Chan Yee-kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 108-131). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / Acknowledgments --- p.v / General Abbreviations --- p.vii / Abbreviation for Chemicals --- p.x / Table of Contents --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Salinity and agriculture --- p.2 / Chapter 1.3 --- Plant adaptation to salinity --- p.4 / Chapter 1.3.1 --- Salt secretion --- p.6 / Chapter 1.3.2 --- Ion transport --- p.8 / Chapter 1.3.2.1 --- Role of H+-ATPase in salt tolerance --- p.8 / Chapter 1.3.2.2 --- Potassium and sodium uptake --- p.13 / Chapter 1.3.2.3 --- Sodium efflux --- p.15 / Chapter 1.3.3 --- Osmotic adjustment --- p.20 / Chapter 1.3.3.1 --- Accumulation of mannitol --- p.21 / Chapter 1.3.3.2 --- Accumulation of proline --- p.23 / Chapter 1.3.3.3 --- Accumulation of glycinebetaine --- p.23 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Plant materials and growth conditions --- p.26 / Chapter 2.1.1 --- Surface sterilization of Arabidopsis seeds --- p.26 / Chapter 2.1.2 --- Determination of sub-lethal inhibitory doses of sodium --- p.27 / Chapter 2.1.3 --- Growth conditions of Arabidopsis seeds for total RNA extraction --- p.27 / Chapter 2.1.4 --- NaCl dosage tests --- p.28 / Chapter 2.1.5 --- Expression kinetic tests --- p.28 / Chapter 2.2 --- Isolation of total RNAs --- p.28 / Chapter 2.3 --- Isolation of genes differentially expressed in NaCl concentration by RAP-PCR --- p.30 / Chapter 2.3.1 --- RNA fingerprinting by RAP-PCR --- p.30 / Chapter 2.3.2 --- PCR reamplificatin of RAP products --- p.31 / Chapter 2.3.3 --- Cloning of differentially expressed genes --- p.33 / Chapter 2.3.3.1 --- Ligation of inserts into pCR-Script vector and transformation --- p.33 / Chapter 2.3.3.2 --- Ligation of inserts into pBluescript II KS (+) T-vector and transformation --- p.36 / Chapter 2.3.3.3 --- Screening of recombinant plasmids --- p.37 / Chapter 2.4 --- Sequencing of differentially expressed genes --- p.39 / Chapter 2.4.1 --- DNA cycle sequencing --- p.39 / Chapter 2.5 --- Northern blot hybridization of NaCl inducible genes --- p.40 / Chapter 2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.40 / Chapter 2.5.2 --- Northern blotting --- p.41 / Chapter 2.5.3 --- Preparation of single-stranded DIG-labeled PCR probes --- p.41 / Chapter 2.5.3.1 --- Isolation of Total RNA --- p.41 / Chapter 2.5.3.2 --- Primer design --- p.42 / Chapter 2.5.3.3 --- PCR amplification of single-stranded DIG PCR probes --- p.43 / Chapter 2.5.4 --- Hybridization --- p.45 / Chapter 2.5.5 --- Stringency washes --- p.46 / Chapter 2.5.6 --- Chemiluminescent detection --- p.46 / Chapter 3. --- Results / Chapter 3.1 --- Determination of sub-lethal inhibitory doses of sodium --- p.48 / Chapter 3.2 --- Isolation of total RNA from A. thaliana treated with sodium chloride --- p.48 / Chapter 3.3 --- Isolation of genes differentially expressed in sodium concentration by RNA arbitrarily primed polymerase chain reaction RAP-PCR --- p.52 / Chapter 3.3.1 --- Differential cDNA fragments identified by RAP-PCR --- p.52 / Chapter 3.3.2 --- PCR reamplification of RAP products --- p.52 / Chapter 3.3.3 --- Cloning of selected RAP-fragments --- p.62 / Chapter 3.4 --- Nucleotide sequence analysis of selected RAP PCR clones --- p.65 / Chapter 3.5 --- Expression pattern analysis of salt inducible genes by northern blot hybridization --- p.75 / Chapter 3.5.1 --- Preparation of single-stranded digoxigenin (DIG)-labeled probes --- p.75 / Chapter 3.5.2 --- Dosage response of NaCl inducible genes --- p.79 / Chapter 3.5.3 --- Expression kinetics of NaCl inducible genes --- p.80 / Chapter 4. --- Discussion / Chapter 4.1 --- Isolation of RAP-PCR targets --- p.93 / Chapter 4.2 --- Expression of NaCl inducible P450 genes --- p.94 / Chapter 4.2.1 --- Cytochrome P450 CYP73A5 --- p.97 / Chapter 4.2.2 --- Cytochrome P450 CYP83A1 --- p.98 / Chapter 4.3 --- NaCl induction gene related to post-transcriptional activities --- p.99 / Chapter 4.3.1 --- Glycine-rich RNA binding protein (BAC F3F19) --- p.100 / Chapter 4.3.2 --- Chloroplast signal recognition particle (54CP) --- p.103 / Chapter 4.4 --- Conclusion --- p.106 / References --- p.108

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The responses of Nicotiana tabacum L. var. Samsun to alterations in the nitrogen (N) supply under saline conditions in vitro were monitored. The aim was to test the hypothesis that nitrate-nitrogen supplementation to salt stressed plants alleviates the deleterious effects of salt on plant growth. Due to its capacity to be maintained under stringent environmental conditions, in vitro shoot cultures were chosen as the system of study. Nicotiana tabacum plantlets regenerated from callus in vitro were excised and rooted on solid MS culture medium containing a range of concentrations of NaCI (0 - 180 mM) and N (0 - 120 mM, as NO3--N, NH4+-N or a combination). A variety of parameters of root and shoot growth, nutrient utilisation and nitrogen metabolism were assessed over a 35 d period. Plant growth on 40 mM NO3--N + 20 mM NH4+-N (standard MS nutrients) was inhibited by the presence of salt, with root growth being more adversely affected by salt than stem growth. Root emergence was delayed from 6 d (0 mM NaCI) to 15 d (180 mM NaCI). Similar suppression of growth for all parameters, except root mass and leaf chlorophyll content, was observed when NaCI was replaced with mannitol at equivalent osmolalities. Root mass and leaf chlorophyll were significantly improved in plantlets supplied with mannitol. The time of root emergence was unaffected by mannitol supply, with all roots emerging after 10 d in culture. Plantlet growth on NH4+-N only (0 - 60 mM) was severely inhibited, even in the absence of NaCI, and was inferior to growth on NO3--N. Nitrate additions to salt stressed plantlets could not match growth in control (0 mM NaCI) plantlets. When plantlets were cultured on NO3--N only (0 mM, 30 mM, 60 mM, 120 mM), the increase in nitrate supply up to 60 mM resulted in a small improvement in growth on 90 mM NaCI, but had almost no effect on growth at 180 mM NaCl. A nitrate supply of 120 mM led to growth inhibition in all parameters, even in the absence of NaCl. Plantlet growth on isosmotic concentrations of mannitol in the presence of 0 - 120 mM NO3--N essentially mimicked that of NaCI, except for leaf chlorophyll content which was improved on mannitol at all NO3-·N levels. Nitrate uptake (measured as depletion from growth medium) by plantlets grown on 0 - 180 mM NaCI was positively correlated to availability of nitrate but negatively correlated to NaCI supply. Similar results were obtained for a mannitol supply except nitrate uptake was enhanced significantly on mannitol compared to NaCl. Sodium and chloride uptake appeared unaffected by nitrate concentration. Leaf protein content responded favourably to an increase in the NO3--N supply up to 60 mM and, in particular, appeared to be stimulated in the presence of 180 mM NaCl. Nitrate reductase (NR) activity was found to be inhibited drastically by salt and NO3--N supplementation to the salt medium had no effect on enzyme activity. A reduction in leaf total RNA content was recorded with an increase in NaCI concentration from 0 - 180 mM. A positive response to an increase in the NO3--N supply from 30 mM to 60 mM was detected in the presence of NaCl. Attempts were made to assess the levels of mRNA for NR in response to the various NaCl and N regimes. The plasmid pBMCI02010 containing a NR cDNA insert was isolated and purified and used in both radioactive and non-radioactive RNA slot blot hybridisation procedures. However, due to problems of non-specific binding of the probe, no quantification of the levels of NR mRNA in response to the various treatments could be made. Nitrate supplementation to plantlets of Nicotiana tabacum growing in vitro did not appear to ameliorate the effects of salinity stress, such that growth of plantlets in the presence of NaCI was always inferior to that in the absence of NaCl. As a large portion of growth inhibition was found in this study to be a result of osmotic rather than ionic effects of salt, it is questioned whether a nitrate supply would have an ameliorating effect on plant growth under field conditions. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

[Truncated abstract] Eucalyptus occidentalis, a timber species from south Western Australia, is highly salt and waterlogging tolerant. Screening identified genotypes tolerant of high salt concentrations and waterlogging. Tolerance at provenance, family and individual level, and how phenotypic performance under salt and waterlogging was inherited was explored to provide a breeding population. Salt and/or waterlogged screening was carried out under controlled conditions up to extreme salt levels to determine tolerance between genotypes. This tank method was shown to produce repeatable results. Seedlings of 30 families from 9 provenances were used for screening. At low salt concentration (up to 300 mM NaCl), differentiation occurred for some traits but in general there was only a slight reduction in growth under salt, and waterlogging alone was not detrimental. At high salt concentration (550 mM) differentiation occurred among genotypes for all traits. Equivalent genotypes were also planted in field trials at three sites, two with medium (583 - 847 mm) and one with low rainfall (372 - 469 mm), in southern Western Australia. Survival was low (<53%) after 9 months due to an exceptional dry season followed by 3 months waterlogging in Kirkwood (38 - 1360 mSm-1), but was high >89% after 33 months in saline fields in Sandalwindy (96 - 976 mSm-1) and Roberts (88 - 1424 mSm-1). Some families were similarly in high rank for height under saline conditions in controlled and field trials. Height had the highest narrow-sense heritability value, especially under controlled saltwaterlogging (0.85) treatment and 20% selection enabled a gain of 8-14% under controlled conditions and in the field. Leaf production under salt was not an inherited trait. Systems were developed to hasten deployment of selected material. Extended daylength (16 h) and paclobutrazol (1 mg a.i/mm stem circumference) stimulated flowering in 2 year-old plants. Clonal propagation was possible. Grafting success varied from 0-100% depending on scion/rootstock provenances. ... There was only a slight reduction in heterozygosity from species level to provenance and family levels, and two superior genotypes maintained high diversity. v Crossing was possible using one stop pollination of cut immature styles and capsule retention varied from 0-34% and germination rate from 2-96%. Genetic distance between parents was correlated with seed set and offspring fitness. Wider genetic distances increased capsule retention, seed germination and seedling survival. Under 500 mM salt-waterlogging, offspring heights were similar when parental genetic distances were similar. High heritability value for height from ANOVA-REML parental screening was confirmed using parent-offspring regression. Screened superior genotypes, which withstood very high salt concentration, provide a breeding population for further breeding and for plantations under saline regions in low-medium rainfall areas in Western Australia and other parts of the world. These trees provide an economic return in areas where no other plants may survive and an environmental service in potentially reducing waterlogging, salinity and its spread.

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.

Bibliography : leaves 197-209 / 210 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1991

Indiana University-Purdue University Indianapolis (IUPUI) / Dehydrins are type II LEA proteins induced in many plants during drought, low temperature, and high salinity to confer stress tolerance. AtERD14 is an Arabidopsis thaliana dehydrin that functions in part of the cold stress pathway. AtERD14 has chaperone-like capabilities that allow it to bind and protect various proteins from dehydration stresses. In order to determine the necessary components for cold induction of AtERD14, AtERD14prom::GFP/GUS and AtERD14prom::AtERD14 in AtERD14 KO constructs were created and stably transformed into A. thaliana. Analysis of the constructs showed the AtERD14 promoter alone was insufficient to respond to cold, and it was necessary to attach the AtERD14 coding region to the promoter to induce a cold response in ERD14. On the other hand, the RD29aprom::GFP/GUS promoter did respond to cold stress, indicating that RD29a does not require its coding region to support an increased amount of reporter activity after cold stress. The protoplast transformation system, while capable of transient expression of introduced constructs in protoplasts, was difficult for use for cold-inducible expression.