Antonio C. Laurena

Amino acid composition, relative nutritive value and in vitro protein digestibility of several Philippine indigenous legumes

This study demonstrates variability in amino acid composition among accessions of several Philippine indigenous legumes. Moreover, two accessions ofD. lablab were identified to have high level of methionine (>2%). Methionine was identified as the first limiting amino acid with leucine, and threonine as the second limiting amino acids for most legumes studies.The IVPDs of the legumes under study ranged from >70 to 79%. Raw mature seeds had relatively low RNVs of 11 to 68% which increased to 68 to 94% and 51 to 89% after boiling and roasting, respectively.

Recent advances in the development of transgenic papaya technology.

Papaya with resistance to papaya ringspot virus (PRSV) is the first genetically modified tree and fruit crop and also the first transgenic crop developed by a public institution that has been commercialized. This chapter reviews the different transformation systems used for papaya and recent advances in the use of transgenic technology to introduce important quality and horticultural traits in papaya. These include the development of the following traits in papaya: resistance to PRSV, mites and Phytophthora, delayed ripening trait or long shelf life by inhibiting ethylene production or reducing loss of firmness, and tolerance or resistance to herbicide and aluminum toxicity. The use of papaya to produce vaccine against tuberculosis and cysticercosis, an infectious animal disease, has also been explored. Because of the economic importance of papaya, there are several collaborative and independent efforts to develop PRSV transgenic papaya technology in 14 countries. This chapter further reviews the strategies and constraints in the adoption of the technology and biosafety to the environment and food safety. Constraints to adoption include public perception, strict and expensive regulatory procedures and intellectual property issues.

Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.)

One prerequisite to reliable molecular biology work is that the genomic DNA of a sample be of good quality. Coconut is quite difficult to work on because of the high lipid and polysaccharide content of its endosperm and the high polyphenol content of its leaves. This study aimed to determine which protocol to use and which part of the coconut tree is most appropriate to extract good-quality genomic DNA. Genomic DNA from the solid endosperm was found to be of poor quality because of high levels of lipid and galactomannan contaminants. By using a modified protocol by Cheung et al. (1993) as modified by Rogers et al. (1996) and by Dellaporta et al. (1983) as modified by Datta et al. (1997), genomic DNA extracted from the young leaves of the first emergent frond provided enzyme-digestible, good-quality DNA. The modification involved the use of a higher salt concentration (2 M instead of 0.5 M) in the extraction buffer and the use of polyvinylpolypyrrolidone. Moreover, this modified protocol did not involve the use of organic solvents.

Effects of soaking in aqueous acidic and alkali solutions on removal of polyphenols and in vitro digestibility of cowpea

Mature dark red seeds of cowpea (Vigna unguiculata (L.) Walp.) UPL Cp 3, were subjected to several soaking treatments to remove their polyphenols. Soaking in water at room temperature for 8 and 24 h resulted in 17% and 21% loss of assayable polyphenols, respectively. Dilute solutions of alkali (Na2CO3, NaHCO3, NH4OH and KOH) and acid (CH3 COOH, HCl and H2 SO4) were more effective in removing polyphenols up to 88% than higher concentrations of alkali and acid solutions.

Effects of heat on the removal of polyphenols and in vitro protein digestibility of cowpea (Vigna unguiculata (L.) Walp.)

Simple wet heat treatments like simple boiling (atmospheric pressure, 100 °C) and pressurized boiling (higher than 100 °C) reduced the polyphenol content of mature dark red seeds of cowpea (Vigna unguiculata (L.) Walp.) cultivar UPL Cp 3 by 61 to 80% and improved in vitro protein digestibility (IVPD) by 6 to 26%. Pressurized steaming (higher than 100 °C) removed 48 to 83% of the polyphenols but increased IVPD by only 1.1 to 4.2%. Dry heat as exemplified by roasting and microwave treatment inactivated 58 to 71% of the tannins but increased IVPD by only 1%. All the heat treatments were effective in removing/inactivating polyphenols though different responses were observed with the resulting in vitro protein digestibilities.

Impaired plant growth and development caused by human immunodeficiency virus type 1 Tat

Previous attempts to express the human immunodeficiency virus 1 (HIV-1) Tat (trans-activator of transcription) protein in plants resulted in a number of physiological abnormalities, such as stunted growth and absence of seed formation, that could not be explained. In the study reported here, we expressed Tat in tomato and observed phenotypic abnormalities, including stunted growth, absence of root formation, chlorosis, and plant death, as a result of reduced cytokinin levels. These reduced levels were ascribed to a differentially expressed CKO35 in Tat-bombarded tomato. Of the two CKO isoforms that are naturally expressed in tomato, CKO43 and CKO37, only the expression of CKO37 was affected by Tat. Our analysis of the Tat confirmed that the Arg-rich and RGD motifs of Tat have functional relevance in tomato and that independent mutations at these motifs caused inhibition of the differentially expressed CKO isoform and the extracellular secretion of the Tat protein, respectively, in our Tat-bombarded tomato samples.

Towards transformation, regeneration and screening of papaya containing antisense ACC synthase gene

The papaya (Carica papaya L.) is usually being harvested when they are about 25% ripe or when a tinge of yellow color appears on the skin. It takes only one to two weeks before the fruit ripens completely from the time of harvest. Losses due to postharvest diseases in some species reach up to about 60% of annual production. The two most important qualities required for efficient marketing of fruits are the taste and the overall appearance. The flexibility in marketing is determined mainly by the rate of fruit ripening. An extended ripening phase would prolong its shelf-life, thus allowing the fruit to be shipped to distant markets without spoilage, thereby increasing the potential target markets, and enabling the fruit to reach the market in better condition. The papaya is a climacteric fruit and ripening is in part, being controlled by the simple hydrocarbon ethylene. The rate-limiting enzyme of the ethylene biosynthetic pathway is ACC synthase. This enzyme has been the target in engineering the ethylene biosynthesis pathway using the antisense technology. This technology for instance has been used to reduce translation of either ACC synthase or ACC oxidase by antisense RNA that blocks ethylene production in tomato, thereby delaying fruit ripening (Oeller et al., 1991; Hamilton et al., 1991). Two ACC synthase genes expressed during fruit ripening have been cloned from papaya (Mason and Botella, 1997). These genes could be used for transforming papaya plants that could constitutively express an antisense copy of the ACC synthase gene. In this study, we aimed to apply antisense technology and the existing transformation protocol to produce transgenic Solo papayas.

P19-16. Potential of an HIV-1 Tat fusion protein expressed in tomato as a plant-based HIV vaccine

Background Utilizing plants to produce pharmaceutical products, including antigens for AIDS, is a new way to produce vaccines. HIV-1 Tat plays a major role in viral replication and is essential for AIDS disease development making it an ideal vaccine target provided that both humoral and cellular immune responses are induced. Previous attempts to express Tat in tomato only reported successful humoral immune responses induction while certain physiological abnormalities were also observed in tomato which would be detrimental to furthering plant-based HIV vaccine development. In this study, an in-depth understanding of the effects of Tat expression in tomato and its immunogenicity was determined.