Teses / dissertações sobre o tema "Callus"

Fracture healing is a complicated coupling of many processes. Yet despite the apparent complexity, fracture repair is usually effective. There is, however, no comprehensive mathematical model addressing the multiple interactions of cells, cytokines and oxygen that includes extra-cellular matrix production and that results in the formation of the early stage soft callus. This thesis develops a one dimensional continuum transport model in the context of early fracture healing. Although fracture healing is a complex interplay of many local factors, critical components are identified and used to construct an hypothesis about regulation of the evolution of early callus formation. Multiple cell lines, cellular differentiation, oxygen levels and cytokine concentrations are examined as factors affecting this model of early bone repair. The model presumes diffusive and chemotactic cell migration mechanisms. It is proposed that the initial signalling regime and oxygen availability arising as consequences of bone fracture, are sufficient to determine the quantity and quality of early soft callus formation. Readily available software and purpose written algorithms have been used to obtain numerical solutions representative of various initial conditions. These numerical distributions of cellular populations reflect available histology obtained from murine osteotomies. The behaviour of the numerical system in response to differing initial conditions can be described by alternative in vivo healing pathways. An experimental basis, as illustrated in murine fracture histology, has been utilised to validate the mathematical model outcomes. The model developed in this thesis has potential for future extension, to incorporate processes leading to woven bone deposition, while maintaining the characteristics that regulate early callus formation.

Thesis (MSc)--Stellenbosch University, 2012.<br>ENGLISH ABSTRACT: Plant growth promoting substances (PGPS) are emerging as useful tools in the investigation of important plant growth traits. Two PGPS, smoke-water derived from burning plant material and a synthetic strigolactone analogue, GR24, have been reported to regulate a wide variety of developmental and growth processes in plants. These PGPS are beginning to receive considerable attention in the area of improving plant biomass yield and production. Variation in growth between plants is a major impediment towards the complete understanding of the intrinsic processes that control biomass production. Callus cultures of the model plant Arabidopsis thaliana could overcome some of these hindrances. However, the suitability of these callus cultures as a model system for plant biomass production must be established first. This study aimed at using A. thaliana callus cultures as a platform to study the plant growth promoting activities of smoke-water and GR24. The first part of this study was conducted to develop an optimal protocol for inducing A. thaliana callus formation. Wild-type A. thaliana Col-O, as well as strigolactone deficient and insensitive mutants (max1-1, max2-1, max2-2, max3-9 and max4-1) were cultured for callus induction. Hypocotyl and leaf explants were cultured onto MS media supplemented with different hormone concentrations of 2,4-D and kinetin (2:2 mg/L 2,4-D:kinetin and 0.5:0.05 mg/L 2,4-D:kinetin). Both media proved suitable for callus induction of all genotypes, with max1-1 showing the highest efficiency (83.33% and 92.22%) of callus induction. Calli were then used as a platform for future investigations into the effects of smoke-water and GR24. Secondly, this study examined the effects of smoke-water and GR24 on wild-type A. thaliana Col-O callus. Basic physiological studies were conducted to determine if these two compounds would positively affect callus growth, as was shown in previous studies using whole plants. Calli cultivated on MS media containing the two different hormone concentrations were transferred onto the same fresh MS medium, supplemented with either smoke-water or GR24. Growth promotion by smoke-water and GR24 in calli was characterized by a significantly increased mass (biomass). Calli were additionally transferred onto MS medium containing either auxin only or kinetin only and supplemented with GR24 or smoke-water. In the auxin only system, increased mass was recorded for both GR24 and smoke-water treatments, while these two compounds seemed to reduce growth in the kinetin only system. The positive growth stimulatory effect observed for the auxin only system could be attributed to the synergistic relationship between auxin and strigolactones, whilst the reduced mass in the latter system could be due to the antagonistic interaction between strigolactones and cytokinins. Finally, this study has discovered a dual role of strigolactones in biomass accumulation and adventitious root formation for Arabidopsis thaliana callus. On an auxin- and cytokinin-free MS medium supplemented with GR24, calli of Arabidopsis thaliana strigolactone deficient mutants (max1-1 and max4-1) and the wild-type Col- O, but not the strigolactone response mutant (max2-2), showed enhanced biomass accumulation. In addition to this, the max4-1 mutant and wild-type Col-O demonstrated enhanced adventitious rooting, which was not apparent in max2-2. Together these data suggested that the biomass accumulation and the adventitious rooting activities of GR24 in Arabidopsis thaliana calli are controlled in a MAX2- dependent manner. The interaction between strigolactone, auxin and cytokinin signalling pathways in regulating these responses appears to be complex. Gene expression profiling showed regulation of stress-related genes such as B-box transcription factors, CALCINEURIN B-LIKE and RAP4.2 Genes encoding hormones associated with stress (ABA, ethylene) and defence mechanisms (JA) were upregulated. Expression of stress related genes indicated clues on some kind of stress mediation that might be involved during the regulation of the rhizogenic response. Conversely, smoke-water treatment could not enhance the biomass of the calli and nor could it induce adventitious rooting in the absence of auxin and cytokinin. This observation strongly emphasized the distinct roles of these two compounds, as well as the importance of the interaction and ratio of auxin and cytokinin in callus growth. This study has demonstrated a novel role of strigolactones in plant growth and development, i.e. enhancement of biomass production in callus cultures. Secondly the enhanced adventitious rooting ability is in agreement with recently published literature on the role of strigolactones in regulating root architecture. In vitro callus production is advantageous to plant sciences. It creates an opportunity for increasing plant material for cultivation and offers the use of cell cultures that accurately mimic specific growth responses. It could greatly contribute to the study of intricate regulatory and signalling pathways responsible for growth and development in plants. Because the regulation of plant biomass production is very complex and the molecular mechanisms underlying the process remain elusive, it is of paramount importance that further work be done in order to gain more in-depth insights and understanding of this aspect and subsequently improve efficiency and returns when applying biotechnology tools on commercially important crop plants.<br>AFRIKAANSE OPSOMMING: Verbindings wat plantgroei bevorder (PGBV) het as nuttige alternatief ontstaan om plant groei te ondersoek. Rook-water, afkomstig van verbrande plant material, en ‘n sintetiese strigolaktoon analoog, GR24, wat ‘n α, β-onversadigde furanoon funksionele groep in gemeen het, is vir die regulering van ‘n wye verskeidenheid ontwikkelings- en groei prosesse in plante verantwoordelik. Tans ontvang hierdie PGBVs aansienlik aandag in die area van die verbetering van plant biomassa opbrengs en -produksie. Die variasie in groei tussen plante is ‘n groot hindernis om die intrinsieke prosesse wat biomass produksie beheer, volledige te verstaan. Deur gebruik te maak van kallus kulture van die model plant Arabidopsis thaliana kan van hierdie hindernisse oorkom word. Tog moet die geskiktheid van kallus kulture as ‘n model sisteem vir plant groei biomass produksie eers gevestig word. Die doel van hierdie studie was om A. thaliana kallus kulture as ‘n platform vir die studie van die plantgroei bevorderingsaktiwiteite van rook-water en GR24 te gebruik. Die eerste deel van die studie is uitgevoer ten einde ‘n optimale protokol vir die induksie van A. thaliana kallus produksie te ontwikkel. Wilde tipe Col-0, asook strigolaktoon afwesige en onsensitiewe mutante (max1-1, max2-1, max2-2, max3-9 en max4-1) is vir kallus induksie gekultiveer. Hipokotiel en blaar eksplante is op MS medium wat verskillende hormoon konsentrasies van 2,4-D en kinetien (2:2 mg/L 2,4-D:kinetien en 0.5:0.05 mg/L 2,4-D:kinetien) bevat, oorgedra. Beide media was geskik vir kallus induksie van al die genotipes, met max1-1 wat die hoogste effektiwiteit (83.33% en 92.22%) van kallus induksie getoon het. Kalli is daarna as ‘n platform vir toekomstige navorsing i.v.m die effek van rook-water en GR24 gebruik. Tweedens ondersoek die studie die effek van rook-water en GR24 op wilde tipe Col-0 kallus. Basiese fisiologiese studies is uitgevoer om te bepaal of die twee verbindings ‘n positiewe effek op kallus groei toon soos aangedui in vorige studies waar intakte plante gebruik is. Kallus wat op MS medium wat die twee verskillende hormoon konsentrasies bevat gekultiveer was, is op dieselfde vars MS medium, wat addisioneel óf rook-water óf GR24 bevat, oorgedra. Die stimulering van groei van kalli deur rook-water en GR24 is deur ‘n merkwaardige toename in massa (biomassa) gekenmerk. Kallus is additioneel op MS medium wat slegs óf ouksien óf kinetin bevat (gekombineer met GR24 of rook-water behandeling), oorgedra. In die sisteem waar slegs ouksien toegedien is, is ‘n toename in massa waargeneem vir beide GR24 en rook-water behandelinge. In teenstelling hiermee, het die twee verbindings in die sisteem waar slegs kinetin toegedien is, ‘n vermindering in groei meegebring. Die positiewe groei stimulerende effek wat waargeneem is vir die sisteem waar slegs ouksien toegedien is, kan toegedra word aan die sinergistiese verhouding tussen die ouksien en strigolaktone; terwyl die verlaagde massa in die laasgenoemde sisteem aan die antagonistiese interaksie tussen strigolaktone en sitokiniene toegedra kan word. Laastens het hierdie studie het ‘n gelyktydige rol van strigolaktone vir biomassa akkumulasie en bywortelvorming in Arabidopsis thaliana kallus ontdek. Kallus van A. thaliana strigolaktoon afwesige mutante (max1-1 en max4-1) en die wilde tipe Col-0 (maar nie die strigolaktoon reagerende mutant (max2-2) het op ‘n ouksien en sitokinien vrye MS medium wat GR24 bevat ‘n verhoogde biomassa akkumulasie getoon. Die max4-1 mutant en wilde tipe Col-0 het verhoogde bywortelvorming getoon, wat nie so opmerklik by max2-2 was nie. Hierdie data het tesame voorgestel dat die biomassa akkumulasie en die bywortelvormingsaktiwiteite van GR24 in Arabidopsis thaliana kallus op ‘n MAX2-afhanklike wyse beheer word. Die interaksie tussen strigolaktoon, ouksien en sitokinien sein transduksie paaie vir die regulering van hierdie reaksies blyk kompleks te wees. Die geen uitdrukkingsprofiel het die regulering van stres verwante gene soos B-boks transkripsie faktore, CALCINEURIN B-LIKE en RAP4.2, getoon. Gene wat vir hormone wat aan stres (ABA, etileen) en verdedigingsmeganismes (JA) verwant is, is opgereguleer. Die uitdrukking van stress verwante gene dui op tekens van ‘n ander tipe stres bemiddeling wat dalk by die regulering van die risogeniese reaksie betrokke kan wees. In teenstelling, rook water behandeling kon nie die kallus biomassa verhoog nie en dit kon ook nie die bywortelingvorming in die afwesigheid van ouksien en sitokiniene induseer nie. Hierdie waarneming is ‘n sterk bevestiging vir die uitsonderlike rol van die twee verbindings, asook die belang van die interaksie en verhouding van ouksien en sitokinine vir die groei van kallus. Hierdie studie toon op ‘n nuwe rol van strigolaktoon in plant groei en ontwikkeling, d.w.s die verhoogde biomassa produksie in kallus kulture. Tweedens, die verhoogde bywortelvormingsvermoë is in ooreenstemming met literatuur wat onlangs gepubliseer is i.v.m die rol van strigolaktone in die regulering van wortel argitektuur. Die in vitro produksie van kallus is voordelig in plant wetenskappe. Dit skep ‘n geleentheid vir die vermeerdering van plant materiaal vir kultivering en bied die gebruik van selkulture wat spesifieke groei reaksies op ‘n merkwaardige wyse akkuraat namaak. Dit kan grootliks bydra tot die studie van die delikate regulatoriese en sein transduksie paaie wat vir groei en ontwikkeling van plante verantwoordelik is. Aangesien die regulering van plant biomassa produksie baie kompleks is en die molekulêre meganismes vir die proses onbekend bly is dit van grootskaalse belang dat meer werk gedoen word om ‘n meer in diepte insig en kennis van die aspekte en gevolglike verbetering van effektiwiteit en wins te kry deur die toepassing van biotegnologiese metodes op die gewas plante wat van kommersiêle belang is.

Orientador : Dr. Carlos Ricardo Soccol<br>Coorientador : André Luis Lopes da Silva<br>Tese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 29/12/2016<br>Inclui referências : f. 45-52<br>Área de concentração<br>Resumo: A cultura de tecidos vegetais propicia em um período curto de tempo a multiplicação de plantas geneticamente idênticas em larga escala, permite a produção de metabólitos secundários em ambiente controlado para aplicação na agricultura e na indústria. Pinhão manso (Jatropha curcas L.) é uma planta usada para produção de biodiesel com alto teor de óleo e apresenta grande quantidade de compostos bioativos que podem ser utilizados para desenvolvimento de novos produtos na indústria. Por essa razão os objetivos desse trabalho foram: (1) Estabelecer curva de crescimento de calos e avaliar o perfil dos ácidos graxos do óleo bruto (pinhão manso); (2) Avaliar a atividade antihelmíntica do extrato aquoso quente do grão e do calo de J. curcas, testar a toxicidade dos extratos em náuplios de Artemia salina e identificar os possíveis compostos bioativos associados à atividade anti-helmíntica. (1) A cinética da curva de crescimento de calos de pinhão manso apresentou padrão de curva sigmoidal com seis fases distintas: lag, exponencial, linear, desaceleração, estationária e de declínio. O perfil lipídico do óleo bruto dos calos apresentaram ácidos graxos de cadeias médias e cadeias longas, onde o ácido graxo de maior percentagem no extrato foi o ácido palmítico (55%) e o perfil lipídico do extrato do calo é diferente do perfil lipídico da semente de J. curcas. (2) Extrato aquoso quente do grão (pHs 5,7 e 9) e do calo (pH 5), 115 dias de cultivo, de J. curcas obtiveram atividade anti-helmítica e os extratos não foram citotóxicos, testados em Artemia salina. Os fitoquimicos nematicidas não foram completamente elucidados. Em diferentes pHs de extração e tipos de tecidos apresentou diferentes compostos nematicidas, como acidos graxos, furfural e ésteres de forbol. Palavras-chave: Cultura de tecidos de plantas, composição lipídica, nematicida natural, ésteres de forbol, calogênese, análises in silico.<br>Abstract: The plant tissue culture allows the multiplication of genetically identical plants on a large scale, allowing the production of secondary metabolites in a controlled environment for application in agriculture and industry. Jatropha curcas L. is a plant used for the production of biodiesel with high oil content and presents a great amount of bioactive compounds that can be used to develop new products in the industry. The purpose of the present study were: (1) To establish a calli growth curve and to evaluate the fatty acid profile of crude oil extracted from callus.; (2) To evaluate the anthelmintic activity of hot aqueous extracts of kernels and calli of J. curcas, the toxicity effects of these extracts against Artemia salina nauplii and to identify the bioactive compounds associated to anthelmintic activity. (1) The Kinetics of the callus growht curve presented a sigmoid standard curve with six distinct phases: lag, exponential, linear, deceleration, stationary and decline. The calli produced medium-chain and long-chain fatty acids and the palmitic acid was the fatty acid with the highest proportion in oil. The lipid profile obtained in callus oil was different from the seed oil profile. (2) The hot aqueous extracts of J. curcas kernel (pHs 5,7 and 9) and callus (pH 5), 115 days of culture was efficient for anthelmintic activity, in lethal concentration, 50%, (LC50), and these extracts were not toxic against Artemia salina. The nematicidal fitochemicals were not completed elucidated. The different pH extraction and tissue type presented different nematicidal compounds, such as fatty acids, furfural and phorbol esters. Keywords Plant Tissue culture o lipid composition o natural nematicide o phorbol esters o callogenesis o in silico analysis

The work described in this thesis focuses on the development of fully reproducible systems of plant regeneration through embryogenic callus and totipotent protoplast cultures of rice (Oryza sativa L. cv. Taipei 309). The calli originated from different explants, including excised anthers, isolated microspores, mature seed-embryos, immature seed-embryos and leaf-base meristems. In the callus culture and plant regeneration studies, excised anther-derived callus produced higher numbers of green (90.5%) and albino (9.5%) plants, both haploids (81.2%) and diploids (18.8%), compared to other explants. The plant regeneration frequency from mature seedembryo-derived callus varied with callus age and embryogenicity of the callus. A maximum of 20% plant regeneration was obtained with approx. 2% of the regenerated plants being albinos. In the case of mature or immature seed-embryos and leaf-base meristems, plant regeneration frequency from callus was dependent on some more specific factors, in addition to callus age and embryogenicity. These factors were the age of seedlings grown in vitro and the age of immature embryos after anthesis for leaf-base meristem and immature embryo cultures, and also the scutellar surface orientation of mature or immature embryos during callus culture initiation. The present study also focused on the plating efficiencies and plant regeneration frequencies obtained from culture of embryogenic cell suspension-derived protoplasts. These varied with the explant source, the embryogenic nature and age of the callus during suspension initiation, the age of the embryogenic suspension culture, the time of enzymatic digestion, the plating density, viability of protoplasts and use of the suspensions at their exponential (actively dividing) growth stage. The results from this study indicated that leaf-base meristem and anther calli-derived suspensions retain their regenerability for a longer time compared to suspensions derived from mature seedscutellum and immature seed-derived embryos. Maximum plant regeneration frequency (45%) was obtained from embryogenic suspensions derived from mature seedembryos. A detailed study of different agronomic characteristics of 57 protoclonal plants (mature seed-scutellum-derived) was performed and the statistical analysis of the data indicated a range of variability among the protoclonal plants and also between the protoclonal and seed-derived plants. A detailed study of the ploidy levels of regenerated plants obtained through different culture systems (callus and protoplasts) was performed by flow cytometry and chromosome counting of meristematic cells. The results showed that anther calliderived plants were mostly haploids, protoplast-derived plants of leaf-base meristem, anther, and mature seed-scutellum origin were mostly tetraploids and in between these two groups, diploids, triploids and aneuploids were also present.

Plant transformation provides a promising methodology of introducing new genes that encode desirable traits to a wide range of crop plants. Success in genetic transformation has been achieved in many of the important crop species, such as soybean, cotton, rice, corn. However, wheat, one of the major crops of the world, has been considered to be difficult to transform via either Agrobacterium or biolistic bombardment (Rakszegi et al., 2001). There have been limited studies on A. tumefaciens-mediated transformation of cereals, including wheat, because of the overall refractory character of host-pathogen interactions between Agrobacterium and the cereal plants (Gould et al., 1991; Hiei et al., 1994; Cheng et al., 1997). While the genetic transformation of rice using Agrobacterium has become routine, only a few successful studies of Agrobacterium- mediated transformation of wheat have been reported, and these involved a model spring wheat, Triticum aestivum cultivar Bobwhite (Cheng et al., 1997). Model genotypes are developed for ease of plant regeneration in tissue culture and both Agrobacterium and biolistic mediated transformation methods require regeneration of plants in tissue culture. More success has been achieved in obtaining fertile transgenic wheat plants by particle bombardment, or biolistics method (Vasil et al., 1992; Weeks et al., 1993; Becker et al., 1994; Zhou et al., 1995; Altpeter et al., 1996). Wheat plants of the model system cultivar Bobwhite were used in most of these studies as well. The primary objective of this study was to use the callus-based transformation procedures mentioned above with a non-model cultivar of hexaploid spring wheat Saratovskaya-29, widely grown in Kazakhstan, to test the genotype dependence of the previously developed transformation protocols with respect to stable transfer of DNA and regeneration of transgenic plants. The spring wheat cultivar Saratovskaya-29 (Albidum-24/ Lutescens-55-11) was chosen for the study as being one of the most widely grown wheat cultivars both in Russia and Kazakhstan. It was bred in early 50??s in the Research Institute of the South-East, Saratov. Because of its drought resistance and good baking quality traits, Saratovskaya-29 reached a peak of nearly 21.2 mln ha in the former USSR in 1996 (Martynov and Dobrotvorskaya, 1996). Economical importance of this cultivar makes it an appropriate candidate for further improvement of economically significant traits. Another objective of the study described was to compare the transformation efficiencies and inheritance in the transgenic plants produced.

Inheritance of androgenetic competence was studied in 10 diploid potato species hybrids and 16 backcross progeny. Ten hybrid families including three reciprocals were generated between competent clones of S. phureja and incompetent clones of S. berthaultii, S. microdontum and S. phureja. The F₁ hybrid families segregated for androgenetic competence with some highly competent and some incompetent genotypes in all families. The expression of androgenetic competence was modified by parents lacking competence. The cytoplasm of species lacking competence exerted a greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. The segregation data of 16 backcross progeny between a highly competent hybrid and its incompetent parent suggested that competence may be under control of a single dominant gene. Androgenetic competence can be transferred among sexually compatible potato species. The transfer of desirable traits to a monoploid background can be expected using an- drogenetically competent selections in hybrid combination with germplasm expressing the desired attributes. In an attempt to determine genetic changes of regenerated potato plants following anther and callus cultures, 20 callilines of two S. phureja clones were examined. Four of 20 callilines selected for fertility and diploidy were morphologically indistinguishable among themselves and from the parental clone that had not undergone a tissue culture cycle. Even though morphologically indistinct from the parental clone, all four callilines exhibited higher seed set as pollen parents in 4x-2x crosses and two of the four exhibited higher recombination frequency between the centromere and the y gene. The estimated increase in map distance of the y locus ranged from 3.4 to 10.0 units. Progeny analysis revealed no significant morphological differences among 4x-2x hybrid families under field conditions, and only a single difference among 2x-2x hybrid families under screenhouse conditions. Hence, variation induced in tissue culture may have occurred without detectable morphological change. Assuming no adverse tissue culture effects, breakage of undesirable linkage groups may be an advantage of caulogenesis before backcrossing.<br>Ph. D.

Gossypium barbadense L. “algodón nativo” es oriundo de la costa norte del Perú y se caracteriza por presentar fibras de colores naturales. La evaluación del efecto de diferentes concentraciones de fitorreguladores de crecimiento en la inducción de callo embriogénico se realizó en explantes de hipocotilo de G. barbadense L. “algodón nativo” color pardo, bajo dos condiciones lumínicas distintas. La desinfección de semillas se llevó a cabo utilizando NaOCl al 2.5% en distintos tiempos de exposición (5-20 min) para obtener plántulas in vitro. Para la iniciación y proliferación de callos, se introdujeron explantes de hipocotilo (con 5 réplicas) en medios Murashige-Skoog (MS) suplementados con diferentes concentraciones de ácido 2,4-diclorofenoxiacético (2,4-D), kinetina (Kin) y agua de coco. Los callos friables de mayor proliferación fueron transferidos a medios MS con distintas concentraciones de Kin, 2,4-D y ácido indol-3-butírico (IBA) para la inducción de callo embriogénico. Los cultivos fueron mantenidos en condiciones de fotoperiodo 16 h luz/ 8 h oscuridad y oscuridad continua. Se logró el 100% de desinfección de semillas en 10 min de exposición al desinfectante. En 85% a 100% de explantes de hipocotilo se obtuvo la formación de callo en todos los tratamientos de iniciación de callo incubados en ambas condiciones lumínicas durante 21 días. La mayor proliferación de callo friable se obtuvo en 82.5% de explantes cultivados en medio MS enriquecido con 0.1 mg/l de 2,4-D y 100 ml/l de agua de coco e incubados en fotoperiodo 16 h luz/ 8 h oscuridad. En los medios MS suplementados con distintas concentraciones de Kin, 2,4-D e IBA no se logró la inducción de callo embriogénico. Sin embargo, en el medio MS sin reguladores de crecimiento y en el suplementado con 0.05 mg/l de Kin y 0.3 mg/l de IBA, en fotoperiodo 16 h luz/ 8 h oscuridad, se inició la organogénesis radical.Gossypium barbadense L. “native cotton” is originally from the northern coast of Peru and is characterized by its naturally colored fibers. The evaluation of the effect of different concentrations of plant growth regulators on embryogenic callus induction was performed in hypocotyl explants of Gossypium barbadense L. “native cotton” brown, under two different lighting conditions. Seed disinfection was carried out using 2.5% NaOCl in different exposure times (5-20 min) in order to obtain in vitro plants. For initiation and proliferation of callus, hypocotyl explants (with 5 replicates) were placed in Murashige-Skoog (MS) medium supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), kinetin (Kin) and coconut water. Friable calli with the highest proliferation were transferred to MS medium with different concentrations of Kin, 2,4-D and indole-3-butyric acid (IBA) for induction of embryogenic callus. The cultures were maintained under conditions of 16 h light/ 8 h dark photoperiod and continuous darkness. 100% of seed disinfection was achieved at 10 min of exposure to disinfectant. In 85% to 100% of hypocotyl explants, callus formation was obtained in all callus induction treatments, incubated in both lighting conditions during 21 days. The highest proliferation of friable callus was obtained in 82.5% of hypocotyl explants cultivated in MS medium enriched with 0.1 mg/l 2,4-D and 100 ml/l coconut water, in 16 h light/ 8 h dark photoperiod. In MS medium supplemented with different concentrations of Kin, 2,4-D and IBA, induction of embryogenic callus was not achieved. However, in MS medium without growth regulators and supplemented with 0.05 mg/l Kin and 0.3 mg/l IBA, in 16 h light/ 8 h dark photoperiod, root organogenesis was initiated.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior<br>Calotropis procera e Cryptostegia grandiflora sÃo plantas laticÃferas. Em seus fluidos laticÃferos, foram encontradas proteÃnas do tipo osmotina. A literatura reporta que osmotinas sÃo proteÃnas relacionadas com mecanismos de defesa vegetal em situaÃÃes de estresse biÃtico e/ou abiÃtico. Entretanto, ainda hà vÃrias inconsistÃncias nessa afirmaÃÃo. Nesse contexto, tÃcnicas in vitro de cultura de tecidos vegetais foram aplicadas como modelo para auxiliar na compreensÃo de como as cÃlulas de calos de C. procera e C. grandiflora respondem ao estresse salino, em termos bioquÃmicos, e se o nÃvel de transcritos para a osmotina seria aumentado em resposta à exposiÃÃo a NaCl. Para induÃÃo desse estresse, NaCl foi adicionado à formulaÃÃo nutritiva de Murashige e Skoog (MS), em concentraÃÃes crescentes (0, 20, 40, 60 e 80 mM). Os resultados mostram que os calos cultivados com NaCl a 80 mM tiveram o crescimento e o teor de umidade reduzidos, respectivamente, em 33% e 10%, em C. procera e de 83% e 39%, em C. grandiflora, em comparaÃÃo ao seu tratamento controle. Nessas mesmas condiÃÃes, foi observado um aumento nas concentraÃÃes dos Ãons Na+ e Cl- de, respectivamente, 98,9% e 98%, em C. procera, e de 98,8% e 96%, em C. grandiflora. Foi tambÃm observada diminuiÃÃo no teor de K+ nos calos tratados com NaCl a 80 mM. Essa reduÃÃo foi de 43%, em C. procera, e de 18% em C. grandiflora, quando comparado ao tratamento controle. Os calos tratados com NaCl a 80 mM, apresentaram uma tendÃncia de acÃmulo de prolina e aÃÃcares solÃveis, alcanÃando, respectivamente valores, 26% e 37% maiores em calos de C. procera, e 55,4% e 45% maiores, em calos de C. grandiflora, que aqueles em condiÃÃes controle. O aumento na atividade das enzimas que degradam H2O2 foi observado em calos de C. grandiflora submetidos a estresse salino, sugerindo um possÃvel dano oxidativo. Esse aumento foi de 73%, para a ascorbato peroxidase, e de 62% para a peroxidase do guaiacol, nos calos tratados com NaCl a 80 mM, em relaÃÃo ao controle. NÃo foi observada qualquer alteraÃÃo significante na atividade das enzimas do sistema antioxidativo em razÃo do estresse salino em calos de C. procera. Em relaÃÃo à transcriÃÃo da osmotina, foi avaliado o perfil de seus transcritos nos intervalos de tempo de 0, 2, 12, 24, 48 horas e de 4, 7, 14 e 28 dias sob estresse. Os transcritos de osmotina foram observados a partir de 12 horas de contato dos calos com NaCl a 80 mM, em ambas as espÃcies. Contudo, nos extratos proteicos dos calos de C. procera e C. grandiflora cultivados em condiÃÃes controle e de 80 mM de NaCl, nÃo foi detectada a presenÃa da proteÃna osmotina quando avaliado pelos ensaios de eletroforese, Dot blotting e espectrometria de massas. Assim, a avaliaÃÃo do estresse salino utilizando como modelo de estudo cÃlulas in vitro foi eficiente, fornecendo informaÃÃes do comportamento celular de duas espÃcies laticÃferas, mostrando suas alteraÃÃes fisiolÃgicas, bioquÃmicas e moleculares. Os resultados sugerem que o estresse salino favoreceu o aumento da transcriÃÃo do gene da osmotina em calos das duas espÃcies em estudo e permite propor uma possÃvel relaÃÃo das osmotinas dessas espÃcies com a tolerÃncia à salinidade. A falha em detectar as proteÃnas correspondentes aos genes propicia a concepÃÃo de vÃrias novas hipÃteses a serem validadas.<br>Calotropis procera e Cryptostegia grandiflora are laticiferous plants. It was found osmotin protein. The literature shows that the osmotinas are associated to plant defence mechanisms in situations of biotic or abiotic stress. However, there are still several inconsistencies in this hypothesis. In this context, it was used in vitro tissue culture techniques as model to assist in the understanding of how the C. procera and C. grandiflora callus cells respond to salt stress in biochemical terms, and whether the transcripts level for osmotin has raised in response to exposure to NaCl. It was added NaCl to the culture medium of Murashige e Skoog (MS) in increasing concentrations (0, 20, 40, 60 e 80 mM). The results show that callus treated with 80 mM NaCl have reduced the growth and the humidity percentage of respectively 33% and 10% in C. procera and 83% and 39% in C. grandiflora compared to the control treatment callus. Under the same conditions, it was seen an increase in ions concentrations of Na+ and Cl-, 98.9% and 98% in C. procera and 98.8% and 96% in C. grandiflora respectively. It was also seen a reduction in K+ level in callus treated with 80 mM NaCl, 43% in C. procera and 18% in C. Grandiflora, when compared to the control. The callus treated with 80 mM NaCl, showed a tendency of the proline accumulation and soluble sugars, increasing 26% and 37% in C. procera callus and 55.4% and 45% in C. grandiflora callus, respectively, when compared to control conditions. The increase in the activity of enzymes that break H2O2 has been observed in C. grandiflora callus under the salt stress, suggesting a possible oxidative damage, this increase was 73% in the ascorbate peroxidase activity and 62% in guaiacol peroxidase activity when compared to the activity of enzymes of the control callus with the treaty in 80 mM NaCl. None connection was seen between changes in the activity of the enzymes of the oxidative system and the salt stress in C. procera callus. It was evaluated the behaviour of osmotin in the osmotin transcription at 0, 2, 12, 24, 48 hours and at 4, 7, 14, 28 days of callus contact under stress. The osmotin transcripts were observed from 12 hours of contact of callus in 80mM NaCl in both species. However, it was not found osmotin by electrophoresis assays, dot blotting and mass spectrometry in the protein extracts of C. procera and C. grandiflora callus grown in control conditions and in 80 mM NaCl. Thus, the salt stress evaluation using in vitro cell model study was effective, providing cellular behaviour information for these two laticifers plants species showing their physiological, biochemical and molecular changes. The results suggest that the induced salt stress has favoured the increase of osmotin gene expression in both cases and suggests a possible relationship between osmotin of these species with the protection to salinity conditions. The failure to detect the proteins corresponding to genes provides the conception of several new hypotheses to be validated.

Axenic cultures of Peridermium pini have been established on modified Shenk &'38 Hilderbrandt's and Harvey &'38 Grasham's media from naturally infected cortex tissues and aeciospore-infected calluses of P. sylvestris and from aeciospores collected from NE Scotland and East Anglia. The cultures occasionally produced immature smooth-surfaced, binucleate spores. Actively growing cultures infected P. sylvestris calluses but not seedlings or trees. In the experiment of fungal nutrition, (NH4)2SO4 appeared essential, sucrose, D-glucose, raffinose and D-sorbitol supported good growth while D-xylose, cellobinose and L-arabinose did not. Opt. medium pH proved to be 5.0-6.0. Axenic cultures were also obtained from 30-40&'37 of single sporelings of some East Anglia spore sources but not from NE Scotland sporelings. When inoculated at a high density, however, all spore sources from both East Anglia and NE Scotland readily formed colonies. Colonies from East Anglia spores mostly appeared smooth at the surface and distinct around margin while those from NE Scotland sources had fluffy surface and irregularly extended periphery. Rapidly expanding hyphal layers developed from both of the colony forms 3 months after inoculation. Callus tissue cultures of P. sylvestris, P. nigra var. maritima and P. mugo vars mughus, rostrata and pumilio were infected by inoculation with aeciospores from NE Scotland. Infections were characterized by formation of aerial hyphae on the callus surface and intercellular hyphae and typical haustoria in the callus tissue. Hyphae from some of the infected calluses penetrated the medium. Seedlings of the pines as above were infected at their cotyledon stage by inoculation with NE Scotland spores. Infections resulted in swelling, death of the seedlings and formation of spermogonia after a year and aecia after two years. Infections of young seedlings of 7 seed sources of P. sylvestris and the UK were examined 6 weeks after inoculation. Discolouration and necrosis of cotyledons were not always related to stem infection.

碩士<br>國立臺灣大學<br>園藝學研究所<br>88<br>Callus of Doritaenopsis ( Dtps. ) Taisuco Ladylip was successfully induced from 26% of the protocorms cultured on 1/2 MS medium ( PC medium ) containing 5 mg/l 2,4-D and 5 mg/l TDZ in the dark condition. For the establishment of Dtps. suspension cells the calli were cultured in liquid medium supplemented with 2000 mg/l of PVP ( polyvinylpyrolidone ). PVP decreased cell necrosis by 83.3 % compared with the control. Suspension cells of Dtps. increased growth weekly by 50 %. Calli and suspension cells of Dtps. subcultured on G7M medium turned green and then produced PLBs in the light. Plantlets was successfully regenerated from calli under illumination for three months. The Agrobacterium-mediated transformation using calli of Phalaenopsis ( Phal. ) Taisuco Kaaladian and Dtps. was established based on the transient expression assay. Acetosyringone ( 100 µM ) could improve the transformation efficiency of Dtps. calli. Phal. calli co-cultivated with Agrobacterium in liquid medium for 2 days had 72.2% of transformation and the transformation efficiency of co-cultivation in liquid medium was higher than co-cultivation on solid medium. Agrobacterium—mediated transformation combined with sonication for 30 seconds using Phal. calli had 40.7% of transformation. GUS enzyme activity assay for the Agrobacterium-mediated transformation using calli of Dtps. confirmed that putative transformants can be selected with antibiotics for 5 months. These results showed the application of Agrobacterium-medium transformation on the gene transformation of Dtps. calli is practicable. Histochemical GUS activity was assayed on the leaves of putative Phal. transgenic plants derived from the introduction of pBI121、pBCH121-1 and pBACH121-1 into protocorms by bombardment-assisted Agrobacterium transformation or Agrobacterium co-cultivation. The expression pattern of GUS activity on the leaves could be classified into four categories by their location : ( 1 ) almost whole leaf； ( 2 ) leaf margin with randomized patch or leaf margin with vein； ( 3 ) randomized patch or randomized spot；( 4 ) no GUS activity. This result demonstrated the elementary evidence of transformation.

碩士<br>國立臺灣大學<br>園藝學研究所<br>87<br>Callus of Phalaenopsis were induced from seed-derived protocorms, leaves and root tips on 1/2 MS (Murashige and Skoog,1962) medium containing 0-1.0 mg/l N-phenyl-N''-1,2,3,-thiadiazol-5-yl urea (TDZ) and 0-10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). It was demonstrated that 2-month old protocorms were better than leaves and root tips for callus inducing. More calli were formed on 1/2 MS supplemented with 0.1-1.0 mg/l TDZ, and 10 mg/l 2,4-D was not suitable for callus induction. There were roots, white and yellow buds appeared while inducing callus. The calli could be maintained by subculturing in the basal medium added 0.5 mg/l TDZ and 0.5 mg/l 2,4-D. Favorable effects of callus proliferation were found in the medium supplemented with 2-3% sucrose or 150 ml/l coconut milk or 50 g/l banana. The calli could be maintaining for long term through month interval subculture. Protocorm-like bodies (PLBs) were formed and regenerated plants resulted from these calli on 1/2 MS medium alone or plus 0.1-1.0 mg/l TDZ. PLBs were also formed on the surface of the white and yellow buds. Plantlets were then potted on sphagum moss in greenhouse and grew well.

碩士<br>長庚大學<br>化工與材料工程研究所<br>92<br>The purpose of this study is to produce human serum albumin by transgenic rice callus. For the past two decades, many scientists used biotechnology to produce biopharmaceutical proteins in transgenic plants. Using plants as expression system for producing biopharmaceutical proteins from human or animals may avoid pollution of unknown viruses’ contamination by traditional extraction methods. In order to enlarge expression of recombinant human serum albumin from the αamy3 promoter in the rice Tainung 67（TNG67）, we carried out suspension culture and immobilization culture to produce human serum albumin. In manufacture, the amount of human serum albumin by suspension culture was larger than by immobilization culture. However, immobilization can protect cell from harm of shear force, and with better cell viability. On the part of immobilization by flask, most calli was immobilizated in the upper section of support. In order to amplify making use of support, we carried out two-way bubble cycle reactor to make calli distribute uniform. Make a comprehensive survey on the result between suspension culture and immobilization culture. Immobilization culture not only can reuse cell but also benefited continue manufacture.

碩士<br>南台科技大學<br>生物科技系<br>92<br>Calla lily (Zantedeschia spp.), a member of Arum family (Araceae), is a tuber flower crop originating from the southern regions of Africa. Two major production problems in calla lily industry are bacterial soft rot caused by Erwinia corotovora and viral disease infected either by cucumber mosaic virus (CMV) or by dasheen mosaic virus (DsMV). Therefore, disease resistance has been an important goal for calla lily breeding programs all over the world. Since hybridization incompatibility usually occurs in interspecific cross in Zantedeschia, it has been a difficulty in traditional breeding that trying to transfer disease tolerance characteristic from the white flower species Z. aethiopica to other species. Gene transformation, therefore, has been considered as a feasible way to circumvent this sexual reproduction barrier; and plantlet regeneration from transformed cell(s) is an essential step for this methodology. The purpose of this research is to investigate optimal conditions for callus induction and organ redifferentiation in calla lily, as well as to provide fundamental information for transgenic plant regeneration when using genetic engineering strategy for this crop improvement. For callus induction, explants excised from petioles of ‘Pacific Pink’ calla lily performed better than those from blades; and half-strength MS is better than full-strength, These is no significant difference between half-strength MS and B5 treatment. The highest percentage of callus induction (80 ± 3.9 %) was obtained on half-strength MS medium supplemented with 1 mg/ L 2,4-D and 2 mg/ L TDZ after dark incubation for two months. Most calli induced by 2,4-D and TDZ is white friable type. They could be maintained and proliferated either on the induction medium mentioned above or on half-strength MS medium supplemented with 2 mg/ L Picloram and 0.5 mg/ L. No organogenesis from this type of callus was observed, even though various treatments have been tried in this research. Most calli derived from petiole explants on half-strength MS medium with 2,4-D and BA showed yellow color and compact texture. The optimal combination of these two plant growth regulators for the callus induction was 1 mg/ L 2,4-D plus 1 mg/ L BA, though the percentage of callus induction only 53 ± 6.7 %. After subculture on half-strength MS medium with 2 mg/ L NAA and 0—0.5 mg/ L BA under light condition for two months, the yellow-compact calli redifferentiated into short-thick roots (diameter 1.5 ± 0.1 mm) with sparse root hairs. On the other hand, slender roots (diameter 0.74 ± 0.1 mm) with dense root hairs were formed if transferred this type of calli to the same medium with 0.05 mg/ L NAA, 1 mg/ L BA, and 0.5-2 mg/ L TDZ was utilized. Incorporation of 1 mM glutamine or 1000 mg/ L yeast extract in the rhizogenesis medium enhanced the number of root induction and their elongation. No shoot formation was found among different treatments, including combining different plant growth regulators, modifying molar concentration of NH4NO3 to KNO3 ratio (15.5: 29.5 and 20.6: 39.4) of half-strength MS basal medium, or adding reduced nitrogen compound (1000 mg/ L casein hydrolysate, peptone, or yeast extract) for two months in light condition. However, yellow-compact calli inoculated on a medium based on half-strength MS plus 0.5 mg/ L 2,4-D, 1 mg/ L BA, and 0.5% sucrose developed green spots, which may have the potential for shoot regeneration.

碩士<br>長庚大學<br>化工與材料工程研究所<br>93<br>Abstract Flavone Glycosides and Terpene Lactones are two main active components of Ginkgo Biloba Leaves. Terpene Lactones contain ginkgolides A, B, C, J, M (abbreviated as GA, GB, GC, GJ and GM) and Bilobalide. The curative effects are due to Ginkgolides. The Ginkgolides characterize in selectively inhibiting the platelet-activating factor (PAF) and posses neuro-protective effects. Especially GB has the strongest potency among these. The Ginkgo Biloba Leaves used in this experiment were provided by Tung Shih Forest District Office. The objective of the present investigation was to establish an efficient ginkgo biloba callus and to analyze the content of ginkgolide B. The suspension cell culture was established to produce ginkgo biloba cell and secondary metabolite. As the cultivation of plant texture requires a lower concentration of MS, 1/2 MS basal medium supplemented with vitamin C, NAA (α-naphthalene-acetic acid), natural supplement (Casein Hydrolysate), 3% sucrose, and 0.5mg/l kinetin was used for callus culture and suspension cell. The 1/2 MS basal medium supplemented with 2 mg/1 NAA, and 0.5 mg/1 kinetin was found to yield the highest induction rate of callus. The induction rate can reach 91.6%. In the 100 cell lines, the highest productivity of GB measured by HPLC was 0.0364% (w/w). The medium controlled at pH 5.7 + 0.1 and inoculated with 3.0 ml suspension cell (PCV) was found to proceed cell suspension culture significantly. The highest productivity of GB measured by HPLC was 0.2329% (w/w). In the suspension culture using stirred tank bioreactor, the primary volume of cell was 50ml, and increased to 92ml after 30 days; the growth rate was 84.0%.The productivity of GB measured by HPLC was 0.0353% (w/w).

博士<br>國立臺灣大學<br>園藝學研究所<br>95<br>Phalaenopsis orchid is one of the most important orchids grown for commercial production of cut flowers and potted plants. It has been chosen by the Council of Agriculture, Executive Yuan, as one of the four flagship speciality products for export. There is substantial interest in the production and improvement of these commercially valuable plants in Taiwan. However, orchids usually have long juvenile periods and reproductive cycles, which restrict genetic improvement using traditional sexual hybridization. Modern genetic engineering techniques provide an effective alternative. A callus-mediated plant regeneration protocol is a critical requirement as it allows exploitation of in vitro selection, somaclonal variation and engineering techniques aimed at genetic improvement of plants. Although several micropropagation methods have been developed for phalaenopsis using shoot tips, leaf segments, root tips and stalk cuttings, establishment of a proliferating callus culture and regeneration from callus is limited to phalaenopsis. The objectives of the project are to develop an efficient plant regeneration system from callus and to establish a platform of gene transformation for phalaenopsis breeding purpose, hoping to lay a solid foundation for export-oriented businesses.

碩士<br>國立高雄大學<br>生命科學系碩士班<br>102<br>The thesis attempts to establish a protocol for callus induction of Coffee arabica L. Firstly, the seeds were sown in in vitro conditions and seedlings obtained were used as donor plants for the subsequent tests. The explants were taken from these donor plants and placed on a modified Murashige and Skoog (MS) medium supplemented with combinations of plant growth regulators (PGRs) in the dark. The calli were proliferated more on the same medium, and their capacities of in vitro morphogenesis were subsequently evaluated. In the seeds germination experiment, 10 min sterilization time with 0.5% sodium hypochlorite gave the highest germination rate (63.16%). The root and leaf explants were taken from 12-week-old seedings and placed on a modified MS medium supplemented with combinations of auxins, (α-naphthaleneacetic acid : NAA or 2,4-dichlorophenoxyacetic acid : 2,4-D) and cytokinins (thidiazuron : TDZ or N6-benzyladenine : BA). In the control treatment, no response was found at all the explants. In contrast, 28 callus lines were obtained from other treatments. These calli could be subcultured on same medium and proliferated more with viable growth. Based on the proliferation rate and their morphology, the callus were classified into 4 types : 1) Yellowish compact callus with a highest proliferation rate; 2) Pale yellowish granular callus with high proliferation rate; 3) Translucent to whitish and friable callus with low proliferation rate; 4) Red to brown callus with lowest proliferation rate. Type 1 callus lines were suggested for inducing in vitro morphogenesis and subsequent plant regeneration.

碩士<br>國立東華大學<br>生物技術研究所<br>87<br>Ginkgo (Ginkgo biloba), a species of ginkgoaceae, was first recorded in Zea-Ung-Pen-Ts''ao-Ching. Its fruit, one of the Chinese medicinal herbs, was used mainly for treating cough. The purpose of this study was to establish the suspension culture of Ginkgo biloba for producing its secondary metabolites, such as ginkgolide B (GKB) and quercetin. Cell suspension culture was established from the leaves- derived callus. The culture conditions for both cell growth and secondary metabolites production were then evaluated. It was found that MS basal medium with the addition of 4 mg/l NAA, 0.5 mg/l kinetin and 3% sucrose was sufficient for the initial establishment of callus from leaves. The callus could be maintained for many generations in WPM basal medium with the addition of 3% sucrose, 4 mg/l NAA, 0.5 mg/l kinetin and 0.4% gelrite. Based on the experimental results, the optimal condition of cultivating Ginkgo biloba callus in suspension state, includes a WPM medium with the addition of 3% maltose or sucrose, 4 mg/l NAA, 0.5 mg/l kinetin, a pH of 5.2, an inoculum amount of 3 ml packed cell volume for a 25 ml culture and a shaking speed of 140 rpm. The cell growth rate showed no difference between the cells cultivated in dark or light conditions. The production of ginkgolide B from callus or suspension cells was found not growth-associated, and rapidly increased in the late log phase. The amounts of ginkgolide B of callus and of suspension cells could reach 65.16 mg/gDCW and 61.91 mg/gDCW, respectively. However, little quercetin was obtained in the callus and suspension cells growing under the conditions tested, and the production of quercetin was decreased with culture time.

碩士<br>國立高雄大學<br>生物科技研究所<br>100<br>This thesis attempts to develop a protocol for induction of callus from explants taken from seedlings of Bupleurum kaoi, and subsequently the in vitro morphogenesis of callus that was induced by different types and concentrations of plant growth regulators (PGRs) were evaluated. It was found that the treatment of 1.25% sodium hypochlorite for 20 min gave the highest germination rate (60%). Segments taken from roots and leaves of 8-week-old seedling were used as explants for callus induction. The results displayed that there were 17 callus lines could be obtained on culture media supplemented with different combinations of auxins (2,4-Dichlorophenoxyacetic acid, 2,4-D or 1-Naphthaleneacetic acid, NAA) with cytokinins (Thidiazuron, TDZ or N6-Benzylaminopurine, BA). These callus showed stable growth and could be subcultured on the same culture medium. They possessed obvious variations on their color and shape among these callus lines. By systematically selection on proliferation rate and callus morphology, yellow or whitish-yellow compact callus, comprising lines BKL-7L, BKL-8R, BKL-10R, BKL-13L, BKL-13R, BKLC-7R, BKLC-12R and BKLC-13L, having high proliferation rate were further selected for in vitro morphogenesis. Then, all of the callus which were cultured in light for induction gradually turned from yellow or whitish-yellow into green. Furthermore, root formations were found in callus lines BKL-8R, of BKL-10R, BKL13R,-7R BKLC, BKLC-12R and BKLC-13R. Additionally, it was found that roots could be induced from callus lines BKL-10R, BKL-13L, BKL-13R, BKLC-7R BKLC-12R and BKLC-13R on media supplemented with different combinations of BA and NAA. The results showed that line BKL-13L at 0.1 mg/L BA+10 mg/L NAA, BKLC-7R at 1 mg/L BA+1 mg/L NAA and BKLC-12R at 0.1 mg/L BA+10 mg/L NAA or at 1 mg/L BA+1 mg/L NAA had a 100% rate of root formation. Callus induced from different combinations of NAA and BA had ability to form roots during callus subculture, and more roots could be obtained when they were transferred onto different combinations of BA and NAA for induction. It was suggested that combinations of NAA and BA could promote root formation from callus cultures in Bupleurum kaoi. Seven callus lines, including BKL-7L, BKL-7R, BKL-12L, BKL-12R, BKLC-2R, BKLC-7R and BKLC-12R, were used to evaluate the ability of induction in vitro morphogenesis under different concentrations of TDZ. After eight weeks, except for line BKLC-2R, all of the callus lines which was cultured in light turned into green. Besides, no roots could be found in all of the treatments. The result showed that cell suspension culture was established from callus of Bupleurum kaoi, the average of monthly proliferation of callus lines BKL-13R in terms of using liquid media by omitting of gelling agent or D2 medium were higher than BKL-8R. The results suggested that callus line BKL-13R should be more conducive to cell suspension culture, transgenic and polyploid research of Bupleurum kaoi in the future.

碩士<br>長庚大學<br>化工與材料工程研究所<br>92<br>Ginkgo biloba is an ancient Chinese medicinal plant. The Ginkgo Biloba leaves contain mainly ginkgolide（ginkgolide A, B, C, J , and M, abbreviated as GA, GB, GC, GJ, and GM）are selective platelet-activating factor（PAF）antagonist and posses the neuro-protective effect. Especially, GB has the most strong potent among these terpenoid and is now used as the antagonist for the prevention and treatment of thrombus , illness of blood vessel of heart and brain arrhythmia , asthma ,bronchitis , senile dementia and allergic reaction, and is also widely used in pharmaceutical, health food industry and others. The objective of the present investigation was to establish an efficient ginkgo biloba callus and to analyze the content of ginkgolide B. The suspension cell culture was established to produce ginkgo biloba cell and secondary metabolite in large scale. The results obtained are summarized as follows. The MS basal medium supplemented with 2.0mg/l NAA 、0.5mg/l kinetin and 3% sucrose was found to yield the highest induction rate of callus. The 1/2MS basal medium supplemented with 3%sucrose 、2mg/l NAA 、0.5mg/l kinetin and 0.75% agar （pH5.7±0.1）incubated of culture in dark at 25±1℃ was found to be optimum for the proliferation of callus. The 1/2 MS basal medium supplemented with 2mg/l NAA 、0.5mg/l kinetin and 3% sucrose （pH5.7±0.1） inoculated with 3ml PCV/125ml initial cells shook with 100rpm was found to proceed cell suspension culture significantly. Moreover，the callus induced from the stem was found to produce the highest content of secondary metabolite ginkgolide B.

碩士<br>國立高雄大學<br>生物科技研究所<br>100<br>Roots of Panax ginseng were used as explants to culture on MS medium supplemented with different concentrations of plant growth regulators. After four weeks, the results showed that callus could be induced by 0.5–2 mg/L of 2,4-D + 0.1–0.5 mg/L of TDZ, but the root explants in PGR–free medium became browning. Callus cultured on MS medium with 2 mg/L 2,4-D + 0.5 mg/L TDZ had highest proliferation rate which had an average of 11.5 folds. We selected the callus which induced by the concentration of 0.5 mg/L 2,4-D + 0.1 mg/L TDZ and 2 mg/L 2,4-D + 0.5 mg/L TDZ with different ratio of BA and NAA medium for in vitro morphogenesis. Under the condition of the callus which induced by the concentration of 0.5 mg/L 2,4-D + 0.1 mg/L TDZ were cultured on MS medium with 0.1 mg/L BA + 10 mg/L NAA, the treatment could promote root formation and had an average of 3.4 roots. In addition, the callus treated with 2 mg/L 2,4-D + 0.5 mg/L TDZ were cultured on MS medium with all different ratio of BA and NAA did not possess the ability of root formation. The result showed that higher NAA/BA ratio favored to promote root induction. 0.5–100 mg/L of colchicines were used to induce polyploidy of callus, the test selected three callus lines comprising 0.5 mg/L 2,4-D + 0.1 mg/L TDZ、2 mg/L 2,4-D + 0.1 mg/L TDZ and 2 mg/L 2,4-D + 0.5 mg/L TDZ﹐respectively. The results showed that at the concentrations of 50 and 100 mg/L colchicine treatment were inhibited callus growth, but other concentrations(0,0.5,1,2,3,4,5 and 10 mg/L)were not affected. It was noteworthy to mention that the callus treated by different concentrations of colchicines became white and soft.

碩士<br>輔仁大學<br>生命科學系碩士班<br>105<br>Scutellaria indica (skullcap), a perennial Lamiaceae herb and a traditional medicinal plant used in East Asia,is genetically close to Scutellaria lateriflora (SL). SL is the American species of SI and is also a commonly used medicinal plant. According to the study of Brock et al. (2012), extracts of SL can relieve anxiety, calm the nervous, and improve the insomnia symptoms; it has been even made into beverages for sale. Although there have some reports about the flavonoids contents of Scutellaria indica, there is no tissue cultures of skullcap has been studied so far. Kumar et al. (2013) showed that flavonoids are directly associated with human health, for example, antioxidative activity, coronary heart disease prevention, anti-inflammatory, and hepatoprotective...etc. Some flavonoids even exhibit anticancer and potential antiviral activities. Because the growth of skullcap is restricted by certain environmental conditions, which make the large scale extraction of skullcap flavoniods become unreachable. Therefore, we aim to establish the skullcap callus culture system, and to compare the total flavonoids contents of callus with its plant counterpart. Our results show that: (1) the MG and NG media are good for callus induction than the others(2) The hormone constituents will affect callus morphology, e.g.callus induced from MG, MB, NG, and NB media were light yellow and loose. Nonetheless, callus induced from MH and NH media were brownish yellow and hard. (3)The optimal conditions for suspension culture are still in study. Addition of MES in suspension culture medium made callus more compact and reduced proliferation rate and browning condition. Addition of MES in suspension culture medium made callus more compact and reduce callus proliferation rate and turning brown. Addition of active charcoal in semi-solid medium did not improve callus proliferation. Total flavonoid contents of callus reduced to certain level in 1/4 MS media. (4)The total flavonoids conents of callus from MG3, NG1, NG3, NG4 media were approximatly equal to the control, i.e. leaves. (5) Infunctionl analysis, Leave, MG, and NG extracts do not showsignificantly effect on damaged N2a cells induced by 6-OHDA. In summary, MG and NG media are the best for callus induction and total flavonoids contents of callus. Among them, the callus from MG3, NG1, NG3, NG4 medium outperform the others and are comparable to the control. The effect of skullcap callus extracts on damaged N2a cells’ is still not clear. It need more study to define if skullcap can be a potential drug for neuron damage.

碩士<br>國立臺灣大學<br>森林環境暨資源學研究所<br>100<br>Two-year-old and four-year-old stem explants of Taiwania cryptomerioides were used to induce shoot multiplication that in vitro cultured in MMS medium containing 1 mg/L BA + 0.1 mg/L NAA + 0.1 mg/L KIN. Each stem of T. cyptomerioides could induce seven shoots. The experimental results indicated that MMS medium supplemented 0.05 mg/L NAA is a well medium for inducting Taiwania root. When root growth about 2 cm long, it was transferred to MMS medium without hormone. In addition, the callus inducing was performed by cutting 0.3 - 0.5 cm stem, shoot tip and leaf of two-year-old and four-year-old T. cryptimerioides. Among different hormone concentrations of callus induction that 3 mg/L 2, 4-D + 0.1 mg/L BA is optimal condition for inducing amount friable callus. To study the gene regulation of callus formation, the PCR-select cDNA subtraction hybridization technology was used. A dirigent like protein named TcDIR1 were cloned from callus in induction medium. There are 585 nucleotides (195 amino acids) with molecular weigh of 21.5 kDa pI 9.57. TcDIR1 expresed highest quality in 2 weeks using by reverse-transcription polymerase chain reaction (RT-PCR) analysis. It indicated that TcDIR1 might play an important role in callus formation. Furthermore, the Agrobacterium-mediated transformation conjugated vacuum infiltration to infect 2 month seedling of T. cryptomerioides. Cultivation in 3 mg/L 2, 4-D + 0.1 mg/L BA antibiotic MMS plate for two month. GUS enzyme activity was estimated to demonstrate the efficiency of genetic transformation.

碩士<br>國立宜蘭大學<br>園藝學系碩士班<br>105<br>There are more than ten thousand species of succulent plants in the world and all of the succulents belong to 50 families in botanical classification. In some families, such as Agavaceae, Aizoaceae, Aloaceae, Apocynaceae, Crassulaceae, Euphorbiaceae, most of their member species are succulents and common in the market. Traditionally they are propagated by seeding, cutting and division of the parent plants. However, the number of offspring is limited and the chances of success with these propagation methods are low due to difficulty in obtaining seeds, low germination rate and damages in cutting propagation. Therefore, in vitro propagation of succulent plants draws a lot of attention. The purpose of this study aims at using shoot apex of Sempervivum tectorum and cluster buds of Haworthia retusa to establish organogenesis clonal propagation system for these species. The results indicated that the basal parts of nodal segment of Sempervivum tectorum cultured in MS medium with different combinations of BA and NAA were successfully induced callus. Callus, under no influence of hormones, protruded shoot formation. The effects of different MS salts concentration, sucrose amount added and gelrite concentration on the bud proliferation were experimented. The medium contain with NAA 0.2 mg/ L; MS 2.2 g/L; 10 g/L sucrose; 5 g/L gelrite resulted in the best proliferation of shoots. MS medium supplemented with IBA 5 mg/L subsequently produced maximum roots number. In Haworthia retusa, after a series experiment, callus were obtained from shoot explant when cultured on MS medium supplemented with BA 2 mg/L and NAA 1 mg/ L. For shoot differentation we tested the effects of different combinations of hormones. Shoots formation was optimal on F1 medium supplemented with NAA 0.2 mg/ L. For shoot propagation it was found when cultured on MS medium supplemented with BA 1 mg/L and NAA 0.5 mg/L result in the best proliferation of buds. Rooting was achieved on MS medium with NAA 0.05 mg/L.This study establish complete regeneration system of Sempervivum tectorum and Haworthia retusa by tissue culture. Morphogenetic studies of callus differentiation, shoot and root regeneration were also analyzed.