Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P <  0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T₀ plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.     

Detection and differentiation of the coconut lethal yellowing phytoplasma in coconut‐growing villages of Grand‐Lahou,Côte d'Ivoire

Surveys for the Côte d'Ivoire lethal yellowing (CILY) phytoplasma were conducted in eight severely CILY‐affected villages of Grand‐Lahou in 2015. Leaves, inflorescences and trunk borings were collected from coconut palms showing CILY symptoms and from symptomless trees. Total DNA was extracted from these samples and tested by nested polymerase chain reaction/RFLP and sequence analysis of the 16S rRNA, ribosomal protein (rp) and the translocation protein (secA) genes. The CILY phytoplasma was detected in 82.9% of the symptom‐bearing palms collected from all the surveyed villages and from all the plant parts. Trunk borings were recommended as the most suitable plant tissue type for sampling. Results indicate that the CILY phytoplasma may have a westward spread to other coconut‐growing areas of Grand‐Lahou. CILY phytoplasma strains infecting coconut palms in the western region of Grand‐Lahou exhibited unique single nucleotide polymorphisms on the rp sequence compared to the strains from the eastern region. Moreover, single nucleotide polymorphisms on the SecA sequence distinguished the CILY phytoplasma from the Cape St. Paul Wilt Disease phytoplasma in Ghana, and the Lethal Yellowing phytoplasma in Mozambique.     

In vitro induction of minitubers in yam (<Emphasis Type="Italic">Dioscorea cayenensis</Emphasis>- <Emphasis Type="Italic">D. rotundata</Emphasis> complex)

Two methods were used to produce yam minitubers from two different yam cultivars (cv. Krengle and cv. Kponan) using in vitro culture techniques. Method 1: Yam microtubers were first initiated in vitro and then transplanted to soil to generate plants from which minitubers were produced. Yam plants were obtained either by directly planting the microtubers to soil, or by inducing the germination of the microtubers using various chemical and physical treatments, before their transfer to soil. Method 2: Yam plantlets were first produced in vitro and then transplanted to soil for further development and tuber production. In both methods, the presence of jasmonic acid (JA) in the culture medium was found to be essential for yam tuberization, as well as for the germination of yam microtubers. In vitro production of yam microtubers was variety dependant. Compared to cv. Krengle, cv. Kponan responded better to microtuberization, and 2.5 μM JA was the optimum concentration resulting in 70 and 90% explants producing microtubers in the MS medium and the Tuberization medium (T-medium), respectively. Germination of the microtubers required treatment of JA at concentrations ranging from 1.0 to 2.5 μM. The overall length of the process to produce minitubers from microtubers took 32 weeks. In contrast, minitubers were obtained within 20 weeks when plantlets were directly transferred to soil. In this case, plantlets were first grown for 8 weeks on medium containing JA (0.1–1.0 μM) and 8% sucrose to initiate plant growth and rooting.     

Suppression of growth and cancer-induced angiogenesis of aggressive human breast cancer cells (MDA-MB-231) on the chorioallantoic membrane of developing chicken embryos by E-peptide of pro-IGF-I

E-peptide of the pro-Insulin-like growth factor-I (pro-IGF-I) is produced from pre-pro-IGF-I by proteolytic cleavage in the post-translational processing. Previous in vitro studies conducted in our laboratory showed that Ea4-peptide of rainbow trout (rt) pro-IGF-I or Eb-peptide of human (h) pro-IGF-I exhibited activities including induction of morphological differentiation, inhibition of anchorage-independent cell growth and suppression of invasion of several well established human cancer cell lines such as MDA-MB-231, HT-29, SK-N-F1, and HepG-2 (Chen et al. [2002] Gen Comp Endocrinol 126:342-351; Kuo and Chen [2002] Exp Cell Res 280:75-89). Seeding of aggressive human breast cancer cells, MDA-MB-231, on the chorioallantoic membrane (CAM) of 5 days old chicken embryos resulted in rapid growth and invasion of the cells and induction of blood vessel formation around the MDA-MB-231 cell mass in the chicken embryos. The invasion of MDA-MB-231 cells in the chicken embryos was further confirmed by immunocytochemistry. The rapid growth and invasion of MDA-MB-231 cells and the induction of blood vessel formation by MDA-MB-231 cells on chicken CAM are inhibited by treatment with a single or multiple doses of rtEa4- or hEb-peptide. Furthermore, a dose-dependent inhibition of angiogenesis by rtEa4- or hEb-peptide was also demonstrated by the chicken CAM assay. Results of microarray analysis of human gene chips (containing 9,500 unique cDNA clones) and confirmation by comparative real-time RT-PCR analysis showed that a group of genes related to cancer cell activities are up- or down-regulated in MDA-MB-231 cells transfected with a rtEa4-peptide gene. Together these results confirm the anti-tumor activity of rtEa4- and hEb-peptides, and further suggest that these peptides could be developed as therapeutics for treating human cancers.     

Morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured zygotic embryos

In this study, we compared the evolution of morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured embryos and from seeds over an 8-yr period. At the end of the second year after planting, the height and root collar diameter of plants originating from in vitro cultured embryos were significantly lower than those originating from seeds. However, palms from both categories had the same number of leaves. When palms originating from in vitro plantlets and from seeds were observed later in their development, they were similar for most morphological characteristics measured, except for minor differences in inflorescence morphology, which were still present 8 yr after planting. The flowering pattern, bunch, and fruit production were similar between the two categories of palms. These results indicate that in vitro culture of zygotic embryos does not adversely affect further development of palms in natural conditions.