Dr. Inez H. Slamet-Loedin

To increase the iron content within the polished rice grains by: Introducing plant iron-storage genes including ferritin from rice, soybean and other commonly consumed fruits and legumes and rice iron transporters and chelators into popular rice cultivars (Note: Expression of iron storage genes, chelators and transporters from rice and soybean have been approved in proposal DOST-BC Ref. No. 2007-0221). Targeted genome mutagenes of regulatory elements from iron-related gene(s) using TALEN and CRISPR technology to improve iron loading to the rice grains (Note: This technology has been approved in proposal DOST-BC Ref. No. 2011-0258). Removal of selectable marker gene from rice transgenic lines obtained from previous research work covered in proposal DOST-BC Ref. No. 2007-0221 using TALEN and CRISPR technology (Note: This technology has been approved in proposal DOST Ref. No. 2011-0258)

Transgenic and untransformed control plants will be grown in CL4 greenhouses and the biosafety screenhouse. Biosafety measures that will be taken: All prepared vectors will be kept in the plasmid collection of Genetic Transformation Lab, PBGB, IRRI Los Banos. The tested plants will be kept in isolation in the CL4 transgenic greenhouse and/or biosafety screenhouse to prevent access of unauthorized personnel. Plant materials for disposal will be placed in sealed plastic bags. Plants grown in CL4 will be destroyed and the pots and soill will be autoclaved at high temperature and high pressure. Plant materials that will be grown and evaluated in biosafety screenhouse will be disposed through appropriate disposal system in compliance with biosafety and related regulations in the Philippines. Access to screenhouse is for authorized staff only. A barcode entry system is applied in the screenhouse to record the entry of personnel; a log book is available for visitors not having IRRI access card system. Screenhouse facilities are adequate to prevent entry of rats and birds. Transgenic seeds harvested from CL4 and the screenhouse will be kept in hermetically-sealed aluminum foil packages and stored in locked refrigerators designated for storage of transgenic seeds in Rice Biotechnology Laboratory, IRRI. Movement of all seeds and plant materials will be done in compliance of all relevant biosafety and phytosanitary requirements of the Philippines.

a) All activities shall be conducted at the Genetic Transformation Laboratory and CL4 Transgenic Greenhouse and biosafety screenhouse of the International Rice Research Institute (lRRl) in Los Baños, Laguna.

b) The proponent shall adhere as closely as possible to the schedule of activities reflected in the submitted Gantt chart.

c) Any modifications in the schedule of activities shall be made with the concurrence of the lRRl-IBC and the DOST-BC. d) A biosafety contingency plan shall be submitted before the conduct of the experiment.

e) The proponent shall inform which activities would require the presence of the IRRI-IBC, DOST-BC and PEQS-BPI personnel.

f) The proponent shall ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities.

g) The DOST-BC and PEQS-BPI personnel should be informed in advance of any visitations by unauthorized persons.

h) The DOST-BC and the PEQS-BPI personnel shall be informed immediately of any intrusion by unauthorized persons.

i) The proponent shall ensure that stray animals are excluded from the experimental facilities while tests are being conducted.

j) In case of undue destruction of experimental materials resulting from unauthorized entry of personnel or breach of containment of experimental facilities, the proponent shall implement measures to prevent the inadvertent escape of any viable material within the facility.

k) The proponent and the supervising IBC shall be held accountable for the undue destruction of the experimental materials and the consequences that their inadvertent escape may cause to the surrounding environment.

l) All viable materials within the experiment area shall be accounted for.

m) The proponent shall strictly observe proper disposal procedures for all materials.

n) Movement of all materials will be done in compliance with all relevant biosafety and phytosanitary requirements of the Philippines.

o) Any additional requirement that the DOST-BC may impose, as necessary, for the duration of the experiment shall be complied with.

p) The proponent shall submit through the IBC a completion report within 90 days after completion of the experiment.

q) The IBC shall submit to the DOST-BC a report on the completion of this project, in the prescribed format.

The World Health Organization (WHO) states that 2 billion people – or about 30% of the world’s population- are anemic, many due to iron deficiency. A recently published study at The Lancet confirmed anemia as a risk factor for maternal death, most likely due to hemorrhage, the leading cause (23%) of maternal deaths.

In developing countries, instances of iron deficiency anemia (IDA) are higher. Certain population groups, particularly women, children, and the elderly, are also more prone to developing IDA. In the Philippines, the National Nutrition Survey of 2008 revealed that IDA affected 19% of the national population. IDA is higher amongst infants (56%), followed by the pregnant women (43%) and the elderly (33%). For women and children, the main cause of IDA is an increased iron requirement coupled with very low iron intake.

This project is a continuation of the previous proposal entitled Biofortification for iron using iron transport related gene(s) submitted by IRRI and approved by DOST Ref. No. 2007-0221. Recently, an IR64 lead event over expressing NAS (iron/metal chelator) and ferritin genes (storage protein) has been developed at IRRI. Iron concentration of 12 ppm in polished seeds (unpublished results) was obtained. Our target to fulfill 30% of estimated human daily need is 14 ppm in polished rice grains. This target was set up by nutritionists based on bioavailability and retention studies in rice. In this proposed study, additional constructs expressing gene(s) related to absorption, translocation and storage of iron within the rice plants will be prepared to further increase the iron content in polished rice grains.

Therefore, the overall objective of the proposed study is to increase the iron content within the polished rice grains by

1. Introducing plant iron-storage genes including ferritin from rice, soybean and other commonly consumed fruits and legumes and rice iron transporters and chelators into popular rice cultivars (Note: Expression of iron storage genes, chelators and transporters from rice and soybean have been approved in proposal DOST-BC Ref. No. 2007-0221).
2. Targeted genome mutagenesis of regulatory elements from iron-related gene(s) using TALEN and CRISPR technology to improve iron loading to the rice grains (Note: This technology has been approved in proposal DOST-BC Ref. No. 2011-0258).
3. Removal of selectable marker gene from rice transgenic lines obtained from previous research work covered in proposal DOST-BC Ref. No. 2007-0221 using TALEN and CRISPR technology (Note: This technology has been approved in proposal DOST-BC Ref. No. 2011-0258).

The proposed 3-years study will be conducted in the previously DOST-BC approved facilities of IRRI, including the Genetic Transformation Laboratory, the CL4 glasshouse and the CS-01 screen house.