Dr. Inez H. Slamet-Loedin

The purpose of the confined trial is to evaluate promising transgenic lines that contain foreign genes that store and enhance the transport of iron in the grain. The confined field test is vital to evaluate high-iron containing lines as compared to ordinary rice. Previous conventional breeding efforts resulted to only 6-8 ppm (parts per million) in the grain, falling short of the targeted level of 13 ppm or even higher to make a significant health impact based on rice-based diet. Again, such objectives can be only achieved with the help of genetic engineering. Rice lines that have iron, based on laboratory analysis, will be tested in what is called the Lead Events Nursery (LEN) and Observation Nursery (ON). LEN requires more seeds planted in four identical replicates; ON, on the other hand, has lesser number of seeds planted with only two replicates. Two promising rice plants that had high iron content from the two previous confined trials at lRRl and one at CIAT will also be planted in an area that is allocated for 'seed increase' or for further propagation of the seeds for future studies.

a) Before commencing with the activities, the DOST-BC and the Post Entry Quarantine Station, Bureau of Plant Industry (PEQS-BPI) shall inspect and approve the proposed site for the confined test (CT) located at Block F1-2 of the IRRI Experimental Station, Los Baños, Laguna

b) Prior to the initiation of the activities, the proponent/s shall discuss the monitoring schedule with the DOST-BC and the PEQS-BPI to identify the activities that will require their presence. The proponent/s shall furnish the DOST-BC and the PEQS-BPI with a copy of the agreed monitoring schedule.

c) The DOST-BC approved Project Information Sheet (PIS) shall be posted for three (3) consecutive weeks in at least three (3) conspicuous places in the barangay and/or city/municipal halls where the proposed activities will be conducted. The PIS should be posted at least 15 days before the actual transplanting of seeded transgenic rice in the confined test site. Proof of posting of the PIS, which may either be a certification from the duly authorized barangay leader(s) or an affidavit executed by the Proponent, should be submitted to the DOST-BC within ten (10) days from the last day of posting.

d) The proponent shall adhere as closely as possible to the schedule of implementation and monitoring activities for this trial.

e) Modifications in the planting schedule and other activities may be made only with the concurrence of the IBC and DOST-BC. f) At least 150 meters physical or temporal reproductive isolation of the GMO shall be observed at all times.

g) All confinement measures should be in place before the actual transplanting of rice seedlings.

h) The proponent shall ensure that only authorized personnel are allowed inside the test site. i) The DOST-BC and the PEQS-BPI shall be informed in advance of any visitations by unauthorized personnel.

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

k) The proponent shall ensure that stray animals are excluded from the test site while trials are being conducted.

l) The proponent shall ensure the security of the trial, the field workers and the monitors.

m) In case of undue destruction of the experimental materials resulting from unauthorized entry of personnel or breach of containment of the CT facility, the proponent must implement measures to prevent the inadvertent escape of any viable material in the site.

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

o) The rice grains or any plant part shall not be eaten or fed to humans, wildlife and livestock.

p) The proponent shall provide a space within the test site for disposal of the used plant materials.

q) The harvested grains shall be properly labeled and stored at the designated Seed Storage Facility where transgenic seeds are kept, while plant materials and other plant debris used within the test site should be buried in a pit within the test site or plowed under.

r) The proponent shall leave the test site fallow for at least 30 days. During the fallow period, the proponent shall strictly monitor emergence of volunteer plants and plant pests/diseases within the site for two (2) consecutive weeks after harvest. Furthermore, the proponent shall coordinate with the PEQS-BPI personnel during this period for the monitoring of the fallow site. If necessary, the proponent may irrigate the test site to induce germination of seeds. Any volunteer plants that will emerge, or any ratoon residues shall be uprooted and disposed either by plowing under or by burying in the pit intended for transgenic plant biomass from this confined field test.

s) The proponent shall comply with additional requirements that the DOST-BC may impose, as necessary, during the experiment period.

t) The proponent shall submit, through the IBC, a completion report 90 days after the official completion of the experiment which shall be determined by the PEQS-BPI monitors after completion of all post-harvest monitoring of volunteers.

u) The IBC shall report on all its monitoring activities upon completion of this project through the submission of the IBC completion report.

Biofortification of rice to increase micronutirent, particularly iron, is considered one of the options in helping solve the problem of micronutrient deficiency affecting a large percentage of the human population. Rice grain contains lower level of iron when milled. Increasing the iron content of the endosperm using iron storage protein and iron transporters could eventually introduce more iron into the grain. We have developed transgenic lines harboring iron storage protein gene, ferritin (NCBP ref. no. 1999-0119) and/or iron transporter genes in rice cultivar IR64 and high iron breeding line IR69428 under the NCBP approved project (NCBP ref no 2007-0221). These evenets have been evaluated in the transgenic screen house and increase in the iron content of several promising events was observed. This proposed confined field trial serves as a confirmation test of the performance of our two best lead events (1-IR64-IRS495-234 and 1-IR64-IRS495-27) selected from 12 transgenic events tested in the previous iron biofortification confined field trials (DOST-BC2011-0264 and DOST-BC2013-027). These will also serve as the benchmark of yield, iron and zinc levels (iron level close to targeted value of 13 ppm Fe was obtained in these events with a bonus of high Zn content). These two events will be included in the LEN (Lead event nursery) with other promising events (shown to have high Fe by ICP analysis and Prussion brlue staining) from other constructs. Testing of other promising events is needed to ensure that the best material will come out of the pipeline for the targeted goal of releasing  high-iron materials in the farmer's field. A total 17 entries which include transgenic events, nulls and controls (wild type and tissue culture will be planted in four replications in the LEN).

The Observation Nursery (ON) will be composed of 58 entries, including transgenic events, nulls and controls planted in two replications. Selection of entries for the ON is based on intensity of grain iron staining using Perl's Prussian blue but have limited amount of seeds. These events are derived from five constructs in the varietal background of IR64 or IR69428. In addition, conventionally-bred materials obtained from the crossing of event 1-IR64-IR495-234 and 1-IR64-IRS495-274 with other popular varieties will be included in observation nursery, including heterozygous and homozygous lines of 1-IR63-IRS495-234 and 1-IR64-IRS495-274 crossed with IR 64 to clean up the tissue culture effect. In all plotes corresponding nulls and controls will also be planted. A portion of the confiend trial site will also be devoted for seed increase of 1-IR64-IRS495-234, 1-IR64-IR495-274 and IR64 wild type to produce sufficient seeds for further research as was done in DOST-BC-2013-0276.

The sire of the confined field test is Blocks F1 & F2, IRRI Experimental Station, Los Baños, Laguna.

The duration of the field trials is from 01 August 2014 to 28 February 2015.

The overall objective is basically the same as the two previous confined test, i.e., to select transgenic event with high dietary Fe and Zn in polished rice, and desirable agronomic performance, suitab le for future multi location trials.

The specific objectives are:

1. To evaluate the agronomic performance and iron content of transgenic events containing ferritin seed storage protein or iron transporter genes or combination in IR64 background from lead events, their crosses and other promising events in observational trial.
2. To identify the best promoter-gene combination to increase iron content in polished rice from the selected events.
3. To increase the seeds of selected events (1-IR64-IRS495-234, 1-IR64-IRS495-274 and IR64 wild type) for future studies.