Seedling Development and Evaluation of Genetic Stability of Cryopreserved Dendrobium Hybrid Mature Seeds

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).

Variability of ricin content in mature seeds of castor bean

The objective of this work was to evaluate ricin concentration in castor bean seeds (Ricinus communis) of 20 accessions from the Banco de Germoplasma de Mamoneira of the Embrapa Algodão, Campina Grande, PB, Brazil, using the Enzyme Linked Immunosorbent Assay. Significant differences were observed among accessions. BRA 3271 had the highest ricin concentration in seeds (32.18 ng µg-1), and BRS Paraguaçu had the lowest (3.53 ng µg-1). There is the possibility of selecting genotypes with different ricin concentrations, which can be used according on the interest of the breeding programs.

Priming and re-drying improve the survival of mature seeds of Digitalis purpurea during storage

Most priming studies have been conducted on commercial seed lots of unspecified uniformity and maturity, and subsequent seed longevity has been reported to both increase and decrease. Here a seed lot of Digitalis purpurea L. with relatively uniform maturity and known history was used to analyse the effects of priming on seed longevity in air-dry storage. Seeds collected close to natural dispersal and dried at 15 % relative humidity (RH), 15 degrees C, were placed into experimental storage (60 % RH, 45 degrees C) for 14 or 28 d, primed for 48 h at 0, -1, -2, -5, -10 or -15 MPa, re-equilibrated (47 % RH, 20 degrees C) and then returned to storage. Further seed samples were primed for 2 or 48 h at -1 MPa and either dried at 15 % RH, 15 degrees C or immediately re-equilibrated for experimental storage. Finally, some seeds were given up to three cycles of experimental storage and priming (48 h at -1 MPa). Priming at -1 MPa had a variable effect on subsequent survival during experimental storage. The shortest lived seeds in the control population showed slightly increased life spans; the longer lived seeds showed reduced life spans. In contrast, seeds first stored for 14 or 28 d before priming had substantially increased life spans. The increase tended to be greatest in the shortest lived fraction of the seed population. Both the period of rehydration and the subsequent drying conditions had significant effects on longevity. Interrupting air-dry storage with additional cycles of priming also increased longevity. The extent of prior deterioration and the post-priming desiccation environment affect the benefits of priming to the subsequent survival of mature seeds. Rehydration-dehydration treatments may have potential as an adjunct or alternative to the regeneration of seed accessions maintained in gene banks for plant biodiversity conservation or plant breeding.

Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and Supercool X1000

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dormancy as exaptation to protect mimetic seeds against deterioration before dispersal

Mimetic seeds simulate the appearance of fleshy fruits and arilled seeds without producing nutritive tissues as a reward for seed dispersers. In this strategy of seed dispersal, seeds may remain attached to the mother plant for long periods after maturity, increasing their availability to naive seed dispersers. The hypothesis that seed coat impermeability in many tropical Fabaceae with mimetic seeds serves as an exaptation to protect the seeds from deterioration and rotting while awaiting dispersal was investigated. Seed coat impermeability was evaluated in five mimetic-seeded species of tropical Fabaceae in south-eastern Brazil (Abarema langsdorffii, Abrus precatorius, Adenanthera pavonina, Erythrina velutina and Ormosia arborea) and in Erythrina speciosa, a `basal` species in its genus, which has monochromatic brown seeds and no mimetic displays. Seed hardness was evaluated as a defence against accelerated ageing (humid chamber at 41 degrees C for 144 h). Seed development and physiological potential of O. arborea was evaluated and the effect of holding mature seeds in pods on the mother plant in the field for a period of 1 year under humid tropical conditions was compared with seeds stored under controlled conditions (15 degrees C and 40 % relative air humidity). All five mimetic-seeded species, and E. speciosa, showed strong coat impermeability, which protected the seeds against deterioration in accelerated ageing. Most O. arborea seeds only became dormant 2 months after pod dehiscence. Germination of seeds after 1 year on the plant in a humid tropical climate was 56 %, compared with 80 % for seeds stored in controlled conditions (15 degrees C, 45 % relative humidity). Seedling shoot length after 1 year did not differ between seed sources. Dormancy acts in mimetic-seeded species as an exaptation to reduce seed deterioration, allowing an increase in their effective dispersal period and mitigating the losses incurred by low removal rates by naive avian frugivores.

Post-abscission, pre-dispersal seeds of Digitalis purpurea remain in a developmental state that is not terminated by desiccation ex planta

Seed quality may be compromised if seeds are harvested before natural dispersal (shedding). It has been shown previously that slow or delayed drying can increase potential quality compared with immediate rapid drying. This study set out to investigate whether or not there is a critical moisture content, below which drying terminates maturation events for seeds harvested after mass maturity but before dispersal. Seeds of foxglove (Digitalis purpurea) in the post-abscission pre-dispersal phase were held at between 15 and 95 % RH for 4 or 8 d, with or without re-hydration to 95 % RH for a further 4 d, before drying to equilibrium at 15 % RH. In addition, dry seeds were primed for 48 h at -1 MPa. Subsequent seed longevity was assessed at 60 % RH and 45 degrees C. Rate of germination and longevity were improved by holding seeds at a wide range of humidities after harvest. Longevity was further improved by re-hydration at 95 % RH. Priming improved the longevity of the seeds dried immediately after harvest, but not of those first held at 95 % RH for 8 d prior to drying. Maturation continued ex planta in these post-abscission, pre-dispersal seeds of D. purpurea dried at 15-80 % RH at a rate correlated positively with RH (cf. ageing of mature seeds). Subsequent re-hydration at 95 % RH enabled a further improvement in quality. Priming seeds initially stored air-dry for 3 months also allowed maturation events to resume. However, once individual seeds within the population had reached maximum longevity, priming had a negative impact on their subsequent survival.

Desenvolvimento inicial e crescimento in vitro de Cattleya violacea (Kunth) Rolfe em diferentes concentrações de sacarose

O presente estudo teve por objetivo analisar os aspectos da germinação e avaliar o efeito de concentrações de sacarose no crescimento in vitro de Cattleya violacea. Sementes provenientes de cápsulas fechadas foram semeadas em meio de cultura Murashige e Skoog (MS) e a morfologia externa da semente à plântula foi fotodocumentada em estereomicroscópio e microscópio eletrônico de varredura. Plântulas com 90 dias após a semeadura foram repicadas em meio de cultura ½ MS (com metade da concentração de macronutrientes) com diferentes concentrações de sacarose (0, 10, 20, 30 e 40 g L-1), incubadas nas mesmas condições in vitro por mais 150 dias e em seguida as plântulas foram avaliadas quanto ao número de raízes, comprimento da maior raiz, número de folhas, comprimento da parte aérea, massa fresca e seca total. Os dados biométricos foram submetidos à análise estatística e a eles ajustadas curvas de regressão. As sementes apresentaram testa reticulada com uma extremidade micropilar (aberta) e calazal (fechada); o embrião originou uma estrutura tuberiforme clorofilada denominada protocormo que pode apresentar rizóides, folíolos e quando provido de raiz é considerado plântula. A ausência de açúcar ou a maior concentração avaliada de sacarose foram prejudiciais ao crescimento da planta. A concentração de 27 g L-1 proporcionou maior crescimento in vitro possibilitando maior eficiência para a propagação massal dessa espécie de elevado potencial ornamental.

The development of seedlings from fragments of monoembryonic seeds as an important survival strategy for Eugenia (Myrtaceae) tree species

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Yield and quality of carrot seeds, cv. Brasilia, as a result of plant population, gibberellic acid and stage of maturity

'Brasilia' is a cultivar of carrot with characteristics suiting cultivation under hot conditions but with problems in seed production, arising from the conflicting requirements of root and seed production. One solution is to select cultivars requiring vernalisation and then to use GA(3) to induce flowering where the climate prevents this. There is, however, little information on plant population, seed maturity and harvesting time on which to base such a procedure. Accordingly this research was carried out to study the physiological quality and production of the seeds in plant populations from 25,000 to 800,000 plants/ha, in the seed-to-seed method of cv. Brasilia in Anapolis, GO, Brazil. In each population, two harvest methods (from first and second orders of umbels, or selected harvest, and remaining orders, or total harvest) and two stages of maturity (brownish, or mature seeds, and yellowish, or immature ones) were also evaluated. Two trials were carried out, with and without gibberellic acid. Seed was evaluated for physical characters, germination, vigour, 1000-seed weight, water content, dry matter and productivity. Seed was produced in both experiments (with or without GA3 spraying). Mature seed showed germination at least 30% greater than immature, and seed from the selected harvest showed germination 16% greater than from the total harvest. In increasing plant population to 200,000 plants/ha, seed quality was not affected, but productivity increased.

Content of essential oil of fennel seeds in two different stages of development

This experiment was carried out at Plant Production Sector, Agronomical Science College-Botucatu, S.P., Brazil, in March, 2000. The aim of this assay was to determine the yield of essential oil of fennel (Foeniculum vulgare Miller) in different stages of development. Essential oils were prepared by hydrodistillation from the seeds using of Clevenger apparatus. The water utilized for the extraction of essential oil was sufficient to cover 100 g of seeds and the mixture was distilled for three hours. The volume of essential oil in the graduated side -arm of Clevenger apparatus was observed. There were no significative difference statistic was observed (Tukey 5%) in percentage (v/m) of oil content, based on dry weight of green seeds compared with dry weight of mature seeds, when they were harvested in two different stages of development. There was significative difference statistic between data obtained of humidity content of green seeds when these were compared with mature seeds. These results shows that others specifics studies about adaptation of fennel in tropical conditions are necessary, because the obtained data were different of data described on literature.

Concentrações de sacarose no desenvolvimento in vitro e na aclimatização de Cattleya loddigesii Lindley

This work aimed to study the influence of sucrose in the culture medium for in vitro growth and acclimatization of the epiphytic orchid Cattleya loddigesii. Five sucrose treatments (absence, 10, 20, 30 and 40g L-1) were used in a randomic experimental design. Mature seeds were sowed in 1/2 MS culture medium and after 90 days the plantlets (1.0 +/-0.2 cm) were inoculated between the treatments, whereby they were remained more 90 days. After 180 days of the beginning of the experiment the plantlets were removed from the flasks and evaluated the number of roots, shoot length, number of leafs, total dry weight and photosynthetic pigments. Survival percentage was evaluated after 75 days of acclimatization. The data of biometric variables were analyzed by Anovaand polynomial regression (p<0.05). Theothers data were submitted to the Anova and the means compared by the Tukey test (p<0.05). The sucrose concentration of 20g L-1 favored the in vitro growth in all evaluated parameters, showed higher production of chlorophyll a, total chlorophyll and carotenoids, in addition to increased survival under ex vitro condition. The sucrose concentration of 20g L-1 in the culture medium was the most efficient among the tested concentrations both for in vitro growth and ex vitro establishment of Cattleya loddigesii.

Cryopreservation, early seedling development, and genetic stability of Oncidium flexuosum Sims

An efficient cryopreservation protocol was developed for mature seeds of Oncidium flexuosum Sims. Seed morphology, protocorm formation, and early seedling development were also assessed. The effects of phloroglucinol and Supercool X-1000® as cryoprotectant additives in the vitrification solution were investigated. Dehydration using the plant vitrification solution 2 (PVS2) for 60 and 120 min prior to immersion in liquid nitrogen promoted the highest frequency of in vitro seed germination 6 weeks following culture on half-strength Murashige and Skoog (1/2 MS) medium. Mature seeds submitted to vitrification for 120 min in PVS2 and 1 % phloroglucinol at 0 °C enhanced germination by 68 %, whereas in PVS2 and 1 % Supercool X-1000® germination was just moderately enhanced (26 %). In vitro-germinating seedlings developed healthy shoots and roots without the use of plant growth regulators. After 6 months of growth, there were no differences between in vitro- and ex vitro-grown seedlings for various phenotypic characteristics, including shoot length, number of leaves, number and length of roots, and fresh and dry weight. Seedlings were transferred to greenhouse conditions and successfully acclimatized, further developing into normal plants with over 90 % survival. Comparative analysis of seedlings from control and vitrified seeds using flow cytometry indicated that no change in ploidy levels occurred as a result of cryopreservation, therefore maintaining seedlings genetic stability. In this study, vitrification with PVS2 for 120 min with the addition of 1 % phloroglucinol offers a simple, safe, and feasible protocol for cryopreservation of O. flexuosum mature seeds. © 2013 Springer Science+Business Media Dordrecht.

Criopreservação, indução de poliploidia e avaliação da estabilidade genética de orquídeas

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

In vitro conservation of Dendrobium germplasm

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation-dehydration, encapsulation-vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation-vitrification and encapsulation-dehydration are the most used protocol, while PLBs represent the main explant explored.