Project Information

Reference Number 20170307
Project Title Establishment of a robust system for haploid induction in rice
Project Type Contained Use (Greenhouse)
Status Terminated
Name of Institution International Rice Research Institute
Cooperating Institution N/A
Supervising IBC International Rice Research Institute (IRRI)
Project Leader(s)

Tobias Kretzschmar
Anindya Bandyopadhyay

Experimental Facility/Site International Rice Research Institute
Purpose / Objectives

The aim of this project is to generate proof of concept for haploid induction in rice. Haploid induction has the potential to reduce the breeding cycle by generating true breeding lines, fixed for all loci within a single generation. MTL/NLD and CENH3, two genes that have been previously demonstrated to induce haploidy in maize, barley and Arabidopis will be targeted using conventional T-TNA-based transformations and CRISPR/CPF1-based genome editing approaches. The optimal product is haploid inducer line that, when crossed with a line of interest produces haploid/doubled-haploid lines of interest at a rate that is suitable for production. Within scope of this project, this line will be of GM origin, generated either via conventional T-TNA-based transformations an/or CRISPR/CPF1-based genome editing.

Biosafety measures

All work is to be conducted inside approved biosafety facilities. Seeds are to be stored in approved facilities. Materials to be disposed of by heat sterilization (autoclaving) and/or deep burial. Institutional contingency plans for accidental release are in place.

Conditions for Approval

a) Activities shall be conducted at the Gene and Genomics Laboratory (GGL), C4 Laboratory, Genetic Transformation Laboratory (GTL), CL 4 glasshouse, and the TG-02 transgenic screen house located at the International Rice Research Institute in Los Ba├▒os, 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 IRRI-IBC and the DOST-BC.

d) Signage should be in place indicating that the facility is restricted/limited access;

e) The proponent must provide a logbook to record access of personnel to the experimental facility.

f) The proponent must take measures to ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities.

g) The experimental facilities must be kept under lock when not in use.

h) 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.

i) 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.

j) The DOST-BC should be informed in advance of any visitations by unauthorized persons.

k) The DOST-BC shall be informed immediately of any intrusion by unauthorized persons.

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

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

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

o) The proponent shall submit through the IBC a completion report within 90 days after completion of the experiment following the prescribed format.

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

Date of Approval (DD-MM-YYYY) 24-06-2017
Date of Completion (DD-MM-YYYY) 08-06-2018
Executive Summary

The aim of this project is to conduct proof of concept work to investigate the utility of CENH3-mediated and/or matrilineal (MTL/NLD)-mediated haploid induction in rice through a variety of complementary approaches. The optimal product is a haploid inducer line that, when crossed with a line of interest produces haploid/doubled-haploid lines of interest at a rate that is suitable for production. Within scope of this project, this line will be of GM origin, generated either via conventional T-TNA-based transformations and/or CRISPR/CPF1-based genome editing.

Haploid induction, where available, empowers and accelerates plant variety development. This novel technology has multiple applications, including, but not limited to, creation of doubled-haploid elite lines and haploid plants for genomics research (containing only one allele of each gene), and creation of cytoplasmic male sterile lines with a desired genotype. The main use of haploids is the rapid production of 100% homozygous inbred lines. Breeders use hybridization of different accessions and secual recombination to combine valuable traits. F1 hybrids require ~8 selfing generations to achieve homozygosity, a necessity for uniform seed production in many crops. This selfing phase can be bypassed by the induction of haploids from F1s, followed by chromosome doubling (spontaneous or colchicine induced) to produce perfectly homozygous dihaploids. Haploids can be produced by culturing and regenerating microspores or by crossing to a Haploid Inducer (HI), but this technology is greatly constrained by genotype and only applicable in selected varieties of certain species. In many, crop sepcies, such as rice, a practical haploid induction system is not available.

Genome elimination refers to selective loss of one parental set of chromosomes and it is one of the processes that result in haploids. In nature, it results from certain plant interspecific crosses in which one of the two parental genomes is lost in the early embryonic cell divisions. Using an Arabidopsis TILLING mutant of CENH3, scientist at UC Davis discovered that complementation with a CENH3 transgene altered in its amino-terminal region marks the genome for elimination when crossed to the wild type. CENH3 is a centromere specific histone variant that is required for establishing centromere identity. The method can transfer paternal chromosomes into maternal cytoplasm. Alternatively, MTL/NLD was characterized in maize knowledge about its exact mechanisms now enables investigation of its putative functionality in rice.

The project is estimated to run for five years. Facilities to be utilized include GTL, GGL, C4 laboratory, CL4 and the new TG-02 screenhouse at the Transgenic Research Facility or TRF, all at the International Rice Research Institute.