Do polyembryonic seeds of Carapa surinamensis (Meliaceae) have advantages for seedling development?

Polyembryony is the differentiation and development of multiple embryos in a single seed. This characteristic can provide advantages, as more than one embryo is produced with the same amount of resources, and the probability of establishment of at least one seedling increases. However, sibling seedlings may also increase competition, affecting development and survival. In the present study, the possible advantages and disadvantages of polyembryony were analyzed in the initial establishment of seedlings of Carapa surinamensis (Meliaceae), a tree species that produces monoembryonic or polyembryonic seeds. In this regard, the development of single seedlings was compared with a pair of seedlings emerging from polyembryonic seeds. We compared the development of seedlings attached to or detached from each other and to the seed resources. We observed two levels of competition: (a) for the seed reserves during germination and initial development, as multiple embryos of C. surinamensis share the same reserves, and (b) for external factors, mostly space for root and shoot development, and also for light. Reducing the competition for external factors by separating the siblings was not enough to reduce the effects of competition for seed reserves in the first six months of development. Nevertheless, viable seedlings were produced in all treatments. Thus, depending on sprout management in the nursery, the number of seedlings per seed can be significantly increased by detaching the seedlings, or more vigorous seedlings can be obtained when only one seedling is maintained.(AU)

Poliembrionia é a diferenciação e o desenvolvimento de múltiplos embriões em uma única semente. Esta característica pode proporcionar diversas vantagens, como aumentar o número de embriões produzidos com a mesma quantidade de recursos, e aumentar a probabilidade de estabelecimento de pelo menos uma plântula de uma única semente. Por outro lado, a competição entre plântulas pode aumentar, afetando seu desenvolvimento e sobrevivência. Neste estudo, foram analisadas as possíveis vantagens e desvantagens da poliembrionia em sementes de andiroba, Carapa surinamensis (Meliaceae), uma espécie arbórea que produz sementes monoembriônicas ou poliembriônicas. Comparamos o desenvolvimento de plântulas únicas com o de pares de plântulas provenientes de sementes poliembriônicas. As plântulas foram mantidas unidas ou separadas entre si e ligadas à ou destacadas da semente. Os resultados revelaram dois níveis de competição: (a) pelas reservas da semente durante a germinação e desenvolvimento inicial da plântula, quando embriões múltiplos de C. surinamensis compartilham as mesmas reservas, e (b) por fatores externos, principalmente espaço para o desenvolvimento da raiz e da parte aérea, e luz. A redução da competição por fatores externos, através da separação das plântulas, não foi suficiente para reduzir os efeitos da competição pelas reservas das sementes nos primeiros seis meses de desenvolvimento. Apesar disso, plântulas viáveis foram produzidas em todos os tratamentos. Assim, dependendo do manejo dos brotos em viveiro, o número de plântulas produzidas pode ser aumentado significativamente por meio da separação entre plântulas, ou o vigor das plântulas pode ser incrementado através de sua manutenção individualizada.(AU)

Reproductive success of Cabralea canjerana (Meliaceae) in Atlantic forest fragments, Brazil / Éxito reproductivo de Cabralea canjerana (Meliaceae) en fragmentos de bosque del Atlántico, Brasil

In Brazil, the Atlantic forest remnants have high biological diversity and a high level of endemism, but very little is known about the reproductive success of native species. Cabralea canjerana is a common tree in the Montane Atlantic forest, and its reproduction is highly dependent on pollinators. In order to contribute with the particular knowledge on this species, we collected data in three fragmented and three continuous forest sites, where the effects of fragmentation on both mutualistic (pollination) and antagonistic (seed predation) interactions were analysed. We determined fruit production and weight of 25 trees per site. The number of seeds and the percentage of predated and aborted seeds were also accessed for seven fruits of 10 trees per site. Pollinator visitation frequencies to flowers were recorded in two forest fragments and in two sites of the continuous forest. Our data showed that plants of C. canjerana produced more fruits (z-value=-8.24; p<0.0001) and seeds per fruit (z-value=-6.58; p=0.002) in the continuous than in the fragmented sites. This was likely due to differences in pollination, because the number of pollinator visits was higher in the continuous forest than in the fragments. Seed abortion (z-value=4.08, p<0.001) and predation (z-value=3.72, p=0.0002), on the other hand, were higher in the fragmented than in the continuous sites. Then, mutualistic and antagonistic interactions were affected by fragmentation, decreasing the reproductive success of the study tree. This study was the first to show a decrease in the reproductive output in forest fragments in an Atlantic forest tree species. This decrease may threaten the population structure and viability of C. canjerana in forest fragments. Rev. Biol. Trop. 63 (2): 515-524. Epub 2015 June 01.

En Brasil, los remanentes de bosque del Atlántico tienen una alta diversidad biológica y un alto nivel de endemismo, pero se conoce muy poco sobre el éxito reproductivo de las especies nativas. Canjerana cabralea es un árbol común en el bosque Atlántico Montano y su reproducción es altamente dependiente de los polinizadores. Con el fin de contribuir con el conocimiento particular de esta especie, se recogieron los datos en tres sitios fragmentados y tres de bosques continuos, donde se analizaron los efectos de la fragmentación tanto en relaciones mutualistas (polinización) como interacciones antagónicas (depredación de semillas). Se determinó la producción de frutos y el peso de 25 árboles. También se calculó el número de semillas y el porcentaje de semillas depredadas y abortadas en 7 frutos de 10 árboles por sitio. Frecuencia de visitas de polinizadores a las flores se registraron en dos fragmentos de bosque y en dos sitios de bosque continuo. Nuestros datos muestran que las plantas de C. canjerana produjeron más frutos (Z=-8.24; p<0.0001) y semillas por fruto (Z=-6.58; p=0.002) en el continuo que en los sitios fragmentados. Esto fue probablemente debido a las diferencias en la polinización, porque el número de visitas de polinizadores fue mayor en el bosque continuo que en los fragmentos. Por otro lado, el aborto de semillas (Z=4.08, p<0.001) y la depredación (Z=3.72, p=0.0002) fueron mayores en los sitios fragmentados que en los continuos. Entonces, las interacciones mutualistas y antagónicas se vieron afectadas por la fragmentación, disminuyendo el éxito reproductivo del árbol estudiado. Este estudio fue el primero en mostrar una disminución en el rendimiento reproductivo en los fragmentos de bosque en una especie de árboles forestales atlánticos. Esta disminución puede poner en peligro la estructura de la población y la viabilidad de C. canjerana en fragmentos de bosque.

Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: challenges and considerations for cryopreservation.

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. However, the variable post-cryo success achieved with the excised zygotic explants traditionally used for cryopreservation has been a concern for some time. Differential drying rates amongst explants of different species, uneven drying amongst explants within a batch of seeds and uneven drying across tissues within individual embryos could be contributory factors to this variable success and these phenomena form the foci of the present study. Using zygotic explants from a range of recalcitrant-seeded species, which included sub-tropical dicotyledonous trees and sub-tropical monocotyledonous geophytes, the study showed that embryo morphology and anatomy are critical determinants of the drying characteristics of the different tissues composing the explant and hence, post-cryo survival. The results suggest that the rates of drying of explants to water contents (WCs) in the theoretically optimal range for successful cryopreservation are species-specific, and that more rapid drying rates may promote post-cryo survival. However, the large variation in WC amongst individual explants in bulk samples challenges the selection of the theoretically optimum WC for cryopreservation. As a consequence of differential drying rates across the different tissues composing explants, either lethal ice crystal damage or desiccation damage may sometimes be likely in tissues responsible for the onwards development of the embryo. Drying times for cryopreservation of such explants should, therefore, be selected on the basis of WC of segments containing root or shoot meristem, rather than embryo bulk WC. Drying intensity and duration also interact with explant morphology and embryo/axis size and anatomy to bring about - or preclude - post-cryo survival.

A novel means for cryopreservation of germplasm of the recalcitrant-seeded species, Ekebergia capensis.

Cryopreserved zygotic embryonic axes offer the best means of genetic diversity conservation of recalcitrant-seeded species, but frequently shoots fail to develop following processing for, and after, cryostorage. The present work offers a means to overcome this, by generating adventitious shoots from seedling roots produced after axis cryopreservation. Embryonic axes of Ekebergia capensis were exposed to cryoprotectants, flash dried, and rapidly cooled in nitrogen slush. Cryoprotection was an essential step, with both glycerol and DMSO permitting survival after cryogen exposure, but sucrose alone, or in combination with glycerol, was deleterious. Adventitious shoots were formed from seedling roots developed by axes germinated after cryogen exposure, after being subjected to intermittent flushing with a BAP-containing medium for 24 h in a temporary immersion system and subsequent culture on a semi-solid BAP-containing medium. After excision, a high proportion of the adventitious shoots produced roots in vitro, with most of these rooted plantlets being subsequently successfully acclimated.

Exploring the use of DMSO and ascorbic acid to promote shoot development by excised embryonic axes of recalcitrant seeds.

Seeds of Trichilia dregeana, T. emetica and Protorhus longifolia are recalcitrant (desiccation-sensitive), hence cryopreservation is the only ex situ means feasible for long-term conservation of these germplasm. For cryopreservation of these species, the excised embryonic axis is the explant of choice due to their small size and higher tolerance to desiccation. However, for many species with seeds having fleshy cotyledons, shoot development fails to occur after excision, which has been attributed to a reactive oxygen species (ROS) burst during excision wounding. This is a critical limiting step in developing cryopreservation protocols for such species. In embryos of T. dregeana, T. emetica and P. longifolia, the cotyledonary insertions are in close proximity to the shoot apical meristem and oxidative stress upon excision of the axis from cotyledons has been consistently associated with shoot tip necrosis, which precludes shoot development. This study tested the effects of dimethyl sulphoxide (DMSO) pre-culture prior to complete removal of the cotyledons, and post-excision soaking in DMSO or in the antioxidant, ascorbic acid, on shoot development by axes of T. dregeana and P. longifolia. These treatments had a significant (P < 0.05) positive effect on shoot production with a 6 h DMSO pre-culture combined with a DMSO post-excision soak being optimal for promoting shoot production in 70 percent of the axes of T. dregeana and 60 percent of those of P. longifolia. Embryonic axes of T. emetica responded best to a 6 h DMSO pre-culture alone, with 55 percent of axes producing shoots. It was further shown that two different post-harvest developmental stages of T. dregeana axes differed significantly initially (P < 0.05) in their response to DMSO and ascorbic acid treatments.

Antifeedant triterpenoids from the seeds and bark of Lansium domesticum cv Kokossan (Meliaceae).

Two tetranortriterpenoids, kokosanolide A (1) and C (2) were isolated from the seeds and three onoceranoid-type triterpenoids: kokosanolide B (3), 8,14-secogammacera-7,14-diene-3,21-dione (4) and a 1.5:0.5 mixture of 8,14-secogammacera-7,14(27)-diene-3,21-dione (5) and compound 4 were isolated from the bark of kokossan (Lansium domesticum). Complete 1H- and 13C-NMR data of the triterpenoids 1-5 are reported. The triterpenoids' structures were elucidated primarily by means of high field 1D- and 2D-NMR, IR and HRMS spectral data. Triterpenoids 1-5 exhibited moderate to strong antifeedant activity against the fourth instar larvae of Epilachna vigintioctopunctata.

Differential drying rates of recalcitrant Trichilia dregeana embryonic axes: a study of survival and oxidative stress metabolism.

Studies to elucidate the biochemical basis of survival of excised embryonic axes (EAs) of recalcitrant seeds of Trichilia dregeana at different drying rates revealed significant differences between slow and rapid drying. Rapid drying allowed these EAs to survive dehydration to much lower water contents (WCs; ca. 0.31 g g⁻¹ dry mass basis with 73% germination) compared with slow drying, where 90% of the EAs lost viability at a WC of ca. 0.79 g g⁻¹. In EAs slowly dried within seeds, the levels of hydroxyl radical (three- to fivefold at WCs > 0.5 g g⁻¹) and lipid peroxidation (50% at similar WC) were significantly higher compared with those dried rapidly to comparable WCs. When EAs were dried slowly, enzymic antioxidant levels were not sustained and declined significantly with prolonged storage. In contrast, sustained activity of enzymic antioxidants was detected in rapidly dried EAs even at relatively low WCs. Furthermore, the greater decline in glutathione (GSH)/GSH disulphide ratio in EAs slowly dried within seeds compared with rapidly dried EAs and a shift in GSH redox potential to relatively more positive values in the EAs slowly dried within seeds was correlated with considerable viability loss. It is apparent from this study that greater retention of viability to lower WCs in rapidly dried EAs from recalcitrant seeds may at least be partly explained by the retention of functional antioxidant status. It is also suggested that the reduction of viability in rapidly dried EAs at very low WCs appears to be a non-oxidative process.

Purification of Azadirachta indica seed cake and its impact on nutritional and antinutritional factors.

Azadirachta indica Juss. (family Meliaceae) is a vital plant with multiple agricultural and medicinal utilities. The seed cake after oil extraction can be a good source of nutrition in animal feed. The limitation to its use is the presence of azadirachtin, salannin, and other bitter constituents. To make it palatable for use as a source of animal nutrition it was detoxified using 50 and 80% methanol and was analyzed for contents of azadirachtin, salannin, and nutritional contents such as total carbohydrates, protein, crude fiber, in vitro protein digestibility, and trypsin inhibitor activity (TIA), prior to and after purification. The contents of azadirachtin and salannin were quantified using HPTLC and HPLC. Various validation parameters were also investigated. A highly significant decrease of antinutritional factor (TIA) was recorded after purification of samples, retaining the contents of protein, carbohydrates, crude fiber, and in vitro protein digestibility. The purified seed cake was found to be free of azadirachtin and salannin contents.

Dehydration kinetics of embryonic axes from desiccation-sensitive seeds: an assessment of descriptive models.

The response of desiccation-sensitive plant tissues to dehydration is significantly affected by dehydration conditions, particularly the rate of drying. Consequently it is important to be able to quantify drying rate. The aim of the study was to assess two models that have been proposed to describe drying kinetics, and thus to provide a quantification of non-linear drying rates, of embryonic axes excised from recalcitrant seeds. These models are an exponential drying time course, and a modified inverse relationship, respectively. For the six species investigated here the inverse function was generally found to fit drying data better than the exponential function under both rapid and slow drying conditions, and so is recommended. The rate of drying, under the conditions used here, was determined by axis size and possibly the nature of the axis outer coverings, rather than the water activity difference between the tissue and surrounding air.

Cryopreservation, encapsulation and promotion of shoot production of embryonic axes of a recalcitrant species Ekebergia capensis, Sparrm.

A study on zygotic axes of the recalcitrant seeds of Ekebergia capensis compared two cryopreservation methods, partial desiccation, and encapsulation-dehydration, and also investigated a method to promote shoot production. High (80 percent) survival (assessed as root production) was obtained after direct immersion into liquid nitrogen of axes rapidly dehydrated by flash drying for 20 min to a water content about 0.4 g water per g dry mass. In contrast, no survival at all was obtained of axes that were first encapsulated, then desiccated for three hours to the same water concentration as those fast-dried, and then placed in a cryovial and immersed in liquid nitrogen. Axes encapsulated after cryopreservation germinated both in vitro and in soil, and could be stored at room temperatures for several weeks while maintaining germinability, thus producing synseeds capable of distribution. However, shoot production after cryopreservation was seldom observed. The inclusion of the plant growth regulator, N6-benzyl adenine (BA) in the MS-based recovery medium promoted vigorous multiple shoot formation. Microscopical examination of embryos of E. capensis revealed that the cotyledonary insertions were contiguous with the shoot apex, leading to the conclusion that injury to, and ultimate necrosis of, the apical meristem following severing of these connections was a primary cause of the observed lack of, or poor, shoot development in excised axes (whether cryopreserved or not). The study demonstrated that it may be possible to resolve two of the problems facing attempts at cryopreservation of axes of recalcitrant seeds; lack of shoot production and difficulty of distribution of cryopreserved material for re-introduction.

Imbibition of Swietenia macrophylla (Meliaceae) seeds: the role of stomata.

BACKGROUND AND AIMS: The occurrence of stomata in seed coats is uncommon and there is limited information about their function(s). The aim of this study was to verify the distribution of stomata in seed coats of Swietenia macrophylla and to relate it to the imbibition process and aspects of the structure of the outer integument layers. METHODS: For the structural and ultrastructural studies, the seeds were processed using the usual techniques and studied under light and scanning electron microscopes. Histochemical tests were employed to identify the cell wall composition in the different seed coat portions. To assess the role of the stomata in the imbibition, non-impervious seeds were compared with partially impervious ones, in which only the embryo, median or hilar regions were left free. Further, the apoplastic pathway marker was employed to confirm the role of the stomata as sites of water passage during imbibition. KEY RESULTS: A positive relationship was observed between seed coat thickness and stomata density. The stomata were devoid of movement, with a large pore. They occurred in large numbers in the embryo region and extended with lower frequency towards the wing. Imbibition rates were related to stomata density, suggesting that the stomata act as preferential sites for water entry in the S. macrophylla seeds. CONCLUSIONS: At maturity, the stomata in the seed coat play a significant role in seed imbibition. The data may also infer that these permanently opened stomata have an important role in gas exchange during seed development, aiding embryo respiration.