Pulse transformation technology transfer

• Indian Institute of Pulses Research, India
• Centre for Legumes in Mediterranean Agriculture, Australia

Production of lentil (Lens culinaris) has doubled in the last decade through increases brought about by improved management practices and higher yielding cultivars. Despite these advances, yield increases are limited by a lack of genetic variation in host plant reaction to some pests and diseases, including pea leaf weevil (Sitona crinitus), Orobanche spp. and grey mould caused by Botrytis cinerea. India is the largest lentil producer and has a classical breeding program that is supported with genetic resources of ICARDA (the Centre also has capacity in tissue culture and molecular biology). CLIMA in Australia has developed a protocol to transform lupins and lentils with alien genes, which has been used to combat pests and diseases.

This project provided assistance in the transfer of the technology to transform lupins and lentils with alien genes from CLIMA to ICARDA, and through training into partner countries. The research had the secondary objective of increasing the efficiency of the transformation methodology.

The CLIMA protocol provided a mechanism to address the problems of pests and diseases. The CLIMA scientists attempted to transfer the transformation technology from CLIMA through training to ICARDA. After establishment of the technology, suitable applications would be defined and tested, such as: 1) Bt-constructs to combat Sitona (Bacillus thuringiensis, an effective and safe biological pesticide is commonly known as Bt); 2) fungal resistance to combat Botrytis and Fusarium wilt.
The project had planned to train national program scientists (of India and/or Turkey) in the technology. This aim was not pursued because of the unclear situation of intellectual property of the transformation technology. Instead lentil transformation and regeneration technology was established at ICARDA/AGERI (Agricultural Genetic Engineering Research Institute) in Egypt.

Bt-toxins of the CLIMA collection were used to screen adult insects and larvae of Sitona at ICARDA. However the Bt strains were only found effective against adult insects, not against larvae.
An ICARDA scientist was trained in the lentil transformation technology at CLIMA, Australia. The scientist then started to set up the technology at AGERI, Cairo, Egypt, and obtained early results suggesting that transformation of lentils with the CLIMA technology was possible.
Experiments undertaken at CLIMA during the training period yielded T0 and T1 seeds. The material still need to be tested at AGERI for BASTA resistance (BASTA is a unique broad spectrum, non-selective herbicide that provides excellent crop safety with a relatively low hazard to the operator). Once it is verified that stable transformants have been obtained, suitable constructs addressing major agricultural problems in lentil (Sitona resistance, herbicide resistance) can be sought.
Within the time frame of the project (2 years) transgenic plants were produced with reporter genes and herbicide resistance genes. Once it is confirmed that the technology is reliably established at AGERI, specific constructs providing biotic/or abiotic stress resistance to lentils will be developed. ICARDA will continue to use the technology at AGERI, and maybe later at ICARDA headquarters and will try to optimise the transformation efficiency. This will provide a basic tool for lentil improvement for the years to come.
The availability of isolated genes for other than research purposes is so far quite limited. Intellectual property in the hands of life science companies is unlikely to become available, or will be very restricted, for ICARDA mandate crops. It will be therefore necessary in the public sector to design and clone plant resistance genes. The availability of plant genome sequence data will ease such development.
No immediate benefit for Australian agriculture can be expected from this research project. However, with the transformation technology available at AGERI, future engineered biotic or abiotic resistance genes will become available for lentil.