Luciana Lopes Fortes Ribas

Micropropagação de Aspidosperma polyneuron (peroba-rosa) a partir de segmentos nodais de mudas juvenis

The objective of the present work was to establish a micropropagation protocol of Aspidosperma polyneuron from juvenile material. Apical shoots from two years old seedlings were collected in a greenhouse and sterilised with NaOCl or HgCl2 to establish aseptic cultures. Multiple shoots induction was evaluated in WPM medium, supplemented with BAP, ZEA or KIN (2.2 - 8.8 µM) in initial culture and two subsequent subcultures. The elongation of shoots was tested with growth regulators combinations: 2.25 µM of BAP, ZEA or KIN with 1.25 µM of IBA. IBA treatments (2.5; 5.0 and 10 mM) were tested with 5 and 15 minutes to induce roots. Plantlets were planted in a greenhouse. Efficient apical shoots sterilization was achieved with NaOCl (0,25% - 10 minutes) or HgCl2 (0.05%-10 minutes); survival rates were 72.89% and 84,10%, respectively. Apical shoots induced 4-5 axillary buds in WPM culture medium, containing ZEA or BAP (4.4 - 8.8 µM) following two subcultures. Reduced concentrations of ZEA or BAP (2.25 µM), combined with IBA (1.25) produced elongated shoots. IBA treatment (10 mM) during 15 minutes induced higher rooting percentages (80%). Plantlets planted in a greenhouse showed higher survival rates (90%).

Micropropagação de Cabralea canjerana

n Cabralea canjerana (Vell.) Mart. (Meliaceae) (icanjaranai) is a native tree of economic importance in Brazil. The storage of seeds is of short duration and it is therefore necessary to establish a protocol for micropropagation of this species. In this work, multiplication experiments were carried out using nodal segments, excised from in vitro germinated plants. The segments were inoculated in MS or WPM culture medium, supplemented with 6-benzylaminopurine (BAP) and/or 2-isopentenyladenine (2-iP) at 2.5 or 5 µM. Micro-cuttings were taken from new shoots developed from the seeds and used in a rooting experiment using a culture medium with half-strength MS medium (MS/2) supplemented with indolbutyric acid (IBA) (0, 2.5 and 5 µM). After 7 days in this medium, they were transferred to MS/2 medium without auxin under light. During the multiplication phase, the MS culture medium was more suitable for the multiplication of C. canjerana than WPM medium. The nodal segments cultured in the presence of 2.5 µM BAP showed the best result, with a multiplication rate of 1.77 per month on MS medium. The rooting of the microcuttings was 87.5% when they were kept in the presence of 5 µM IBA for 7 days. An acclimatization rate of 90% was achieved after 30 days in the greenhouse. In conclusion, the micropropagation of C. canjerana from nodal segments of plantlets is possible for this species.

Anatomia comparada das folhas e raízes de Cymbidium Hort. (Orchidaceae) cultivadas ex vitro e in vitro

ABSTRACT – (Comparative leaf and root anatomy of ex vitro and in vitro cultured Cymbidium Hort. plants). During in vitro cultureplants are kept in an atmosphere with high relative humidity , low light intensity and reduced gas exchange, resulting in low transpirationrates. Therefore, when these plants are exposed to ex vitro conditions, they suffer stress, which can induce mortality. The purpose ofthis study was to compare the anatomical structure of Cymbidium ‘Joy Polis’ plants from ex vitro (mother plant and acclimatized plants)and in vitro cultures and to verify if the anatomical structure of in vitro cultured plants affects acclimatization. The ex vitro plants werekept in a greenhouse in pots containing a mixture of coconut-fiber powder and coconut fiber . The in vitr o plants were kept in MS culturemedium. For the qualitative anatomical analysis, samples of leaves and roots were collected from ex vitro and in vitro plants. Theacclimatized plants presented morphological and anatomical structure similar to the mother plant. The anatomical structure of

INFLUENCE OF CULTURE MEDIUM, EXPLANT LENGTH AND GENOTYPE ON MICROPROPAGATION OF Pinus taeda L.

This work aimed to establish a micropropagation protocol for Pinus taeda L. Apical shoots from 5-day seedlings, of different genotypes (F27, B05 and PC), were cultured on WV5 medium supplemented with 44 µM 6-benzylaminopurine (BAP) and 0.05 µM α-naphtaleneacetic acid (NAA), for 14 days, followed by subcultures on growth regulator-free medium. Explants were sectioned into apical shoots and nodal segments for multiplication. Different lengths of explants, BAP concentrations and cultivation periods were tested. Rooting was induced on WV5/2, WV3/2, GDm/2 and agar-water culture media supplemented with 2.68 µM NAA and 0.44 µM BAP for nine days, followed by a 4-week subculture on growth regulator-free medium. It was verified that genotype influenced shoot formation and rooting. The length of 1.0 cm is recommended for nodal segment explants to obtain a high number of axillary shoots. For apical shoots, 0.5 cm explant and WV5 medium formulation allowed the best results for elongation. The best period of subculture was eight weeks, both for nodal segments and for apical shoots. The higher percentage of nodal segments with shoots (99.2%) and the higher average number of shoots per explant (4.0) were obtained with F27 genotype in medium culture containing 2.5 µM BAP. For apical shoots, the best result for elongation was observed for the shorter explants (0.5 cm) on WV5 culture medium (218.3%). The maintenance of in vitro clonal micro garden of vigorous shoots was obtained for two years of 8-week subcultures on WV5 culture medium. The best rooting rate (55.6%) was obtained when shoots were inoculated in agar-water medium with 2.68 μM NAA and 0.44 μM BAP for nine days. Plantlets were successfully acclimatized (85% of survival), so a micropropagation protocol was established.

Germinação in vitro de eixos embrionários zigóticos de imbuia (ocotea porosa (NEES EX MARTIUS) Liberato Barroso)

Imbuia has recalcitrant seeds with strong tegumental dormancy, irregular and low germination, which impair natural propagation. The objective of this study was to evaluate the in vitro germination of imbuia embryonic axes by testing the effect of different concentrations of sucrose, activated charcoal and salt formulations in order to accelerate the production of plants. Zygotic embryonic axes were inoculated in MS/2 culture medium with different concentrations of sucrose (15, 30, 60 or 90 g L -1 ), of activated charcoal (0, 1, 2 or 3 gL -1 ) and in different salt formulations (MS, WPM, MS/2 or WPM/2). The results indicated that the addition of 15 g L -1 sucrose was not enough to promote germination of embryonic axes and the other concentrations promoted between 45.5 and 59.4% germination. The best results were observed with MS and MS/2 salt formulations obtained 67.7 and 74.2% of germination respectively. The addition of activated charcoal to the media favored the in vitro germination of zygotic embryonic axes, while oxidation of the embryo tissues occurred in media without activated charcoal and germination did not happen. The in vitro germination of imbuia can be achieved with the MS/2 salt formulation supplemented with 30 g L -1 sucrose and 1 g L -1 of activated charcoal.

Micropropagation of Pinus taeda L. via axillary buds

Pinus taeda stands for productivity and quality of its timber [1]. Researches using biotechnology are of great importance and have been applied to the improvement of its timber and plantation [2]. The main method of Pinus propagation is by seeds, once the minicuttings depends on the season of the year or depends of juvenile material [3-5]. Thus, researches on micropropagation of Pinus taeda are currently a priority in Brazil [6]. Micropropagation is the best method for mass production of superior genotypes and represents a strategy for tree improvement and capture of genetic gains [7]. Studies on Pinus taeda micropropagation by axillary bud proliferation are quite few. The purpose of this study was to develop a protocol for micropropagation of this species from juvenile material.

SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germination and that makes its natural propagation difficult. The aim of this study was to establish a protocol of regeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculated on WPM culture medium supplemented with 2.4-D (200 µM) combined or not with hydrolyzed casein or glutamine (0.5 or 1 g l-1), during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D (22.62 µM) combined with 2-iP (2.46 µM) followed by transfer to culture medium with hydrolyzed casein or glutamine (1 g l-1) during 90 days. The maturation of somatic embryos was tested in culture medium containing NAA (0.5 μM) and 2-iP (5; 10 and 20 μM). The highest percentage of somatic embryos induction (8.3%) was observed in WPM culture medium containing 200 µM 2.4-D and 1 g L-1 hydrolyzed casein and the development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promoted in WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containing hydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. During the maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages. The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histological studies.