Project Information

Reference Number 20150288
Project Title Localization and expression studies of rice non-symbiotic hemoglobins through a GUS promoter reporter gene system to understand their role in tolerance of anaerobic conditions caused by different kind of floods
Project Type Contained Use (Greenhouse)
Status Ongoing
Name of Institution International Rice Research Institute
Cooperating Institution University of Manitoba, Canada
Supervising IBC International Rice Research Institute (IRRI)
Project Leader(s)

Abdelbagi M. Ismail
Inez H. Slamet-Loedin

Experimental Facility/Site Genetic Transformation Laboratory (GTL), Plant Breeding, Genetics and Biotechnology Division, DOST-BC approved facility for genetic transformation and tissue culture.Transgenic plants are grown under controlled conditions in CL4 greenhouse (KJ Lampe Laboratory). This facility has been approved for transgenic plant growth with standard measures for pest and disease control.
Purpose / Objectives

Non-symbiotic hemoglobins (nsHb) genes have been thoroughly studied in plants over the last 30 years (Appleby 1985; Arredondo-Peter et al. 1998). Despite existing knowledge in their ability to enhance tolerance of anaerobic conditions, their effectiveness in conferring tolerance of any type of flood has not been established in rice. Non-symbiotic hemoglobins have been found to function in seed development and germination, flowering, root development and differentiation, abiotic stresses like submergence responses, pathogen invasion and symbiotic bacterial associations. While progress has been made towards understanding their physiological role, a numbe of unanswered questions remain with respect to their biological function (Hill 2012). The aims of this study are to:

1) generate transgenic plants in rice backgound;

2) validate gene expression and localization changes of 5 known nsHb under different flooding stresses i.e. submergence during germination, flash flooding, complete submergence at vegetative stage and stagnant flooding up to reproductive stage;

3) validate tissue expression differences of 5 known nsHb genes under the above mentioned flooding stresses;

The generated transgenic lines will be used for destructive sampling and also for further studies. Part of the developed materils will be shared for testing with the collaborating institution, the University of Manitoba, Canada.

Biosafety measures

Transgenic and untransformed control plants will be grown in CL4 grenhouses and the biosafety screenhouse.

Biosafety measures that will be taken:

  • All prepared vectors will be kept in the plasmid collection of Genetic Transformation Lab, PGBG, 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 soil will be autoclaved at high temperature and high pressure.
  • 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.
Conditions for Approval

a) All activities shall be conducted at the Genetic Transformation Laboratory and CL4 Greenhouse 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 BPI-PEQS 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.

Date of Approval (DD-MM-YYYY) 27-06-2015
Date of Completion (DD-MM-YYYY) N/A
Executive Summary

Rice is a staple food for millions. As population and human activities rapidly increase, the concomitant deterioration of the environment threatens rice adaptability and productivity. The development of rice varieties with submergence tolerance during germination and vegetative stages is particularly important in Asia, where early foods are common or fields have poor leveling. At IRRI, current approach for generating transgenics is based on high-throughput integration through Agrobacterium tumefaciens, which can produce knock-outs of non-target genes and other undesirable effects because of its random nature. Obviously, site-specific integration method is highly desirable to obviate these effects.

Rainfed lowland and flood-prone rice areas in Asia cover about 47 million ha or about 35% of the total global rice area. There are various types of floods that currently affect rice growing areas and greatly reduce rice production (Ismail et al. 2012). Apparently each of these types of floods requires specific adaptive traits, which necessitates the development of unique varieties (Ismail et al. 2013). Major progress has been achieved with the SUB1 gene has been deployed in breeding but it confers tolerance of only short duration complete flooding (Ismail et al. 2013). This gene was functionally characterized through transgenic approaches. Several new genes need to be characterized to develop varieties with sufficient tolerance to the other submergence stresses.

Non-symbiotic hemoglobins (nsHb) genes are known to be effective in enhancing tolerance of anaerobic conditions, however, their effectiveness in conferring tolerance of any type of flood have not been established in rice. These nsHb have been an active research topic for over 30 years (Appleby 1985; Arredondo-Peter et al. 1998), during which time a considerable portfolio of knowledge has accumulated relative to their chemical and molecular properties, and their presence and mode of induction in plants (Hill 2012). Non-symiotic hemoglobins have been found to function in seed development and germination, flowering, root development and differentiation, abiotic stresses like submergence responses, pathogen invasion and symbiotic bacterial associations. While progress has been made towards understanding their physiological role, there remain a number of unanswered questions with respect to their biological function (Hill 2012).

The proposed project aims to study the function of the 5 known nsHb (Ross 2004; Garrocho-Villegas et al. 2007) in submergence tolerance of rice. To achieve the objective, plasmid with the constructs will be imported from Manitoba, Canada through an approved MTA. Five promoter:GUS constructs (one for each rice nsHb promoter) were prepared by Prof. Robert Hill at the Department of Plant Sciences, University of Manitoba, Canada. These promoter-reporter constructs will be evaluated for expression and localization in the seeds and plant parts in different flooding stresses: during germination, flash flooding and complete flooding at vegetative and stagnant flooding until reproductive stage. Their effectiveness in enhancing flood tolerance of rice during germination and vegetative stage will be checked. The duration of the project is expected to be about 3 years, after which the information gained from will be used to identify which nsHb genes or gene combinations will be effective in rice, which will then be used in breeding and for allele mining at IRRI. Part of the transgenic seeds generated will also be shared with the group of Prof. Hill in Canada, where appropriate biosafety procedures will be applied.