A protocol for cauliflower micro-propagule production was developed and optimised for both micropropagation and artificial seed production techniques using meristematic tissues from cauliflower curd. All steps in the protocol were empirically optimised including: blending, sieving, culture methods, liquid culture media composition and plant growth regulator combinations and concentrations. The cost of the micro-propagules could be reduced by as much as 50% on the initial costings reported previously since treatments doubled the number of microshoots produced per culture unit. The research confirmed the suitability of cauliflower microshoots to be encapsulated as artificial seeds and an effective protocol for microshoot encapsulation was designed through the optimization of 1) the production of cauliflower microshoots suitable for encapsulation, 2) encapsulation procedures, 3) artificial seed artificial endosperm structure, 4) conversion materials. The possibility of culturing cauliflower artificial seeds in commercial substrates such as perlite, sand, vermiculite and compost was confirmed. The use of plant preservative mixture (PPM) for the control of contamination in cauliflower culture media and artificial seeds was optimised and the effect of this material on the development of plant material was assessed. It was confirmed that cauliflower artificial seed could be stored in a domestic refrigerator for up to 6 months which could have a great impact in cauliflower breeding programmes. The huge number of cauliflower microshoots that could be produced using this protocol and the homogeneity of the culture system, provided a tool for the molecular analysis of cauliflower microshoots (and artificial seed) abiotic stress tolerance analysis. Various treatments were conducted to improve microshoot cold tolerance and the up-regulation of the CBF/DREB1 transcription factor including low temperature acclimation, mannitol, ABA (abscisic acid) and Mo (molybdenum). Microshoots were confirmed to acclimate successfully using low temperature. Mo was shown to improve the cold tolerance of cauliflower microshoots and to up-regulate CBF/DREB1 in the absence of low temperature acclimation. Acclimation did not increase the accumulation of dehydrin proteins and it is concluded that dehydrins do not play a significant role in the cold tolerance of cauliflower microshoots. Since cauliflower breeding and seed multiplication protocols make extensive use of micropropagation, the studies reported in this research could make a significant impact by decreasing the cost of micropropagation and increasing its reliability. It also opens new perspectives for further research for cauliflower artificial seed production and the possibility of sowing these seeds directly in the field. Furthermore, this research helps to facilitate cauliflower breeding programmes by improving the understanding of abiotic stress tolerance mechanisms and the relationship between different types of abiotic stresses such as cold and drought.

Orientador: Paulo Mazzafera / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T08:03:59Z (GMT). No. of bitstreams: 1 Santos_AdrianaBrombinidos_M.pdf: 1079927 bytes, checksum: 028eeeefe5616e233dd57be085df965f (MD5) Previous issue date: 2009 / Resumo: Plantas das cultivares Catuaí Vermelho e Mundo Novo de Coffea arabica L. e da cultivar Apoatã de C. canephora Pierre ex Froehner, e plantas de Mundo Novo enxertadas sobre Apoatã foram avaliadas quanto a possíveis estratégias utilizadas para suportar a baixa disponibilidade de água no solo. Em paralelo, foi conduzido um ensaio com suspensões celulares de Catuaí Vermelho sob estresse osmótico induzido por PEG. Assim, este estudo teve por objetivo examinar alguns mecanismos de tolerância à seca em café através da avaliação de aspectos morfológicos (sistema radicular), fisiológicos (ajuste osmótico) e moleculares (expressão de deidrinas e aquaporinas) em plantas jovens e cultura de células. A análise de expressão do gene CaDh1 (deidrina) em suspensão celular de café tratada com PEG (15% m/v), por 8, 24 e 72 h, mostrou aumento na atividade sob estresse osmótico, o que poderia estar associado a um possível mecanismo de proteção de membranas e macromoléculas contra a dessecação. Não foi verificada alteração na resposta dos genes PIP1, PIP2 e TIP1(aquaporinas) sob condição de estresse nas células de café. Plantas de café cultivadas em vasos (4,0 L) foram submetidas a estresse hídrico moderado por suspensão da irrigação, sob condições de casa de vegetação, até que o potencial hídrico da folha no "predawn" atingisse cerca de -2,0 MPa. Verificou-se que as taxas de crescimento nos genótipos de café arábica, avaliada pelo acúmulo de biomassa total (folhas e raízes), foram intensamente afetadas pela seca, com redução em torno de 26,5% em Catuaí e 29,0% em Mundo Novo. Por outro lado, as plantas enxertadas não apresentaram redução significativa de crescimento em relação às plantas irrigadas, sugerindo uma contribuição positiva da enxertia para melhorar a eficiência na captação de água pelo porta-enxerto Apoatã, sob condição de baixa disponibilidade de água no solo. O conteúdo de açúcares solúveis totais nas folhas das plantas irrigadas foi maior que o observado para as plantas sob déficit hídrico. A maior redução foi constatada entre os genótipos de café arábica (23,0% em Catuaí e 43,8% em Mundo Novo), enquanto as reduções em Apoatã e MN/Apoatã não foram significativas. Verificou-se maior acúmulo de aminoácidos totais nas folhas e raízes de plantas de Catuaí sob estresse hídrico comparado a Mundo Novo. Apoatã e MN/Apoatã não mostraram aumentos significativos em relação às respectivas plantas irrigadas. Em todos os genótipos analisados, os teores de prolina (Pro) nas folhas e raízes de plantas sob estresse foram aumentados e, nas raízes, esse acúmulo foi bastante acentuado, sendo 6 vezes maior nos genótipos de café arábica, 4 vezes em Apoatã e 16 vezes na enxertia MN/Apoatã em relação às plantas irrigadas. Entretanto, tais aumentos não parecem estar diretamente associados a maiores graus de tolerância à seca em café. Os conteúdos dos aminoácidos asparagina (Asn), ácido ?- aminobutírico (Gaba), glutamato (Glu) e alanina (Ala) também foram alterados, observandose, de modo geral, aumento nos teores de Asn e Gaba e redução de Glu e Ala nas folhas e raízes das plantas sob estresse. Em conclusão observou-se que os genótipos com sistema radicular mais vigoroso como Apoatã e Catuaí foram menos afetados pela seca, provavelmente porque foram mais hábeis em explorar o perfil do solo para a captação de água. / Abstract: Seedlings of the cultivars Catuaí Vermelho and Mundo Novo of Coffea arabica L., of the cultivar Apoatã of C. canephora Pierre ex Froehner, and plants of Mundo Novo grafted on Apoatã were evaluated for possible strategies to withstand low water availability in the soil. In parallel, an assay with cellular suspension culture (Catuaí Vermelho) was carried out under osmotic stress induced by PEG. Thus, this study aimed to examine morphological (root systems), physiological (osmotic adjustment) and molecular (expression of dehydrins and aquaporins) traits in coffee plants and coffee cell suspension cultures as possible mechanisms of drought tolerance. The expression analysis of CaDh1 (dehydrin) in coffee cell suspensions grown with PEG (15% w/v) for 8, 24 and 72 h, showed an increase in activity under osmotic stress indicating a possible protection mechanism against desiccation. No changes were observed in the response of PIP1, PIP2 and TIP1 genes (aquaporins) under stress condition in coffee cells. Under greenhouse conditions, coffee seedlings were cultivated in 4.0 L pots submitted to moderate drought stress by suspending irrigation until leaf water potential at predawn reach about -2.0 MPa. Among the genotypes of arabica coffee subjected to water stress there was reduction in total dry weight, around 26.5% in Catuaí and 29.0% in Mundo Novo when compared with irrigated control plants. On the other hand, the grafted plants were not affected, suggesting a positive contribution of the grafting due to bigger efficiency in the water acquisition by the rootstock Apoatã under low water availability in the soil. The content of total soluble sugars was greater in leaves of irrigated plants than plants under water stress. The greatest reduction was verified among the genotypes of arabica coffee (23.0% in Catuaí and 43.8% in Mundo Novo) while the reduction in Apoatã and MN/Apoatã was not statistically significant. The accumulation of total amino acids was higher in leaves and roots of water stressed Catuaí than Novo Mundo. Apoatã and MN/Apoatã did not show significant increase in the content of amino acids in relation to irrigated control plants. In all genotypes, water stress induced an increase in the levels of proline (Pro) in leaves and roots. In the roots this increased was about 6 times in the genotypes of arabica coffee, 4 times in Apoatã and 16 times in the grafting MN/Apoatã in relation to irrigated control plants. However this increase in the levels of Pro does not seem to be directly associated to higher degrees of drought tolerance in coffee. The content of amino acids asparagine (Asn), ?-aminobutiric acid (Gaba), glutamate (Glu) and alanine (Ala) was also changed in the leaves and roots of stressed plants. In general, there was increase in levels of Asn and Gaba while was observed reduction in content of Glu and Ala. In conclusion it was found that the genotypes with more vigorous root system as Apoatã and Catuaí were less affected by drought, probably because they were more efficient in exploring the soil profile for the water absorption. / Mestrado / Biologia Vegetal / Mestre em Biologia Vegetal

Changing climate in combination with growing world populations mean that there is growing need for plants to be grown on land that is currently considered marginal for agriculture. Sorghum is a C4 plant that serves as an important food crop in Africa and India. It is also known to be highly drought tolerant but the mechanisms responsible for this tolerance are unclear. The overall aim of this study was to understand the drought tolerance mechanisms that enable the plant to maintain leaf function for a long time during water deficit. In Chapter 2 of this thesis, I studied the underlying physiological mechanisms for tolerating drought in two sorghum varieties with differing degrees of drought tolerance compared to a closely related species, Zea mays. During progressive drought, the more tolerant sorghum variety Samsorg 17 maintained net CO2 assimilation and photochemistry longest relative to the less tolerant Samsorg 40 and Zea mays. Differences were also seen in stomatal aperture, stomatal density, total chlorophyll content, chl a:b and A/Ci curve responses with maize more affected than the sorghum varieties. In Chapter 3, I identified novel drought tolerance mechanisms in the sorghum varieties. The less tolerant Samsorg 40 lost PsbA (D1) and Rubisco proteins and reengineered its photosynthetic apparatus to accumulate amino acids and sugars in order to maximise survival under drought. Samsorg 17 maintained photosynthetic proteins notably PsbA (D1) and Rubisco and accumulated high constitutive sugar content allowing for the maintenance of transpiration and photosynthesis. The two sorghum varieties had strikingly contrasting approaches of tolerating drought as demonstrated in Chapter 3. In Chapter 4, the aim was to characterise biochemical and metabolic changes that occur in response to drought. In particular, to identify sugars that are accumulated constitutively in Samsorg 17 and nitrogen sinks for lost N in Samsorg 40. My findings indicated a contrasting response in terms of sugar content in Samsorg 17 but support for amino acids as N sinks in Samsorg 40 as reported earlier. Sugars, sugar alcohols, lipids, organic acids, heat shock proteins and dehydrins were generally higher or more induced in Samsorg 17 relative to Samsorg 40. Samsorg 40 rather made amino acids. The implications of my findings and future work arising from this study were discussed in detail in the final chapter. In conclusion, in this thesis, it was demonstrated that closely related plants can have mechanistically different physiological and biochemical mechanisms for responding to drought.

- Ph.D. thesis - Milan Urban, 2017 In some years, the agricultural production of oilseed rape, an important crop in the Czech Republic, is - besides biotic stress - facing the problem of damage caused by frost or drought. Together with special attention paid to proteins revealing responses between crop genotypes with differential abiotic stress tolerance levels we reviewed possible applications of proteomic results in crop breeding programs aimed at an improvement of crop stress tolerance (paper 1). For first original result, cold temperature was imposed upon non-vernalized plants in the stage of leaf rosette. The article (paper 2) shows a significant correlation between frost tolerance (FT), dehydrin (DHN) accumulation, and photosynthetic acclimation in five cultivars (cvs). Newly, the specific DHN D97 was shown to accumulate and other DHNs were shown to have qualitative differences in accumulation. These results imply that proper FT assessment is based on rapid photosynthetic acclimation together with higher accumulation of protective compounds. Drought stress (paper 3) was imposed in the water- demanding stem prolongation phase before flowering, because late-spring drought before and during flowering decreases the yield and seed quality significantly. This paper newly describes two water-uptake...

Indiana University-Purdue University Indianapolis (IUPUI) / Strawberry (Fragaria spp.) is an agricultural crop grown often in temperate regions that has high variability in its susceptibility to freezing injury. To breed cultivars for frost and freezing tolerance, identification of molecular markers associated with low temperature tolerance is advantageous. In this work, I investigated a high-throughput method for protein assays and western blotting. Success in streamlining these processes saves an immense amount of time and allows for the processing of more samples and obtaining larger datasets. Thirty-three octoploid varieties were tested for their accumulation of five different potential biomarkers in response to cold exposure. It was found that total dehydrin content, has the strongest potential to be reliable biomarkers for breeding programs. Previous work identified seven putative dehydrins in Fragaria, where two were purified and positively identified by mass spectrometry and determined to be COR47-like (SKn) and XERO2-like (YnSKn). This work demonstrated that cold tolerance positively correlated with dehydrin protein expression levels. To understand the cold-regulated expression of dehydrins as a function of cold exposure time, the levels of transcripts and corresponding proteins were examined in strongly cold tolerant (Alta) and lesser cold tolerant (FDP817, NCGR1363) Fragaria diploid genotypes. The COR47-like (SKn) and XERO2-like (YnSKn) dehydrins both had higher transcript accumulation and protein levels in the more cold tolerant line in comparison to the two less cold tolerant lines. Lack of correlation between transcript and resulting COR47 protein level in Alta were observed at several different timepoints, where protein accumulation preceded an increase in RNA. This trend was not seen with XERO2. This initiated an investigation to discover at what level COR47 is being regulated. First, the COR47 coding region was sequenced for all the genotypes to confirm against the predicted sequence. In addition, since two isoforms of the COR47 gene exist, and could possibly explain the discrepancy in transcript counts, primers were designed for both isoforms and RT-qPCR was performed to examine the transcripts of COR47 more closely. Through examination of the non-congruence of COR47 transcripts and protein, it was found that transcriptional mechanisms of regulation are not involved, and that post transcriptional and post-RNA splicing mechanisms are likely to be responsible for the observed trend in Alta. Conclusions from this work demonstrate that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low temperature tolerance in diploid strawberry.

The group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization. / NSERC