The objectives of this study were to develop an in vitro direct adventitious bud induction and an organogenic callus induction and shoot regeneration system via floral organ segments culture for bromeliads, moreover, explore the effect of auxin on plantlet elongation of Guzmania. And further, apply the above micropropagation system to physical and chemical methods to induce somaclonal variances of bromeliad plantlets in vitro for mutation breeding. The explant sources of bromeliads and the components of culture medium were studied to develop a micropropagation system for bromeliads. The results indicated that the 1/3MS basal medium supplemented with a combination of 1.0 mg l-1 BA + 0.5-1.0 mg l-1 NAA, or a combination of 3.0 mg l-1 BA + 0.5 mg l-1 NAA, showed the highest frequency of direct initiation of adventitious buds derived from shoot apex and lateral bud explants of Aechmea fulgens var. fulgens and Guzmania 'Focus'. The best results of adventitious buds induction of the both species were found in the lower lateral bud explants, at 47.5% and 35%, respectively. In addition, the adventitious buds began to form on day 16 after the G. 'Focus' decapitated plantlets had been cultured in medium supplemented with 3.0 mg l-1 BA + 0.5 mg l-1 NAA. However, this phenomenon did not occur in case of undecapitated explants, where only protruding nodules appeared. Petal- and ovary-derived calli of A. fasciata and G. 'Hilda' were induced on 1/2MS basal medium supplemented with 1.0-1.5 mg l-1 2,4-D in combination with 1.0 or 0.5 mg l-1 NAA. Organogenic calli were cultured on medium with 1.0 mg l-1 NAA and 0.5 mg l-1 TDZ could be induced to differentiate and regenerate the adventitious buds. Furthermore, the number of adventitious buds proliferating at the base of the plantlets derived from G. 'Hilda' floral organs, cultured in media with different concentrations of IAA, IBA, NAA, and 8-azaadenine, was only 1-2 adventitious buds individually. This result shows that auxin can indeed suppress cytokinin-effects. The influence on plantlet elongation was greatest in the treatments using 0.5 mg l-1 NAA and 1.0 mg l-1 NAA. After 4 months culture, plantlets grew to 5.73 and 5.62 cm in height, that was 2.22 and 1.95 cm higher than the control, respectively. Plantlets of A. fasciata hardened under the middle (50 £gmol m-2s-1) light intensity condition had a higher survival rate, 95%, than that hardened at a low light intensity (1 £gmol m-2s-1; 17.5%). The maximum number of newly developing roots, up to 4.15 per shoot, was also observed at the same light intensity treatment. During transplantation, plantlets growing in coir fiber showed the best results in terms of plant growth within 6 months ex vitro culture. The average length of the plantlets was 22.0 cm, and an average of 19.3 leaves per plantlet was achieved. When calli of G. 'Hilda' treated by sodium azide, the survival rate was 0%. The survival rate of decapitated plantlet explants treated with 0.5 mM sodium azide for 60 minutes was 51.3%, about half-lethal dose. In addition to the survival rates of decapitated plantlet explants of A. fasciata, G. 'Hilda', G. 'Cherry', G. 'Luna' and G. 'Focus' irradiated by £^-ray showed 74.2-100% with the exception of the G. 'Focus' irradiated by 15 Gy, which dropped to 45.0%. At present, mutant plantlets showed a great deal of chimeras in leaf and were transplanted to potting media.

The objective of the study was to investigate the optimum conditions for in vitro regeneration of common juniper (Juniperus communis L.) by using indirect organogenesis approach. Throughout the study / callus induction, organogenesis, improved organogenesis and root induction experiments were performed sequentially. It was found that explant position, genotype, gender, treatments and sampling time had significant effects on callus induction rate in common juniper. The results of treatments indicated that IBA (indole-3-butyric acid) at concentration range 0.5-4.0 mg/l combined with MS medium supplemented with 0.1 mg/l BAP (benzylaminopurine), 3 % sucrose and 0.7% agar was the best one among the treatments to induce callus formation from common juniper explants collected as Spring buds. Also, a two-month culture was adequate period for the callus induction of common juniper regardless of position, before transferring the explants into organogenesis media. After a two-month culture in callus induction media, explants were transferred to organogenesis treatments in order to investigate adventitious bud development from callus tissues. There were significant differences among genotypes, treatments and explant-sampling times in initiation of organ development in common juniper. Additionally, it was found that excluding the auxin components while maintaining 1.0-2.0 mg/l BAP concentration in culture media, as refreshing after a month, stimulated the formation and development of adventitious buds and shoots. Among the treatments tested, it was found that 1.0 mg/l BAP plus 0.5 mg/l 2,4-D was the optimum culture media with adventitious bud formation capacity of 37.5% was though ageing of callus significantly affected the frequency of adventitious bud formation. Finally, rooting experiments were performed to investigate rooting efficiency of adventitious shoots. In the adventitious rooting experiments, no rooting was observed in any of the treatments used with common juniper explants. Although whole plantlet development from callus tissues could not be achieved as indirect organogenesis, the results of the study could aid to future studies dealing in vitro regeneration and production of secondary chemicals from common juniper.

A transformação genética possibilita a introdução de genes de interesse agronômico no genoma das plantas e pode ser empregada na tentativa de obter plantas resistentes a doenças. No entanto, para se obter uma planta transgênica é necessário primeiramente estabelecer um procotolo eficiente de regeneração de plantas in vitro. Assim, o objetivo desse trabalho foi estudar a organogênese in vitro e a transformação genética de limão Volkameriano e laranja azeda com um fragmento do gene da capa protéica do CTV. Para a organogênese in vitro utilizou-se, como explante, segmento internodal, obtido de planta cultivada em casa-de-vegetação, segmento de epicótilo, coletado de plântula cultivada in vitro e segmento de cotilédone associado ao hipocótilo obtido de semente introduzida in vitro. Esses explantes foram mantidos em meio de cultura EME suplementado com 6-benzilaminopurina (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L- 1), sendo incubados sob fotoperíodo de 16 h de luz ou em condições de escuro por 30 dias e então transferidos para fotoperíodo de 16 h de luz. A avaliação foi realizada após 45 dias de cultivo, determinando-se o número de explantes responsivos e o número de gemas por explante. A caracterização anatômica do processo de regeneração foi realizada por meio de cortes histológicos. Pela análise dos dados foi possível verificar que a organogênese in vitro ocorreu a partir dos três tipos de explantes testados, sendo que, nas duas espécies em estudo, os melhores resultados foram obtidos com o cultivo de segmento de cotilédone associado ao hipocótilo. As concentrações de BAP que estimularam as melhores taxas de regeneração foram de 1,0 e 1,5 mg L-1, para limão Volkameriano, e 0,5 e 1,0 mg L-1 para laranja azeda. A incubação dos explantes em ausência de luz favoreceu a regeneração in vitro. Pela análise histológica foi possível observar que o processo de regeneração, a partir dos três tipos de explantes testados, ocorreu por meio de organogênese indireta. O protocolo de desenvolvimento estabelecido durante os experimentos de organogênese in vitro foi utilizado para a transformação genética dessas espécies via Agrobacterium, contendo o plasmídeo pCAMBIA 2201, com um fragmento do gene da capa protéida do CTV, em uma construção gênica do tipo hairpin. As gemas de limão Volkameriano e laranja azeda identificadas como transgênicas pelo teste histoquímico GUS foram enxertadas in vitro em citrange Carrizo. A confirmação da transformação genética foi realizada pela análise de PCR, a qual mostrou a amplificação de um fragmento de 671 pb, correspondente a parte do gene amplificada. / Genetic transformation permits the introduction of agronomically important genes in plant genome and can be utilized in order to produce disease resistant plants. However for the recovery of transgenic plants is required to establish an efficient in vitro plant regeneration protocol. In this work the aim was to study an in vitro organogenesis and the genetic transformation of Volkamer lemon and sour orange with a sequence of the CTV coat protein gene. For in vitro organogenesis explant internodal segments collected from plants cultivated in greenhouse, epicotyl segments obtained from in vitro cultivated seedlings and cotyledon fragment with hypocotyl attached obtained from in vitro germinated seed were used as explant. These explants were cultured in EME medium supplemented with benzilaminopurine (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L-1). Cultures were maintained under a 16 h photoperiod or in the dark for 10 d and then transferred to a 16 h photoperiod. The evaluation was performed 45 d after the incubation determining the number of responsive explant and the number of buds per explant. The anatomical characterization of in vitro regeneration process was carried out through histological analyses. The in vitro organogenesis occurred in the three types of explant tested, however cotyledon fragment with hypocotyl attached showed higher morphogenetic potential in both species. The best responses of regeneration were obtained when the medium was supplementation with 1,0 e 1,5 mg L-1 BAP for the Volkamer lemon and 0,5 e 1,0 mg L-1 BAP for the sour orange. The incubation in darkness favored the in vitro regeneration. The histological analyses showed that the regeneration process occurred through indirect organogenesis in the three types of explants tested. The developed protocol was use for genetic transformation of Volkamer lemon and sour orange with Agrobacterium, containing pCAMBIA 2201 plasmid with a sequence of CTV coat protein gene, in a hairpin construction. Volkamer lemon and sour orange shoots identified as transgenic by histochemical test GUS were micrografted into Carrizo citrange. PCR analysis were performed after micrografted showing the presence of the 671 pb fragment of the transgene.

A transformação genética possibilita a introdução de genes de interesse agronômico no genoma das plantas e pode ser empregada na tentativa de obter plantas resistentes a doenças. No entanto, para se obter uma planta transgênica é necessário primeiramente estabelecer um procotolo eficiente de regeneração de plantas in vitro. Assim, o objetivo desse trabalho foi estudar a organogênese in vitro e a transformação genética de limão Volkameriano e laranja azeda com um fragmento do gene da capa protéica do CTV. Para a organogênese in vitro utilizou-se, como explante, segmento internodal, obtido de planta cultivada em casa-de-vegetação, segmento de epicótilo, coletado de plântula cultivada in vitro e segmento de cotilédone associado ao hipocótilo obtido de semente introduzida in vitro. Esses explantes foram mantidos em meio de cultura EME suplementado com 6-benzilaminopurina (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L- 1), sendo incubados sob fotoperíodo de 16 h de luz ou em condições de escuro por 30 dias e então transferidos para fotoperíodo de 16 h de luz. A avaliação foi realizada após 45 dias de cultivo, determinando-se o número de explantes responsivos e o número de gemas por explante. A caracterização anatômica do processo de regeneração foi realizada por meio de cortes histológicos. Pela análise dos dados foi possível verificar que a organogênese in vitro ocorreu a partir dos três tipos de explantes testados, sendo que, nas duas espécies em estudo, os melhores resultados foram obtidos com o cultivo de segmento de cotilédone associado ao hipocótilo. As concentrações de BAP que estimularam as melhores taxas de regeneração foram de 1,0 e 1,5 mg L-1, para limão Volkameriano, e 0,5 e 1,0 mg L-1 para laranja azeda. A incubação dos explantes em ausência de luz favoreceu a regeneração in vitro. Pela análise histológica foi possível observar que o processo de regeneração, a partir dos três tipos de explantes testados, ocorreu por meio de organogênese indireta. O protocolo de desenvolvimento estabelecido durante os experimentos de organogênese in vitro foi utilizado para a transformação genética dessas espécies via Agrobacterium, contendo o plasmídeo pCAMBIA 2201, com um fragmento do gene da capa protéida do CTV, em uma construção gênica do tipo hairpin. As gemas de limão Volkameriano e laranja azeda identificadas como transgênicas pelo teste histoquímico GUS foram enxertadas in vitro em citrange Carrizo. A confirmação da transformação genética foi realizada pela análise de PCR, a qual mostrou a amplificação de um fragmento de 671 pb, correspondente a parte do gene amplificada. / Genetic transformation permits the introduction of agronomically important genes in plant genome and can be utilized in order to produce disease resistant plants. However for the recovery of transgenic plants is required to establish an efficient in vitro plant regeneration protocol. In this work the aim was to study an in vitro organogenesis and the genetic transformation of Volkamer lemon and sour orange with a sequence of the CTV coat protein gene. For in vitro organogenesis explant internodal segments collected from plants cultivated in greenhouse, epicotyl segments obtained from in vitro cultivated seedlings and cotyledon fragment with hypocotyl attached obtained from in vitro germinated seed were used as explant. These explants were cultured in EME medium supplemented with benzilaminopurine (BAP 0,0; 0,5; 1,0; 1,5; 2,0 mg L-1). Cultures were maintained under a 16 h photoperiod or in the dark for 10 d and then transferred to a 16 h photoperiod. The evaluation was performed 45 d after the incubation determining the number of responsive explant and the number of buds per explant. The anatomical characterization of in vitro regeneration process was carried out through histological analyses. The in vitro organogenesis occurred in the three types of explant tested, however cotyledon fragment with hypocotyl attached showed higher morphogenetic potential in both species. The best responses of regeneration were obtained when the medium was supplementation with 1,0 e 1,5 mg L-1 BAP for the Volkamer lemon and 0,5 e 1,0 mg L-1 BAP for the sour orange. The incubation in darkness favored the in vitro regeneration. The histological analyses showed that the regeneration process occurred through indirect organogenesis in the three types of explants tested. The developed protocol was use for genetic transformation of Volkamer lemon and sour orange with Agrobacterium, containing pCAMBIA 2201 plasmid with a sequence of CTV coat protein gene, in a hairpin construction. Volkamer lemon and sour orange shoots identified as transgenic by histochemical test GUS were micrografted into Carrizo citrange. PCR analysis were performed after micrografted showing the presence of the 671 pb fragment of the transgene.