Dissertations / Theses on the topic 'Recalcitrants seeds'

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The genus Campomanesia spp. has great economic potential unexploed. How its seeds not tolerate desiccation its propagations has been made by seeds extraction, when they are in germinate at appropriate physiological conditions. Several problems such as dormancy, recalcitrance, seasonality, difficulty of propagation has difficult he rational exploration of various species. Environmental conditions appropriate for the germination process may be provided in laboratories by in vitro multiplication. In this work techniques were applied to study some factors that affect ex vitro and in vitro germination of gabirobeira seeds and anther and ovaries culture through micropropagation. The results indicated that ex vitro germination the test substrates (Sand, Bioplant®, Tri-mix®, Bioplant®+Sand (1:1 v/v), Tri-mix®+Sand) for all substrates were superior to Tri-mix® substrate for germination percentage and for emergency speed of index. For the in vitro germination was found that all the culture medium tested in the absence of sucrose had a satisfactory germination rate. Evaluating the effects of accelerated aging and GA3 in the in vitro germination conclude that premature aging interfered the gabirobeira seeds ability of germination and GA3 is unnecessary for the germination stage. In the anthers and ovaries of culture, no callus formation in any of the treatments. However, the PVP was effective as an antioxidant. Keywords: sucrose,
A gabirobeira pertence ao gênero Campomanesia spp. possui grande potencial econômico não explorado. Como suas sementes não toleram a dessecação, sua propagação tem sido realizada logo após a extração, quando estas estão em condições fisiológicas apropriadas para germinar. Vários problemas como dormência, recalcitrância, sazonalidade e dificuldades de propagação têm inviabilizado a exploração econômica racional de várias espécies. Condições ambientais apropriadas para o processo de germinação podem ser fornecidas em laboratórios por meio da multiplicação in vitro. Neste trabalho, foram aplicadas técnicas para estudar alguns fatores que interferem na germinação ex vitro e in vitro de sementes de gabiroba e na micropropagação através da cultura de anteras e ovários. Os resultados indicaram que ao testar os substratos (Areia, Bioplant®, Tri-mix®, Bioplant®+Areia (1:1 v/v), Trimix ®+Areia)todos os substratos foram superiores ao substrato Tri-mix® para a porcentagem de germinação e vigor. Para a germinação in vitro verificou-se que todos os meios de cultura testados (MS, MS meia força, WPM e WPM meia força) na ausência de sacarose tiveram uma taxa de germinação satisfatória. Avaliando os efeitos do envelhecimento acelerado e de GA3 na germinação in vitro, o envelhecimento acelerado interferiu na capacidade germinativa das sementes de gabiroba e verificou-se que a aplicação de GA3 exógeno é desnecessária para a etapa de germinação. Na cultura de anteras e ovários, não houve calogênese em nenhum dos tratamentos testados. No entanto, o PVP foi eficiente como antioxidante.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O elevado metabolismo das sementes recalcitrantes de Inga vera Willd. subsp. affinis (DC.) T.D. Pennington, é um dos principais fatores que dificultam sua conservação. Uma forma de diminuir os efeitos do intenso metabolismo dessas sementes poderia ser a redução da temperatura do ambiente, pois a temperatura determina a velocidade das reações enzimáticas, afetando as taxas respiratórias. Contudo, as sementes recalcitrantes são intolerantes à dessecação e frequentemente não podem ter seu teor de água reduzido até níveis que permitam a redução da temperatura sem que ocorram danos pelo congelamento da água. Devido à falta de informações sobre o metabolismo respiratório dessas sementes em diferentes condições de estresse, o presente trabalho teve como objetivo verificar as taxas respiratórias desses embriões com diferentes níveis de hidratação e após aplicação de ácido abscísico e estresse osmótico em diferentes temperaturas. Os resultados possibilitaram observar a presença de reações oxidativas que não às da respiração, devido ao consumo elevado de oxigênio, e que as alterações nos níveis de hidratação e temperatura de incubação assim como a aplicação de ácido abscísico e estresse osmótico promoveram alterações nesse metabolismo oxidativo. Os resultados obtidos trazem novas perspectivas para a conservação das sementes recalcitrantes
The high metabolism of recalcitrant seeds of Inga vera Willd. subsp. affinis (DC.) T.D. Pennington is one of the main factors that complicate their conservation. One way to lessen the effects of the intense metabolism of these seeds could be the reduction of temperature of the environment, because temperature determines the speed of enzymatic reactions, affecting respiration rates. However, recalcitrant seeds are desiccation intolerant and often can not have their water content reduced to levels that allow a reduction in temperature without damage by the freezing of water. Due to lack of information on the respiratory metabolism of these seeds in different conditions of stress this study aimed to determine respiration rates of embryos with different levels of hydration and after application of abscisic acid and osmotic stress at different temperatures. The results allowed to observe the presence of oxidative reactions than those of breath due to high consumption of oxigen, and that changes in moisture levels and incubation temperature as well as the application of abscisic acid and osmotic stress promoted changes in oxidative metabolism. The results provide new perspectives for the conservation of recalcitrant seeds

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The objective of this work to determine effective treatment for breaking dormancy of seeds of Ocotea puberula, check the behavior of the seed during drying and storage and evaluate the appropriate method for detecting pathogens, asepsis, seeds prior to detection of pathogens and genera of fungi which infest seeds of this species. The seeds were collected in five counties of the State of Santa Catarina (Fraiburgo Joaçaba, Curitibanos, Ponte Alta and Brunópolis), and each collection site was considered a lot. To break dormancy were tested four treatments: 1- control, 2- seeds without tegument; 3- sulfuric acid for 5 minutes and 4- drying the seeds at 25 °C for 12 hours. After performing the tests break dormancy, seeds were subjected to germination test gerboxes substrate with blotting paper in BOD at 30 oC with 4 replicates of 20 seeds per treatment/lot. Regarding the types of drying seeds with an initial moisture content of 38% was dried to 18% with gradient of 2% in oven (35 °C) in a desiccator with silica gel (25 °C). After drying, was determined the water content and viability (tetrazolium and germination tests). In the armazenament study, the seeds were stored with and without fruit, in a dry camera (40% RH and Temp. 10 ± 2 oC) for periods of 0, 3, 6 and 9 months. At each time interval were determined water content and the viability of the seeds by germination, tetrazolium and DNA integrity testing. Determination of seed health was held in PDA culture medium, medium culture V8 and "Blotter Test". In each test were used seeds With and without disinfection (disinfection with sodium hypochlorite and alcohol) totaling 80 seeds for each test. Incubation of seeds was realized in a chamber with controlled temperature of 22 °C ± 3 °C, with a photoperiod of 12 hours for seven days when the assessment and identification of fungi occurred. It was observed that seeds without tegument started germination after 14 days of the start of the test, with stabilizing stand at 36 days, with average scores of 71% germination. Not was observed seed germination for other treatments and control in all lots used. Regarding drying, it was observed that up to 32% humidity no change in seed quality, regardless of the type of drying and verified significant loss of germination after this value. Seeds of Ocotea puberula lost their viability after 3 months of storage, with or without fruit. Nine genera of fungi were observed: Penicillium sp., Phomopsis sp., Epicocum sp., Curvularia sp., Colletotrichum sp., Aspergillus sp., Alternaria sp., Fusarium sp. and Trichoderma sp.. It was concluded that the method of seed coat removal was effective in breaking dormancy of seeds of Ocotea puberula and the type of drying does not affect the quality of the seed; however, reducing the water content below 32% decreased germination. Seeds of Ocotea puberula lose their viability after 3 months of storage in dry chamber and agarized media were more sensitive for the detection of fungi in weed seeds and seed disinfection with sodium hypochlorite and alcohol reduces the incidence of these fungi, is indicated when sanity test is performed with seeds of this species
Objetivou-se com este trabalho determinar tratamento eficiente para superação de dormência de sementes de Ocotea puberula, verificar o comportamento das sementes durante a secagem e o armazenamento e avaliar o método adequado para detecção de patógenos, da assepsia, das sementes antes da detecção dos patógenos e quais gêneros fúngicos infestam sementes desta espécie. As sementes foram colhidas em cinco municípios do Estado de Santa Catarina (Fraiburgo, Joaçaba, Curitibanos, Ponte Alta e Brunópolis), e cada local de coleta foi considerado um lote. Para a superação da dormência, foram testados quatro tratamentos: 1- testemunha, 2- sementes sem o tegumento; 3- ácido sulfúrico por 5 minutos e 4- secagem das sementes a 25 oC por 12 horas. Após a realização dos testes de superação da dormência, as sementes foram submetidas ao teste de germinação em caixas gerbox com substrato de papel mata borrão, em BOD a 30 oC, com 4 repetições de 20 sementes por tratamento/lote. Em relação aos tipos de secagem, sementes com teor de água inicial de 38% foram secas até 18%, com gradientes de 2%, em estufa (35 ºC) e em dessecador com sílica-gel (25 ºC). Após cada secagem, foram determinados o teor de água e a viabilidade (testes de tetrazólio e germinação). No estudo de armazenamento, as sementes foram armazenadas com e sem fruto, em câmara seca (UR 40% e Temp. 10 ± 2 oC), pelos períodos de 0, 3, 6 e 9 meses. A cada intervalo de tempo, foram determinados o teor de água e a viabilidade das sementes, pelos testes de germinação, tetrazólio e a integridade do DNA. A determinação da sanidade das sementes foi realizada em meio de cultura BDA, meio de cultura V8 e Blotter Test . Em cada teste, foram utilizadas sementes com e sem assepsia (desinfestação com hipoclorito de sódio e álcool), totalizando 80 sementes para cada teste. A incubação das sementes foi realizada em câmara com temperatura controlada a 22 °C ±3 oC, com fotoperíodo de 12 horas, durante sete dias, quando ocorreu a avaliação e identificação dos fungos. Foi observado que sementes sem o tegumento iniciaram a germinação após 14 dias do início do teste, com estabilização do estande aos 36 dias, com resultados médios de 71% de germinação. Não foi observada germinação para sementes dos demais tratamentos e testemunha, em todos os lotes utilizados. Em relação à secagem, foi observado que até 32% de umidade não houve alteração na qualidade das sementes, independente do tipo de secagem, sendo verificada significativa perda de germinação após esse valor. Sementes de Ocotea puberula perderam sua viabilidade após 3 meses de armazenamento, com ou sem fruto. Foram observados nove gêneros fungícos: Penicillium sp., Phomopsis sp., Epicocum sp., Curvularia sp., Colletotrichum sp., Aspergillus sp., Alternaria sp., Fusarium sp. e Trichoderma sp. Conclui-se que o método de retirada do tegumento foi eficiente na superação da dormência de sementes de Ocotea puberula e que o tipo de secagem não influencia na qualidade dessas sementes; porém, a redução do teor de água abaixo de 32% prejudica a germinação. Sementes de Ocotea puberula perdem sua viabilidade após 3 meses de armazenamento em câmara seca e os meios agarizados foram mais sensíveis para a detecção de fungos infestantes nas sementes e a assepsia das sementes com hipoclorito de sódio e álcool reduz a incidência desses fungos, sendo indicada quando se realiza teste de sanidade com sementes dessa espécie

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Sementes recalcitrantes sempre apresentam dificuldades em seu armazenamento, tornandose um problema para produtores de mudas que muitas vezes não têm como realizar a semeadura logo após a colheita das sementes ou necessitam produzir mudas em diferentes épocas do ano, além de dificultar a conservação de germoplasma. As sementes de ingá, depois de colhidas, perdem sua viabilidade rapidamente, geralmente não ultrapassando 15 dias. As espécies de sementes recalcitrantes normalmente não utilizam bancos de sementes como estratégia de regeneração em condições naturais. Observações ecológicas têm mostrado que é mais comum sua regeneração natural ser por meio de bancos de plântulas. O objetivo do trabalho foi avaliar o armazenamento de sementes pré-germinadas de Inga vera Willd., provenientes de frutos em dois estádios diferentes de maturação, em três condições diferentes de umidade de substrato e em baixa temperatura. Os frutos de Inga vera Willd. colhidos foram separados em duas categorias, de acordo com sua coloração de casca, entre verdes e maduros. Após seu beneficiamento, as sementes foram prégerminadas e armazenadas em substrato sem adição de água (testemunha), com 30 e 60% de umidade sob baixa temperatura (10°C), por um ano. O armazenamento feito a 30 e 60% de umidade mostrou-se com melhor eficiência, tanto para plântulas provenientes de frutos verdes, como as de frutos maduros. Entre os dois tipos de frutos, observou-se que as plântulas obtidas de vagem verde, obtiveram melhor desempenho sob as condições de armazenamento, em relação àquelas obtidas de vagem madura. Podem ser armazenadas por até quatro meses, mantendo 50% de sobrevivência das plântulas do lote, quando o armazenamento é feito sob baixa temperatura (10 ± 5°C) e em substrato com 30 e 60% de umidade. Após um período de armazenamento...
Recalcitrant seeds always present difficulties for storage procedures, becoming a problem for seedling producers that often have no choice to sow them immediately after seed harvest or when they need to produce seedlings through different times by the year. Inga seeds, once harvested, lose quickly their viability, often not exceeding a period of 15 days. Species of recalcitrant seeds do not use seed banks as regeneration strategy into the natural environment. Ecological observations have shown that is common occur their natural regeneration through seedling banks. The objective of this work was to evaluate pregerminated seed storage of Inga vera Willd. from two fruit maturation stages, under different conditions of substrate humidity, and under low temperature. The collected fruits of I. vera 9were splited into two types, according to their color, between unripe and ripe. After the processing of those seeds, they were pre-germinated on substrate and stored in substrate with no water addition (control), 30, and 60% humidity under low temperature (10°C) for a year. The storage made under 30 and 60% humidity showed to be the best for both, seedlings from unripe and ripe fruits. Between two types of fruits, we observed that the seedlings from unripe fruits, presented better performance under storage conditions, when compared to those obtained from mature fruits. They can be stored for until four months, with 50% of seedling survivals when stored under low temperature (10 ± 5°C) and with 30 and 60% of substrate humidity. After a year of seedling storage it was possible to get until 10% of survival... (Complete abstract click electronic access below)

Ocotea odorifera é uma espécie nativa da Mata Atlântica, com características recalcitrantes, que se encontra em risco de extinção devido a sua exploração econômica. Técnicas baseadas na biotecnologia podem ser utilizadas para o processo de conservação e reintrodução no seu ambiente natural de espécies ameaçadas de extinção. Dentre as técnicas biotecnológicas, a embriogênese somática (ES) vem sendo utilizada com bastante sucesso em programas de melhoramento genético e conservação de germoplasma de espécies arbóreas. Contudo, para o estabelecimento de protocolos eficientes de ES são necessários estudos básicos de fisiologia e bioquímica do desenvolvimento da semente e da germinação, para que as condições in vivo possam ser recriadas de forma eficiente in vitro. O objetivo deste trabalho foi avaliar as variações no conteúdo endógeno de aminoácidos, PAs, AIA, ABA e estabelecer marcadores protéicos durante a germinação de sementes de O. odorifera. As sementes foram germinadas em substrato de vermiculita em fotoperíodo de 16h a 27°C. A seguir, coletadas após 0, 15, 30 e 45 dias de semeadura. Foi observado que: a) o conteúdo de aminoácidos aumentou durante o processo germinativo. A asparagina foi observada em maior quantidade e predominou durante todo o período analisado. A metionina e a ornitina, precursores das PAs, e o triptofano, precursor do AIA, aumentaram ao longo do processo germinativo; b) o conteúdo total de PAs decresceu nos últimos 15 dias de avaliação. Neste período, as poliaminas conjugadas apresentaram aumento indicando possível relação entre conjugação e disponibilidade de PAs nas células; c) a relação Put/(Spm+Spd) diminui ao final do período analisado enquanto no mesmo período a Spm foi a PA livre mais abundante indicando relação entre Spm e alongamento celular; d) O AIA apresentou aumento ao logo do processo germinativo, ao mesmo tempo em que a queda no conteúdo de ABA pode ser observada; e) o conteúdo de proteínas decresceu ao longo do processo germinativo. Os perfis protéicos diferiram pouco entre os estádios analisados. Entretanto, foi observada variação significativa na abundância (% de volume) em 39 spots, que foram definidos como marcadores do processo germinativo. Dentre esses spots incluem-se 26 que reduziram e 2 spots que aumentaram suas abundâncias ao longo do processo germinativo, 10 que apresentaram tanto aumento quanto diminuição durante o período observado e apenas 1 exclusivo da semente madura. Estes resultados abrem perspectivas para a avaliação de parâmetros fundamentais para a otimização de protocolos de ES e permitiram uma maior compreensão das variações endógenas dos diferentes compostos citados no processo germinativo das sementes de O. odorifera.
Ocotea odorifera is a native tree from the Atlantic Forest with recalcitrant features that is in risk of extinction because its economical exploration. Techniques based on biotecnology can be used for the conservation and reintroduction process of species in risk of extinction in their natural environment. Between these biotecnologycal techniques, the somatic embryogenesis (SE) have been used sucessfully in programs of genetic breeding and tree germoplasm conservation. However, basic studies of physiology and biochymical of the seed development and germination are necessary for the establishment of efficient protocols of SE. Thus, these condictions in vivo can be recriated in vitro. The aim of this work was to evaluate the variations in the endogenous content of amino acids, polyamines, IAA, ABA and protein markers throughout the seed germination of O. odorifera seeds. The seeds were germinated on vermiculite substrate at 27°C and 16h of photoperiod and collected after 0, 15, 30 e 45 days of sowing. It was observed that: a) the amino acids content increased troughout the germination process. Asparagine was the highest content sighted and prevailed during the period studied. Methionine and ornithine, polyamines precursors, and tryptophan, IAA precursor, increased troughout germination process; b) the total PAs content decreased in the last 15 days of evaluation. At the same period, conjugated PAs showed an increase, indicating a relation between conjugation and avalability of polyamines inside the cells; c) the Put/(Spm+Spd) relation decreased while the Spm was the most abundant free PA observed at the end of the period analyzed indicating a relation between Spm and celular elongation; d) IAA increased during all the process while a decrease of ABA was sigthed; e) protein content decreased troughout the period observed. The proteic profile didnt change a lot between the stages. Os perfis protéicos diferiram pouco entre os estádios analisados. Entretanto, foi observada variação significativa na abundância (% de volume) em 39 spots, que foram definidos como marcadores do processo germinativo. Among them, could be include 26 that decreased and 2 that decreased its abundances troughout the germinative process, 10 that both increased and decreased throughout the period analyzed and just 1 exclusive from mature seed. These results open perspectives to avaliation of fundamentals parameters to optmization of ES protocols and allow a better comprehension about the edogenous variation of the differents compounds citeds in the process of O. odorifera seeds germination.

Orientador: Claudio José Barbedo
Banca: Danilo da Cruz Centeno
Banca: João Domingos Rodrigues
Banca: João Nakagawa
Banca: Patrícia Pinho Tonini
Resumo: O elevado metabolismo das sementes recalcitrantes de Inga vera Willd. subsp. affinis (DC.) T.D. Pennington, é um dos principais fatores que dificultam sua conservação. Uma forma de diminuir os efeitos do intenso metabolismo dessas sementes poderia ser a redução da temperatura do ambiente, pois a temperatura determina a velocidade das reações enzimáticas, afetando as taxas respiratórias. Contudo, as sementes recalcitrantes são intolerantes à dessecação e frequentemente não podem ter seu teor de água reduzido até níveis que permitam a redução da temperatura sem que ocorram danos pelo congelamento da água. Devido à falta de informações sobre o metabolismo respiratório dessas sementes em diferentes condições de estresse, o presente trabalho teve como objetivo verificar as taxas respiratórias desses embriões com diferentes níveis de hidratação e após aplicação de ácido abscísico e estresse osmótico em diferentes temperaturas. Os resultados possibilitaram observar a presença de reações oxidativas que não às da respiração, devido ao consumo elevado de oxigênio, e que as alterações nos níveis de hidratação e temperatura de incubação assim como a aplicação de ácido abscísico e estresse osmótico promoveram alterações nesse metabolismo oxidativo. Os resultados obtidos trazem novas perspectivas para a conservação das sementes recalcitrantes
Abstract: The high metabolism of recalcitrant seeds of Inga vera Willd. subsp. affinis (DC.) T.D. Pennington is one of the main factors that complicate their conservation. One way to lessen the effects of the intense metabolism of these seeds could be the reduction of temperature of the environment, because temperature determines the speed of enzymatic reactions, affecting respiration rates. However, recalcitrant seeds are desiccation intolerant and often can not have their water content reduced to levels that allow a reduction in temperature without damage by the freezing of water. Due to lack of information on the respiratory metabolism of these seeds in different conditions of stress this study aimed to determine respiration rates of embryos with different levels of hydration and after application of abscisic acid and osmotic stress at different temperatures. The results allowed to observe the presence of oxidative reactions than those of breath due to high consumption of oxigen, and that changes in moisture levels and incubation temperature as well as the application of abscisic acid and osmotic stress promoted changes in oxidative metabolism. The results provide new perspectives for the conservation of recalcitrant seeds
Doutor

Orientador: Edson Seizo Mori
Banca: Marcia Balistiero Figliolia
Banca: Edvaldo Aparecido Amaral da Silva
Resumo: Sementes recalcitrantes sempre apresentam dificuldades em seu armazenamento, tornandose um problema para produtores de mudas que muitas vezes não têm como realizar a semeadura logo após a colheita das sementes ou necessitam produzir mudas em diferentes épocas do ano, além de dificultar a conservação de germoplasma. As sementes de ingá, depois de colhidas, perdem sua viabilidade rapidamente, geralmente não ultrapassando 15 dias. As espécies de sementes recalcitrantes normalmente não utilizam bancos de sementes como estratégia de regeneração em condições naturais. Observações ecológicas têm mostrado que é mais comum sua regeneração natural ser por meio de bancos de plântulas. O objetivo do trabalho foi avaliar o armazenamento de sementes pré-germinadas de Inga vera Willd., provenientes de frutos em dois estádios diferentes de maturação, em três condições diferentes de umidade de substrato e em baixa temperatura. Os frutos de Inga vera Willd. colhidos foram separados em duas categorias, de acordo com sua coloração de casca, entre verdes e maduros. Após seu beneficiamento, as sementes foram prégerminadas e armazenadas em substrato sem adição de água (testemunha), com 30 e 60% de umidade sob baixa temperatura (10°C), por um ano. O armazenamento feito a 30 e 60% de umidade mostrou-se com melhor eficiência, tanto para plântulas provenientes de frutos verdes, como as de frutos maduros. Entre os dois tipos de frutos, observou-se que as plântulas obtidas de vagem verde, obtiveram melhor desempenho sob as condições de armazenamento, em relação àquelas obtidas de vagem madura. Podem ser armazenadas por até quatro meses, mantendo 50% de sobrevivência das plântulas do lote, quando o armazenamento é feito sob baixa temperatura (10 ± 5°C) e em substrato com 30 e 60% de umidade. Após um período de armazenamento... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Recalcitrant seeds always present difficulties for storage procedures, becoming a problem for seedling producers that often have no choice to sow them immediately after seed harvest or when they need to produce seedlings through different times by the year. Inga seeds, once harvested, lose quickly their viability, often not exceeding a period of 15 days. Species of recalcitrant seeds do not use seed banks as regeneration strategy into the natural environment. Ecological observations have shown that is common occur their natural regeneration through seedling banks. The objective of this work was to evaluate pregerminated seed storage of Inga vera Willd. from two fruit maturation stages, under different conditions of substrate humidity, and under low temperature. The collected fruits of I. vera 9were splited into two types, according to their color, between unripe and ripe. After the processing of those seeds, they were pre-germinated on substrate and stored in substrate with no water addition (control), 30, and 60% humidity under low temperature (10°C) for a year. The storage made under 30 and 60% humidity showed to be the best for both, seedlings from unripe and ripe fruits. Between two types of fruits, we observed that the seedlings from unripe fruits, presented better performance under storage conditions, when compared to those obtained from mature fruits. They can be stored for until four months, with 50% of seedling survivals when stored under low temperature (10 ± 5°C) and with 30 and 60% of substrate humidity. After a year of seedling storage it was possible to get until 10% of survival... (Complete abstract click electronic access below)
Mestre

As jabuticabeiras são árvores tipicamente brasileiras, de origem subtropical e tem como principal distribuição o centro sul do país. Dentre elas está a Sabará, a qual é a mais cultivada em todo o país. Sua propagação é realizada basicamente através de sementes, pois métodos de propagação vegetativa ainda são pouco utilizados. Contudo, a enxertia tem se mostrado promissora e a necessidade de porta enxertos bem formados torna-se essencial para o sucesso desta técnica. As sementes de jabuticaba são classificadas com recalcitrantes por serem sensíveis à dessecação o que dificulta seu armazenamento. As jabuticabeiras Sabará (Myrciaria jaboticaba Berg) são frutas nativas do Brasil, cuja distribuição concentra-se no centro sul do país. Trata-se de uma espécie subtropical com frutos pequenos, de coloração escura e muito doces, sendo utilizados principalmente para a confecção de geleias, licores e vinhos, pois o fruto in natura possui vida de prateleira muito curta, de até dois dias. Fatores externos e de cultivo limitam a produção de frutos e um deles poderia ser a presença de luz solar na maturação do fruto. O objetivo deste trabalho foi avaliar a influencia da luz solar na maturação do fruto de jabuticaba, através da divisão da planta em quadrantes e se a germinação da semente diminui durante o armazenamento em dois ambientes, geladeira e temperatura ambiente. Para a primeira etapa dividiu-se cinco plantas em quadrantes Norte, Sul, Leste e Oeste, onde foram coletados frutos para a análise físico-química e os parâmetros avaliados foram: o diâmetro, peso do fruto, Brix, acidez titulável, o índice de maturação e pH. Foi observado que apenas para o parâmetro Brix e, consequentemente para o índice de maturação, houve diferença estatística entre o quadrante Sul dos demais. A partir disso pode-se concluir que há influência da luz solar sobre alguns parâmetros de qualidade do fruto de jabuticabeira. Para o armazenamento sementes de jabuticaba Sabará foram coletadas e armazenadas em geladeira (5°C) e temperatura ambiente (~25°C) durante 105 dias em saquinhos de polietileno. Foram postas para germinar aos 7, 14, 21, 28, 42, 56, 70 e 105 dias e foram feitas avaliações semanais. Foi possível perceber que não houve diferença estatística entre os dois ambientes, mostrando que se trata de uma semente tolerante a temperaturas baixas para o armazenamento. A partir do teste de secagem foi possível observar o comportamento recalcitrante da semente, pois esta perdeu o poder germinativo com a perda gradual de umidade. Conclui-se, então, que a semente de jabuticaba é recalcitrante, porém classificada como minimamente recalcitrante já que tolerou armazenamento em temperaturas baixas.
Jabuticaba trees are typical Brazilian trees and are mainly distributed in the country\'s center- south. Among them there is Sabará, which is the most spread specie through Brazil. Its propagation is basically made with seeds since the vegetative propagation is still less used. However, grafting seems to be efficient and the necessity of good rootstocks becomes essential to the technique\'s success. Jabuticaba seeds are classified as recalcitrant because they are drying sensible turning the storage difficult. Jabuticaba Sabará trees (Myrciaria jaboticaba Berg) are brazilian native fruits, which are mainly distributed in the country\'s center-south. It is subtropical specie with small fruits, dark colored and very sweet, being used to make jellies, liquors and wines, since in natura fruits are very perishable not lasting two days. This work aimed to evaluate the influence of the sun light in jabuticaba\'s maturation and if the germination decrease during the storage in two different environments. It was possible to notice that during jabuticaba\'s development it was possible to notice that TSS, and consequently the TSS/TTA ratio, was influenced by the position of the fruit on the tree. The south side had lower results compared to the other quadrants, showing that there should be a sunlight influence in some quality parameters in jabuticaba\'s fruits. For storage test jabuticaba\'s seeds were collected and stored in fridge (5°C) and in environmental temperature (~25°C) during 105 days in polyethylene bags. Germination tests were taken at 7, 14, 21, 28, 42, 56, 70 e 105 days and weekly evaluations were made. It was possible to notice that there were no differences between both environments, showing that this seed tolerates low storage temperatures. From the drying test it was possible to observe the recalcitrant behavior of the seed, since it lost its germination power with the humidity loss. Therefore, it is possible to conclude that jabuticaba\'s seeds are recalcitrant, however it could be classified as minimally recalcitrant due the fact that it could be stored in low temperatures.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The gabirobeira (Campomanesia adamantium) is a fruit species of the Cerrado, its fruit presents commercial potential in the in natura consumption and in the food industry. However, gabiroba seeds have recalcitrant behavior. Species with this type of behavior can be conserved by means of tissue culture techniques of plants, such as, for example, synthetic seeds. The objective of this work was to establish the protocol for the production of synthetic seeds of Campomanesia adamantium from true seeds. For the encapsulation of the seeds, 4 concentrations of sodium alginate (0.1.5, 2 and 3%) and 3 complexation times (15, 20 and 30 minutes) were tested, with no significant interaction between the studied factors, however. As the alginate concentration increases the germination time increases and the germination capacity decreases. Synthetic seeds with the same concentrations of alginate were produced in 20 minutes of complexation and stored at 4 ° C for 1, 7, 15, 30 and 60 days in order to evaluate the storage capacity of the same. The concentrations of 2% sodium alginate were able to preserve the integrity of the membranes of the encapsulated seeds regardless of storage time, however this was not enough to promote germination.
A gabirobeira (Campomanesia adamantium) é uma espécie frutífera do Cerrado, seu fruto apresenta potencial comercial no consumo in natura e na indústria alimentícia. No entanto, sementes de gabiroba possuem comportamento recalcitrante. Espécies com esse tipo de comportamento podem ser conservadas por meio de técnicas de cultura de tecidos de plantas, como por exemplo, as sementes sintéticas. Diante disso, o objetivou-se com este trabalho o estabelecimento do protocolo para produção de sementes sintéticas de Campomanesia adamantium a partir de sementes verdadeiras. Para o encapsulamento das sementes foram testados 4 concentrações de alginato de sódio (0,1,5, 2 e 3%) e 3 tempos de complexação (15, 20 e 30 minutos), não havendo interação significativa entre os fatores estudados, no entanto a medida que se aumentou a concentração de alginato aumentou o tempo de germinação e a capacidade germinativa diminuiu. Sementes sintéticas com as mesmas concentrações de alginato foram produzidas em 20 minutos de complexação e armazenadas a 4ºC por 1, 7, 15, 30 e 60 dias a fim de avaliar a capacidade de armazenamentos das mesmas. As concentrações de 2% de alginato de sódio conseguiu preservar a integridade das membranas das sementes encapsuladas independentemente do tempo de armazenamento, no entanto isso não foi o suficiente para a promoção da germinação.

Hazelnuts are mostly non-dormant at harvest but develop seed dormancy after a few days of storage. The seeds have been classified as recalcitrant since they cannot be stored for more than one year under ambient conditions. Cryopreservation has not been satisfactory so an alternative protocol is required. To test for recalcitrance, chilled non-dormant seeds (control) were compared with gibberellic acid (GA3) treated seeds during 6 weeks storage at 5°C or at ambient temperature. Control seed moisture content (MC) was 14-15% compared with 20% for GA3 treated seeds. No change in viability was noted until the end of 6 weeks at ambient temperature, when infection proliferated. Reduced germinability, associated with increased leachate conductivity, was noted on all treatments and controls, with ambient temperature storage most harmful for seed viability. This supports classification of hazel seeds as recalcitrant. However, orthodox behaviour could be induced by reducing seed moisture to <6%, showing survival for more than 3 years at -20°C with acceptable germinability and producing healthy seedlings. Pathogen tests show that 6 weeks chilling to break seed dormancy may activate the seeds’ internal protective mechanisms, thereby reducing infection and enabling germination and healthy seedling establishment. The link between seed viability and protection from free radicals and pathogens was examined. Antioxidant activity in hazelnut seed associates (such as endocarp, funiculus and testa) was found to be much higher than in the seed embryo, perhaps indicating that hazel seeds have natural protective mechanisms within the pericarp. Antioxidant activity of seed associates increased during chilling, indicating their role in protecting the seed. Nevertheless, TTC test revealed that seeds acclimatised to ii < 6% MC and stored at 5°C for 45 weeks showed viability loss due to damage of the embryonic axes, probably caused by free radicals. Initial tests to stabilise seed moisture content showed that reduction in seed moisture did not impose dormancy and seed moisture content (MC) stabilisation resulted in > 80% germination but many abnormal seedlings. Dormancy reversibility was tested by treatments T1 (one period at 15°C) and T2 (two periods at 15°C), designed following a consideration of the natural environment. Both resulted in reduced germination, delayed seedling emergence, increased abnormal seedlings, reduced seedling height and decreased internode numbers. To test the role of temperature in reduced seed performance, non-dormant hazelnuts were held at either 5°C or at ambient temperature for up to 6 weeks. Seeds from both sets exhibited high viability, but germinability was significantly decreased in the ambient temperature set, associated with increases in leachate conductivity and infection. Work in this thesis has confirmed that dormancy was broken by chilling, with gradually increasing germination as chilling time was increased. Germination increased with increase in chilling and reduction in infection. No infection was recoreded after 6 weeks chilling. It is most likely that protective agents are produced causing suppression of infection. In these experiments it was observed that not all germinated seeds produced healthy seedlings, suggesting that germination tests without observation of seedlings may give an incomplete assessment of germination success. Assessment using the Tetrazolium test (TTC) was found to be much more dependable and it was also possible to detect damage to specific tissues that might result in unhealthy seedlings.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
To produce rubber tree (Hevea brasiliensis) seedlings in nursery, the collection is limited by the short fall of seeds, which have a rapid loss of their germinal power and no guarantee their viability. This forces the nurseryman to acquire a much larger seeds than really necessary. With the adoption of standards for the quality of seed, the minimum parameters established 70% viability being suggested for such confirmation the tetrazolium test, according Rules for Seed Analysis (RSA) and/or a practical test done from the visual analysis of the seed endosperm color named in this work of "visual test". Despite tetrazolium test be scientifically based, its realization is made improbable by many financial and temporal reasons. Thus, the visual test points as a low-cost alternative, high reliability and immediate result, since both tests are considered trusted, but only the test tetrazolium test is recognized. The subject proposed to verify the credibility of the "visual test" legitimizing or not its use in the field. The field is composed of rubber seed, formed with the adult clone GT1. The seeds were homogenised and stored on two conditions: braided polypropylene bags and transparent plastic bags with minimum thickness of 0.02 mm microdrill at top. In each bag, in three treatments were placed ten kilograms of seed and these were stored in natural conditions of temperature and humidity (25° C ± 5 °C). Was pulled randomly a bag of seeds of rubber tree of each storage type, to zero and fourteen days of storing lots. The tests: visual, sprouting in sand and tetrazolium, besides the determination of the degree of moisture. Tetrazolium and visual tests were highly correlated, and the visual test results before had as the best way to analyze the seeds, for its significant correlation in General with the tetrazolium test, and simplicity in its execution and instant result, which gives good lot projection nurseryman to be acquired by him.
A produção de mudas de seringueira (Hevea brasiliensis) em viveiro, é limitada pelo curto período de produção, queda e coleta das sementes, que possuem uma rápida perda do seu poder germinativo e nenhuma garantia da sua viabilidade. Isto obriga o viveirista a adquirir uma quantidade de sementes maior do que aquela realmente necessária. Com a aprovação de normas para o padrão de qualidade de semente, se estabeleceram parâmetros mínimos de 70% de viabilidade sendo sugerido para tal confirmação o teste de tetrazólio, segundo as Regras para Análise de Sementes (RAS) e, ou, um teste prático feito a partir da análise visual da cor do endosperma da semente nomeado no presente trabalho de “teste visual”. Apesar do teste de tetrazólio ser embasado cientificamente, sua realização é inviabilizada por diversos motivos de ordem financeira e temporal. Sendo assim, o teste visual aponta como uma alternativa de baixo custo, alta confiabilidade e resultado imediato, já que ambos os testes são considerados confiáveis, mas apenas o teste de tetrazólio é reconhecido. Nesta pesquisa objetivou-se verificar a credibilidade do “teste visual” legitimando ou não seu uso no campo. As sementes de seringueira foram coletadas no município de Goianésia, estado de Goiás. O campo de sementes é constituido de seringal adulto, formado com o clone GT1. As sementes foram homogeneizadas e armazenadas em duas condições: sacos de polipropileno trançado e sacos plásticos transparentes com espessura mínima de 0,02 mm, microperfurados na parte superior. Em cada saco, nos dois tratamentos, foram colocados dez quilogramas de sementes e estes foram armazenados em condições naturais de temperatura e umidade (25° C ± 5 °C). Foi retirado aleatoriamente um saco de sementes de seringueira de cada tipo de armazenamento, no tempo zero e aos quatorze dias de armazenamento dos lotes de sementes coletados. Foram realizados os testes: visual, de germinação em areia e de tetrazólio, além da determinação do grau de umidade. Os testes visual e de tetrazolio foram altamente correlatos. O teste visual apresentou a melhor forma de análise das sementes, por sua significativa correlação no geral com o teste de tetrazólio, além da simplicidade em sua execução e resultado instantâneo, o que confere ao viveirista uma boa projeção do lote de sementes a serem adquiridas para a produção de mudas de seringueiras

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As espécies invasoras são reconhecidas como um dos mais importantes e difíceis fatores que influenciam a conservação dos ecossistemas nativos. Na restauração de ambientes degradados, estas espécies podem afetar profundamente a trajetória do recobrimento vegetal pelas espécies nativas e, conseqüentemente, a composição do ecossistema. Um fator importante para o sucesso de algumas espécies vegetais invasoras é a plasticidade fenotípica, dandolhes grande capacidade de aclimatação em diversas condições ambientais. O conhecimento dos fatores ambientais e das características da planta que contribuem para o sucesso das espécies invasoras é de grande importância para predizer a habilidade de invasão e para esforços de manejo. Neste contexto, o objetivo principal deste estudo foi conhecer algumas características fisiológicas e ecológicas de Clausena excavata Burm. f., uma espécie de árvore exótica reconhecida como invasora em algumas regiões no mundo, no intuito de compreender o sucesso da espécie como invasora e contribuir com informações relevantes para possíveis tentativas de controle. Primeiramente, foram avaliados os efeitos da luz e da temperatura em sua germinação (condição controlada) e o efeito da luz na emergência de suas plântulas (condição natural, a pleno sol e sob a copa das árvores). As sementes germinaram tanto na presença como na ausência de luz, nas temperaturas de 20 a 35°C, sem diferença entre a porcentagem de sementes germinadas nas diferentes temperaturas. Sementes mantidas a 20°C, em ambas as condições de luz, germinaram mais lentamente em relação às demais temperaturas. Independente da temperatura, na presença de luz as sementes apresentaram uma germinação muito mais sincronizada do que aquelas mantidas no escuro. Tais resultados mostraram que as sementes de C. excavata são fotoblásticas neutra. Em campo, a emergência...
The invasive species are recognized one of more important and difficult factors that prejudice the conservation of native ecosystems. In the restoration of degraded environment, these species can to affect profoundly the trajectory of recovery by native species and, therefore, the ecosystems composition. An important factor to the success of invasive species is their phenotypic plasticity, which gives them a great ability to adjust in several environmental conditions. The knowledge of environmental factors and plant characteristics that contribute to the success of invasive species is very important to predict the invasion capacity and to manage efforts. In this context, the main objective of this study was to know some physiological and ecological characteristics of Clausena excavata Burm. f., a nonnative tree species recognized as invasive somewhere of the world, with intention to understand the success of this species as invasive and to contribute with relevant informations for a possible management. At first, were evaluated the effects of light and temperature on their seeds germination (controlled condition) and the effect of light on seedling emergence (natural condition, at full sun and shade). The seeds germinated in presence and absence of light, from 20 to 35°C, without difference among the germination percentage at different temperatures. Seeds maintained in 20°C, at both light conditions, germinated slowly than others temperatures. Independent of temperature, in light the seeds showed a germination more synchronized than the seeds kept in darkness. Those results showed that C. excavata have neuter photoblasics seeds. On the field, the emergence occurred at both environments, but at full sun all seeds produced a seedling. The seedling emergence occurred with same rate and synchronization index on two tested conditions. Thereafter, the seedlings were observed with purpose... (Complete abstract click electronic access below)

Orientador: Massanori Takaki
Banca: Victor José Mendes Cardoso
Banca: Sergius Gandolfi
Banca: Débora Leonardo dos Santos
Banca: Dalva Maria Silva Matos
Resumo: As espécies invasoras são reconhecidas como um dos mais importantes e difíceis fatores que influenciam a conservação dos ecossistemas nativos. Na restauração de ambientes degradados, estas espécies podem afetar profundamente a trajetória do recobrimento vegetal pelas espécies nativas e, conseqüentemente, a composição do ecossistema. Um fator importante para o sucesso de algumas espécies vegetais invasoras é a plasticidade fenotípica, dandolhes grande capacidade de aclimatação em diversas condições ambientais. O conhecimento dos fatores ambientais e das características da planta que contribuem para o sucesso das espécies invasoras é de grande importância para predizer a habilidade de invasão e para esforços de manejo. Neste contexto, o objetivo principal deste estudo foi conhecer algumas características fisiológicas e ecológicas de Clausena excavata Burm. f., uma espécie de árvore exótica reconhecida como invasora em algumas regiões no mundo, no intuito de compreender o sucesso da espécie como invasora e contribuir com informações relevantes para possíveis tentativas de controle. Primeiramente, foram avaliados os efeitos da luz e da temperatura em sua germinação (condição controlada) e o efeito da luz na emergência de suas plântulas (condição natural, a pleno sol e sob a copa das árvores). As sementes germinaram tanto na presença como na ausência de luz, nas temperaturas de 20 a 35°C, sem diferença entre a porcentagem de sementes germinadas nas diferentes temperaturas. Sementes mantidas a 20°C, em ambas as condições de luz, germinaram mais lentamente em relação às demais temperaturas. Independente da temperatura, na presença de luz as sementes apresentaram uma germinação muito mais sincronizada do que aquelas mantidas no escuro. Tais resultados mostraram que as sementes de C. excavata são fotoblásticas neutra. Em campo, a emergência... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The invasive species are recognized one of more important and difficult factors that prejudice the conservation of native ecosystems. In the restoration of degraded environment, these species can to affect profoundly the trajectory of recovery by native species and, therefore, the ecosystems composition. An important factor to the success of invasive species is their phenotypic plasticity, which gives them a great ability to adjust in several environmental conditions. The knowledge of environmental factors and plant characteristics that contribute to the success of invasive species is very important to predict the invasion capacity and to manage efforts. In this context, the main objective of this study was to know some physiological and ecological characteristics of Clausena excavata Burm. f., a nonnative tree species recognized as invasive somewhere of the world, with intention to understand the success of this species as invasive and to contribute with relevant informations for a possible management. At first, were evaluated the effects of light and temperature on their seeds germination (controlled condition) and the effect of light on seedling emergence (natural condition, at full sun and shade). The seeds germinated in presence and absence of light, from 20 to 35°C, without difference among the germination percentage at different temperatures. Seeds maintained in 20°C, at both light conditions, germinated slowly than others temperatures. Independent of temperature, in light the seeds showed a germination more synchronized than the seeds kept in darkness. Those results showed that C. excavata have neuter photoblasics seeds. On the field, the emergence occurred at both environments, but at full sun all seeds produced a seedling. The seedling emergence occurred with same rate and synchronization index on two tested conditions. Thereafter, the seedlings were observed with purpose... (Complete abstract click electronic access below)
Doutor

Control of seed-associated micro-organisms is vital in reducing losses of plants of economic importance. Recalcitrant seeds being metabolically active and able to be stored only under conditions of high relative humidity makes it more difficult to control contaminants. Nevertheless, means need to be developed to eliminate, or at least curtail, seed-associated fungi and bacteria. The use of biological control is a highly recommended alternative to chemical control for reducing the risk of killing beneficial organisms, as well as in terms of health and environmental hazards. Furthermore, when working with seed-derived tissues, it is extremely important to optimise a method or methods to control contamination without compromising the viability or further development of the explants. The original aim of the present study was to determine whether the biocontrol agents, EcoT® and Eco77® (commercial products of the spores of Trichoderma harzianum) would effectively control/eliminate micro-organisms from the embryonic axes of Trichilia dregeana, while promoting growth under in vitro conditions. Other means were also tested for their efficacy in controlling contaminants; these were application of Benlate®, Nipastat® (a mixture of parabens), anodic water (the anodic fraction of an electrolysed dilute solution of calcium and magnesium chloride), sodium dichloro-isocyanurate (Medi-Chlor®[NaDCC]) and alginate gel encapsulation of the embryonic axes. Prior to the experiments, fungal contaminants from the embryonic axes were isolated on potato dextrose medium and identified using light microscopy. EcoT and Eco77 were initially individually tested by co-culture as conidial suspensions with the embryonic axes. A further approach used liquid culture (potato dextrose broth) as well as solid culture medium (based on sugarcane bagasse) in/on which the strains of Trichoderma harzianum had been grown. This was aimed at testing for the possible presence of compounds released by T. harzianum into the media, which might prove to be effective in curtailing/eliminating the axis-associated microflora. Among the different treatments tested, the best method was utilised to decontaminate the embryonic axes prior to minimal-growth storage (hydrated axes encapsulated in alginate gel ‘beads’). Penicillium spp. were predominant among the different fungi isolated, which included Fusarium spp., Rhizopus spp., Aspergillus niger and Aspergillus flavus. Co-culturing with T. harzianum for 24 h was successful in terms of the survival of the embryonic axes, although the roots produced were shorter than when axes were cultured alone, but had no effect in eliminating the contaminants. Longer periods of co-culturing with T. harzianum affected the germination of the embryonic axes of T. dregeana compared with axes germinated in the absence of the biocontrol agent (control). The culture filtrate negatively affected germination of the T. dregeana embryonic axes, although it was effective against the associated contaminants. Nipastat was effective in reducing the contamination, and, depending on the concentration, did not affect germination adversely. Medi-Chlor was highly effective in eliminating all the contaminants from axes in vitro. Both these treatments were therefore used to decontaminate axes before minimal-growth storage. All the NaDCC-treated, encapsulated axes examined after 14 d hydrated storage [in Magenta boxes] and after 14-42 d in polythene bags survived; however the axes stored in aluminium foil-lined bags and Eppendorf tubes soon lost viability. The recommendation is therefore made that the decontamination treatment based on use of NaDCC (or other preparations of sodium dichloro-isocyanurate) be tested on embryonic axes of a range of recalcitrant-seeded species, and, if successful, the procedure be introduced into cryopreservation protocols. The use of NaDCC has emerged as a promising method of eliminating contaminating microflora which otherwise compromise in vitro procedures, from seed-derived explants. Furthermore, containment of decontaminated encapsulated axes in sealed polythene bags offers an apparently ideal means of temporary storage and dissemination. The results should find considerable applicability when excised embryonic axes representing the germplasm of recalcitrant seeds, are cryoconserved.
Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

The seeds of Trichilia dregeana, Trichilia emetica, Podocarpus henkelii and Syzygium cuminii display the characteristics typical of recalcitrant seeds. It is the phenomena of ongoing metabolic activity and desiccation sensitivity that render them unsuitable for storage by the conventional methods used for orthodox seeds. Investigations on the storage responses of 'sub-imbibed' (partially dried) and fully hydrated seeds of all four species were carried out to study the effects of partial drying on viability and subsequent storage lifespan; i.e. to assess whether 'sub-imbibed' storage is feasible for these species. The outcome of this investigation was proposed to contribute to the resolution of the argument that storing recalcitrant seeds at lowered water contents might extend their longevity; i.e. storage at a relatively high water content but below the fully hydrated level, might prevent germination but would not be sufficient to be injurious to the seed. Seeds of T. dregeana, T. emetica, P. henkelii and S. cuminii were dried to various target moisture contents (which were determined for each species in the initial drying experiment) and then subjected to storage for 3-22 weeks at 6, 16 and 25°C (in sealed containers). In parallel, seeds of each species were stored at the shedding water content. The seeds were periodically removed for sampling, and assessed for water content, germination, respiration, electrolyte leakage and microscopical features. Storage temperature appeared to affect viability of seeds of T. emetica and T. dregeana which displayed characteristics of chilling sensitivity. Storage at 6°C was detrimental (when compared with seeds stored under the same conditions at 16 and 25°C), but regardless of whether the seeds were undried or partially dried prior to storage. The seeds of P. henkelii did not demonstrate chilling sensitivity, the viability not being compromised at 6°C compared with those seeds stored at 16 and 25°C. Syzygium cuminii seeds were not subject to storage at 6°C because previous work indicated that they would be chilling-sensitive. Storage of 'sub-imbibed' seeds of T. dregeana, T. emetica, P. henkelii and Syzygium cuminii does not to confer any benefit over seeds stored in the fully hydrated state; rather it appears to be deleterious to seed survival during storage. This was apparent from the assessment of viability, electrolyte leakage and respiration. Vigour and viability of the 'sub-imbibed' seeds of all species declined more rapidly than the fully hydrated seeds. The only exception was P. henkelii seeds stored at 25°C, the fully hydrated seeds showed no survival after 11 weeks in storage, while 88% of the 'sub-imbibed' seeds survived this period. These results were, however, attributed to the proliferation of fungi on the fully hydrated seeds at 25°C. Although ultrastructural observations were made only on the T. emetica seeds, it was apparent that the cells from the 'sub-imbibed' seeds (after storage at 16 and 25°C) showed extensive degradation, with the intra-cellular components being largely unrecognisable. The cells from the seeds stored in the fully hydrated condition at 16 and 25°C maintained integrity and appeared metabolically active. In keeping with the suggestion that T. emetica seeds are chilling sensitive, the ultrastructure of the cells from both the 'sub-imbibed' and fully hydrated seeds showed deteriorative changes. All the results of the present study indicated that storage in the 'sub-imbibed' state is deleterious to seed survival. It is apparent that the removal of water, however small a proportion, accelerates seed deterioration during storage. Thus 'sub-imbibed' storage has no practical application for the storage of recalcitrant seeds.
Thesis (M.Sc)-University of KwaZulu-Natal, 2007.

Achieving long-term storage of germplasm is critical for the conservation of plant biodiversity. Seed storage practices require that degradative reactions causing ageing be limited. By reducing the water content, cytoplasmic viscosity is increased to levels that minimise deteriorative reactions. Reducing the storage temperature additionally increases the storage lifespan by further reducing the rate at which such deleterious processes occur. Two broad categories of seeds can be distinguished based on their storage behaviour. Orthodox seeds are desiccation-tolerant; generally shed in the dry state and are metabolically quiescent. Such seeds are usually stored at low water contents (e.g. 5%), and their high cytoplasmic viscosity prevents freezing damage during cooling to subzero temperatures. On the other hand, desiccation-sensitive (recalcitrant) seeds do not undergo a maturation-drying phase, they are metabolically active at shedding, and sensitive to extreme or prolonged drying. Accordingly, recalcitrant seeds cannot be stored under conventional conditions because they do not survive drying to low water contents and are damaged by sub-zero temperatures, even when dried to the lowest water content tolerated. Therefore, procedures that facilitate harmless drying and cooling to low temperatures are required to achieve long-term storage of recalcitrant germplasm. Recalcitrant seeds that are dried rapidly can attain relatively lower water contents without injury. However, these seeds are usually large and this limits the drying rates that can be achieved even under favourable conditions. Isolating embryonic axes from the rest of the seed facilitates faster drying, and a consequent reduction in the water content at which damage occurs. In axes of many species, the level of drying attained before lethal desiccation damage occurs is sufficient to limit freeZing damage during cryogenic exposure and facilitate survival in vitro. However, many others are damaged when dried to water contents that preclude freezing, and also are killed if cooled to sub-zero temperatures at higher water contents. In such instances, the window of permissible water contents leading to survival may be small or nonexistent. A basic premise explored in this thesis is that by restricting the growth of intracellular ice crystals using increasingly rapid cooling rates, the range of permissible water contents can be widened, facilitating survival of axes at higher water contents. An overview of the problems associated with the long-term storage of recalcitrant germplasm, and the rationale behind such rapid cooling approach are presented in Chapter 1 of the present thesis. Subsequent chapters report investigations on the effects of variables required to dry and cryopreserve embryonic axes with minimum damage, in keeping with this approach. Collectively, those studies aimed at establishing a robust cryopreservation procedure for the conservation of recalcitrant germplasm with broad applicability across species. The approach presently adopted entailed manipulating the water content of excised axes using rapid drying to discrete water content ranges, and also using different methods to cool axes to cryogenic temperatures at various rates. The calorimetric properties of water in axes were investigated for Camellia sinensis (L.) O. Kuntze using differential scanning calorimetry (DSC). For all species, the effect of any drying or cooling treatment tested was determined by assessing the survival of axes in vitro, which provided the most reliable indicator of cellular damage. Additionally, the effects of different treatments upon the structural and functional integrity of axes were assessed using light and electron microscopy as well as measurement of electrolyte leakage. The studies undertaken are presented in a similar sequence to that in which they took place during the course of the experimental phase of this work. These are summarised below. Partial drying plays a pivotal role in the approach developed, and microscopy has contributed towards increasing present understanding of desiccation damage. Microscopy was used to determine the effects of drying rate upon the ultrastructure of recalcitrant axes. It was necessary to find reliable protocols to prepare specimens for light and electron microscopy that did not alter the architecture of the cells in the dry state. Freeze-substitution and conventional aqueous fixation were compared in Chapter 2 using variously dried material from three species. The results obtained revealed that an unacceptably high extent of artefactual rehydration occurs during aqueous fixation, and highlight the need for anhydrous processing of dehydrated samples. Significantly, that study also revealed that many cellular events commonly associated with desiccation damage (e.g. withdrawal, tearing and/or vesiculation of the plasmalemma) are not seen in freeze-substituted preparations, and are likely artefacts of aqueous fixation. Freeze-substitution was used subsequently (Chapter 3) to assess the effects of slow drying (2 - 3 days) or rapid drying (min) upon the survival of embryonic axes of jackfruit (Artocarpus heterophyllus Lamk.) Results confirmed the beneficial effects of rapid drying, and also provided insights into ultrastructural changes and probable causes underlying cellular damage that occur during a drying/rehydration cycle. Efforts subsequently focused on determining the effect of cooling rate upon survival of recalcitrant axes at various water contents. The study on embryonic axes of recalcitrant camellia sinensis (tea; Chapter 4) tested the hypothesis that rapid cooling facilitates survival of axes at higher water content by restricting the growth of ice crystals to within harmless dimensions. The presence of sharp peaks in DSC melting thermograms was indicative of decreased survival in vitro. These peaks were attributed to the melting of ice crystals sufficiently large to be detected by DSC as well as to cause lethal damage to axes. Increasing the cooling rate from 10°C min-1 to that attained by forcibly plunging naked axes into sub-cooled nitrogen increased the upper limit of water content facilitating survival in vitro from c. 0.4 to 1.1 - 1.6 g H20 g-1 (dry mass [dmb]). Subsequent studies tested whether a similar trend occurred in other recalcitrant species cooled under similar conditions. In order to investigate further the relationship between water content, cooling rate and survival it was necessary to achieve cooling rates reproducibly, and to quantify these reliably. The plunging device required to achieve rapid cooling, and instruments required to measure the cooling rates attained, are described in Chapter 5. That study investigated the effects of cryogen type, depth of plunge and plunging velocity on the cooling rates measured by thermocouples either bare or within tissues of similar size and water content as encountered in cryopreservation experiments. This plunger was used in subsequent studies to achieve the fastest cooling conditions tested. Favourable cooling conditions were selected, and the efficacy of this procedure to cryopreserve relatively large axes was tested (Chapter 6) using embryonic axes of horse chestnut (Aesculus hippocastanum L.) Axes at water contents above c. 0.75 g g-1 could not be cooled faster than c. 60°C S-1, but cooling rates of axes below this water content increased markedly with isopentane, and to a lesser extent with subcooled nitrogen. Axes were killed when cooled at water contents above 1.0 g g-1 but survived fully (albeit abnormally) when cooled in isopentane between 1.0 and 0.75 g g-1. Complete survival and increasingly normal development was attained at water contents below 0.75 g g-1, especially if isopentane was used. The study on horse chestnut axes emphasised that water content and cooling rate are co-dependent during non-equilibrium cooling. Accordingly, that study could not determine whether survival at lower water contents increased because of the corresponding increase in cooling rates measured, or because of the higher cytoplasmic viscosity that resulted from drying. That uncertainty was addressed by the study discussed in Chapter 7, using axes of the trifoliate orange (Poncirus trifoliata [L.] RAF.) That study investigated the effect of cytoplasmic viscosity upon survival of axes cooled and warmed at different rates. Survival and normal development was high at lower water contents, and seemingly independent of cooling rate at about 0.26 g g-1. At higher water contents the range of cooling rates facilitating survival became narrower and displaced towards higher cooling rates. This study revealed two conspicuous inconsistencies that questioned the beneficial effect of rapid cooling. Firstly, the fastest cooling rates did not necessarily facilitate the highest survival. Secondly, survival of fully hydrated axes was higher when cooled under conditions that encouraged - rather than restricted - the growth of intracellular ice crystals. These inconsistencies were explored further using embryonic axes of silver maple (Acer saccharinum L.). Freeze-fracture replicas and freeze-substitution techniques provided valuable insights into the way in which ice crystals were distributed in cells cooled using different methods at rates ranging between 3.3 and 97°C S-1. Extensive intracellular freezing was common to all treatments. Unexpectedly, fully hydrated axes not only survived cryogenic exposure, but many axes developed normally when cooled using the relatively slower methods (77 and 3.3°C S-1) if warming was rapid. The most conspicuous ultrastructural difference between plunge cooling and the relatively slower methods was the exclusion of ice from many intracellular compartments in the latter. It is possible that even the fastest warming cannot prevent serious cellular damage if ice crystals form within such 'critical' compartments. It is proposed that the intracellular location of ice is a stronger determinant of survival that the size attained by ice crystals. The study of A. saccharinum also investigated the recovery of axes cooled fully hydrated either rapidly (97°C S-1) or slowly (3.3°C S-1). This facet of the study showed that cell lysis became apparent immediately after warming only where damage was most extensive. In other cells damage became apparent only after 2.5 to 6 h had elapsed, thus cautioning against inferring survival from the ultrastructural appearance of cells immediately after warming. Microscopy enabled cell repair as well as the pattern of growth of cryopreserved tissues to be appraised at the cellular, tissue and organ levels. Similar studies are required to understand further the nature of freezing damage, and how those events affect cell function. The salient trends observed in previous chapters are brought together in Chapter 9.
Thesis (Ph.D.)-University of Natal, Durban, 2002.

One of the factors that affect the post-harvest behavior of seeds is provenance. Different geographical locations are characterised by different environmental conditions, hence, plant materials harvested from different locations may respond differently to laboratoryinduced stresses. The aim of the present study was to assess the role that provenance plays on the degree of recalcitrance of a single species – a factor that needs to be taken into consideration when choices are to be made for plant germplasm conservation. In this study, seeds of Trichilia dregeana, which have been shown to display recalcitrant post-harvest behavior, were harvested from four locations that were slightly different in climatic conditions: Mtunzini (MTZN), Durban (DBN), Pietermaritzburg (PMB) and Port Edward (P.ED), all within the KwaZulu-Natal province of South Africa. Clean seeds, whose aril and seed coat have been removed, were subjected to different drying and chilling stresses and their responses to the stresses are examined. Excised embryonic axes of the seeds were also subjected to flash (very rapid) drying and their responses to the stress are shown. Although the vegetation of these locations can be categorized as being sub-tropical, the present study suggests that there may be ‘degrees’ in the sub-tropical nature based on the rainfall and maximum temperature data. The ‘degree of sub-tropicality’ is suggested to be in the order MTZN > DBN > P.ED > PMB. Mean seed size assessed as seed length, width and fresh mass varied significantly across the four regions, with seeds from MTZN being the smallest and those from P.ED being the largest. The shedding water content of the embryonic axes of the seeds for 150 min. However, shoot production, root and shoot length and dry mass accumulation showed that axes of seeds from MTZN were the most adversely affected by rapid drying while those from PMB were the least sensitive. When clean whole seeds were dried slowly by burying in silica gel for 36 h, all seeds of the four provenances still retained more than 50% of their shedding water. MTZN seeds lost viability completely from the 24 h of slow drying while seeds from the other three locations retained some viability after 36 h. The order of survival of slow drying was MTZN < DBN < P.ED = PMB. Storage of cleaned whole seeds at chilling temperatures (3oC, 6oC and 16oC) caused increase in the water content of the embryonic axes of the seeds across the four locations. All seeds of the four provenances had lost more than 50% survival after they had been stored at 3oC for 10 weeks. Survival was completely lost from the 12th week for MTZN seeds, from the 14th week for DBN and P.ED seeds and from the 16th week for PMB seeds. There was no survival recorded for seeds across the four regions at the 18th week. The seeds were able to tolerate storage at 6oC for a little longer than those stored at 3oC. Only MTZN seeds completely lost viability at the 18th week; seeds from the other three locations retained some viability throughout the 18 weeks of storage. Storage at 16oC was the most tolerable, as all seeds from the four provenances maintained some viability until the 18th week in storage, with MTZN having the least survival and those from P.ED having the highest survival. Phylogenetic analysis of the internal transcribed spacer (ITS) region of the DBN and PMB samples showed intraspecific levels of genetic variation, and were separated by a genetic distance of 0.9%, an indicator that differences in storage behavior, chilling and desiccation sensitivity between seeds obtained from different collection localities may be genetically based. This study showed that MTZN seeds were the most sensitive, while seeds from PMB and P.ED were the most tolerant, to drying and chilling treatments, respectively. The prevailing climatic conditions may have a bearing on the responses observed in this study.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Three questions are considered in the context of the possible effects of seedassociated mycoflora, typified by Fusarium moniliforme, during hydrated storage of recalcitrant seeds of the tropical species, Avicennia marina. These are: 1) whether fungal infection reduces storage lifespan; 2) whether seeds become more susceptible to fungal attack during storage and whether they posses defence mechanisms that might suppress fungal proliferation in hydrated storage (production of antifungal compounds and 13-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14)] and 3) whether it is possible to discriminate ultrastructurally between inherent deteriorative changes and those that are fungally-induced. 1) The data indicate unequivocally that if fungal activity is curtailed, then the hydrated storage lifespan of A. marina seeds can be considerably extended. 2) When inoculated immediately with F. moniliforme, newly harvested seeds were extremely susceptible to the adverse effects of the fungus, while seeds that had been wet-stored for 4 days showed a considerably heightened resilience to the effects of the fungus prior to inoculation. The enhanced resilience, although declining, persisted in seeds stored hydrated for up to 10 days prior to inoculation, being lost after 12 days. This finding was supported by significant increase in 13-1,3-glucanase and chitinase and in antifungal compound production during 10 days of wet storage. After 14 days of wetstorage, seeds become more susceptible to the effects of fungusthanthose in the newly harvested condition. 3) The resilience of seeds that had been stored in the short-term was associated with ultrastructural changes indicative of enhanced metabolic activity associated with the onset of germination (e.g. increase in vacuolation, well-developed mitochondria and endomembrane system [ER and Golgi bodies]). However, with sustained stress associated with wet-storage IV conditions, the seeds became increasingly badly affected by the fungus, showing some ultrastructural fungally-induced abnormalities (e.g. nuclear lobing, presence of lipid bodies and prevalence of Golgi bodies that had many associated vesicles) and a decrease in 13-1,3-glucanase and chitinase activity. It is suggested that the decreased susceptibility of A. marina seeds during short-term storage relies on the ability to create an antifungal environment prior to infection (through synthesis and accumulation of pre-formed and induced antifungal compounds and antifungal enzymes), which would also be an effective strategy during germination in the natural environment.
Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004

Seeds remain the most convenient and successful way for storing the genetic diversity of plant species and for producing new plants routinely for agriculture and horticulture. The importance of seed storage and the ability to predict seed longevity must therefore not be underestimated. To be successful, storage conditions must maintain seed vigour and viability and ensure that normal seedlings are subsequently established under field conditions. Seed quality is best retained when deteriorative events are minimised, which is achieved by storage of low moisture-content seeds under cool to cold, or even sub-zero, temperatures. Such conditions are employed for 'orthodox' seeds, which are desiccation tolerant and able to survive at sub-zero temperatures in the dehydrated state for extended periods. It is seeds referred to as 'recalcitrant' that cannot be dehydrated and often not stored at low temperatures because they are desiccation sensitive and may not tolerate chilling. According to almost anecdotal records chilling temperatures for such seeds are those below 15°C down to 0°C, depending on the species. The limited storage lifespan of recalcitrant seeds presents a problem even for short-term storage, and as most research on chilling sensitivity has been conducted on vegetative tissue, relatively little data exist for seeds, especially recalcitrant types. The purpose of this study was to gain an understanding of the chilling response of recalcitrant seeds, as reduced temperature could have the potential to extend, rather than curtail, storage lifespan, depending on the species. Selected physiological, biochemical and ultrastructural responses of recalcitrant seeds of Avicennia marina and Ekebergia capensis were characterised. Seeds of the two species were stored at 25, 16 and 6°C. Germination, water content (determined gravimetrically), respiration (measured as CO2 production) and leachate conductivity (tissue electrolyte leakage over time) were assessed at regular intervals. Chilling response at the subcellular level was examined using transmission electron microscopy (TEM). Changes in sugar metabolism and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were assessed for A. marina seeds, which were severely affected by the chilling temperature of 6°C, losing viability after 1 week. In contrast, the seeds of E. capensis retained viability after 12 weeks of storage at 6°C, indicating the marked difference in chilling response between seeds of the two recalcitrant species, despite their common tropical provenance. However, when E. capensis seeds were stored at 3°C viability decreased significantly after 8 weeks, thus indicating how critically temperature must be controlled if such conditions are to be profitably employed. Ultrastructural studies revealed that in both E. capensis and A. marina seeds vacuole formation was initiated more rapidly at lower temperatures than at higher temperatures, indicating that this was a response specific to the chilling stress imposed. Once again, 'lower temperatures' differed relative to the species concerned. In the E. capensis seeds, nucleolar morphology was affected and the extent of chromatin patches in the nuclei increased as the storage temperature was reduced. Other ultrastructural findings could not be linked specifically to the chilling stress imposed on the E. capensis and A. marina seeds. Activity of the antioxidant enzymes SOD and GR was detected in the A. marina seeds. No measurable CAT activity was detected. Glutathione reductase activity increased in response to chilling stress, the rate of the increase depending upon the severity of the chilling stress imposed. Other than when the A. marina seeds were placed directly at 6°C, there were no notable increases in SOD activity. Interestingly, SOD and GR activity was not the same in the axes as in the cotyledons. Superoxide dismutase activity was found to be higher in the axes and GR activity higher in the cotyledons. It would have been beneficial to determine the extent of antioxidant enzyme activity in the E. capensis seeds as well if this had been possible. Generally, chilling of recalcitrant seeds seems to evoke a response similar to that of dehydration below a critical water content. This could lead to the conclusion that recalcitrant seeds do not possess the genetic ability to cope with dehydration or chilling stress, if it were not for the existence of recalcitrant seed species that are more chilling tolerant.
Thesis (M.Sc.)-University of Natal, Durban, 2002.

Genetic diversity of cultivated species and their wild relatives, as well as of wild species encompasses plant genetic resources or germplasm, the ex situ preservation of which embodies a critical aspect of biological conservation. While seed storage affords an efficient ex situ conservation method, recalcitrant seeds are intolerant of desiccation and cannot be stored conventionally in seed banks. Seeds of the three indigenous tree species investigated in this study, viz. Trichilia emetica, T. dregeana and Protorhus longifolia are recalcitrant, with the species considered to be endangered. Cryopreservation, which involves storage at ultra-low temperatures of selected tissue(s) from which plants are subsequently able to be generated, is currently the only method available for long-term ex situ conservation of recalcitrant-seeded species and affords significant potential for the future. Many protocols that have been applied for the cryopreservation of the germplasm of recalcitrant zygotic embryonic axes excised from seeds of tropical/sub-tropical species have resulted in survival post-cryo which has been recorded only as root development or callus formation, with shoot formation seldom occurring. Successful cryostorage of genetic resources cannot be achieved until post-cryopreservation recovery facilitates normal seedling development, i.e. the formation of both a fully functional root and a shoot. Cryopreservation requires the utilisation of the smallest explant possible (greatest surface area to volume ratio), the most suitable for recalcitrant seeds in general being the zygotic embryonic axis. Based on preliminary studies it was demonstrated that shoot production by axes is inhibited in association with a burst of reactive oxygen species (ROS), produced in response to wounding upon excision of the axis from the cotyledons, when these are attached close to the shoot apical meristem. It was postulated that a combination of the oxidative burst at the site of excision coupled with inadequate antioxidant machinery within the recalcitrant axis tissue, precludes shoot production. It was further considered highly probable that each subsequent stressful manipulation throughout the cryopreservation process would be accompanied by a surge of uncontrolled oxidative activity within the tissue, in response to the stress. Therefore, the primary aim of the study was to investigate the underlying causes of failure of shoot production after procedures associated with cryopreservation and to focus on ways to ameliorate the consequences of unbalanced oxidative metabolism. Additionally, studies were carried out to optimise each step of the cryopreservation procedure, viz. cryoprotection, dehydration, rehydration and cooling, and subsequent recovery, in conjunction with assessment of oxidative responses, ultimately to achieve successful cryopreservation of the embryonic axes of these species. The experimental work conducted to achieve this aim assessed changes in various biomarkers of injury, those selected for this study being three ROS, viz. superoxide, the hydroxyl radical and hydrogen peroxide, after axes were exposed to various pre-treatments, cryopreservation and recovery. Concomitantly, the elicited responses of endogenous antioxidant systems accompanying these steps were assessed. Changes in the levels of ROS and antioxidant activity were determined using various biochemical assays, and these parametres, together with assessment of shoot development, were investigated after each step of the cryopreservation process. The effect of stress on oxidative metabolism was tested after exposure to pre-treatments with and without the provision of various antioxidants, viz. DMSO, ascorbic acid and cathodic water, so as to determine the efficacy of selected ROS scavengers and, in general, to develop the best protocol for cryopreservation of embryonic axes of the three species. Significant results, in terms of shoot development and regulated ROS generation, were obtained after three major processes of the cryopreservation procedure. The production of roots and shoots by excised axes of T. emetica, T. dregeana and P. longifolia after excision (75%, 80% and 75%, respectively), and by 40% of excised axes of T. dregeana after each of the two further stages, cryoprotection and desiccation, were major achievements towards cryopreservation of the recalcitrant germplasm. The modulation of ROS by ascorbic acid and cathodic protection significantly improved survival of axes of both Trichilia species. In its entirety, the present study made significant advancements towards cryopreservation of recalcitrant germplasm and also towards understanding oxidative events associated with cryogenic processing and exposure to cryogenic conditions. This study concludes that unregulated metabolism is one of the underlying causes of failure of recalcitrant germplasm represented by zygotic axes, to survive cryopreservation. The application of antioxidants and cathodic protection during cryopreservation facilitated survival that has been previously unattainable. The outcomes of this study provide an informative platform for further optimising cryopreservation procedures for the germplasm of the species investigated, and extending the work to other recalcitrant-seeded species, especially those of tropical/sub-tropical provenances.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

The conservation of germplasm of indigenous plant species is vital not only to preserve valuable genotypes, but also the diversity represented by the gene pool. A complicating factor, however, is that a considerable number of species of tropical and sub-tropical origin produce recalcitrant or otherwise non-orthodox seeds. Such seeds are hydrated and metabolically active when shed and cannot be stored under conventional conditions of low temperature and low relative humidity. This poses major problems for the longterm conservation of the genetic resources of such species. Presently, the only strategy available for the long-term conservation of species that produce recalcitrant seeds is cryopreservation. Ekebergia capensis is one such indigenous species that produces recalcitrant seeds. The aim of the present study was to develop methods for the cryopreservation of germplasm of this species. Different explant types were investigated for this purpose, viz. embryonic axes (with attached cotyledonary segments) excised from seeds, and two in vitro-derived explants, i.e. ‘broken’ buds excised from in vitro-germinated seedlings and adventitious shoots generated from intact in vitro-germinated roots. Suitable micropropagation protocols were developed for all explant types prior to any other experimentation. Before explants could be cryopreserved it was necessary to reduce their water content in order to limit damaging ice crystallisation upon cooling. All explants tolerated dehydration (by flash drying) to 0.46 – 0.39 g gˉ¹ water content (dry mass basis) with survival ranging from 100 – 80%, depending on the explant. In addition, penetrating and non-penetrating cryoprotectants were used to improve cryo-tolerance of explants. The cryoprotectants tested were sucrose, glycerol, DMSO and a combination of sucrose and glycerol. Explant survival following cryoprotection and dehydration ranged from 100 – 20%. Cryoprotected and dehydrated explants were exposed to cryogenic temperatures by cooling at different rates, since this factor is also known to affect the success of a cryopreservation protocol. The results showed that ‘broken’ buds could not tolerate cryogen exposure. This was likely to have been a consequence of the large size of explants and their originally highly hydrated condition. Adventitious shoots tolerated cryogenic exposure slightly better with 7 – 20% survival after cooling in sub-cooled nitrogen. Limited shoot production (up to 10%) was obtained when axes with attached cotyledonary segments were exposed to cryogenic temperatures. In contrast, root production from axes cooled in sub-cooled nitrogen remained high (67 – 87%). Adventitious shoots were subsequently induced on roots generated from cryopreserved axes by applying a protocol developed to generate adventitious shoots on in vitrogerminated roots. In this manner, the goal of seedling establishment from cryopreserved axes was attained. Each stage of a cryopreservation protocol imposes stresses that may limit success. To gain a better understanding of these processes the basis of damage was investigated by assessing the extracellular production of the reactive oxygen species (superoxide) at each stage of the protocol, as current thinking is that this is a primary stress or injury response. The results suggested that superoxide could not be identified as the ROS responsible for lack of onwards development during the cryopreparative stages or following cryogen exposure. The stresses imposed by the various stages of a cryopreservation protocol may affect the integrity of germplasm. Since the aim of a conservation programme is to maintain genetic (and epigenetic) integrity of stored germplasm, it is essential to ascertain whether this has been achieved. Thus, explants (axes with cotyledonary segments and adventitious shoots) were subjected to each stage of the cryopreservation protocol and the epigenetic integrity was assessed by coupled restriction enzyme digestion and random amplification of DNA. The results revealed little, if any, DNA methylation changes in response to the cryopreparative stages or following cryogen exposure. Overall, the results of this study provided a better understanding of the responses of germplasm of E. capensis to the stresses of a cryopreservation protocol and two explant types were successfully cryopreserved. Future work can be directed towards elucidating the basis of damage incurred so that more effective protocols can be developed. Assessment of the integrity of DNA will give an indication as to the suitability of developed protocols, or where changes should be made to preserve the genetic (and epigenetic) integrity of germplasm.
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Recalcitrant unlike orthodox seeds do not show a sharp border between maturation and germination and remain highly hydrated and desiccation-sensitive at all developmental and post-harvest stages. In contrast with recalcitrant seeds, orthodox types retain viability for predictably long periods in the dry state and hence can be stored under low relative humidity and temperature conditions. Storage of recalcitrant seeds under conditions allowing little to no water loss, at moderate temperatures, allows for short- to medium-term storage but only facilitates viability retention for a matter of a few weeks to months, at best, because the seeds are metabolically active and initiate germination while stored. Cryopreservation, i.e. storage at ultra-low temperatures (usually in liquid nitrogen [LN] at -196°C), is a promising option for the long-term germplasm conservation of recalcitrant-seeded species but their seeds present some unavoidable difficulties in terms of the amenability of their germplasm to cryopreservation. Pre-conditioning treatments can reduce the amount of ‘free’ water available for freezing and may increase the chances of cells or tissues surviving exposure to cryogenic temperatures. Such conditioning may be imposed by physical dehydration or cryoprotection, i.e. exposure to compounds that depress the kinetic freezing point of water and so reduce the likelihood of lethal ice-crystal formation during cooling (i.e. exposure to LN at -196°C or sub-cooled LN at -210°C) and subsequent thawing. Partial dehydration is presently a standard pre-treatment for the cryopreservation of recalcitrant zygotic germplasm and explant cryoprotection has been shown to improve postthaw survival in some recalcitrant-seeded species. However, there is a paucity of information on the physiological and biochemical basis of post-thaw survival or death in recalcitrant seeds, and this is the major focus of the current contribution. Additionally, in light of the lack of understanding on how cryo-related stresses imposed at the embryonic stage are translated or manifested during subsequent seedling growth, this study also investigated the effects of partial dehydration and the combination of partial dehydration and cooling of recalcitrant zygotic embryos on subsequent in and ex vitro seedling vigour. All studies were undertaken on the zygotic embryos of two recalcitrant-seeded members of the Amaryllidaceae, viz. Amaryllis belladonna (L.) and Haemanthus montanus (Baker); both of which are indigenous to South Africa. Studies described in Chapter 2 aimed to interpret the interactive effects of partial dehydration (rapidly to water contents > and <0.4 g g-1), cryoprotection (with sucrose [Suc; nonpenetrative] or glycerol [Gly; penetrative]) and cooling rate (rapid and slow) on subsequent zygotic embryo vigour and viability, using three stress markers: electrolyte leakage (an indicator of membrane integrity); spectrophotometric assessment of tetrazolium chloride-reduction (an indicator of respiratory competence); and rate of protein synthesis (an indicator of biochemical competence). These studies showed that in recalcitrant A. belladonna and H. montanus zygotic embryos, stresses and lesions, metabolic and physical, induced at each stage of the cryopreservation protocol appear to be compounded, thus pre-disposing the tissues to further damage and/or viability loss with the progression of each step. Maximum post-thaw viability retention in both species appeared to be based on the balance between desiccation damage and freezing stress, and the mitigation of both of these via Gly cryoprotection. Post-thaw viabilities in both species were best when Gly cryoprotected + partially dried zygotic embryos were rapidly, as opposed to slowly, cooled. However, the rate at which water could be removed during rapid drying was higher in A. belladonna and this may explain why the optimum water content range for post-thaw survival was <0.40 g g-¹ for A. belladonna and >0.40 g g-¹ for H. montanus. These results suggest that to optimise cryopreservation protocols for recalcitrant zygotic germplasm, attention must be paid to pre-cooling dehydration stress, which appears to be the product of both the ‘intensity’ and ‘duration’ of the stress. Cryoprotection and dehydration increased the chances of post-thaw survival in A. belladonna and H. montanus zygotic embryos. However, transmission electron microscopy studies on the root meristematic cells from the radicals of these embryos (described in Chapter 3) suggest that their practical benefits appear to have been realised only when damage to the sub-cellular matrix was minimised: when (a) pre-conditioning involved the combination of cryoprotection and partial dehydration; (b) the cryoprotectant was penetrating (Gly) as opposed to non-penetrating (Suc); and (c) embryos were rapidly cooled at water contents that minimised both dehydration and freezing damage. The ability of A. belladonna and H. montanus embryos to tolerate the various components of cryopreservation in relation to changes in extracellular superoxide (.O2 -) production and lipid peroxidation (a popular ‘marker’ for oxidative stress) was investigated in studies featured in Chapter 4. Pre-conditioning and freeze-thawing led to an increase in oxidative stress and the accompanying decline in viability suggests that oxidative stress was a major component of cryoinjury in the embryos presently investigated. Post-thaw viability retention in Gly cryoprotected + partially dried embryos was significantly higher than noncryoprotected + partially dried embryos, possibly due to the relatively lower post-drying lipid peroxidation levels and relatively higher post-drying and post-thawing enzymic antioxidant activities in the former. Exposure of certain plant tissues to low levels of oxidative or osmotic stress can improve their tolerance to a wide range of stresses. In contrast, exposure of H. montanus zygotic embryos to low levels of oxidative stress provoked by exogenously applied hydrogen peroxide (H2O2) or exposure of A. belladonna embryos to low levels of osmotic stress provoked by low water potential mannitol and polyethylene glycol solutions (in studies featured in Chapter 5) increased their sensitivity to subsequent dehydration and freeze-thaw stresses. Exposure of Gly cryoprotected and non-cryoprotected amaryllid embryos to such stress acclimation treatments may pre-dispose A. belladonna and H. montanus embryos to greater post-drying and post-thaw total antioxidant and viability loss than untreated embryos. To assess the vigour of seedlings recovered from partially dried H. montanus embryos, seedlings recovered from fresh (F) and partially dried (D) embryos in vitro were hardened-off ex vitro, and subsequently subjected to either 42 days of watering or 42 days of water deficit (in studies described in Chapter 6). In a subsequent study (described in Chapter 7), seedlings recovered from fresh (F), partially dried (D) and cryopreserved (C) A. belladonna embryos were regenerated in vitro, hardened-off ex vitro and then exposed to 12 days of watering (W) or 8 days of water stress (S) followed by 3 days of re-watering. Results of these studies suggest that the metabolic and ultrastructural lesions inflicted on A. belladonna and H. montanus zygotic embryos during cryopreservation may compromise the vigour (e.g. development of persistent low leaf water and pressure potentials and reduced photosynthetic rates) and drought tolerance of recovered seedlings, compared with seedlings recovered from fresh embryos. While the adverse effects of freeze-thawing were carried through to the early ex vitro stage, certain adverse effects of partial drying were reversed during ex vitro growth (e.g. the increased relative growth rate of seedlings from partially dried embryos). The reduced vigour and drought tolerance of seedlings recovered from partially dried and cryopreserved embryos in the present work may therefore disappear with an extension in the period afforded to them for hardening-off under green-house conditions, and in the field. The results presented in this thesis reinforce the notion that each successive manipulation involved in the cryopreservation of recalcitrant zygotic germplasm has the potential to inflict damage on tissues and post-thaw survival in such germplasm relies on the minimisation of structural and metabolic damage at each of the procedural steps involved in their cryopreservation. The results also highlight the need to design research programmes aimed not only at developing protocols for cryopreservation of plant genetic resources, but also at elucidating and understanding the fundamental basis of both successes and failures.
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2010.

The two species investigated in this study were Theobroma cacao and Barringtonia racemosa. Theobroma cacao has worldwide economic importance, as cocoa (the main ingredient in chocolate) is produced from the seeds of this tree; while B. racemosa has several applications in herbal medicine. The seeds of both T. cacao and B. racemosa are highly recalcitrant and therefore not amenable to storage for any significant periods. The long-term conservation of the germplasm of these species may only be feasible via cryopreservation. The aims of the present study were to: 1) optimize in vitro regeneration protocols for different types of explants that have the potential to be cryopreserved while maintaining the genetic integrity of these two species; and 2) develop cryopreservation protocols for selected explants. For T. cacao, protocols were established for bud-break and multiplication for both in vitro - and greenhouse-derived nodal explants, as well as a rooting medium for shoots derived from axillary buds. Parameters investigated towards the cryopreservation of axillary shoots, from greenhouse nodal segments, and nodal segments from in vitro plantlets, included the size of the explant and pre-treatments for cryopreservation. Nodal segments (6 - 7 mm) and axillary shoots (2 - 4 mm) needed to be soaked in 0.5% (w/v) ascorbic acid for 10 min to minimise phenolic production and subsequent tissue death, and surface-sterilized by soaking in 1% Ca(OCl)2 solution for 5 min to reduce microbial contamination. Subsequent cryopreservation attempts involved only in vitro nodal segments because of the lack of success in achieving elongation of excised axillary buds. Vitrification and slow freezing methods, with or without the application of cryoprotectants, did not achieve successful cryopreservation. Attempts to establish a protocol for producing somatic embryos, as an alternate to axillary shoots and in vitro nodal segments, resulted in the production of globular embryogenic callus for both leaf and cotyledon explants. Cryopreservation of these explants was not investigated in the scope of this study. The study on B. racemosa focused on the development of a somatic embryogenesis protocol. Segments of embryonic axes produced globular-stage embryos when placed on MS medium supplemented with 30 g 1-1 sucrose, 1.0 g 1-1 casein hydrolysate, 2.0 mg 1-1 2,4-D, 0.1 mg 1-1 BAP and 8.0 g 1-1 agar. Various strategies were employed to obtain embryo germination, which included 1) different time intervals on callus initiation medium; 2) the use of different auxins (IAA, NAA and 2,4-D) in combination with the cytokinins BAP and kinetin; 3) desiccation and 4) cold treatments. Although somatic embryo germination was not achieved, globular embryos proceeded with development to the cotyledonary stage when cold-treated for 8 h at 4°C. This study provides some fundamental bases for further investigation towards achieving long-term conservation for both T. cacao and B. racemosa. However, the use of meristems as explants for cryopreservation is suggested to be the way forward for the cryopreservation of both species.
Thesis (M.Sc.)-University of KwaZulu-Natal, 2008.

Cryopreservation is the most promising method for the long-term conservation of germplasm of plants producing desiccation-sensitive seeds. While such seeds are generally termed recalcitrant in the context of conventional storage practices, the term ‘cryo-recalcitrant’ is used for germplasm which is not readily amenable to cryopreservation. Cryo procedures usually involve a sequential combination of steps which must be optimised to limit the stresses experienced by specimens, thus promoting their survival. The present contribution reports on the effects of some of the steps involved in cryopreservation on the survival of the embryos of the amaryllids, Ammocharis coranica, Brunsvigia grandiflora and Haemanthus albiflos, with the ultimate aim of developing a protocol(s) for the successful cryopreservation of the germplasm of these species. The main foci of the investigations were the effects of rapid (flash) drying, the use of the cryoprotectant additives, glycerol (5 & 10%) and DMSO (0.1 & 0.25%), and employment of different cooling rates on the zygotic embryos of the selected species, which are known to be recalcitrant as well as being cryo-recalcitrant. Furthermore, this study reports on attempts at improving the rapidity of dehydration during flash drying by applying a vacuum, and also of providing cathodic protection (via highly reducing cathodic water and/or direct exposure to a static {negatively-charged} cathodic field during flash drying) to the explants at various stages in the protocol. These techniques were employed in attempts to ameliorate the adverse effects of reactive oxygen species associated with stresses imposed by the procedures during the cryopreservation process. The embryos of Ammocharis coranica, Brunsvigia grandiflora and Haemanthus albiflos were initially at water contents (WCs, dry mass basis) of 3.28±0.52, 2.55±0.22, 4.48±0.92 g g-1, respectively, after harvest. These embryos proved to be tolerant to moderately rapid water loss in the short term, with >60% retaining germinability at water contents ≥0.5 g g-1. The results from this study confirmed that dehydration to water contents below 0.5 g g-1 (dry mass basis) compromised survival, and that this effect was exacerbated if the embryos were cryoprotected prior to drying. Interestingly, the rate of water loss in embryos of these species differed, with A. coranica and H. albiflos drying at a (comparably) much slower rate than those of B. grandiflora. Subsequent rapid cooling yielded promising results when compared with slow cooling, as 30% of glycerol cryoprotected, rapidly cooled A. coranica embryos that had been flash-dried to 0.36±0.10 g g-1 generated normal seedlings. It was clear, however, that the effects of these procedures were exacerbated when all the steps of the cryo procedure were applied sequentially. However, the work also showed that these adverse effects may be ameliorated if each step of the cryopreservation protocol is optimised on a species-specific basis, thus promoting the chances of survival after cryopreservation and facilitating subsequent seedling establishment. This was evident in the 30% germination obtained when embryos of A. coranica, which had been cryoprotected with glycerol prior to flash drying before exposure to rapid cooling, while those that had not been cryoprotected or were cryoprotected with DMSO before drying did not survive. The incorporation of cathodic protection during flash drying appeared promising as it promoted the survival of 10% of H. albiflos embryos dehydrated to WCs between 0.37 and 0.26 g g-1 (whereas no survival was achieved without the inclusion of this step), and 70% of A. coranica embryos that were dehydrated to 0.35±0.21. In addition, the reduction of the explant size, from a whole 6 mm embryo to a 3-4 mm excised axis, promoted survival by up to 30% for A. coranica and H. albiflos, even at higher WCs. However, survival in these cases was based on observations of abnormal development, i.e. the development of roots or shoots, or calli. No surviving embryos were obtained from B. grandiflora after cooling, regardless of the preconditioning treatment or rate of cooling, and this was accredited to the greater degree of sensitivity of these embryos to the cryo procedures than those of the other two species. The use of cathodic water to re-hydrate explants after dehydration and of applying a vacuum during flash drying did not result in any observable benefits, and require further investigation for optimisation. The very limited success towards establishing a cryopreservation protocol for the species investigated in this study reinforces the difficulties associated with the cryopreservation of recalcitrant germplasm, which informs the cryo-recalcitrance of some explants. However, the results obtained have helped to identify a number of intervention points that could be used to minimise the damage incurred during the various procedural steps involved in cryopreservation.
Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.