In this study, optimization of a transformation and regeneration system via indirect organogenesis in cotyledonary petiole tissue of lentil (Lens culinaris Medik.) was investigated. Eight different medium types differing in their plant growth regulator compositions were employed to examine the callus induction potency of cotyledonary petiole. Except two, all other tested medium yielded more than 80% callus induction. Nine different medium types were studied to test the potencies of callus structures for shoot induction. Only the callus induced in medium H (1 mg/L Zeatin riboside + 1 mg/L Naphthalane acetic acid) yielded shoots at 8 to 40 % frequency. The most responsive medium was MS basal medium with no growth regulators. Also five and three different medium types were employed to examine callus induction potency of epicotyl tissues respectively. Each medium type yielded 90% callus induction. Only the callus induced in medium H yielded shoots At 6 to 26% frequency. Preliminary studies were carried out for somatic embryogenesis in cotyledonary petiole. Effects of salicylic acid on somatic embryogenesis were also investigated. Salicylic acid at 200&micro / M was found to enhance the percentage of somatic embryos by 25 % and reduce the necrosis 24 %. However none of the globular and heart shape embryos were able to regenerate. Transient GUS expression efficiencies of roots, shoot tips, and cotyledonary petioles were tested after Agrobacterium-mediated transformation. Transformation frequencies were 26, 74, and 38 % for cotyledonary petiole, shoot tips, and roots respectively.

In vitro micropropagation of saffron (Crocus sativus L.) by using direct and indirect organogenesis was the aim of this study. Also, the effect of plant growth regulators on growth parameters, such as corm production, sprouting time and germination ratio were investigated in ex vitro conditions. For in vitro regeneration of saffron, the effects of 2,4-D (2,4-dichlorophenoxyacetic acid) and BAP (6-benzylaminopurine) were tested initially. It was observed that 0,25 mg/L 2,4-D and 1 mg/L BAP combination was superior for indirect organogenesis while 1 mg/L 2,4-D and 1 mg/L BAP combination was favorable for direct organogenesis. During the improvement of direct organogenesis experiments, BAP (1 mg/L) without 2,4-D stimulated further shoot development. For adventitious corm and root induction, NAA (naphthaleneacetic acid) and BAP combinations were tested. Although a few corm formations were achieved, root development was not observed with these treatments. Further experiments with the culture medium supplemented with 1 mg/L IBA (indole-3-butyric acid) and 5% sucrose was effective on obtaining contractile root formation and increasing corm number. As a result, the overall efficiency was calculated as 59.26% for contractile root formation, 35.19% for corm formation and 100% for shoot development. In ex vitro studies, 50 mg/L IAA (indole-3-acetic acid) , 50 mg/L kinetin and 200 mg/L GA3 (gibberellic acid) were used. These applications were not as efficient as expected on assessed growth parameters.

In this study, optimization of a transformation and regeneration system via indirect and direct organogenesis in cotyledon, hypocotyl, petiole, leaf and shoot base tissues of sugar beet (Beta vulgaris L. cv. ELK 345 and 1195) was investigated. Two different germination, three different callus induction and shoot induction medium was used for indirect organogenesis of sugar beet cultivar ELK 345. Except cotyledon, other explants (hypocotyl, petiole and leaf) produced callus. However no shoot development was observed from callus of these explants. Shoot base tissue of sugar beet cultivar 1195 was employed for direct organogenesis. Shoot development was achieved via direct organogenesis using 0.1 mg/L IBA and 0.25 mg/L BA. Root development and high acclimatization rate were accomplished from shoot base tissue. Different concentrations of kanamycin and PPT were applied to leaf blade explants to find out optimum dose for selection of transformants. Kanamycin at 150 mg/L and PPT at 3 mg/L totally inhibited shoot development from leaf blades. Moreover, an Agrobacterium mediated transformation procedure for leaf explants of ELK 345 was also optimized by monitoring transient uidA expression 3rd days after transformation. Effects of different parameters (vacuum infiltration, bacterial growth medium, inoculation time with bacteria, Agrobacterium strains and L-cysteine application in co-cultivation medium) were investigated to improve transformation procedure. Vacuum infiltration and Agrobacterium strains were significantly improved transformation procedure. Percentage of GUS expressing areas on leaves increased three folds from the beginning of the study.

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.

Submitted by Jadson Francisco de Jesus SILVA (jadson@uefs.br) on 2018-01-30T22:51:06Z No. of bitstreams: 1 Tese Definitivo ZERGurgel.pdf: 2392487 bytes, checksum: 97870ae444449d037535f874c18b1d37 (MD5) / Made available in DSpace on 2018-01-30T22:51:06Z (GMT). No. of bitstreams: 1 Tese Definitivo ZERGurgel.pdf: 2392487 bytes, checksum: 97870ae444449d037535f874c18b1d37 (MD5) Previous issue date: 2017-07-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Comanthera mucugensis Giul. subsp. mucugensis, an endemic species of the municipality of Mucug?-BA is threatened with extinction. To reduce extractivism in natural populations, it is necessary to develop efficient multiplication protocols; In this sense somatic embryogenesis and organogenesis may be viable and preservation cryopreservation may be a strategy for its long-term. The objectives of this study were: (1) to analyze the embryogenic competence and the indirect organogenesis of C. mucugensis; (2) to evaluate the cryopreservation of seeds and different methods of cryopreservation of C. mucugensis plants and (3) to identify the best way to store seeds in the long term. Experiments were performed to induce somatic embryogenesis with different concentrations of 2,4-D X BAP and Picloran X BAP and for indirect organogenesis BAP and ANA were used. Seeds were cryopreserved for 0 (control), 1, 7, 30, 360 and 540 days and other seeds were stored at different temperatures to verify the best form of storage. Plants were cryopreserved with the technique of vitrification and encapsulation-desiccation. Plants from cryopreserved seeds were acclimatized in sand and vegetal soil (1: 1) for 60 days. The work showed that the plant regulators Picloran and 2,4-D are promising in the induction of callus with embryogenic potential and that the plant regulator ANA at 4.9 ?M is efficient in indirect organogenesis. The seeds of C. mucugensis can be cryopreserved without compromising their physiological quality, but the techniques for cryopreservation of plants have not been efficient. / Comanthera mucugensis Giul. subsp. mucugensis, possui flor de interesse comercial que devido ao extrativismo excessivo encontra-se amea?ada de extin??o. Para suprir a demanda do mercado e evitar o decl?nio populacional, faz-se necess?rio desenvolver protocolos para sua multiplica??o, podendo a embriog?nese som?tica e a organog?nese serem alternativas vi?veis. Al?m disso, ? importante investir em estudos de conserva??o a longo prazo como, por exemplo, a criopreserva??o. O trabalho teve como objetivos: realizar estudos para tornar a micropropaga??o mais eficiente e avaliar a conserva??o de C. mucugensis em diferentes temperaturas como estrat?gia para a conserva??o do seu germoplasma. Foram testados para a indu??o da embriog?nese som?tica 2,4-D x BAP e Picloram x BAP e para organog?nese indireta BAP e ANA. Na criopreserva??o foram avaliadas sementes mantidas em nitrog?nio l?quido (-196?C) por 0, 1, 7, 30, 360 e 540 dias e as plantas inteiras foram submetidas a duas t?cnicas: vitrifica??o e encapsulamento-desidrata??o. Para avaliar o armazenamento, as sementes foram mantidas em temperatura ambiente, 4?C, -20?C, -80?C e -196?C por 30, 90 e 180 dias. Visando observar se a criopreserva??o das sementes interfere no desenvolvimento, plantas oriundas da germina??o in vitro de sementes criopreservadas foram aclimatizadas em areia e terra vegetal (1:1) por 60 dias. O trabalho demonstrou que os reguladores vegetais Picloram e 2,4-D s?o promissores na indu??o de calos com potencial embriog?nico e que o regulador vegetal ANA (4,9 ?M) ? eficiente na organog?nese indireta. As sementes de C. mucugensis podem ser criopreservadas sem comprometer sua qualidade fisiol?gica, entretanto, n?o foram eficientes as t?cnicas para criopreserva??o de plantas inteiras.