Sample size for collecting seeds in germplasm conservation: the case of the Lima bean (Phaseolus lunatus L.)

 The design of optimum sampling strategies integrating criteria of efficiency relevant to multilocus models and many target populations has been investigated with respect to the number of plants and the number of seeds per plant to be sampled for a Lima bean (Phaseolus lunatus L.) gene pool. This study, using five populations and six polymorphic enzyme loci, shows that the number of plants rather than the number of seeds collected per plant primarily determines the success of seed sampling, suggesting that plant number plays an essential part in maintaining the allelic multiplicity of predominantly selfing species like Lima bean. According to the results, it appears that among Lima bean populations an efficient sampling procedure is achieved by collecting 1–4 seeds from 200 to 300 plants. These sample sizes will retain 8–10 alleles, regardless of their frequencies. When we consider polymorphism at the 5% level, it is expected that sampling 10–80 plants will collect combinations of 4–8 alleles. Based on data from genetic and demographic studies, we suggest an efficient sampling scheme for Lima bean germplasm at both population and geographical levels. Received: 10 March 1998 / Accepted: 1 April 1998     

Factors Influencing Tree Phenology in Taï National Park,Côte d'Ivoire1

Entrained phenology patterns of tropical trees are expected to be sensitive to short‐term fluctuations in typical rainfall and temperature. We examined 47 mo of data on the flowering, fruiting, and new leaf phenology for 797 trees from 38 species in the Taï National Park, Côte d'Ivoire. We determined the timing of the phenology cycles in relation to seasonal rainfall, temperature, and solar radiation. Regression analysis was used to examine how variations in rainfall and temperature influenced deviations in the peaks and troughs of phenology cycles. We also investigated whether populations that fruit during periods of community‐wide fruit scarcity were those populations with relatively long‐ or short‐fruiting duration. Flower, fruit, and leaf‐flushing phenophases all exhibited 12‐mo cycles. The broad peak in flowering began with the northward zenithal passing in April and ended with the southward zenithal passing in September. Fruiting peaks occurred in the long dry season, and leaf flushing peaked in the long dry season but continued into the wet season. Deviations from phenology cycles were largely attributable to short‐term fluctuations in rainfall and/or temperature. Fruiting durations of species were related to the mean diameter at breast height. Species with long‐ and short‐fruiting durations contributed equally to fruit abundance during periods of community‐wide fruit scarcity.     

Reproductive biology of the andromonoecious Cucumis melo subsp. agrestis (Cucurbitaceae)

Background and Aims

Cucumis melo subsp. agrestis (Cucurbitaceae) is cultivated in many African regions for its edible kernels used as a soup thickener. The plant, an annual, andromonoecious, trailing-vine species, is of high social, cultural and economic value for local communities. In order to improve the yield of this crop, the first step and our aim were to elucidate its breeding system.

Methods

Eight experimental pollination treatments were performed during three growing seasons to assess spontaneous selfing, self-compatibility and effects of pollen source (hermaphroditic vs. male flowers). Pollination success was determined by pollen tube growth and reproductive success was assessed by fruit, seed and seedling numbers and characteristics. The pollinator guild was surveyed and the pollination distance determined both by direct observations and by indirect fluorescent dye dispersal.

Key Results

The species is probably pollinated by several Hymenoptera, principally by Hypotrigona para. Pollinator flight distances varied from 25 to 69 cm. No evidence for apomixis or spontaneous self-pollination in the absence of insect visitors was found. The self-fertility index (SFI = 0) indicated a total dependence on pollinators for reproductive success. The effects of hand pollination on fruit set, seed number and seedling fitness differed among years. Pollen tube growth and reproductive success did not differ between self- and cross-pollinations. Accordingly, a high self-compatibility index for the fruit set (SCI = 1·00) and the seed number (SCI = 0·98) and a low inbreeding depression at all developmental stages (cumulative δ = 0·126) suggest a high selfing ability. Finally, pollen origin had no effect on fruit and seed sets.

Conclusions

This andromonoecious species has the potential for a mixed mating system with high dependence on insect-mediated pollination. The selfing rate through geitonogamy should be important.     

Assessment of genetic diversity and structure in cocoa trees (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) in Côte d’Ivoire with reference to their susceptibility to Cocoa swollen shoot virus disease (CSSVD)

Resistance to Cocoa Swollen Shoot Virus disease (CSSVD) is becoming an increasingly important criterion for selection of new cocoa cultivars in Côte-d’Ivoire, where the disease resurfaced since 2003. This virus can seriously affect the yield of trees with a loss of 25%, 1 year after infection, to around 100% 3 years after. In order to find tolerant plant material, 337 farm accessions have been collected on fields affected by CSSVD, according to the status of accessions potentially tolerant (APT) or susceptible (APS). Both phenotypic groups were genotyped using 30 microsatellite markers (SSR) in the presence of representative clones of the ten genetic groups of cocoa. This study revealed 214 alleles with the set of primer pairs used. The number of alleles per locus was between 3 and 16 with an average of 7.13 alleles per locus. The results showed a high contribution of genetic diversity within population (H_s?=?0.51) to the total genetic diversity (H_t?=?0.53) for the two studied groups. There was no significant difference between tolerant and susceptible groups (Fst?=?0.05). These results suggest that APT could be a potential genetic reservoir for other traits of interest associated with virus resistance. The phylogenetic tree, as the STRUCTURE analysis of Ivorian cocoa population, showed a distribution of individuals following four groups marked by a high contribution of group 4 (Nanay, Maranon, Guiana) followed by group 2 (Criollo), and group 1 (Amelonado), and a lower contribution of group 3 (Iquitos, Purus, Nacional, Curaray, Contamana).     

Genetic structure of a Lima bean base collection using allozyme markers

 Genetic diversity and structure within a Lima bean (Phaseolus lunatus L.) base collection have been evaluated using allozyme markers. The results obtained from the analysis of wild and cultivated accessions confirm the existence of Andean and Mesoamerican gene pools characterised by specific alleles. Wild and cultivated accessions of the same gene pool are grouped. The Andean natural populations have a very limited geographic distribution between Ecuador and northern Peru. The Mesoamerican wild form extends from Mexico up to Argentina through the eastern side of the Andes. Andean and Mesoamerican cultivated accessions of pantropical distribution contribute substantially to the genetic diversity of the Lima bean base collection. Population genetic parameters, estimated from allozymes, confirmed the predominant selfing mating system of the Lima bean. The selfing mating system, the occurrence of small populations, and low gene flow lead to an interpopulation gene diversity (D_ST=0.235) higher than the intrapopulation gene diversity (H_S=0.032). On the basis of the results, guidelines are given to preserve and exploit the genetic diversity of this threatened species. The results also confirm the independent domestication of the Lima bean in at least two centres, one of which is located at medium elevation in the western valleys of Ecuador and northern Peru. Received: 3 June 1997 / Accepted: 17 June 1997     

Regenerative medicine bioprocessing: Concentration and behavior of adherent cell suspensions and pastes

Regenerative medicines based on human cells demand their harvesting, culture, and processing. Manufacturing processes are likely to include cell concentration and subsequent controlled dosing of concentrates, for example, to the patient or tissue construct. The integrity and functionality of the cells must be maintained during these processing stages. In this study the performance of two different cell concentration protocols (involving centrifugation and resuspension) are compared and consideration given to possible causes of cell loss. Further studies examine cell size and rheological behavior of anchorage‐dependent mammalian cell suspensions, and the effect of capillary flow stress (0.5–15 Pa, laminar flow regime) on cell number and membrane integrity as quantified by flow cytometry. The cell concentration protocols achieved maximum cell volume fraction of around 0.3 and the improved protocol exhibited intact cell yield of 80 ± 13%, demonstrating proof‐of principle for achieving tissue‐like cell concentrations by a process of centrifugation and orbital shaking. Volume mean cell diameter (cell diameter at the mean cell volume) for the rat aortic smooth muscle cells (CRL‐1444) used in this study was 22.4 µm. Concentrated cell suspension rheology approximated to power law behavior and exhibited similar trends to reports for plant and yeast cells. Capillary transfer at 2–15 Pa (wall shear stress) did not significantly affect cell number or membrane integrity while losses observed at low shear (0.5, 1.0 Pa) were probably due to surface attachment of cells in the apparatus. Biotechnol. Bioeng. 2009;103: 1236–1247. © 2009 Wiley Periodicals, Inc.     

Design and development of a new ambr250® bioreactor vessel for improved cell and gene therapy applications

The emergence of cell and gene therapies has generated significant interest in their clinical and commercial potential. However, these therapies are prohibitively expensive to manufacture and can require extensive time for development due to our limited process knowledge and understanding. The automated ambr250® stirred-tank bioreactor platform provides an effective platform for high-throughput process development. However, the original dual pitched-blade 20 mm impeller and baffles proved sub-optimal for cell therapy candidates that require suspension of microcarriers (e.g. for the culture of adherent human mesenchymal stem cells) or other particles such as activating Dynabeads® (e.g. for the culture of human T-cells). We demonstrate the development of a new ambr250® stirred-tank bioreactor vessel which has been designed specifically to improve the suspension of microcarriers/beads and thereby improve the culture of such cellular systems. The new design is unbaffled and has a single, larger elephant ear impeller. We undertook a range of engineering and physical characterizations to determine which vessel and impeller configuration would be most suitable for suspension based on the minimum agitation speed (N_JS) and associated specific power input (P/V)_JS. A vessel (diameter, T, = 60 mm) without baffles and incorporating a single elephant ear impeller (diameter 30 mm and 45° pitch-blade angle) was selected as it had the lowest (P/V)_JS and therefore potentially, based on Kolmogorov concepts, was the most flexible system. These experimentally-based conclusions were further validated firstly with computational fluid dynamic (CFD) simulations and secondly experimental studies involving the culture of both T-cells with Dynabeads® and hMSCs on microcarriers. The new ambr250® stirred-tank bioreactor successfully supported the culture of both cell types, with the T-cell culture demonstrating significant improvements compared to the original ambr250® and the hMSC-microcarrier culture gave significantly higher yields compared with spinner flask cultures. The new ambr250® bioreactor vessel design is an effective process development tool for cell and gene therapy candidates and potentially for autologous manufacture too.

     

Inheritance and relationship between key agronomic and quality traits in an interspecific cross between Coffea pseudozanguebariae Bridson and C. canephora Pierre

The inheritance and relationships between four traits of agronomic and quality interest??fructification time, caffeine, and heteroside contents and 100-bean weight??were analyzed in the first backcross hybrids derived from an interspecific cross between Coffea pseudozanguebariae and Coffea canephora. We showed that short vs. long fructification time was governed by one major gene with two co-dominant alleles ft1 and ft2. Absence vs. presence of both caffeine and heteroside was also controlled by one major gene. The allele responsible for the presence of caffeine (caf2) dominated over the absence one (caf1) whereas both alleles controlling heteroside, het1 and het2, were co-dominant. The fructification time and the heteroside content were additive while the caffeine content seemed multiplicative. The 100-bean weight was additive and under a polygenic control. The two genes ft and caf were linked, separated by 30.8?cM, and were independent from the het gene. The relationships between the four traits were not strong enough, except between 100-bean weight and fructification time (r?=?0.43) or caffeine content (r?=?0.41). Recombination occurred between the genes controlling the four traits suggesting that new introgressed Robusta varieties, characterized by short, medium, or long fructification time depending on demand, bigger seeds with low or no caffeine content, and being heteroside-free, could be produced.     

Morphological and allozyme variation in a collection of Cucumeropsis mannii Naudin (Cucurbitaceae) from Côte d'Ivoire

To set up a rational collecting strategy for germplasm of the edible-seeded cucurbit Cucumeropsis mannii, a study was conducted using 24 morphological and seven putative enzyme markers to determine the intra-specific variability from 16 and 22 accessions (representing three cultivars), respectively. The analysis of variance, showed a significant difference between the three cultivars. Principal component analysis pointed out a variation among individuals, mainly on the basis of flower, fruit, and seed size. Dendrogram with UPGMA method allowed clustering of the cultivars. Genetic diversity indices estimated equalled: 9.96% for the proportion of polymorphic loci (P), 1.10 for the number of alleles (A) and 0.023 for observed heterozygosity (H_o). The level of the within accessions genetic diversity (H_S = 0.078) was higher than among accessions (D_ST = 0.042). Nei's genetic distances between the three cultivars were also low (0.079–0.147), indicating a high degree of similarity of the analysed cultivars.