium tumefaciens mediated gene transfers. One construct possessed 12 nucleotides of the untranslated leader sequence 5' to the putative coat protein gene start codon. The other construct, which was also inserted in the reverse orientation to produce negative-sense RNA, had 192 nucleotides from this leader sequence. When these sequences were introduced as chimaeric genes under the control of a duplicated cauliflower mosaic virus 35S (CaMV) promoter, transcription levels were high. Both positive-sense transcripts produced potato leafroll coat protein which accumulated to maximum levels of approximately 0.5% and 0.01% of total leaf protein in tobacco and potato, respectively. Results show that significant levels of inoculum concentration-independent sustained resistance were obtained with each construct, resulting in PLRV titres as low as 1% of the level observed in untransformed plants, as determined by enzyme-linked immunosorbent assays. Virus transmission from PLRV-inoculated transgenic 'Russet Burbank' was reduced substantially and was correlated with virus titre. The pattern and level of protection were the same for constructs producing positive- and negative-sense RNA, suggesting a similar mechanism of resistance. Virus levels were negatively correlated to transcript levels within the transgenic plants. This resistance will have practical applications for the control, of PLRV. Elucidation of the mechanism of resistance may also help understand the mechanisms of virus infection. / Land and Food Systems, Faculty of / Graduate

Field experiments were conducted in two different growing environments to evaluate the spread and movement of Dickeya dadantii. A procedure to inoculate seed potatoes with Dickeya dadantii was developed to use during this study. Spread of Dickeya dadantii from inoculated potato seed to healthy potato seed during the handling, cutting and planting procedures was not detected at either location. Spread of Dickeya dadantii from inoculated seed to surrounding progeny tubers in the field was documented in both locations. In Florida, 33% of progeny tubers tested positive for Dickeya using PCR, and in North Dakota, 13% of the progeny tubers tested positive. Stunting was observed in plants grown from Dickeya dadantii inoculated seed tubers in North Dakota, but not in Florida. These results indicate that Dickeya dadantii may spread during the seed handling and cutting processes and can spread in the field from infected seed tubers to progeny tubers.

No description available.

No description available.

No description available.

No description available.

No description available.

Thirty-two monoploids (<i>2n = x = 12</i>) derived by anther culture of ten diploid clones of <i>Solanum phureja</i> were used to generate doubled monoploids through in vitro shoot regeneration. Doubled monoploids were compared to the anther donor and progenitor monoploids for morphological characteristics, and were evaluated for fertility in the greenhouse and progeny performance under field conditions. Monoploids varied for frequency and earliness of shoot regeneration, number of shoots formed per explant and frequency of chromosome doubling among regenerated shoots. Regeneration was greater when stock plantlets were frequently subcultured (2- or 4-week intervals) and maintained under a 16 h photoperiod, and when explants were incubated at 20°C compared to 25°C. In addition, leaf explants regenerated at higher frequencies than stem explants. Significant high correlations between monoploids and their doubled monoploids were observed for 14 of 17 characters in the greenhouse. Doubled monoploids were significantly greater than monoploids for 15 characters, indicating a positive effect of increasing gene dosage from monoploid to diploid. The anther donor was not significantly greater than the mean of doubled monoploids for 10 characters; therefore, for specific characters, doubled monoploids without homozygote depression can be obtained. Doubled monoploids varied for nurnber of days to flower, duration of flowering, abundance of flowers, flower quality, fruit set and seed set; they had lower fruit and seed set than the anther donor. A few clones produced low levels of stainable pollen which had high 2n pollen frequency but did not germinate in vitro. Therefore, they were considered male-sterile for practical purposes. Used as female parents, doubled monoploids were able to transmit the 2n pollen trait to their progenies. Two of four doubled monoploids exhibited superior general combining ability over the anther donor under field conditions. This demonstrates the potential of passage of a heterozygous genotype through the monoploid sieve. The advantage of the monoploid sieve may be more or less evident depending on the combining ability of the crossing partner and variable performance can be expected among doubled monoploids from an unselected anther donor. The performance of unselected doubled monoploids demonstrates the potential for their utilization in breeding and warrants further research in the area. / Ph. D.