Genetic transformation of Populus nigra X P. deltoides (black poplar, clone Gradizka)

Aim. To carry out genetic transformation of poplar Populus nigra x P. deltoides clone Gradizka with the model gene construct pCB002 carrying selective gene of kanamycin resistance and marker gene of β-glucuronidase. Methods. Genetic transformation was performed with the using leaf, stem and petiole poplar explants. Transformants were selected on the medium with kanamycin, and transgene was identified by polymerase chain reaction (PCR) and histochemical GUS assay. Results. Successful transformants selected on kanamycin media were confirmed by the presence of PCR-product for the gene nptII with the length 700 bp, and gus gene expression was also observed. Conclusions. Protocol for genetic transformation of P. nigra x P. deltoides clone Gradizka established here will be used for poplar genetic modification to create new clones with commercially important traits. Keywords: genetic transformation, Populus sp., microclonal propagation.

Transformation of Coffee arabica using chitinase gene and regeneration of planlets from transformed-zygotic embryos Transformasi Coffea arabica menggunakan gen kitinase dan regenerasi planlet dari embrio zigotik-transforman

RingkasanRekayasa genetika kopi arabika tahan penyakit cendawan dapat dilakukan dengan cara memasukkan gen kitinase (gen chi) ke dalam genom tanaman tersebut. Penelitian ini bertujuan untuk mengintroduksikan gen chi pada kopi arabika serta meregenerasi eksplan yang ditransformasi menjadi plantlet. Gen chi disubkloning dari pBS G11 ke dalam plasmid pCAMBIA2301. Melalui Agrobacterium tumefaciens, plasmid rekombinan pCAMBIA2301/35s-chi kemudian dimasukkan ke dalam eksplan daun dan embrio zigotik kopi arabika. Eksplan daun transforman ditumbuhkan pada media seleksi yang mengandung kanamisin untuk induksi kalus embriogenik . Beberapa kombinasi 2,4-D dan dicamba serta kinetin, BAP dan 2-iP diuji kemampuannya untuk menginduksi terbentuknya kalus embriogenik. Embrio zigotik transforman ditumbuhkan pada media MS modifikasi yang mengandung kanamisin. Hasil penelitian menunjukkan bahwa perbedaan tipe sitokinin dan kombinasinya dengan 2,4-D atau dicamba. menyebabkan terjadinya variasi persentase pembentukan kalus embriogenik tahan kanamisin. Penambahan 100 mg/L kanamisin dalam media seleksi cukup efektif untuk menghambat pertumbuhan eksplan daun nontransforman. Persentase tertinggi induksi kalus embriogenik pada eksplan daun non transforman maupun transforman diperoleh pada media yang mengandung 5 mM 2,4- D dengan 5 mM of kinetin atau 5 mg/L dicamba dengan 5 mM BAP. Sedangkan dalam media dengan penambahan 5 mM kinetin, 100 mg/L asam sitrat dan 100 ppm asam askorbat, jumlah eksplan yang membentuk kalus mencapai optimum pada konsentrasi 0 dan 1 ppm dicamba untuk eksplan transforman dan 10 mg/L dicamba untuk non transforman. Pada eksplan embrio zigotik transforman, peningkatan konsentrasi kanamisin dari 100 mg/L hingga 500 ppm menurunkan persentase pengecambahan embrio dari 80.5 % menjadi 49%, persentase perakaran, dari 34 % menjadi 16%, jumlah akar, panjang akar dan tinggi tunas dari 7 mm menjadi 4 mm. Pada semua perlakuan kanamisin, embrio zigotik non transforman tidak membentuk akar dan pada umur kultur yang sama tunas yang dihasilkan lebih pendek dibandingkan dengan embrio-zigotik transforman. Hasil tersebut membuktikan bahwa gen ketahanan terhadap kanamisin (NPTII) telah terinsersi dan terekspresi dengan baik pada plantlet kopi arabika yang berasal dari eksplan embrio-zigotik transforman. Karena gen chi dikonstruksi dalam satu vektor dengan NPTII, maka diharapkan gen tersebut juga telah terinsersi ke dalam genom tanaman kopi.SummaryGenetic engineering of arabica coffee resistant to fungal diseases might be done by introducing a chitinase-encoding gene (chi) into genome of this plant. This research was aimed to introduce chi construct into arabica coffee and regenerate plantlets from the transformed explants. The chi gene was previously subcloned from pBS G11 into pCAMBIA2301 plasmid. With Agrobacterium tumefaciens,the recombinant plasmid pCAMBIA2301/35s-chi was then introduced into leaf and zygotic embryos explants of arabica coffee. The transformed leaf explants were cultured on the selection media containing kanamycin in the presence of several combinations of 2,4-D and dicamba with kinetin, BAP and 2-iP to induce the formation of embryogenic callus. The transformed zygotic embryos were cultured on the media of modified MS containing kanamycin. The results showed that the several types of cytokinin used in combination with 2,4-D or dicamba caused the percentage of kanamycin resistant-embryogenic calli was varied. The addition of 100 mg/L kanamycin in the selection media was effective for inhibiting the growth of untransformed explants. Among the several combinations of auxin and cytokinin tested, the highest percentage of embryogenisis for untransformed and transformed leaf explants were achieved on the media containing 5 mM 2,4-D and 5 mM kinetin or 5 mg/L dicamba and 5 mM BAP. However in the presence of 5 mM kinetin together with antioxidants of 100 mg/L citric acid and 100 mg/L ascorbic acid, the explants calluses was optimum at 0 - 1 mg/L dicamba for transformed explants and 10 mg/L dicamba for untransformed explants. In the explants of transformed-zygotic embryos, increasing kanamycin from 100 mg/L up to 500 mg/L decreases the percentage of embryo germination from 80.5 % to 49%, rooted-shoots from 34 % to 16%, number of roots, root length and shoot length from 7 mm to 4 mm. At all the kanamycin treatments, root was not developed from the untransformed-zygotic embryos and the lenght of shoots were shorter compared to the transformed-zygotic embryos. This result demonstrates that the kanamycin-resistant gene (NPTII) has been inserted and well expressed in the plantlets of arabica coffee derived from transformed-zygotic embryos. Since the chi gene was constructed in one vector with NPTII, this gene might also been inserted in the genome of coffee.

Transformation of Coffee arabica using chitinase gene and regeneration of planlets from transformed-zygotic embryos Transformasi Coffea arabica menggunakan gen kitinase dan regenerasi planlet dari embrio zigotik-transforman

RingkasanRekayasa genetika kopi arabika tahan penyakit cendawan dapat dilakukan dengan cara memasukkan gen kitinase (gen chi) ke dalam genom tanaman tersebut. Penelitian ini bertujuan untuk mengintroduksikan gen chi pada kopi arabika serta meregenerasi eksplan yang ditransformasi menjadi plantlet. Gen chi disubkloning dari pBS G11 ke dalam plasmid pCAMBIA2301. Melalui Agrobacterium tumefaciens, plasmid rekombinan pCAMBIA2301/35s-chi kemudian dimasukkan ke dalam eksplan daun dan embrio zigotik kopi arabika. Eksplan daun transforman ditumbuhkan pada media seleksi yang mengandung kanamisin untuk induksi kalus embriogenik . Beberapa kombinasi 2,4-D dan dicamba serta kinetin, BAP dan 2-iP diuji kemampuannya untuk menginduksi terbentuknya kalus embriogenik. Embrio zigotik transforman ditumbuhkan pada media MS modifikasi yang mengandung kanamisin. Hasil penelitian menunjukkan bahwa perbedaan tipe sitokinin dan kombinasinya dengan 2,4-D atau dicamba. menyebabkan terjadinya variasi persentase pembentukan kalus embriogenik tahan kanamisin. Penambahan 100 mg/L kanamisin dalam media seleksi cukup efektif untuk menghambat pertumbuhan eksplan daun nontransforman. Persentase tertinggi induksi kalus embriogenik pada eksplan daun non transforman maupun transforman diperoleh pada media yang mengandung 5 mM 2,4- D dengan 5 mM of kinetin atau 5 mg/L dicamba dengan 5 mM BAP. Sedangkan dalam media dengan penambahan 5 mM kinetin, 100 mg/L asam sitrat dan 100 ppm asam askorbat, jumlah eksplan yang membentuk kalus mencapai optimum pada konsentrasi 0 dan 1 ppm dicamba untuk eksplan transforman dan 10 mg/L dicamba untuk non transforman. Pada eksplan embrio zigotik transforman, peningkatan konsentrasi kanamisin dari 100 mg/L hingga 500 ppm menurunkan persentase pengecambahan embrio dari 80.5 % menjadi 49%, persentase perakaran, dari 34 % menjadi 16%, jumlah akar, panjang akar dan tinggi tunas dari 7 mm menjadi 4 mm. Pada semua perlakuan kanamisin, embrio zigotik non transforman tidak membentuk akar dan pada umur kultur yang sama tunas yang dihasilkan lebih pendek dibandingkan dengan embrio-zigotik transforman. Hasil tersebut membuktikan bahwa gen ketahanan terhadap kanamisin (NPTII) telah terinsersi dan terekspresi dengan baik pada plantlet kopi arabika yang berasal dari eksplan embrio-zigotik transforman. Karena gen chi dikonstruksi dalam satu vektor dengan NPTII, maka diharapkan gen tersebut juga telah terinsersi ke dalam genom tanaman kopi.SummaryGenetic engineering of arabica coffee resistant to fungal diseases might be done by introducing a chitinase-encoding gene (chi) into genome of this plant. This research was aimed to introduce chi construct into arabica coffee and regenerate plantlets from the transformed explants. The chi gene was previously subcloned from pBS G11 into pCAMBIA2301 plasmid. With Agrobacterium tumefaciens,the recombinant plasmid pCAMBIA2301/35s-chi was then introduced into leaf and zygotic embryos explants of arabica coffee. The transformed leaf explants were cultured on the selection media containing kanamycin in the presence of several combinations of 2,4-D and dicamba with kinetin, BAP and 2-iP to induce the formation of embryogenic callus. The transformed zygotic embryos were cultured on the media of modified MS containing kanamycin. The results showed that the several types of cytokinin used in combination with 2,4-D or dicamba caused the percentage of kanamycin resistant-embryogenic calli was varied. The addition of 100 mg/L kanamycin in the selection media was effective for inhibiting the growth of untransformed explants. Among the several combinations of auxin and cytokinin tested, the highest percentage of embryogenisis for untransformed and transformed leaf explants were achieved on the media containing 5 mM 2,4-D and 5 mM kinetin or 5 mg/L dicamba and 5 mM BAP. However in the presence of 5 mM kinetin together with antioxidants of 100 mg/L citric acid and 100 mg/L ascorbic acid, the explants calluses was optimum at 0 - 1 mg/L dicamba for transformed explants and 10 mg/L dicamba for untransformed explants. In the explants of transformed-zygotic embryos, increasing kanamycin from 100 mg/L up to 500 mg/L decreases the percentage of embryo germination from 80.5 % to 49%, rooted-shoots from 34 % to 16%, number of roots, root length and shoot length from 7 mm to 4 mm. At all the kanamycin treatments, root was not developed from the untransformed-zygotic embryos and the lenght of shoots were shorter compared to the transformed-zygotic embryos. This result demonstrates that the kanamycin-resistant gene (NPTII) has been inserted and well expressed in the plantlets of arabica coffee derived from transformed-zygotic embryos. Since the chi gene was constructed in one vector with NPTII, this gene might also been inserted in the genome of coffee.

Eficiência de transformação genética de citrange 'carrizo' com duas construções gênicas

Transformação genética é considerada uma importante ferramenta auxiliar no melhoramento genético de plantas cítricas. Entretanto, a eficiência de transformação pode variar em função de diversos fatores, incluindo a própria construção gênica utilizada. Este trabalho buscou avaliar a eficiência de transformação genética de plantas de citrange 'Carrizo' [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck] com duas construções gênicas diferentes contendo o gene uidA (GUS) sob o controle dos promotores Arabidopsis thaliana phloem protein 2 (AtPhP2) e Arabidopsis thaliana sucrose transporter 2 (AtSuT2). Segmentos de epicótilo de plântulas germinadas in vitro foram utilizados como explantes. O gene nptII, que confere resistência ao antibiótico canamicina, foi utilizado nas construções gênicas como agente de seleção para regeneração de plantas transgênicas. O ensaio histoquímico com X-GLUC foi realizado em todas as brotações regeneradas para verificar a expressão do gene uidA. Dos 4.790 segmentos de epicótilo utilizados, registrou-se a regeneração de 366 brotações com reação positiva no ensaio histoquímico, as quais foram enxertadas em porta-enxertos cultivados in vitro. Cinco dessas brotações, de cada construção gênica, foram selecionadas para análise da PCR, com primers específicos para amplificação da sequência do gene uidA. A inserção do transgene foi confirmada por PCR em todas as brotações selecionadas. A eficiência de transformação e o número de brotos escapes, avaliada pelo teste histoquímico, variaram em função das construções gênicas utilizadas.

Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression

This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression. In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%) was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and, depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein.

ORGANOGÊNESE E TRANSFORMAÇÃO GENÉTICA DE MARACUJÁ AMARELO (Passiflora edulis f. flavicarpa DEG.) COM OS GENES CMe-ACO1 AS E nptII VIA Agrobacterium tumefaciens

O Brasil é o principal produtor mundial de maracujá – amarelo (Passiflora edulis f. flavicarpa Deg.). Seus frutos, de grande valor nutricional e farmacêutico são comercializados in natura e industrializados, garantindo o abastecimento do mercado interno e exportação. Contudo, seus frutos perdem grande quantidade de água durante o processo de maturação, conferindo à eles um aspecto de murcha em poucos dias, além de aumentar a fragilidade durante o transporte. Assim, o presente trabalho teve como principal objetivo, introduzir em tecidos desta espécie o gene que codifica para a enzima ACCO, em orientação antisense, via Agrobacterium tumefaciens. Esta enzima é responsável pela conversão do ácido 1-carboxílico -1 - aminociclopropano (ACC) em etileno, promovendo a maturação dos frutos. Como controle do processo de transformação, foi utilizado o gene nptII, que confere resistência ao antibiótico canamicina e que foi introduzido com sucesso nesta espécie em trabalhos anteriores. A transformação genética só é obtida com sucesso, se os explantes potencialmente transgênicos adquirirem aptidão para regenerar uma nova planta. Neste sentido, dois experimentos de organogênese foram realizados. Neles avaliou-se o efeito da idade fisiológica dos explantes (jovem e adulto); da posição de cultivo dos explantes (abaxial e adaxial) e o efeito da citocinina BAP: 1,0; 1,5 e 2,0 mg.L-1 na porcentagem de explantes com gemas, no número médio de gemas por explante, número médio de folhas por explante, comprimento médio das folhas, comprimento da haste principal e porcentagem de explantes que formaram calos. Os experimentos foram realizados sob delineamento experimental inteiramente casualizado, com quatro repetições de cada tratamento e dez explantes por parcela. A concentração 1,5 mg.L-1 de BAP promoveu os melhores resultados para todas as variáveis avaliadas, em ambas as posições de cultivo do explante e para tecidos jovens e adultos. Foi observada formação excessiva de calos na concentração de 2,0 mg.L-1 de BAP. Explantes foliares jovens, oriundos de sementes, regeneraram gemas adventícias em 50,22% dos casos após 30 dias de cultivo, em ambas as posições do explante no meio de cultura. Já os genótipos IAPAR 309, 156 x 123 e 352 (material adulto), apresentaram maior porcentagem de explantes regenerados quando estes foram cultivados na posição adaxial. O alongamento e enraizamento dos brotos foram obtidos em ambos os tratamentos: MS e MS/2 + 1 mg.L-1 de GA3. No entanto, o segundo tratamento resultou em maior comprimento de haste (3,73 cm), número médio de raízes por plântula (2,08) e comprimento de raízes (6,64 cm) após 60 dias de cultivo. Nos ensaios com Agrobacterium, apenas uma planta de seis regeneradas e enraizadas em meio seletivo apresentou o gene antisense da ACCO, com uma eficiência de transformação de 1,43%. Para o gene nptII esta eficiência foi de 4,3%. Estes resultados foram confirmados pela técnica de PCR. Uma análise colorimétrica de frutos comerciais foi realizada e permitiu a classificação destes frutos em três grupos: predominantemente verde, predominantemente colorido e totalmente colorido. A produção de etileno e atividade enzimática da ACCO foi avaliada, mostrando que o maracujá – amarelo é produtor intermediário de etileno e que a atividade enzimática da ACCO é limitada, necessitando de cofatores para sua atividade máxima.