1. Enriching cassava through Bio-fortification and effects of environment on gene expression in Kenya Introduction Cassava food diets are mainly carbohydrate staples that provide low cost and poor quality nutrition to approximately 30% of rural poor in Western Kenya. Such foods results in ‘hidden hunger ‘ among vulnerable groups such as children under the age of five years. Vitamin A deficiency affects 30% children in Kenya leaving them with night blindness, severe eye infections such as Xeropthalmia and Keratitis, and low immunity which may lead to death. Supplementation, Food based approaches and Bio-fortification are important management approaches. Bio-fortification of a staple food allows poor courtesy on-line resource. people to consume Vitamin A daily, naturally at no extra costs. The Bio-Cassava plus project targets a 30 fold increase in the cassava staple. Aim: Evaluation of transgenic lines in different locations will help to predict the performance and stability of the trait. Materials and Methods: BSL II screen house: The BC+ tissue culture cassava plantlets were transferred to the screen house at KARI- Kakamega on 10 th April, 2011. They were unpacked, their condition checked and the number of plants verified. There were five (5) entries consisting of four (4) transgenic events and one (1) non-transgenic entry (Wild Type). Plants were hardened in the screenhouse over eight weeks Confined Field Trial: The Trial was planted on 22 nd June 2011 by the Kenya and DDPSC teams in the presence of the regulatory supervisory of the Kenya Plant Health Inspectorate Service (KEPHIS). The trial layout was designed using the ARM software and laid out in a Randomized complete block design. The total CFT area was 53 m x 38 m= 2014 m2 while the trial area measured 25 m x 12.5 m = 312.5 m 2. There were six entries planted : four events (DXS/PS 2; DXS/PS 20, DXS/PS 37, DXS/PS 73), the wild type (Cv 60444) and a local variety of yellow flesh, Nyaboda cv. The experiment was replicated three times with experimental plots measuring 6 m x 1.5 m and the rows spaced at 1.5 m. The replicates were separated by 2 m wide. Two guard rows of the wild type 60444 surrounded the experimental trial. Throughout the trial, the team maintained high stewardship quality. Enriching cassava through Bio-fortification and effects of environment on gene expression in Kenya Joyce Maling’a 1, Gichuki S.T. 1, Mallowa S 1., Obiero H.M. 1, Ndungu J.N 1., Solomon, F 2.,Gaitan-Solis E. 2, Solomon F. 3, Cahoon E. 3, Fregene M 2, and Anderson P 2. 1. Kenya Agricultural Research Institute, P.O. Box 57811-00100, Nairobi, Kenya. Email: joycemalinga@yahoo.com. 2. Donald Danforth Plant Science Center, P.O. Box Donald Danforth Plant Science Center (DDPSC). 975 N Warson Rd, St. Louis, MO 63132, USA. 3. University of Nebraska, E518, Beadle Center 1901, Vine St Lincoln, Nebraska.NK68588, USA Abstract: Cassava is a starchy food with carbohydrates accounting for 80% of the nutritive value. The levels of protein and micro nutrient are significantly low or absent in the majority of varieties preferred by farmers in various African countries. This is important during periods of food scarcity when most poor households eat cassava starch based diets devoid of any other supplementation. In rural and dry land western and coastal Kenya, cassava is eaten as gruel, ‘ugali’ or boiled from fresh roots. During dry seasons, children under five years of age depend on cassava gruel. In these areas, approximately 30% children suffer from mild to severe vitamin A deficiency (VAD). An ambitious project to enrich cassava meal has successfully introgressed DXS and PSY genes into a cassava line 60444. Evaluation of a confined field trial of four different events show that the genes have been over-expressed in the roots and significantly differ from that of the local check and the wild type. There are also differences between the events when plants are harvested at different plant ages. The results indicate that under AEZ’s similar to Kenya, Beta-carotene levels will be improved when these genes are introgressed into commercial cassava cultivars. DXS/PSY 73 Partial harvesting at 6, 9 and 12 months after planting The trial has undergone partial harvesting at 6 and 9 months. The final harvest is planned shortly. At each harvest, 3 root samples per plant and per plot have been taken to the laboratory for molecular and biochemical analysis. After the final harvest a stake trial will evaluate the stability of the beta-carotene trait in two subsequent clonal generations. Data Analysis The data is being analyzed with ANOVA and means differentiated with Student- Newman-Keuls test at P<0.05 using ARM 8.1 software. Preliminary results of partial harvest at 6 and 9 Months after planting (MAP) : At both 6 and 9 MAP, all the transgenic lines and the wild type were infected by Cassava anthracnose disease while the local check Nyaboda remained disease free. There no agronomic differences between the transgenic lines, the wild type and the local check at 6 MAP, although Event 37, performed moderately better in root numbers and weights. This performance remained consistently better than the wild type and the check, at 9MAP making it a promising line for selection. Table1: Means ± S.E per plant of Cassava Anthracnose disease (CAD) scores, Plant height (PHT), First Branch height (BHT), Plant Vigor, Total Root Number (Rt No), Marketable Root Number (Mkt Rt No), Total Root weight (Rt Wt) and Marketable weight(Mkt Wt) of Four events, a Wild type (WT) and a local yellow cassava check, Nyaboda at 6 MAP (Table IA) and 9 MAP (Table 1B) at Busia Alupe, Kenya in 2012. The CFT at Busia, Kenya The Biosafety level II Screenhouse at Kakamega, Kenya The plants being hardened in the screenhouse Conclusion Although the level of beta-carotene present in the transgenic lines root is yet to be determined, there is phenotypic expression of the orange color associated with beta- carotenoids. The high cost of investment for the development of this cassava will enable rural poor, and disadvantage persons such as children, productive mothers and HIV infected groups access daily ration of high quality but low cost staple food of high nutrition. Acknowledgement: USAID for travel grant, Kenya Agricultural Research Institute, Donald Danforth Plant Science Center and Nebraska State University for funds and GM technology capacity building. Event CAD scores (1-5)PHT(cm)BHT(cm) Plant Vigor (Sore 1-5)Total Rt NoMkt Rt No Total Rt Wt(kg)Mkt Wt(kg) DXS-PS 21.3 ab152 ± 8.168.3 + 8.3310 ± 1.21.0 ± 1.00.5 ± 0.70.2 ± 0.1 DXS PS 202.0 a168 ±16.863.7 + 9.7414.7 ± 3.24.0 ± 2.11.9 ± 0.71.2 ± 0.6 DXS-PS 372.0 a158.0 11.757.7 + 7.9417.3 ± 3.57.0 ± 3.81.7 ± 0.81.2 ± 0.8 DXS-PS 731.3 ab144.7 15.364.3 + 8.139.7 ±2.34.3 ± 2.21.3 ± 0.61.0 ± 0.7 WT1.7 ab142.7 ± 7.262.3 + 3.3318.3 ± 3.55.3 ± 1.31.3 ± 0.30.8 ± 0.2 Check1.0 b160.0 ± 5.738.7 +3.2515.3 ± 1.95.3 ± 2.21.1 ± 0.30.7 ± 0.3 Wild type (60444) DXS/PSY 20 L. check (Nyaboda) DXS/PSY 37 DXS/PSY 2 Event CAD scores (1-5)PHT(cm)BHT(cm) Plant Vigor (Sore 1-5)Total Rt NoMkt Rt No Total Rt Wt (kg)Mkt Wt(kg) DXS-PS 21.3 ab139.3 ± 15.636.7 ± 17.1315 ± 1.54.0 ± 1.01.1 ± 0.20.6 ± 0.1 DXS PS 202.0 a164.0 ± 8.652.0 ± 3.2514.7 ± 2.76.0 ± 1.52.2 ± 0.51.7 ± 0.4 DXS-PS 372.0 a161.3 ± 16.450.7 ± 5.2519.7 ± 2.78.3 ± 3.82.3 ± 0.72.3 ± 1.2 DXS-PS 731.3 ab146.7 ± 12.054.3 ± 4.5311.3 ± 2.26.7 ± 1.32.1 ± 0.31.8 ± 0.4 WT1.7 ab154.7± 6.067.3 ± 5.0315.0 ± 3.27.0 ± 0.62.5 ± 0.22.0 ± 0.1 Check1.0 b162.7 1.328.0 ± 3.5414.7 ± 3.48.0 ± 1.31.9 ± 0.61.4 ±0.4 Table IB (9 months after planting) Table IA (6 months after planting) Key: CAD scores (1- clean, 5- very infected; Plant Vigor scores, 1-weak, 5- very healthy BELOW: ROOTS HARVESTED AT 9MAP FROM THE FIELD RIGHT.