The possibility of manipulating calcium (Ca2+) and potassium (K+) levels in seeds of Brassica rapa by altering parent plant nutrition and investigating the potential for increased salinity tolerance during germination, given that considerable amounts of literature imply that greater amounts of available exogenous Ca2+ and K+ can ameliorate the effects of salinity on both whole plant growth and germination, was evaluated. The investigation consisted of four growth trials. Two preliminary growth trials suggested that seed ion manipulation was possible without affecting the overall growth and vigour of the plant. After developing suitable high and low Ca2+ and K+ nutrient solutions for growth, a trial was carried out in a growth room and greenhouse, with various substrates and the seed of a certain size category was collected for subsequent ion and salinity tolerance analysis. Seed Ca2+ and K+ was significantly affected by growth substrate and nutrient solution and data showed that a significant negative regression relationship existed between seed Ca2+, K+ and Ca2+ + K+ levels and salinity tolerance. Further experimentation using hydroponic culture attempted to remove any possible effects of substrate and also to compare size categories of seed with a view to elucidating localisation of Ca2+ and K+. Seed Ca2+ was found to be significantly altered by nutrient solution in the two different sizes tested and higher Ca2+ nutrient solution was found to increase salinity tolerance in daughter seed. One significant negative regression correlation between salinity tolerance and seed K+ concentration existed in smaller seed, but disregarding seed size in a regression analysis of seed ion content and salinity tolerance, a significant negative relationship existed between seed Ca2+, K+ and Ca2++ K+. The results, especially in terms of Ca2+ nutrition, contradict much previous research that suggests increased salinity tolerance at germination can arise with the increased presence of Ca2+ and/or K+. Salinity tolerance was greater in seeds of larger size across all nutritional treatments and the smaller size range exhibited increased Ca2+ and K+ per μg seed. Ca2+ concentration in smaller seeds with greater surface area:volume ratios provided a clue to the potential localisation of Ca2+. Cross sectional staining showed that a greater proportion of seed Ca2+ may reside in the coat. This was confirmed by analysis which showed an approximate 50% split of total extractable seed Ca2+, regardless of size, between coat and embryo within a seed; the majority of which, per μg, resides in the coat. Further work looked at the relative solubility of the Ca2+ and K+ in these tissues and whole seed to look at the potential bioavailability of Ca2+ during germination from various parts of the seed. Most water soluble Ca2+ exists in the embryo and most insoluble Ca2+ exists in the coat, but coat Ca2+ was found to be ionically exchangeable and therefore bioavailable. K+ appeared mostly water soluble in embryo and coat. In line with previous whole plant research in this species, most Ca2+ is readily water soluble or ionically exchangeable in form and the possible negative effects of how increasing bioavailable Ca2+ may reduce salinity tolerance was discussed.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Soil salinity is a major limiting factor of plant and crop growth, because the absorption of water and nutrients is such a complex process while low and moderate salinity are omnipresent. Plant growth is affected negatively if a specific ion concentration exceeds its threshold and becomes toxic. The detrimental effect of soil affected by salt on crop production is increasing worldwide (Tanji, 1990). The level to which plants can tolerate high salinity levels is genetically controlled with several physiological and genetic mechanisms contributing to salt tolerance (Epstein & Rains, 1987). The most effective way of addressing the limitations of crop productivity in saline areas, is the development of salt tolerant varieties. Understanding the genetics of salt tolerance is, therefore, necessary for the development of an effective breeding strategy for salt tolerance. The department of Genetics (US) conducts a wide crosses research programme aiming to transfer genes for salt tolerance to wheat and triticale. The donor species, Thinopyrum disticum, an indigenous coastal wheat grass, adapted to high concentrations of salt, was crossed with cultivated rye (Secale cereale) in an attempt to study the genetics of salt tolerance (Marais et al., 1998). The primary goal of this study was to find molecular markers (RAPD and AFLP) which associate with chromosomes promoting salt tolerance for later attempts to transfer the genes to triticale. Seventy clones of secondary hybrids (Th disticum /4x-rye 1/2x-rye) were tested for salt tolerance and showed different levels of salt tolerance. RAPD-marker analyses were used to identify polymorphisms between salt tolerant and salt sensitive plants. Twelve RAPD primers produced clear, analyzable and repetitive polymorphic . fragments that can be used as useful markers. Different AFLP-primer combinations were tested against the genotypes of 15 clones (Marais & Marais 2001, unpublished data) and produced approximately 2000 clearly distinguishable AFLP fragments, of which 54 (3%) were polymorphic fragments. Two RAPD fragments and 4 AFLP fragments that can be used as possible markers for the presence of chromosomes that contribute to salt tolerance were identified. The interpretation of the markers was complicated by heterogeneity among plants with regard to the origin of their chromosomes and the genetic diversity of the rye genome. It is also possible that chromosome re-arrangement took place during backcrossing, which could have complicated the data. / AFRIKAANSE OPSOMMING: Versouting is een van die groot beperkende faktore op plant- en gewasgroei, omdat die opname van water en voedingstowwe so In ingewikkelde proses is en die effek van lae of matige versouting so alomteenwoordig is. Plantgroei word nadelig geaffekteer as 'n spesifieke ioonkonsentrasie sy drempelwaarde oorskry en toksies word. Die nadelige effek van soutgeaffekteerde grond op gewasproduksie, is wêreldwyd aan die toeneem (Tanji, 1990). Die vlak waartoe plante hoë konsentrasies sout kan hanteer is onder genetiese beheer met verskeie fisiologiese en genetiese meganismes wat 'n bydrae maak tot soutverdraagsaamheid (Epstein & Rains, 1987). Die mees effektiewe manier om die beperkinge op gewas produktiwiteit in versoute gebiede te oorkom, is die ontwikkeling van soutverdraagsame variëteite. Begrip van die genetika van soutverdraagsaamheid is dus noodsaaklik vir die ontwikkeling van In effektiewe telingsstrategie. Die departement Genetika (US) bedryf tans 'n wye-kruisings navorsingsprogram waarmee gepoog word om gene vir soutverdraagsaamheid na korog en koring oor te dra. Die skenkerspesie, Thinopyrum disticum, In inheemse strandkoringgras wat aangepas is by hoë konsentrasies sout, is gekruis met verboude rog (Secale cereale) in 'n poging om die oorerwing van soutverdraagsaamheid te bestudeer (Marais et al., 1998). Die hoofdoel van hierdie studie was om molekulêre merkers (RAPD en AFLP) te vind, wat assosieer met chromosome wat soutverdraagsaamheid bevorder en om nuttige merkers daar te stel vir latere pogings om die gene na korog en koring oor te dra. Ongeveer 70 klone van sekondêre hibriede (Th distichum I 4x-rog /I 2x-rog) is onderwerp aan souttoetse en het verskillende grade van soutverdraagsaamheid getoon. RAPDmerker analise is gebruik om polimorfismes te identifiseer tussen soutverdraagsame en soutsensitiewe plante. Twaalf RAPD inleiers het duidelike, ontleedbare en herhalende polimorfiese fragmente opgelewer en moontlike nuttige merkers uitgewys. Verskillende AFLP-inleier kombinasies, wat getoets is teen die genotipes van 15 klone (Marais & Marais, 2001 ongepubliseerde data) het ongeveer 2000 duidelik onderskeibare AFLP fragmente geproduseer, waarvan 54 (3%) polimorfiese fragmente was. Twee RAPD fragmente en 4 AFLP fragmente is geïdentifiseer wat as moontlike kandidaat merkers gebruik kan word vir die identifisering van chromosome wat bydra tot soutverdraagsaamheid . Die interpretasie van die merkers is bemoeilik deur heterogeniteit tussen die plante wat betref die agtergrond van chromosome wat hulle besit en die genetiese diversiteit van die rog genoom. Dit is ook moontlik dat chromosoom herrangskikking plaasgevind het tydens terugkruising, wat die data verder kon kompliseer.

Two accessions of tepary (phaseolus acutifolius Gray var. latifolius) and navy (P. vulgaris L. 'Fleetwood') beans were studied for salt tolerance at several• developmental stages. Genotypes were germinated at 0.0 through -2.5 MPa NaCl at 25°C and 35°C for nine days. Tepary accessions had higher germination percentages and rates than navy for ≤ - 2.0 MPa at 250C and ≤ - 1.5 MPa at 35°C. Fresh weights of root plus hypocotyl decreased severely with the first increment of NaCl (-0.5 MPa) for all genotypes. Fresh weight of navy was reduced more at 35°C than at 25°C. Genotypes were stressed in vermiculite-filled trays with 0.0 through -1.5 MPa NaCl for 14 days. Final growth stage and rates of emergence were reduced at salinities ~ -0.6 MPa NaCl, and were higher in tepary than navy at -1.2 MPa. Tepary beans tended to maintain higher water and osmotic potentials, and at -0.9 MPa had less reduction in leaf area than navy beans. Fresh weights, dry weights and root:shoot ratios declined in all genotypes with increasing salinities. Plants grown hydroponically were stressed with -0.10, -0.25, and -0.50 MPa NaCl during either vegetative or reproductive stages. Navy had equal or greater fresh and dry weights of leaf, stem, and pods at -0.10 MPa, but tepary beans had equal or greater weights at the highest salinity relative to navy. Tepary had the greatest pod weight with -0.50 MPa NaCl applied during the reproductive stage. Carbon exchange rates (CER) were lower in navy than one or both tepary beans at some sampling times. Tepary beans tended to have higher leaf water and osmotic potentials than did navy. Transpiration and stomatal resistance values were similar in all genotypes, while leaf temperatures were different in white tepary versus navy. Tepary beans yielded higher than navy when grown in low and high salinity fields. Transpiration rates, leaf water and osmotic potentials, and CERs were similar or higher, while stomatal resistance and leaf temperatures were similar or lower in tepary than in navy. Plant height and stand count also were measured. Tepary was more salt tolerant than navy, exhibiting greater tolerance to NaCl at every growth stage.

Shoot and root fresh and dry weight, shoot length, leaf area, leaf area index and relative growth rate of 14 day old tepary bean (Phaseolus acutifolis Gray) and navy bean (Phaseolus vulgaris L.) seedlings were reduced following treatment with NaCl solution exhibiting osmotic potential of either -0.25, 0.50, and -0.75 MPa. Salinity reduced the growth of navy bean more than tepary bean. The physiological basis of the adaptive response of tepary bean seedlings to salt stress was explored by determining the water and osmotic potentials, relative water content, free amino acid and sugar concentrations, distribution and levels of inorganic ions within the seedlings and ATPase activity of the root plasma membrane. Salinity led to an osmotic adjustment in the leaves and the proximal part of the root of tepary bean. Turgor remained almost constant whereas osmotic and water potential and relative water content declined following the salt treatments. The osmotic adjustment of the leaves and proximal part of the roots was -1.7 MPa and -1.2 MPa, respectively, in seedlings treated with -0.75 MPa NaCl solution. Free amino acids and sugars increased under salinity stress in both species but they increased more in the tepary bean. Glucose was the most abundant free sugar. The nonstructural carbon solutes contributed -0.15 MPa to the seedling's osmotic adjustment whereas Na, Cl, K and Ca ion levels contributed -0.85 MPa. However, the levels of these solutes were not large enough to account for the total osmotic adjustment observed in the salt treated seedlings. This study shows that tepary bean has specific strategies to overcome the impact of salinity through osmotic adjustment and exclusion of Na and Cl ions from the stems and leaves by retaining these ions in the proximal part of root and stem base. (Abstract shortened with permission of author.)

Tissue culture and radicle excision techniques were employed to evaluate salt tolerance in alfalfa (Medicago sativa L.). Plant suspension cultures of either seedling root or shoot origin were studied in media with or without supplemental NaCl (3.54 g liter⁻¹). In most cases, the growth rates of root-derived cultures were stimulated by this low level of supplemental NaCl while most shoot-derived cultures were not stimulated by NaCl. Excised radicles of three populations of alfalfa which possessed widely differing ranges of germination salt tolerance were screened in four salts (NaCl, KCl, Na₂SO₄, and K₂SO₄) at six varying concentrations. As was observed in the tissue culture experiments, low levels of NaCl (7.09 g liter⁻¹) stimulated radicle elongation of all populations as compared to the elongation levels of the control solutions (no supplemental salts). In general, for NaCl, the population that posessed the highest degree of germination salt tolerance (Az-St 1982) also displayed the greatest rates of radicle elongation especially in the highest salt concentrations. Additionally, this population along with the moderately germination salt tolerant population (Az-ST 1979) maintained higher rates of elongation in KCl, K₂SO₄ and Na₂SO₄ than did the control germplasm which has little germination salt tolerance (Mesa Sirsa Control). Examinations of each individual population in all four salts simultaneously, indicated that the sulfate salts reduced radicle elongation to a greater extent than did the chloride salts. Evaluation of both osmotic effects and specific ion effects showed that the specific ion effects attributed to the anions were more detrimental to radicle elongation than were the osmotic effects.

Soil salinity affects over 20% of the worlds irrigated land. Potato (Solanum tuberosum L.), the most important vegetable crop worldwide, is relatively salt sensitive. However, relatively little work has been done on salt tolerance of the potato plant. This thesis investigated the methodology of treatment application and scion/rootstock effects on subsequent salt stress responses of four contrasting potato genotypes: Norland, 9506, 9120-05 [ABA-deficient mutant], and 9120-18 [ABA-normal sibling] grown hydroponically in sand. The effect of incremental salt stress were studied, using NaCl, CaCl2 and combined NaCl + CaCl2 pre- treatments as well as varying methods of ABA application with a specific focus on the role of rootstock and scion. Physiological responses of various potato genotypes to salt stress differed depending on how the salt stress was applied. An incremental salt stress regime was able to more effectively differentiate genotypes based on salt stress resistance and greater salt tolerance compared to a sudden salt shock. Generally, the ability to produce ABA was positively related to the degree of salt stress resistance, with higher ABA levels induced under incremental salt stress treatments compared to salt shock. The method of ABA application also had a marked effect on potato responses to salt stress. Slowly increasing concentrations of exogenous ABA maintained growth rates, enhanced root water content and induced more lateral shoot growth compared to a single ABA dose. The degree of salt tolerance induced by the grafted rootstock was primarily modulated by salt acclimation and was manifested in the scion as increased water content, stem diameter, dry matter accumulation, stomatal conductivity, and osmotic potential and was associated with reduced leaf necrosis. Using the salt-resistant 9506 line as a scion also significantly increased root fresh and dry weight and stem diameter as well as root water content of salt-sensitive ABA-deficient mutant rootstocks. Exogenous ABA appeared to enhance plant water status via the roots under salt stress beyond that of grafting alone. This was verified by more positive stomatal conductivity and greater upward water flow in ABA treated grafted and non-grafted plants as compared to the absence of upward water flow in non-treated grafted plants as measured via micro NMR imaging. NaCl pre-treatment produced greater salt stress resistance compared to pre-treatment with CaCl2 and was associated with a specific Na+ ion effect rather than a non-specific EC-dependent response. However, the presence of both ABA and CaCl2 appears to be necessary in order to enhance Na+ exclusion from the shoot and increases the K+/Na+ ratio.

Soil salinity affects over 20% of the worlds irrigated land. Potato (Solanum tuberosum L.), the most important vegetable crop worldwide, is relatively salt sensitive. However, relatively little work has been done on salt tolerance of the potato plant. This thesis investigated the methodology of treatment application and scion/rootstock effects on subsequent salt stress responses of four contrasting potato genotypes: Norland, 9506, 9120-05 [ABA-deficient mutant], and 9120-18 [ABA-normal sibling] grown hydroponically in sand. The effect of incremental salt stress were studied, using NaCl, CaCl2 and combined NaCl + CaCl2 pre- treatments as well as varying methods of ABA application with a specific focus on the role of rootstock and scion. Physiological responses of various potato genotypes to salt stress differed depending on how the salt stress was applied. An incremental salt stress regime was able to more effectively differentiate genotypes based on salt stress resistance and greater salt tolerance compared to a sudden salt shock. Generally, the ability to produce ABA was positively related to the degree of salt stress resistance, with higher ABA levels induced under incremental salt stress treatments compared to salt shock. The method of ABA application also had a marked effect on potato responses to salt stress. Slowly increasing concentrations of exogenous ABA maintained growth rates, enhanced root water content and induced more lateral shoot growth compared to a single ABA dose. The degree of salt tolerance induced by the grafted rootstock was primarily modulated by salt acclimation and was manifested in the scion as increased water content, stem diameter, dry matter accumulation, stomatal conductivity, and osmotic potential and was associated with reduced leaf necrosis. Using the salt-resistant 9506 line as a scion also significantly increased root fresh and dry weight and stem diameter as well as root water content of salt-sensitive ABA-deficient mutant rootstocks. Exogenous ABA appeared to enhance plant water status via the roots under salt stress beyond that of grafting alone. This was verified by more positive stomatal conductivity and greater upward water flow in ABA treated grafted and non-grafted plants as compared to the absence of upward water flow in non-treated grafted plants as measured via micro NMR imaging. NaCl pre-treatment produced greater salt stress resistance compared to pre-treatment with CaCl2 and was associated with a specific Na+ ion effect rather than a non-specific EC-dependent response. However, the presence of both ABA and CaCl2 appears to be necessary in order to enhance Na+ exclusion from the shoot and increases the K+/Na+ ratio.

Thesis (Ph.D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references (leaves 97-114). / Microfiche. / xiii, 114 leaves, bound ill. 29 cm

Thesis (Ph.D.)--Flinders University, School of Biological Sciences. / Typescript bound. Includes bibliographical references. Also available online.

Thesis (MSc.)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The detrimental effect of soil salinity on crop production is a growmg problem worldwide (Tanji, 1990b). The degree to which plants can tolerate high concentrations of salt in their rooting medium is under genetic control with different genetic and physiological mechanisms contributing to salt tolerance at different developmental stages (Epstein & Rains, 1987). Only limited variation exists for salt tolerance in the cultivated cereals. This has prompted attempts to select tolerant progeny following hybridisation of cultivated species and wild, salt-tolerant species. Thinopyrum distichum, an indigenous wheatgrass that is naturally adapted to saline environments (McGuire & Dvorak, 1981), was crossed with triticale (x Triticosecale) in an attempt to transfer its salt tolerance and other hardiness characteristics (Marais & Marais, 1998). The aims of this study were to (i) identify Thinopyrum chromosomes carrying genes for salt tolerance and to identify molecular markers for these chromosomes, (ii) identify a number of diverse monosomic and disomie addition plants. Bulked segregant analysis (BSA), in combination with AFLP, RAPD and DAF marker analysis was implemented to screen for polymorphisms associated with salt tolerance. Five putative AFLP markers and two RAPD markers were detected using bulks composed of salt tolerant plants and bulks composed of salt sensitive plants. The distribution of the markers in these bulks suggests that more than one Thinopyrum chromosome carry genes for salt tolerance. Salt tolerant monosomic and disomie addition plants were characterised for AFLP, RAPD and DAF polymorphisms in an attempt to find markers associated with the chromosome(s) conditioning salt tolerance. One salt tolerant monosomic and one disomie addition plant was identified. One AFLP and two RAPD markers were identified for the Thinopyrum chromosome( s) present in the monosomic addition plant, while three AFLP and three RAPD markers were identified for the disomie addition plant. An attempt was also made to identify diverse chromosome addition plants having complete or near complete triticale genomes plus an additional random Thinopyrum chromosome. Plants with 2n = 43 /44 were identified and characterised for molecular markers (AFLP and RAPD). Cluster analysis was used to group the putative monosomic or disomie addition plants according to the specific Thinopyrum chromosomes they retained. Seventeen AFLP and RAPD markers could be used to group the 24 putative addition plants into six broadly similar groups with different additional Thinopyrum chromosomes. While the members of each group are likely to carry the same additional Thinopyrum chromosomes, this may not necessarily be the case as the interpretation of the marker results is complicated by heterogeneity among plants with regard to the triticale background chromosomes they possess. It is also likely that chromosome translocations occurred during backerossing which may further complicate data. Nonetheless, it is now possible to select disomie addition plants from each group that are likely to represent different Thinopyrum chromosomes. The data will also be useful in future attempts to find further addition plants carrying the remaining Thinopyrum chromosomes. / AFRIKAANSE OPSOMMING: Die skadelike effek van grond versouting op gewasproduksie neem wêreldwyd toe (Tanji, 1990b). Die mate waartoe plante hoë konsentrasies sout in die wortelstelsel kan hanteer is onder genetiese beheer en verskillende genetiese en fisiologiese meganismes dra by tot die soutverdraagsaamheid tydens verskillende ontwikkelingstadia (Epstein & Rains, 1987). Slegs beperkte variasie bestaan vir soutverdraagsaamheid in verboude grane. Dit het aanleiding gegee tot pogings om soutverdraagsame nageslag te selekteer na hibridisasie van verboude spesies en wilde, soutverdraagsame spesies. Thinopyrum distichum, 'n inheemse koringgras, wat aangepas is by brak omgewings (McGuire & Dvorak, 1981), is met korog (x Triticosecale) gekruis in 'n poging om die gene vir soutverdraagsaamheid en ander gehardheidseienskappe oor te dra (Marais & Marais, 1998). Die oogmerke van hierdie studie was om (i) Thinopyrum chromosome te identifiseer wat gene bevat vir soutverdraagsaamheid en molekulêre merkers te vind vir hierdie chromosome, (ii) 'n aantal diverse monosomiese en disomiese addisieplante te identifiseer. Bulksegregaatanalise (BSA), gekombineer met AFLP-, RAPD- en DAF-merkeranalise, is gebruik om polimorfismes geassosieerd met soutverdraagsaamheid op te spoor. Vyf moontlike AFLPmerkers en twee RAPD-merkers is geïdentifiseer met gebruik van bulks bestaande uit soutverdraagsame plante en bulks bestaande uit soutgevoelige plante. Die verspreiding van die merkers in soutverdraagsame bulks dui daarop dat meer as een Thinopyrum chromosoom bydra tot soutverdraagsaamheid. Soutverdraagsame, monosomiese en disomiese addisieplante is gekarakteriseer vir AFLP- en RAPD-polimorfismes in 'n verdere poging om merkers te vind vir chromosome betrokke by soutverdraagsaamheid. Een soutverdraagsame monosomiese en een disomiese addisieplant is geïdentifiseer. Een AFLP- en twee RAPD-merkers is geïdentifiseer vir die Thinopyrum chromosoom(e) teenwoordig in die monosomiese addisieplant, terwyl drie AFLP- en drie RAPDmerkers geïdentifiseer is vir die disomiese addisieplant. 'n Poging is ook gemaak om diverse addisieplante te identifiseer met 'n volledige koroggenoom plus 'n addisionele Thinopyrum chromosoom. Plante met 2n = 43 / 44 is geïdentifiseer en gekarakteriseer met molekulêre merkers (AFLP en RAPD). Tros-analise is gebruik om die vermoedelik monosomiese of disomiese addisieplante te groepeer volgens die spesifieke Thinopyrum chromosome wat hulle behou het. Sewentien AFLP- en RAPD-merkers is gebruik om die 24 vermoedelike addisieplante in 6 groepe met verskillende Thinopyrum chromosome te groepeer. Alhoewel dit voorkom of die verskillende plante in 'n groep dieselfde addisionele Thinopyrum chromosoom het, is dit nie noodwendig die geval nie aangesien die interpretasie van die merkers bemoeilik word deur die heterogeniteit tussen die plante wat betref die agtergrond korogchromosome wat hulle besit. Dit is ook moontlik dat chromosoom herrangskikkings plaasgevind het gedurende die terugkruisings, wat die data verder kan bemoeilik. Nietemin, dit is nou moontlik om disomiese addisies te selekteer uit elke groep wat moontlik verskillende Thinopyrum chromosome bevat. Die data kan ook gebruik word om in die toekoms verdere addisieplante te identifiseer wat die oorblywende Thinopyrum chromosome bevat.