Marguerite Quoirin

Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis

Breeding methods based on genetic transformation techniques need to be implemented for Eucalyptus camaldulensis to shorten the long breeding cycles and avoid manipulation of adult trees; that requires the development of plant regeneration protocols enabling development of plants from transformed tissues. The present work aimed to optimise the regeneration process already established for the species. Cotyledonary leaves of E. camaldulensis were cultured in MS medium supplemented with naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) combinations. The most efficient treatment for bud indirect regeneration (2.7 µmol L-1 NAA and 4.44 µmol L-1 BAP) was used for further experiments. When explants were kept in the dark during the first 30 days, the percentage of explants forming calluses increased and explant necrosis was reduced in comparison with light-cultured explants. Mineral medium modifications were compared and half-strength MS mineral medium turned out to be as efficient as full-strength medium, producing 54% and 47% of explants with buds, respectively. For shoot elongation, MS medium with half-strength nitrate and ammonium salts, and 0.2% activated charcoal yielded rooted shoots 1 to 8 cm high after one month. The procedure is an efficient protocol for E. camadulensis plant regeneration, reducing the stages necessary for the obtention of complete plants.

Organogenesis and Agrobacterium tumefaciens -mediated transformation of Eucalyptus saligna with P5CS gene

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.

Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis dehn and histological study of organogenesis in vitro

The present work aimed at regenerating plants of Eucalyptus camaldulensis from the cotyledonary explants and describing the anatomy of the tissues during callogenesis and organogenesis processes, in order to determine the origin of the buds. The cotyledonary leaves of E. camaldulensis were cultured in Murashige and Skoog (MS), WPM and JADS media supplemented with 2.7 µM NAA and 4.44 µM BAP. The best results for bud regeneration were obtained on MS and WPM media (57.5 and 55% of calluses formed buds, respectively). Shoot elongation and rooting (80%) were obtained on MS/2 medium (with half-strength salt concentration) with 0.2% activated charcoal. Acclimatization was performed in the growth chamber for 48 h and then the plants were transferred to a soil:vermiculite mixture and cultured in a greenhouse. Histological studies revealed that the callogenesis initiated in palisade parenchyma cells and that the adventitious buds were formed from the calluses, indicating indirect organogenesis.

Multiplication of juvenile black wattle by microcuttings

The influence of mineral formulation, growth regulators and activated charcoal on micropropagation of juvenile black wattle (Acacia mearnsii) was studied. Nodal segments of one-month-old seedlings were used as starting material. After 30 to 45 days, they developed into shoots, which were divided into microcuttings and transferred to fresh media. The addition of 2 g l−1 of activated charcoal to basal medium (3/4 strength MS salts and MS vitamins) improved the multiplication phase, reducing leaf chlorosis and raising the percentage of elongated shoots. The multiplication rate varied between 1.7 and 3 every month. Rooting percentage too was higher in the presence of charcoal, even when auxin was not added to the culture medium. The addition of 8.87 μM of benzyladenine and/or 1.44 or 2.88 μM of gibberellic acid did not influence significantly the multiplication, nor modifications in the FeSO4, MnSO4, CaCl2 content of the medium. The system described allows the multiplication, elongation and rooting of black wattle in one step.

IN VITRO MULTIPLICATION OF Swietenia macrophylla KING (MELIACEAE) FROM JUVENILE SHOOTS

Big-leaf mahogany (Swietenia macrophylla King) is an important species for timber production that is considered the most valuable in the world. For this reason its exploitation is indiscriminate and leads this species to the risk of extinction. Moreover, mahogany is difficult to regenerate naturally and, when used in reforestation programs, plants are severely damaged by the shoot-borer (Hypsipyla grandella Zellar). This work aimed at developing the multiplication stage of micropropagation of Swietenia macrophylla King using juvenile material. After desinfestation, seeds were germinated in MS solid culture medium. Shoot formation from seeds occurred during five months, giving 5.54 nodal segments per seed. These explants were excised, each containing one axillary bud, and transferred on multiplication media. Four experiments with cytokinins were conducted, using media supplemented with 6-benzylaminopurine (BAP) (2.5 to 50.0 µM), 2isopentenyladenine (2-iP) (0; 1.1 to 8.8 µM), combinations of BAP (0; 2.5 to 50.0 µM) and 2-iP (2.2 µM). For the first treatments the basal culture medium was MS medium and in the last one MS and QL media were used in separate experiments. When BAP was tested alone, the maximum point of multiplication rate average was obtained on medium containing 23.61 µM, while 2-iP did not induce bud multiplication. On QL culture medium supplemented with the combinations of BAP (0; 2.5; 5.0; 10.0 e 20.0 µM ) and 2-iP (2.2 µM), there was no multiplication. The maximum point of multiplication rate average was 5.7 µM, obtained when the MS culture medium was supplemented with 18.51 µM BAP and 2.2 µM 2-iP.

Organogenesis and transient genetic transformation of the hybrid Eucalyptus grandis × Eucalyptus urophylla

The hybrid Eucalyptus grandis x Eucalyptus urophylla presents high levels of productivity and potential for use in paper, cellulose and fiber industries. The bud organogenesis from leaf explants of two clones of E. grandis × E. urophylla was studied in order to verify the effect of several factors: subculture duration on multiplication medium, type of explants, entire and half leaves: basal and apical portions, and duration of the culture on a regeneration medium. Differences in organogenic capacity of the two clones tested were observed. The explant most recommended for organogenesis is the basal section of the leaf collected from shoot clusters subcultured every 17 days. Moreover, the leaf explants must be transferred to a fresh bud induction medium every five days. This study also aimed at evaluating factors affecting the genetic transformation of leaf explants with the uidA gene, via co-cultivation with Agrobacterium tumefaciens, such as the pre-culture of the explants on a specific medium, the duration of their co-culture with the bacteria and the addition of acetosyringone to the culture media. The best conditions for the expression of the uidA gene were two days of pre-culture of the leaf tissues, three days of co-culture with the bacteria and the addition of acetosyringone in pre- and co-culture media.

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

Blends of Agar/Galactomannan for Marubakaido Apple Rootstock Shoot Proliferation

Galactomannans (GMs) extracted from seeds of a native Brazilian specie designated Cassia fastuosa (cassia) and from Cyamopsis tetragonolobus (guar gum - a commercial GM) were mixed with agar, in the proportion of 3/3 g.L -1 (w/w), and used as a gelling agent in Marubakaido apple rootstock (Malus prunifolia Borkh) micropropagation. The rheological measurements of the gels showed a good interaction between both hidrocolloids. The micropropagation results on these media were compared with behaviour of standard medium containing agar only (6 g.L -1 ). Results from in vitro experiments indicated enhanced proliferation of apple shoots and also the occurrence of hyperhydric shoots was lower in the modified gel-medium.

USE OF SUGARCANE BAGASSE AS AN ALTERNATIVE LOW-COST SUPPORT MATERIAL DURING THE ROOTING STAGE OF APPLE MICROPROPAGATION

SummaryStudies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.

Calogênese e rizogênese em explantes de mogno ( Swietenia macrophylla King) cultivados in vitro.

The indiscriminate exploitation of tropical trees in a search for economically valuable species leads to the risk of extinction of several species. This is the case of mahogany (Swietenia macrophylla King) in Brazil. The establishment of a method of direct or indirect bud regeneration could help to produce a great number of plantlets and could constitute an alternative to sexual propagation. The latter is limited by the fact that mahogany seeds lose their germinative power soon after harvest. In this work, two kinds of explants were used: leaf and root fragments from in vitro cultured plants. After disinfection, the explants were cultured in petri dishes containing modified Murashige and Skoog (1962) culture medium, with three-quarters of salt concentration, vitamins, 30 g.L -1 sucrose and 7 g.L -1 agar. The combinations of growth substances were: naphthaleneacetic acid (ANA 0.11 μM and 0.54 μM) and one type of cytokinin, kinetin (CIN 1.2 μM, 2.3 μM, 4.7 μM and 9.3 μM), 6-benzylaminopurine (BA 2.2 μM, 4.4 μM and 8.8 μM) or 2-isopentenyladenine (2-iP 2.5 μM). The variables were the concentration and combinations of the growth regulators and the explant origin.The cultures were evaluated every 30 days, the number of explants forming calluses or roots was recorded and the callus consistency was observed. Calluses were formed in both kinds of explants. In leaf explants, 90% of explants formed callus when culture medium contained 4.4 μM BA with 0.54 μM ANA and 8.9 μM BA with 0.11 or 0.54 μM ANA. For root explants, the combination that gave the highest number of calluses was 2.2 μM BA and 0.54 μM ANA and 55% of them formed callus. Adventitious roots were regenerated from leaf calluses or directly from leaf lamina cultured in media containing CIN and ANA.