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Thesis (MScAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Potatoes is the fourth most important food source in the world. To provide in the ever increasing demand by the growing world population for this staple food, alternative ways will have to be found to produce a cheaper and a healthier end product. The tubers mainly consist of carbohydrates and starch, although 9% of the dry weight of the tubers consist of proteins. In its natural state the potato tuber is low in sodium and high in potassium and vitamin C. These proteins can provide in all the amino-acid requirements of humans and make it an outstanding balanced diet. The most important factor by which the production and quality of potatoes can be increased, is to provide better propagation material to producers. Minituber production is a relatively new method of producing better quality propagation material for producers. It is, however, very expensive because of the high capital investment needed. By producing more tubers per plant, or more tubers per square meter, the unit price per tuber may be lowered. The system commonly used for minituber production is where in vitro plants are planted in a greenhouse, usually in a vermiculite/peat mixture as medium. As in a hydroponic system, nutrients are added to the irrigation water. As possible strategy to increase the production of mini-tubers, the effect of nutrition was investigated. No significant differences were found in tuber production with small deviations in phosphorous and calcium nutrition. Researchers who reported significant differences in tuber production with nutrition, either corrected extreme shortages or induced it. By subjecting the root and tuber medium of potato plants to a pH-shock (nutrient solution with a pH of 3.5 for 10 hours), large amounts of tubers were initiated. A new production technique was developed where plants were grown in a hydroponic way without any root medium. In this way the right sized tubers could be removed without damaging the plant. Potato plants do not initiate tubers easily where stolons are not subjected to mechanical resistance. This problem was solved with the help of a pHshock, where significantly more tubers were formed per plant. The action of the pH-shock could be explained by a temporary inaccessibility of nutrients to the plant. Both calcium and magnesium absorpsion are hampered at a low pH, but calcium is also poorly translocated in the plant. Considering that a nutrient solution without calcium produced the same results as the pH-shock treatment in this study, one may make the assumption that an induced calcium shortage may be responsible for the low pH effect. The same effect may even be induced by giving the plants clean water for some time. Such a treatment was, however, not tested but should receive attention in further studies. The correct timing for a low pH-shock may need more research. A precise number of days after transplanting can not be prescribed since many factors such as light intensity, daylength and cultivar difference affect plant growth and tuber initiation. Environmental conditions, the physiological age and the biomass of the plant may affect the timing of the shock. A low pH-shock could not at any stage induce tubers in potato plants where unfavorable environmental conditions occurred. It seems as if the plants need to be ready for tuber initiation before the pH-shock can be successfully applied. By removing/harvesting the tubers at the required weight, the burden on the plant may be relieved enabling more tubers to form. It was experienced that the reproductive system of the potato plant is complex with a number of factors affecting it. The new production system has potential, but more developmental work is still needed. / AFRIKAANSE OPSOMMING: Aartappels is die vierde belangrikste voedelsoort in die wereld, Om te kan voorsien aan die ewigdurende verhoogde vraag na hierdie stapelvoedel deur die groeiende wereld-bevolking, sal daar maniere gevind moet word om die eindproduk goedkoper en in 'n gesonder toestand op die mark te plaas. Die knolle bestaan hoofsaaklik uit koolhidrate en stysel, alhoewel 9% van die droe gewig van knolle uit proteiene bestaan. Die aartappelknol is in sy natuurlike toestand laag in natrium en hoog in kalium en vitamien C. Hierdie protei'ene kan in al die aminosuur-behoeftes van mense voorsien wat dit dus 'n uitstekende gebalanseerde dieet maak. Die belangrikste faktor waarmee die produksie en kwaliteit van aartappels verhoog kan word is om beter voortplantingsmateriaal aan produsente te verskaf. Miniknolproduksie is 'n relatief nuwe manier om gesonde en groeikragtige voortplantingsmateriaal vinnig by die produsente te kry. Dit is egter 'n duur metode omdat die kapitaal-investering groot is. Deur meer knoUe per plant, of meer knolle per vierkante meter te produseer kan die eenheidsprys per moer verlaag word. Die sisteem wat algemeen vir miniknolproduksie gebruik word is om in vitro plantjies in 'n kweekhuis, gewoonlik in 'n vermikulietldennebas mengsel te plant. Voedingstowwe word dan soos in 'n hidrokultuurstelsel saam met die besproeiingswater toegedien. As moontlike strategie om die produksie van miniknolle by aartappels te verhoog, is die invloed van voeding daarop ondersoek. Daar is gevind dat daar nie groot verskille in knolproduksie met klein afwykings in fosfaat- en kalsium-voeding veroorsaak is nie. Navorsers wat wel betekenisvolle verskille III knolproduksie met voedingsbehandelings verkry het, het ekstreme tekorte reggestel of gemduseer. Deur die wortel- en knolmedium van aartappelplante aan 'n pH-skok (voedingsoplossing met 'n pH van 3.5 vir 10 ure) bloot te stel, is daar gevind dat 'n groot hoeveelheid knolle geinisieer word. Verder is 'n nuwe produksietegniek ontwikkel waar die plante met 'n suiwer waterkultuur metode geproduseer word, sodat knolle wat oesgereed is verwyder kan word. Aartappelplante inisieer nie maklik knolle onder hierdie omstandighede waar stolons geen meganiese weerstand ervaar nie. Met behulp van die pH-skok het daar betekenisvol meer knolle per plant gevorm. Die werking van die pH-skok kan moontlik aan 'n tydelike ontoeganklikheid van voedingstowwe toegeskryf word. Beide kalsium en magnesium word moeilik by 'n lae pH opgeneem maar kalsium word boonop moeilik deur die plant getranslokeer. Aangesien 'n voedingsoplossing sonder enige kalsium dieselfde resultate as die pHskok gelewer het, lyk dit asof 'n geinduseerde kalsium-gebrek vir die lae pH effek verantwoordelik mag wees. Die skok kan makliker en veiliger toegepas word deur vir 'n tydperk net skoon water aan die plante te gee. So 'n behandeling is egter nie beproef nie maar behoort in opvolgwerk aandag te ontvang. Die optimum tyd waarop so 'n skok toegedien moet word is nog onduidelik. Daar kan nie op 'n eksakte aantal dae na uitplant gewerk word nie, aangesien daar te veel faktore soos ligintensieteit, daglengte en kultivar is wat plante se groei en knolinisiasie kan beinvloed, Omgewingstoestande, die fisiologiese ouderdom van die plant en die hoeveelheid biomassa wat gevorm het mag bepaal wanneer die plante geskok moet word. Waar ongunstige omgewingstoestande geheers het, kon 'n lae-pHskok op geen stadium knolinisiasie induseer nie. Dit wil voorkom asof die plant vir 'n knolinisiasie fase gereed moet wees voordat 'n lae-pH-skok doeltreffend is. Dit verhoog dan bloot die aantal knolle wat per plant ontwikkel. Deur die knolle op die verlangde grootte te pluk kan die las op die plante verlig word sodat meer knolle mag vorm. Daar is ervaar dat die reproduktiewe stelsel van die aartappelplant kompleks is en dat daar baie faktore is wat dit mag beinvloed. Die nuwe produksiestelsel toon potensiaal, alhoewel verdere ontwikkelingswerk nog nodig is.

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Being the third most cultivated crop in South Africa, potatoes are of great economic importance. As potatoes originated from cooler areas in the world, they do not easily adapt to South African conditions. The main objective of potato breeding is, therefore, to extend the crop's limited genetic base. Progress in crop improvement is slow due to dominance, segregation and other factors caused by the tetraploid character of cultivated potatoes. A new breeding program for rapid progress has been initiated at the Vegetable and Ornamental Plant Institute, Roodeplaat, South Africa, which comprises the combination of conventional and unconventional breeding techniques. The program is based on the reduction of the ploidy level from the tetraploid to the dihaploid level to facilitate crossings with diploid wild species. Anther culture is the preferred technique for the rapid reduction of the ploidy level and has been successfully applied on different members of the Solanaceae. Cultivated potato, Solanum tuberosum is, however, an important exception. In this study various potato genotypes (tetraploid cultivars, dihaploid breeding lines and a diploid wild species) were used in experiments concerning microtechniques, alternative culture methods and medium manipulation. The main objectives were to evaluate and compare the androgenetic ability of the various genotypes used and to try and identify the factors limiting their in vitro response. Regarding microtechnique, the study focussed on the investigation of the frequency of androgenesis - as a function of plant age - and the determination of defined flower bud lenqths representative of the correct microspore developmental stage for optimal androgenetic response. Combined with an extensive histological study on the microspore development within anthers, from the time of flower selection, after a cold-pretreatment and at various time-intervals during the culture period of 42 days, the following conclusions were reached: In vitro androgenetic response proved optimal when flowers of responsive genotypes were selected during the first seven to 21 days of the flowering period. Both microspore derived embryoid- and callus development were visible within responsive anthers after a culture period of only seven days. The flower bud length required for anthers to be in the optimal stage of microspore development, e.g. the uninucleate stage, varied between the different genotypes but could readily be determined with the DAPI (4,6-diamidino-2- phenylindole) technique. It was also concluded that anthers of the tetraploid cultivar Atzimba should be selected later, between the late-uninucleate and the early-binucleate developmental stages. This suggested a limited selection period for Atzimba anthers, as starch depositioning - which prevent embryogenesis - occurs within anthers during the binucleate stage. Histologically, Atzimba showed limited embryoid development with no embryoid release, while the diploid wild species, S. canasense, proved androgenetically unresponsive. Alternative culture methods were applied to study the effect of different culture phases (liquid, double layered and agar solidified) and anther orientations (lateral, dorsal and ventral) on the androgenetic response of the potato genotypes used. Liquid cultures, based on the so-called shed-pollen technique, enhanced the androgenetic response of the tetraploid cultivar Atzimba. Optimal embryogenesis was obtained for responsive breeding line 87.2002/3 with the utilization of agar solidified media, with maximal response when anthers were cultured in the lateral orientation. No response was observed from S. canasense. The effect of medium manipulation on the androgenetic response of the three genotypes was investigated. The utilization of various combinations of different concentrations of indole-3-acetic acid (1M) and benzyladenine (BA), the alteration of the initial time of incubation of anthers on the initiation media and the use of media without growth regulators compared to that containing gibberellic acid (GA[3]), were investigated. BA had to be present in the initiation media and had a major, though not exclusive, effect on embryogenesis compared to 1M. The optimal BA concentration varied between the two trials. IAA also had an increasing effect on anther response, both in the absence of BA and, especially, in addition with relatively high BA concentrations. In this experiment, only breeding line 87.2002/3 responded. The initial culture of anthers, during the first seven to 21 days of the culture period, on media containing growth regulators proved essential for microspore derived embryoid production in the tetraploid cultivar Atzimba. As these growth regulators are metabolized in the culture media, the regular transfer at shorter, two-weekly intervals to media containing metabolically active substances, proved important. GA[3] had no enhancing-effect on embryogenesis in any of the three tetraploid cultivars. The results obtained in this study suggest that the first 21 days is the critical stage in the anther culture period in terms of the optimal time for flower selection, embryoid induction and the increase in embryogenetic response due to growth regulator influence. It is important to pre-determine the developmental stage when most microspores were in the uninucleate stage of development and to correlate this stage with a specific flower bud length. This would assure maximum response of those genotypes amenable to anther culture. It also implies a more practical and economical starting pOint to anther culture experiments. Following the determination of microspore developmental stage and pollen fertility, flowers should be selected from the donor plants only during the first three weeks of the flowering period. The composition of the nutrient media used for potato anther cultures were sufficient with respect to growth regulators. The growth regulators SA, IAA and the amines glutamine and asparagine had to be present in the initiation media, especially during the first three weeks of the culture period. As microspore development within anyone anther was found to be asynchronous, the regular transfer of anthers to fresh media is recommended to assure proper development of all microspores. The use of a slightly higher IAA concentration could be considered, but care should be taken as too-high concentrations would induce callus production. Microspore derived embryoid production is preferred, as the ploidy level of callus derived plantlets normally varies and somaclonal variation can occur. Liquid media should be considered for anther culture of tetraploid genotypes, while embryoid production can be increased by culturing the anthers of responsive genotypes on agar solidified media on the lateral orientation. Finally, the diploid wild species S. canasense seemed androgenetically unresponsive, or the media and culture conditions used did not satisfy the specific requirements of this genotype. Androgenetic amenability should first be transferred by means of interspecific crossings with a responsive dihaploid genotype, such as the breeding line 87.2002/3. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

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

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.

Androgenic processes in diploid potato (2n=2x=24) were studied in three interspecific hybrids. Five incubation temperature treatments were examined. Temperature shock (35°C) during the first 12 h of anther culture and elevated incubation temperature (30°C 16h/20°C 8h) (hereafter 35°C-30°C/20°C) enhanced androgenic embryo production. Variation among experiment dates was highly significant. Temperature treatment (35°C-30°C/20°C) during anther culture did not influence the subsequent conversion rate of androgenic embryos, thus providing a simple and effective way to enhance androgenic embryo yield. Repeated experiments were conducted to study extended anther culture by replacing anthers into medium following the usual harvest 6 weeks after culture initiation. Embryos continued to be produced after the first harvest. Embryo yield at the first harvest was significantly correlated with that at the second harvest (P <0.01). Significantly more embryos were produced when anthers were put back into the same medium compared to fresh medium in extended anther culture. Although relatively high embryo yield was produced in extended anther culture, high contamination and low regeneration rate eliminate any practical use unless a better regeneration protocol is developed. Randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the genetic composition of anther-derived plants, whose ploidy level was predetermined by flow cytometry. The RAPD fragments amplified from various anther derived diploid plants (2n=2x=24) were compared with those from anther donor (2n=2x=24) and anther-derived monoploids (2n=1x=12). Anther donor and anther-derived monoploids were distinguished by scoring segregating bands as well as total number of scorable bands that exhibited polymorphism. Thus RAPD has the potential to separate homozygous from heterozygous diploids, since the frequency of RAPDs present in homozygous diploids is expected to be the same as in a group of known monoploids, whereas heterozygous ones will be similar to the anther donor. / Master of Science

Competence for leaf disc regeneration, anther culture, and protoplast culture was tested in the parental, F₁, and F₂ generations of a diploid cultivated primitive potato, S. phureja (2n=2x=24). The parental pair consisted of AM3-8, an anther culture derived homozygous diploid, and NBP2, a heterozygous, field selected line. AM3-8 produced embryos in anther culture, and shoots on cultured leaf discs, but its cells did not divide after protoplast isolation. Cells of NBP2 divided to form calli and shoots in protoplast culture, but the clone did not respond to anther culture or leaf disc regeneration. All the individual plants in the F₁ generation were responsive to both anther culture and protoplast culture; however, there was segregation for the ability to regenerate shoots from leaf discs. The F₂ population, the result of a sib-cross, segregated for all three tissue culture competencies. Segregation data fit a one gene model for anther culture competence with the homozygous dominant genotype expressing the highest response, the heterozygous resulting in a marginal response, and the homozygous recessive resulting in no response. A two-gene model applied to the protoplast culture data, with a dominant allele at both loci required for division to occur after protoplast isolation. Leaf disc regeneration data could only be explained by a two gene model with recessive alleles at each locus required for the highest response, a dominant allele at either of the loci resulting in a marginal response, and dominant alleles at both loci resulting in no response. No significant correlation was found among these traits, implying three separate genetic mechanisms which segregate independently. Several temperature regimes were used in an attempt to enhance caulogenesis following protoplast isolation during the p-callus growth and regeneration phase of a single F₂ clone from this S. phureja population. Each of eight treatments was applied to 120 p-calli in six replications of 20 each. Shoot regeneration was scored at 94, 105, 121, and 131 days after protoplast isolation. P-calli cultured at 30°C days and 20°C nights produced significantly more shoots than those cultured at a constant 25°C. Therefore, the standard 25°C used for p-calli regeneration in potato may not be optimal; elevated temperatures or simply a diurnal temperature fluctuation may enhance morphogenesis. / Master of Science

Inheritance of androgenetic competence was studied in 10 diploid potato species hybrids and 16 backcross progeny. Ten hybrid families including three reciprocals were generated between competent clones of S. phureja and incompetent clones of S. berthaultii, S. microdontum and S. phureja. The F₁ hybrid families segregated for androgenetic competence with some highly competent and some incompetent genotypes in all families. The expression of androgenetic competence was modified by parents lacking competence. The cytoplasm of species lacking competence exerted a greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. The segregation data of 16 backcross progeny between a highly competent hybrid and its incompetent parent suggested that competence may be under control of a single dominant gene. Androgenetic competence can be transferred among sexually compatible potato species. The transfer of desirable traits to a monoploid background can be expected using an- drogenetically competent selections in hybrid combination with germplasm expressing the desired attributes. In an attempt to determine genetic changes of regenerated potato plants following anther and callus cultures, 20 callilines of two S. phureja clones were examined. Four of 20 callilines selected for fertility and diploidy were morphologically indistinguishable among themselves and from the parental clone that had not undergone a tissue culture cycle. Even though morphologically indistinct from the parental clone, all four callilines exhibited higher seed set as pollen parents in 4x-2x crosses and two of the four exhibited higher recombination frequency between the centromere and the y gene. The estimated increase in map distance of the y locus ranged from 3.4 to 10.0 units. Progeny analysis revealed no significant morphological differences among 4x-2x hybrid families under field conditions, and only a single difference among 2x-2x hybrid families under screenhouse conditions. Hence, variation induced in tissue culture may have occurred without detectable morphological change. Assuming no adverse tissue culture effects, breakage of undesirable linkage groups may be an advantage of caulogenesis before backcrossing. / Ph. D.

The ultimate goal of the· breeding scheme, of which the present study is a part, is to introduce exotic germplasm into existing cultivars of Solanum tuberosum through· 4x X 2x crosses using the South American diploid potato species Solanum phureja as the pollen parent. The first phase of this program includes the 'reconstruction' of a highly heterozygous diploid, pollen parent by .first : reducing the chromosome number of the S. phureja clones to the monoploid level and subsequently fusing genomes of two unrelated monoploid plants either by somatic hybridization or by cross-pollination between fertile doubled monoploids. Within this framework, the objectives of this research were to analyze variation among anther-derived plants of Solanum phureja regarding their: 1) ploidy level and morphology, 2) net photosynthesis and its biochemical components, and 3) nuclear genomic structure, particularly with regard ·to the amplification of rRNA genes as influenced by the anther-culture process. Based on the analysis of several morphological characters of the anther-derived plants by canonical discriminant function, four characters (anther length, number of chloroplasts/pair of guard cells, leaf width, corolla width) were selected for most effective assignment of plants to their ploidy groups by clustering procedures. Clustering of the anther-derived plants proved to be an efficient means of separating monoploids from higher ploidy levels. To assess the impact of the process of anther-culture on the physiology of the resulting plants and to evaluate the possibility of selecting anther-derived genotypes for further breeding efforts, monoploid, diploid and tetraploid anther-derived plants were studied regarding their net photosynthesis and its component characteristics. Leaf area, net photosynthesis and chlorophyll content increased with increasing ploidy' Among .the. monoploids I. Rubisco activity and concentration displayed a. significant genotypic effect; whereas in the diploid group variation among genotypes was significant for total protein content and maximum specific activity of ribulose bisphosphate carboxylase. Among the tetraploid genotypes, significant differenc.es were found with respect to net photosynthesis and specific leaf weight. Two exceptional genotypes were identified: a monoploid with an increase of 28% fcfr maximum activity of ribulose bisphosphate carboxylase and a tetraploid with an increase of 30% for net photosynthesis over the anther-donor plant. To evaluate DNA variation among the anther-derived plants, the nuclear genomes of anther-derived monoploid and diploid plant were studied by DNA reassociation kinetics. It was found that the nuclear genome of the monoploid has undergone differential replication resulting in an increase of sequences consisting of highly· repetitive DNA. Free solution RNA-DNA hybridization showed that the monoploid DNA contained 30% more rDNA sequences than the diploid. Southern blot analysis using rRNA as the probe revealed variation for copy number of certain restriction fragments and for restriction enzyme cleavage sites. / Ph. D.