Optimization of a protocol for the micropropagation of pineapple

The present work aimed at maximizing the number of plantlets obtained by the micropropagation of pineapple (Ananas comosus (L.) Merrill) cv. Pérola. Changes in benzylaminopurine (BAP) concentration, type of medium (liquid or solidified) and the type of explant in the proliferation phase were evaluated. Slips were used as the explant source, which consisted of axillary buds obtained after careful excision of the leaves. A Sterilization was done in the hood with ethanol (70%), for three minutes, followed by calcium hypochlorite (2%), for fifteen minutes, and three washes in sterile water. The explants were introduced in MS medium supplemented with 2mg L-1 BAP and maintained in a growth room at a 16h photoperiod (40 mmol.m-2.s-1), 27 ± 2ºC. After eight weeks, cultures were subcultured for multiplication in MS medium. The following treatments were tested: liquid x solidified medium with different BAP concentrations (0.0, 1.5 or 3.0 mg L-1), and the longitudinal cut, or not, of the shoot bud used as explant. The results showed that liquid medium supplemented with BAP at 1.5 mg L-1, associated with the longitudinal sectioning of the shoot bud used as explant presented the best results, maximizing shoot proliferation. On average, the best treatment would allow for an estimated production of 161,080 plantlets by the micropropagation of the axillary buds of one plant with eight slips and ten buds/slips, within a period of eight months.

In vitro culture of Spondias mombin L. nodal segments

Spondias mombin L. shoot cultures were initiated from nodal explants taken from plants propagated by seeds. Explants coming from 4-6 months old plants, previously disinfected, were cultivated on WPM medium supplemented with a wide range of concentrations of BAP (0.0, 0.22, 0.44, 2.22 and 4.44 muM) and NAA (0.0, 0.27 and 2.70 muM). After four weeks, the responses obtained were axillary shoot and root formation. The first response were preferentially induced with the medium containing only BAP, regardless of the BAP concentration. The addition of NAA on medium reduced significantly axillary shoot formation and induced rhizogenesis. Roots were formed on nodal explant basis, preferentially on medium supplemented with 4.44 muM NAA. The medium supplemented with BAP reduced significantly root formation.

Induction of somatic embryogenesis in immature seeds of guavatree cv. Paluma

The biotechnological techniques may help solve many problems of guava culture, such as the high perishability of fruits. Somatic embryogenesis can generate highly multiplicative cell cultures and with high regenerative potential, serving as basis for genetic transformation. The aim of this work was to obtain somatic embryogenesis of guava (Psidium guajava L.) cv. Paluma. Immature seeds were used, and they were inoculated in MS environment containing 400 mg L-1 of L-glutamine, 100 mg L-1 myo-inositol, 60 g L-1 sucrose, 100 mg L-1 ascorbic acid and supplemented with different types and concentrations of growth regulators. Embryogenic callus appeared after 37 days of culture in an environment containing 1.0 mg L-1 2.4-D + 2.0 mg L-1 2-ip, in 7% of the explants. After 65 days of culture, the treatment containing 0.5 mg L-1 CPA showed 20% of explants with direct embryos, while the treatment with 1 mg L-1 had 14% of explants with direct embryos and 7% of explants with embryogenic callus. In 66.6% of embryos regenerated with 0.5 mg L¹ CPA there was the formation of secondary embryos. The use of IASP and BAP, aiming embryogenesis proliferation, led to an increase in the cellular proliferation, but calli apparently lost their embryogenic potential.

Sour orange bud regeneration and in vitro plant development related to culture medium composition and explant type

In order to evaluate the formation of adventitious buds and in vitro regeneration of sour orange plants (Citrus aurantium L.) two organogenesis-inducing experiments were conducted. In the first experiment, the induction and in vitro regeneration of adventitious buds were tested on epicotyl and internodal segments under the influence of BAP or KIN associated with NAA. The second experiment evaluated the in vitro regeneration of sour orange plants related to different explant types (epicotyls segments, internodal segments of in vitro germinated plantlets and internodal segments of greenhouse cultivated plants). Data collected on both experiments included the percentage of responsive explants (explants that formed buds), and the number of buds per explant. The addition of BAP showed the best organogenic response. In vitro germinated epicotyl segments and internodal segments are recommended as explants for sour orange in vitro organogenesis. Rooting of regenerated shoots was achieved without the need of auxin in the medium.

In vitro organogenesis in some citrus species

In vitro organogenesis of Citrus was studied for the genotypes Citrus sinensis cv. 'Natal', C. limonia, C. volkameriana, and C. aurantium, with the use of epicotyl segments-derived explants, cultured in MT salts and vitamins medium supplemented with different concentrations of 6-benzylaminopurine (BAP - 0.0; 0.5; 1.0; 1.5 or 2.0 mg L-1). For the recalcitrant genotypes C. limonia and C. aurantium the in vitro organogenesis was also studied with internodal segments-derived explants, cultured in MT salts and vitamins medium supplemented with 0; 0.5; 1.0; 2.0, or 4.0 mg L-1 of BAP. The efficiency of culture medium supplementation with the combination of BAP (0.0; 1.0, or 2.0 mg L-1) and NAA (1-naphthaleneacetic acid - 0.0; 0.3, or 0.5 mg L-1) in the development of adventitious shoots was evaluated for C. aurantium. Culture medium supplementation with BAP is not essential for the adventitious shoots development in the four genotypes studied when epicotyl segments-derived explants are used. In general, culture media supplementation with BAP decreased the percentage of responsive explants excepted for C. sinensis cv. 'Natal' and C. limonia when the concentrations of 1.5 and 2.0 mg/L were used. The presence of cytokinin, in concentrations up to 2 mg/L, stimulated the in vitro organogenesis when internodal segments-derived explants were used for C. limonia and C. aurantium. For C. aurantium no adventitious shoots developed in explants (internodal segments) cultured in basal culture medium, without BAP supplementation. Although no statistic differences could be detected, culture media supplementation with the combination of BAP and NAA favored the development of adventitious shoots in C. aurantium. The best concentration of NAA varied according to BAP concentration. The results presented herein, show that Citrus in vitro organogenesis depends on the interaction of culture medium composition, explant differentiation level, and genotype.

In vitro organogenesis of rangpur lime

Rangpur lime (Citrus limonia Osbeck) in vitro organogenesis was studied based on explant type and cytokinin culture media supplementation. Four explants types collected from epicotyl or hypocotyl regions of in vitro germinated seedlings were evaluated. The epicotyl-derived explants consisted of epicotyl segments and the hypocotyl-derived explants consisted of the entire hypocotyl segment, the hypocotyl segment attached to a cotyledon fragment, and the hypocotyl segment divided longitudinally. The explants were cultured on EME culture medium supplemented with benzylaminopurine (0, 0.5, 1.0, or 1.5 mg L-1). The evaluation was performed after 6 weeks. Best results considering both the explant responsiveness and number of shoots developed per explants were obtained when epicotyl segments-derived explants were evaluated. Considering the explant responsiveness of hypocotyl segments-derived explants no difference was detected between the entire hypocotyl segment and the hypocotyl segment attached to a cotyledon fragment. Moreover, the percentage of responsive explants decreased when hypocotyl segments divided longitudinally were tested. No difference was detected for the number of shoots developed per explant considering the three types of hypocotyl-derived explants. Culture media supplementation with BAP was not essential for Rangpur lime in vitro organogenesis. However, adventitious shoot development was stimulated in concentrations between 0.5 - 1.0 mg L-1.

In vitro multiplication of Tabernaemontana fuchsiaefolia L. (Apocynaceae)

This study describes a simple and promising for in vitro multiplication of Tabernaemontana fuchsiaefolia, a species abundantly found in southern Brazil utilized for medicinal purposes and as a source of compounds that may be used to develop new synthetic drugs. Apical and hypocotyl explants were cultured in MS medium containing different concentrations of the cytokinins benzylaminopurine (BA) and 6-furfurylaminopurine (kinetin), supplemented with phloroglucinol (1, 3, 5-hydroxybenzene) to stimulate growth and shoot proliferation. Cytokinin added to the culture media positively influenced the micropropagation of T. fuchsiaefolia.and kinetin induced more shoots per explant than BA cytokinin. A favorable effect of phloroglucinol on apical and lateral buds from hypocotyls was also achieved in medium containing no kinetin or in all kinetin concentrations tested. Short pulses of auxin 3-indolebutyric acid (IBA) 5.0 mg/l resulted in satisfactory rooting in apical microcuttings. The addition of phloroglucinol to MS medium induced rhizogenesis in 29% of the nodal segments transferred to MS medium in the absence of IBA and in 50% of the nodal segments transferred to MS medium containing 0.5 mg/l IBA and in nodal segments previously submitted to short pulses of IBA.

Micropropagation of Maclura tinctoria L.: an endangered woody species

Some native species produce seeds with low germination percentage and in most cases with dormancy, which makes the appearance of new individuals by sexual propagation difficult. The Maclura tinctoria has been considered an endangered species due to the indiscriminate use of its wood and low rate of seed germination. In this context, the objective of the present study was to establish an in vitropropagation methodology for this species. Combinations of NAA + BAP, different concentrations of GA3 and combinations IBA + activated charcoal were evaluated for shoot induction, shoot growth and root formation, respectively. The results indicated that the maximum shoot formation was obtained when 5.37 µM NAA + 4.45 µM BAP was used. The use of 5.48 µM GA3 promoted shoot growth. Root formation was observed on explants inoculated in WPM with a pH adjusted to 7.0 and supplemented with 23.62 µM IBA + 4.7 g L-1 activated charcoal. The use of a 70% light screen for 7 days followed by the use of 50 and 30% light screens also for 7 days each provided 97% plantlet survival.

CLONAL PROPAGATION OF NEEM (Azadirachta indica A. Juss.) VIA DIRECT AND INDIRECT IN VITRO REGENERATION

ABSTRACTThe study was conducted with shoot tip explants of neem (Azadirachta indica A. Juss) to identify a viable regenerative process. Shoot tips were obtained from neem embryos cultured alternatingly in DKW medium supplemented with BAP and medium without hormones. Initial shoot development was influenced by cotyledon presence. Basal callus, excised from in vitro stem base, also presented organogenic potential. In some cases, plant lines, obtained from each seed, presented different characteristics. The most common characteristic observed in vitro was callus formation at the stem base. However, the rarest characteristics were stem callus formation and leaf senescence. The regenerated shoot tips were further subculture and rooted on a medium supplemented with IBA so that complete plants could be obtained. The rooted plants were transplanted to a greenhouse and successfully acclimatized. No significant differences in in vivo development were observed between neem plants from callus and from shoot tip propagation.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831)

Abstract: The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Sinningia allagophylla(Gesneriaceae): in vitro cultivation of a native plant of the Brazilian cerrado

We used axillary buds as initial explants for hormone interaction studies required for in vitro cultivation of S. allagophylla. Callus production was achieved on gelled Murashige & Skoog medium (MS) supplemented with indole-3-acetic acid (IAA= 0.1 and 0.5 mg.l­1 alone or combined with 6 benzylaminopurine) (BA= 0.01 and 0.1 mg.l-1). A hormone balance between IAA and BA that would encourage shoot bud development was not found. Nodal segments from axenic cultures grown in the presence of cytokinin (0.1 mg.1­1 of BA) without any auxin on MS medium with half-strength macronutrients were used as a standard explant source for subsequent experiments on optimum mineral culture media composition for S. allagophylla in vitro cultivation. We found that explants kept in vitro on gelled Gamborg et al. (B5) mineral composition culture medium showed better shoot and specially root growth than on MS medium. Comparisons of the ammonium and nitrate ratios of MS and B5 media indicate that B5 medium has a substantial reduced ammonium ion when compared to MS medium, as well as a lower total nitrogen level. The growth response pattern obtained in vitro may be evidence of the adaptation of this species to soils of poor mineral composition as found in the Brazilian cerrado, as well as an indication that nitrogen levels play a key role for S. allagophylla growth.

In vitro culture of Phyllanthus stipulatus (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus stipulatus (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication rates (8-9 shoots per explant) was achieved on MS media supplemented with either 2.5-5.0 muM IBA. The best basal media for axillary shoot proliferation when 0.62 muM BA was supplemented were MS, MS/2 and AR (4-5 shoots per explant). Rooting was achieved with 100% of the microshoots on MS medium without growth regulators. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed in 81% of the ex vitro grown plantlets after 12 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated either in the vertical position, in the light on MS medium supplemented with 5.0 muM NAA or horizontally oriented, in the dark on MS supplemented with 5.0 muM NAA or 1.25-5.0 muM BA or 2iP. Root cultures were successfully established on MS medium containing 1.1 muM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/organ culture techniques for vegetative propagation and secondary metabolism studies.

In vitro somatic embryogenesis and adventitious root initiation have a common origin in eggplant (Solanum melongena L.)

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

Anomalous somatic embryos in Acca sellowiana (O. Berg) Burret (Myrtaceae)

Somatic embryogenesis represents a valuable tool for the studies on the basic aspects of plant embryo development. Today this process is used as a potencial technique for large-scale plant micropropagation although, so far, it has been applied to only a small number of species. However, when somatic embryos are malformed they are considered economically useless. In Acca sellowiana (O. Berg) Burret, an important fruit-producing crop, large amounts of anomalous somatic embryos (76.3%) were found just after 40 days of culture of explants in a 2,4-D containing medium. Among the anomalous forms found in the cotiledonary stage, 12.2% consisted of fused embryos, 40.4% displayed fused cotyledons, 13.0% presented supernumerary cotyledons, and 10.7% showed absence or poorly developed cotyledons, including those without the shoot apical meristem. Histological analyses indicated that the altered embryos were formed either directly from cotyledons, hypocotyl and radicle of the zygotic embryos used as explants, or indirectly from calli formed from these tissue parts. It is suggested that the formation of anomalous somatic embryos, as well as a low frequency of conversion into emblings reflect physiological and/or genetic disturbances triggered by the presence of 2,4-D in the medium. In vitro experimental alternative approaches are discussed in order to lessen the occurrence of malformed somatic embryos.

Callus induction and plant regeneration by Brazilian triticale and wheat genotypes

In order to determine the in vitro behavior of Brazilian triticale, 16 triticale genotypes, and three wheat genotypes used as checks, were sown in June 1994. The explants used were immature embryos. In addition to the genotype tests, two culture media for callus induction were also evaluated, i.e., MS (Murashige and Skoog, Physiol. Plant. 15: 473-497, 1962) medium containing 2.0 mg 2,4D/l, and MS medium containing 4.0 mg 2,4D/l. The plant regeneration protocol used was the one employed at the Laboratório de Cultura de Tecidos, Departamento de Plantas de Lavoura, Universidade Federal do Rio Grande do Sul, for wheat. Differences in plant regeneration were observed both among triticale and wheat genotypes, with triticale usually showing better regeneration than wheat. No differences were observed between the callus induction media.