2010. Sustainable energy crop- An analysis of ethanol production from cassava in Thailand

2010. Sustainable energy crop- An analysis of ethanol production from cassava in Thailand

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ABSTRACT

The first essay formulates a dynamic general equilibrium optimal control model model attempts to allocate consumption, production, and factors of production to achieve  is used for both utility and production functions in the model. We calibrate the model with Thailand data. The selected model is used to generate the stationary state solution and to simulate the optimal policy function and optimal time paths. Two methods arc used: a linear approximation method and the Runke-Kutta reverse shooting method. The model provides numerical results that can be used as information for decision makers and stakeholders to devise an economic plan to achieve sustainable development goals.

The second essay studies the effect of international trade and changes in labor
supply, land supply, and the price of imported energy on energy crop production for bio fuel and food, as well as impacts on social welfare. We develop a dynamic general equilibrium model to describe two baseline scenarios, a closed economy and an open economy. We find that international trade increases welfare and decreases the energy price. Furthermore, resources are allocated to produce more food under the open economy scenario than the quantities produced under a closed economy assumption. An increase in labor supply and land supply result in an increase in social welfare. An increase in imported energy price leads to a welfare loss, higher energy production, and lower food production.

The third essay develops a partial equilibrium econometric model to project the impacts of an increase in ethanol production on the Thai agriculture sector over the next ten years. The model is applied to three scenarios for analyzing the effect of government ethanol production targets. The results from the baseline model and scenario analysis indicate that an expansion in ethanol production will result in a significant increase in cassava production, price, and land use. The increase in cassava production will shift land use from maize and sugar cane, thus increasing in price of maize  .

2010. Genetic diversity and differentiation of cassava accessions in Puerto Rico

2010. Genetic diversity and differentiation of cassava accessions in Puerto Rico

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ABSTRACT

Cassava is consumed daily by 600 million people living in the poorest places in Africa, Asia and South America. Natural genetic variation that exists among different cultivars of cassava has been studied in order to assist in the reduction of constraints present in cassava. In Puerto Rico little is known about the genetic diversity of cassava. In order to access genetic diversity, 162 samples were collected from different townships of the island and were analyzed by 33 SSR markers on polyacrylamide gels (detected using a fluoresce nee-based technique using an automated DNA sequencer), and compared with the Puerto Rican cassava collection using NTSYS, GENSURVEY, FSTAT and GEAIE software programs. Our results showed that genetic diversity (He) of the unknown cassava samples (0.7242) is higher than in the Puerto Rican cassava collection (0.7026). Furthermore the observed proportion of hctcrozygotcs (Ho) was higher in Puerto Rican cassava collection compared to the unknown samples. The proportion of inbreeding coefficient (Fis) showed a larger proportion of heterozygotes in the unknown samples than in the Puerto Rican cassava collection. The genetic diversity evaluated across all loci and all populations (Ht) was high (0.7416). In general the highest genetic diversity was present within populations (0.7134). There was no genetic differentiation between populations (Dst= 0.0235) and there is no gene differentiation of populations (Gst= 0.0375 ± 0.0466). The UPGMA analysis showed that most of the samples clustered with the varieties of the Puerto Rican cassava collection. However there was one cluster with only unknown cassava. From this cluster the unknown sample SpL-27 presented the highest genetic dissimilarity (71%). At least 4 pairs of unknown cassava gathered from different regions showed the same identity (Fal071-129 and FalO 17-130; Fal041-121 and Fal067-124; Fal017-131 and FalO 17-132; FalO-19 and Fal0-20). The high levels of heterozygosity found in Puerto Rican cassava can be due to three reasons. First, the high levels of heterozygosity in the local accessions could be due to the high heterozygosity inherited from cassava ancestors. Second, sexual reproduction occurring in vegetative propagated crops have been shown to increase heterozygosity due to farmer practices which allow sexually reproduced seedlings to germinate and to be subsequently incorporated in to the next planting. This new genotype might then be named based on an existing morphologically similar variety. Third, the accumulation of fixed somatic mutations, the frequency of mutations depends on both the velocity of those changes occurring and the selection of the individuals which have them. The levels of genetic diversity found in this study, revealed a high potential for local Puerto Rican cassava to be part of a genetic improvement program in the future  .

2009. Pre-absorptive metabolism of carotenoids

2009. Pre-absorptive metabolism of carotenoids 

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ABSTRACT

Vitamin A deficiency (VAD) is still a major public health issue in developing countries. Biofortification of staple crops with pro-VA carotenoids is proposed as a complementary and sustainable approach to alleviate VAD. However, it is unknown if the additional pro-VA carotenoids accumulated in the staple crops will be bioavailable. First, I focused on screening the relative bioaccessibility of /7-carotene (BC) from ten cultivars of cassava using the coupled in vitro digestion/Caco-2 cell model. All-/r<my-BC, 9-cA-BC, and 13-cA-BC were the most abundant carotenoids in boiled cassava and recoveries after digestion exceeded 70%. Efficiency of micellarization of total BC was 30% for various cultivars with no significant difference among isomers and was linearly proportional to concentration in cooked cassava. Accumulation of a 11-/raws-BC by Caco-2 cells incubated with the diluted micelle fraction for 4 h was proportional to the quantity present in micelles. In addition to boiling, cassava is also traditionally prepared by fermentation and roasting. These different methods of preparation have the potential to affect both the retention and bioaeccssibility of BC. So I next focused on the effects of traditional preparation of cassava on retention and bioaeccssibility of BC in boiled cassava, gari (fermentation and roasting), and fufu (fermentation and boiling the paste). Apparent retention of BC was approximately 90% for boiled cassava and fufu. In contrast, roasting fermented cassava at 195°C for 20 min to prepare gari decreased BC content by 90%. Retention was increased to 63% when temperature was decreased to 165°C and roasting was limited to 10 min. The efficiency of micellarization of all-tram and cis isomers of BC during simulated digestion was 25-30% for boiled cassava and gari and independent of cultivar. However, miccllarization of BC isomers during digestion offufit was only 12-15%. These differences in retention and bioaccessibility of BC from cassava products prepared according to traditional processing methods suggest that gari and fufu may provide less retinol activity equivalence (RAE) than isocaloric intake of boiled cassava.

It is noteworthy that cassava has only BC as the primary carotenoid in the tuber. However, maize, another staple crop targeted for biofortification also has /7-cryptoxanthin (B-CX) and lutein (LUT) and zeaxanthin (ZEA). Studies have demonstrated interaction between carotenoids, therefore primary aim of my next study was to examine if LUT and ZEA attenuate the bioacccssibility of pro-VA carotenoids at amounts and ratios present in maize. BC incorporation into micelles during chemical preparation and during in vitro digestion of carotenoid-enriched oil was slightly increased when the concentration of LUT was 6-fold or greater than BC. Likewise, the efficiency of BC micellarization was slightly increased during simulated small intestinal digestion of white maize porridge supplemented with oil containing 9-fold molar excess of LUT to BC. Mean efficiencies of micellarization of BC, BCX, LUT and ZEA were 16.7, 27.7, 30.3 and 27.9%, respectively, and independent of the ratio of LUT plus ZEA to pro-VA carotenoids during simulated digestion of maize porridge prepared from flours containing 0.4—11.3 pg/g endogenous pro-VA carotenoids. LUT attenuated uptake of BC by differentiated cultures of Caco-2 human cells from medium containing micelles in a dose-dependent manner with inhibition reaching 35% when the molar ratio of LUT to BC was 13. Thus, bioaccessibility of pro-VA carotenoids in maize is likely to be minimally affected by the relative levels of xanthophylls present in maize.

Manipulation of intracellular retinoic acid (RA) concentration is needed to examine the impact of inadequate VA status on the transport and metabolism of dietary carotenoids and other activities of enterocytes. Here, I examined whether citral induces RA deficiency in the TC-7 clone of Caco-2 cells. 1 assessed RA status by monitoring the expression of several genes, and particularly ^-carotene 15, 15' monooxygenase 1 (BCOl), regulated by this ligand for retinoid acid receptor/retinoid X receptor. Differentiated monolayers of TC-7 cells first were exposed to various concentrations of citral for 16h to define non-cytotoxic doses. Lower concentrations (0.1-10 pM) of citral were not toxic for Caco-2 cells. The relative expression of BCOl dose dependently increased in cultures treated overnight with 0.1-3 pM citral; increased time of exposure did not further elevate BCOl mRNA. Citral mediated changes were blocked by simultaneous presence of 10 pM retinoic acid in medium. BCOl activity in cell lysates and intact cells likewise significantly increased after pre-treatment with 1.0 pM citral for I6h. However, there was no significant difference in the amount in cellular accumulation and transepithelial transport of BC by control and citral treated cells. Relative expression of several other genes involved in retinoid and carotenoid metabolism showed that exposure to citral increased scavenger recepeptor type B class I and retinaldehyde dehydrogenase 1 mRNA and decreased cellular retinoic acid binding protein 1 and cytochrome p450 26A1 compared to control. Collectively, the results suggest that citral induced a moderate deficiency of RA in Caco-2 cells. Thus, citral represents an effective tool for the future investigation of the impact of VA and therefore RA deficiency on various activities of enterocytes  .

2009. Application of simple sequence repeat (SSR) markers for genotype differentiation of 24 cassava (Manihot esculenta Crantz) accessions

2009. Application of simple sequence repeat (SSR) markers for genotype differentiation of 24 cassava (Manihot esculenta Crantz) accessions

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ABSTRACT

The genetic diversity and differentiation of 24 cassava (Manihot esculenta Crantz) accessions from the Puerto Rican collection are assessed in this study. In addition, 63 cassava DNA samples from the CIAT (Centro Internacional de Agricultura Tropical) germplasm collection, representing Latin America (Colombia, Costa Rica, Guatemala, Mexico, and Brazil) and Africa (Benin, Cameroon, Ghana, Nigeria, and Sierra Leone) were included from previous cassava diversity studies. Using simple sequence repeat (SSR) markers, variation in allele frequency at 37 unlinked loci was used to estimate genetic diversity and differentiation and to find the relationships between these 24 accessions and their cultivated relatives from primary and secondary diversity centers. The SSR markers were chosen because they represent a broad coverage of the cassava genome with moderate to high polymorphism information content (PIC) and robust amplification. For all the tested SSR loci studied there were on average 96.85 % ± 6.86 polymorphic loci across all country samples with an average of 4.58 ± 1.83 alleles per locus, and a mean observed heterozygosity of 0.7 ± 0.055. Despite the low level of differentiation [FSt (theta) = 0.047± 0.005] found between country samples, sufficient genetic distance (Pairwise Nei’s and Fst distances) existed between individual accessions to separate them according to their country of origin. UPGMA analysis of country samples revealed that Puerto Rican accessions clustered together with South American landraccs. The SSR markers detected two genetically identical accessions in the Puerto Rican collection and led us identify the possibly closest cultivated relatives from Brazil and Colombia for some of the collection’s accessions.

Finally, we evaluate the embryogenic capacity of the accessions present in the in vitro collection using the conventional solid induction medium system with 8 mg/1 of 2,4-D. The explants used were young leaf lobes isolated from in vitro grown plants. Somatic embryogenesis was achieved in 58 % of the accessions, but only SG804, CM3064, Tremesiana, PI 12900, PI 12903 and Seda produced germinated somatic embryos. Differences found among the explants for their capacity to form embryogenic structures and germinate could be due to their intrinsic genetic characteristics. Our results support the thought that the capacity to produce embryogcnic tissue in cassava varies among different accessions. Furthermore, the solid system used could be forming gradients in the uptake of nutrients and hormones promoting variation and reducing the growth of the embryogenic structures  .

2003. Somatic embryogenesis and transformation of cassava for enhanced starch production

2003. Somatic embryogenesis and transformation of cassava for enhanced starch production

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ABSTRACT

Cassava (Manihot esculenta C'rantz), a member of the family Euphorbiaceae, is one of the most important food crops of sub-Saharan Africa. It is grown throughout the tropics including Asia and Latin America. The five main producers are; Nigeria, Brazil, Thailand, Zaire and Indonesia, which together account for 63% of the total world production of 120-130 million tons of fresh roots per year. The roots are the primary plant part consumed and store abundant amounts of starch. They provide a valuable source of cheap calories for about 500 million people, many of whom are subsistence farmers. In addition, cassava leaves and tender shoots are eaten as a vegetable in many parts of Africa and are an excellent source of vitamins, minerals and protein. Although most cassava is consumed by humans, it is also used in the production of ethanol for fuel, for animal feed, and as a raw material for the starch industry.

Cassava’s high photosynthctic rate, ability to grow on poor soils and its resistance to many pests and herbivores due to the presence of cyanogens make it an ideal crop for subsistence farmers. Furthermore, cassava is largely propagated clonally making it an ideal plant for improvement through genetic engineering.

One of the requirements for the generation of genetically engineered cassava is an efficient and reproducible plant regeneration and transformation system. One of my research objectives was to improve upon current technologies used for cassava transformation. We describe here improved methods for plant regeneration of recalcitrant African cassava cullivars. This technology will allow us to increase the range of cultivars that can potentially be engineered using recombinant DNA technologies.

One of the constraints for cassava starch production is the long growing season. Cassava typically takes 9-12 months to yield a good harvest. This is longer than other major starch-producing crops such as com and potatoes. The longer growing season of cassava also means that it may need more maintenance in the field than other crops. One objective of my research program was to increase the starch biosynthesis capacity of cassava by enhancing the enzyme activity of ADP-glucose pyrophosphorylase (AGPase), the rate-limiting enzyme in starch biosynthesis. To do this, we transformed cassava with a modified E. coli glgC gene that encodes AGPase. The glgC gene was modified by site-directional mutagenesis (K296E/G336D) to remove the allosteric regulation (enhancement by fructose-1, 6-P and inhibition by adenine monophosphate (AMP) sites and to increase the velocity of the enzyme. Root-specific expression of the glgC gene product was achieved using the tuber-specific patatin promoter of potato. We obtained antibiotic-resistant putative transformed plants which have been shown to have integrated and expressed the transgene by PCR, Southern blot, RT-PCR, and enzyme activity analyses. AGPase enzyme activity in transformed plants was increased by more than 65%. Transgenic plants expressing the bacterial glgC gene had two-fold greater stem and root biomass than wild-type plants.

Cassava has a high efficiency of photosynthetic conversion of carbon dioxide into assimilates. We also postulated that we could also enhance starch biosynthesis by increasing sucrose biosynthesis in cassava leaves. This was done by transforming cassava with maize sps gene that encodes sucrose phosphate synthase, the enzyme that catalyzes sucrose synthesis in leaves. Expression of the sps gene in leaves was driven by the CAB1 promoter which is leaf specific. Southern blotting, RT-PCR and sucrose phosphate synthase enzyme assays were used to demonstrate enhanced expression of the maize sps gene and increased SPS enzyme activity (58% to 82%). Given the long-term nature of field trials it is estimated that growth analysis studies will extend an additional
year from the current time  .

1997. Genetic engineering approaches to improve agronomic traits in cassava (Manihot esculenta Crantz)

1997. Genetic engineering approaches to improve agronomic traits in cassava (Manihot esculenta Crantz)

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Cassava (Manihot esculenta Crantz) is a tropical tuber crop that is grown for its starchy, thickened roots. The cassava roots are used mainly as a food source or for animal feed. Due to the presence of cyanogenic glycosides, however, cassava is potentially toxic. This and other aspects of cassava are potentially amenable to improvement through genetic manipulation. Cassava is a highly heterozygous plant with low natural fertility which makes genetic manipulation via traditional breeding methods very long and difficult. Genetic engineering is an alternative approach to circumvent this problem and modify some aspects of cassava such as: starch quality and quantity, resistance to pests and diseases, and reduction of cyanogenic potential.

Linamarin, a cyanogenic glycoside, is stored in the root, stems and leaves of cassava and upon tissue damage (such as food preparation) is broken down by linamarase to produce acetone cyanohydrin. Acetone cyanohydrin can break down to produce acetone and hydrogen cyanide either spontaneously or by the action of hydroxynitrile lyase (HNL). Of these three cyanogens (linamarin, acetone cyanohydrin, and hydrogen cyanide) acetone cyanohydrin is the main contributor to consumer cyanide exposure.

Here, we report for the first time the stable transformation of cassava via Agrobacterium-mediated system with a gene of agronomic interest. We have cloned a HNL cDNA into an Agrobacterium binary vector under the control of a double CaMV 35S promoter. The modified binary vector was transformed into two different strains of Agrobacterium, LB A 4404 and EHA105 which were used for stable transformation of cassava. In vitro apical leaves and germinated somatic embryos of a cassava cultivar, Mcol 2215, were used to regenerate transgenic cassava plants resistant to paromomycin after co-cultivation with Agrobacterium. The overall efficiency of transformation was approximately 2.8%, however, when only apical leaves were cocultivated with the modified LBA4404, the transformation efficiency increases to 5.5%. All plant DNA evaluated so far by PCR amplification of specific introduced genes indicates integration of the selectable marker, April, and the gene of interest,
HNL.

Hydroxynitrile lyase from leaves and stems of untransformed plants had an activity of 1.7 mmol HCN/mg protein/h versus 2.4, 3.8, 4.0 mmol HCN/mg protein/h from three different transformed plants. Western blots of untransformed and transformed leaf-stem total protein support the higher activity of hydroxynitrile lyase in at least one of the transformed plants. However, HNL has not yet been detected in root tissues of transformed plants  .

1996. Characterization, purification, and molecular cloning of hydroxynitrile lyase from cassava

1996. Characterization, purification, and molecular cloning of hydroxynitrile lyase from cassava


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ABSTRACT

Cassava (Manihot esculenta Crantz) is a staple food crop in Africa. The cyanogenic glycoside, linamarin, is stored in cassava cells and probably functions as an herbivore deterrent. When cassava roots are prepared for human consumption, linamarin and its degradation products, acetone cyanohydrin and hydrogen cyanide, can remain in the food products. Exposure to these cyanogens has been linked to epidemics of neurological disorders in Africa. Acetone cyanohydrin is the main contributor to cyanide exposure from cassava consumption.

We purified hydroxynitrile lyase, which converts acetone cyanohydrin to acetone and hydrogen cyanide, from cassava leaf apoplast extracts and whole leaves. The enzyme was a homodimer of 29 kD subunits. The pH and temperature optima were 5.0 and 30°C, respectively. The enzyme displayed typical Michaelis-Menten kinetics. Activity was saturated by 20 mM acetone cyanohydrin and the Km was 0.925 mM. Hydroxynitrile lyase maintained over 50 percent of its optimal activity at pH 4.0-5.5 and temperatures 4-45°C. Also, the enzyme was not permanently inactivated by 24 hours of exposure to low pHs (4.0-6.0).

Anti-hydroxynitrile lyase antibodies were raised and used to localize the enzyme. Using FITC-labeled secondary antibodies, hydroxynitrile lyase was found in the cell walls of leaf epidermal, parenchyma, and mesophyll cells. Western blots demonstrated the presence of hydroxynitrile lyase in leaves and not stems or roots.

The gene encoding hydroxynitrile lyase was isolated from a cassava leaf cDNA library. The 1.1 kb cDNA clone encoded a 258 amino acid protein. The derived amino acid sequence had 78 percent identity to rubber tree (Hevea brasiliensis) hydroxynitrile lyase and 30 percent identity and 56 percent similarity to two pathogen induced response proteins from rice (Oryza sativa). Southern blot analysis supported the presence of one copy of the hydroxynitrile lyase gene in the cassava genome. Little expression of the hydroxynitrile lyase gene (1.3 kb mRNA) was found in cassava stems and roots  .