New Technologies in Biotech Cotton Dr. Neil Forrester, Director Neil Forrester Consulting Pty. Ltd. Sydney Australia,
[email protected] Abstract Since the first introduction of transgenic cotton in 1995, there have been an increasing number of biotech traits commercialised. So far these have been all input traits for bollworm control and herbicide tolerance, mainly from Monsanto but in the past few years, both Dow and Bayer and some local Indian and Chinese companies have entered the market. The current commercial cotton biotech products will be outlined followed by a discussion of likely near-term future biotech cotton products from Monsanto, Dow, Bayer, DuPont, Syngenta and some smaller national companies. Longer term possible biotech products will then be discussed as well as the potential contribution of new biotechnologies to facilitate cotton breeding. Current commercial biotech products The first cotton biotech product to be commercialised was BXN cotton from Calgene in 1995 (technology now with Bayer). This was a herbicide tolerant cotton resistant to bromoxynil, a photosystems II photosynthesis inhibitor (HRAC [Herbicide Resistance Action Committee] mode of action class C3). This was followed in 1996 by Monsanto’s Bollgard 1 (an insect tolerant cotton utilising Cry 1Ac protein from Bacillus thuringiensis) and in 1997, Monsanto’s Roundup Ready cotton resistant to glyphosate, an aromatic amino acid inhibitor at EPSP synthase (HRAC mode of action class G). This first version of glyphosate tolerant cotton had full vegetative (that is pre-squaring) tolerance but only limited reproductive (that is squaring, flowering, boll development and maturation) tolerance to glyphosate resulting in glyphosate use in this initial technology being restricted to 2 over-the-top sprays before the 4 true leaf stage followed by up to another 2 possible post-directed sprays to the base of the plant until row closure. Full vegetative and reproductive tolerance to glyphosate was not achieved until the release of Monsanto’s improved 2-gene Roundup Ready Flex technology in 2006 which allowed growers much greater flexibility in glyphosate spray applications (up to 3 over-the-top sprays to 16 nodes and one post-directed spray from 16-22 nodes plus 1 end-of-season spray if required). The only other herbicide tolerant biotech trait commercialised so far is Bayer’s Liberty Link cotton back in 2004, which is resistant to glufosinate, a glutamine synthesis inhibitor (HRAC mode of action class H). So far this technology has not been out-licensed in cotton but cross-licensing agreements have been announced in other crops. In regards to insect control technology, Monsanto released their Bollgard 2 stacked 2-gene technology in 2002 which included a new Cry 2Ab protein from Bacillus thuringiensis, alongside the previous Bollgard 1 Cry 1Ac gene. This certainly improved efficacy longevity and the range of pests controlled, as well as greatly enhancing the potential durability of the technology from a Resistance Management perspective. Monsanto has broadly out-licensed their biotech cotton technologies. Dow brought competition to the cotton insecticidal transgene market with the commercialisation of their Widestrike technology in 2004. Widestrike is also a 2-gene stacked technology utilising Cry 1Ac and Cry 1F genes from Bacillus thuringiensis but this technology has not yet been actively out-licensed. Dow has announced a cross-licensing agreement with Monsanto. Also from 1997, Biocentury from China has commercialised both single and 2-gene insecticidal technologies in China. These technologies were developed by the CAAS (Chinese Academy of Sciences) with the single gene product a ‘fused’ Cry 1Ac/Cry 1Ab gene from Bacillus thuringiensis and the 2-gene stacked product also containing CpTI (a trypsin inhibitor from cowpea). It is unclear if the 2-gene product is still being sold but there are claims that the single Cry
1Ac/1Ab-fusion gene technology accounts for around 80% of Bt cotton sales in China. However, Monsanto’s Bollgard 1 technology was also commercialised at the same time as the CAAS technology so it is almost impossible to accurately determine the surviving technology mix in China’s current varieties. Biocentury has also just received permission to commercialise their Cry 1Ac/Cry 1Ab-fusion technology in Indian cotton hybrids and are actively pursuing other markets and licensees. Also in India, the local Indian seed company JK Agrigenetics has commercialised a single gene insect tolerant cotton utilising a modified Cry 1Ac protein from Bacillus thuringiensis developed by the Indian Institute of Technology at Kharagpur. Near term developing biotech products There are still a number of new insect control and herbicide tolerant traits being developed by both current technology providers and some new players. rd
Monsanto has announced they are developing a 2-gene 3
generation Bollgard 3 insect control technology,
which will most likely be based on hybrid Bt proteins from Bacillus thuringiensis. This should improve the potential durability of the technology from a Resistance Management perspective and may also enhance the efficacy and range against some lepidopteran pests. Monsanto has also announced that they are developing a second herbicide tolerance trait to stack with Roundup Ready Flex. This new cotton technology will confer resistance to dicamba, a synthetic auxin growth regulator (HRAC mode of action class O). This should certainly help address the developing concerns over the increasing number of glyphosate resistant weeds which will be further exacerbated by the wider and more liberal application window afforded by the introduction of Roundup Ready Flex cotton in 2006. However, there may be volatility and drift issues with dicamba herbicide which will have to be closely watched and managed if required. Monsanto have also announced their work to develop Lygus bug tolerance which, if successful, will be the first transgenic technology to work on non-lepidopteran sucking insects. However, highly mobile insects such as the heteropteran mirids which have damaging adult as well as juvenile stages, will present a real challenge for insecticidal transgenes which have to be ingested first to work. Also some researchers have questioned whether the resistance should be directed against the insect itself or the secondary pathogens that they vector. Bayer are also developing their own Twin Link 2-gene insect control technology based on Cry 1Ab and Cry 2Ae from Bacillus thuringiensis. This will be stacked with their proposed 2-gene stacked Glytol / Liberty Link herbicide tolerant technology. Glytol will be Bayer’s own glyphosate resistance technology which will be stacked with their current Liberty Link (glufosinate resistant) technology. Dow have also announced the development of their own 2-gene stacked DHT herbicide tolerant technology. DHT stands for Dow Agrosciences Herbicide Tolerance Trait and is based on resistance to 2 separate herbicide classes: the synthetic auxin growth regulators (HRAC mode of action class O) and the aryloxy phenoxy propionate or “fop” herbicides (HRAC mode of action class A). Once again, this should help to manage potential herbicide resistance problems but the auxinic herbicides do present potential volatility and drift management issues. Syngenta are also close to commercialisation of their 2-gene stacked VipCot insect control technology based on Cry 1Ab and Vip 3A from Bacillus thuringiensis. The Cry 1Ab endotoxin protein is very similar to the Cry 1Ab and Cry 1Ac proteins used by all the other companies but the Vip 3a exotoxin has a unique mode of action and will be a very useful component for future resistance management programmes for insecticidal transgenic cottons. However, the recent acquisition of Syngenta’s VipCot commercilisation partner Delta and Pine Land Company, leaves some uncertainity as to the commercial future of this technology. Syngenta is also developing their own glyphosate tolerant cotton technology (Touchdown Tolerance).
Dupont has also recently announced the development of their new OPTIMUM GAT herbicide tolerance technology for cotton and other field crops. OPTIMUM is an umbrella brand name and GAT stands for Glyphosate ALS Tolerant which is based on resistance to 2 separate herbicide classes: glyphosate and the acetolactate synthase (ALS) inhibitor herbicides such as the sulfonylureas and imidazolinones (HRAC mode of action class B). The OPTIMUM technology is based on DuPont’s proprietary “gene shuffling” technique to optimise expression/activity of candidate transgenes. Once again, this will certainly help in the management of potential herbicide resistance problems. There are also a number of other companies/institutes developing various insecticidal transgenic cottons based on Cry proteins derived from Bacillus thuringiensis, including the Central Institute for Cotton Research in India (for Gossypium arboreum as well as G. hirsutum) and NIBGE and CAMB in Pakistan. An Australian company Hexima is also developing insecticidal transgenic cotton based on 2-gene stacked proteinase inhibitors from ornamental tobacco and potatoes. The first output traits which are likely to be commercialised in the near term are drought tolerance genes from Monsanto and possibly Bayer which could prove very timely for any future potential issues with climate change. Longer term potential biotech products There are quite a range of new biotech technologies being studied in cotton, mostly output traits but also some new input traits for disease and nematode control. Some of the more important ones are:- Disease tolerance, especially Fusarium tolerance in Australia and CLCV tolerance in India and Pakistan - Nematode tolerance in the US - Yield enhancement (including improved photosynthetic ability) - Improved nutrient use efficiency - Tolerance to high temperatures - Chilling tolerance - Salt tolerance - Waterlogging tolerance - Improved oil quality (eg. healthier high oleic cottonseed oils) - Improved fibre quality (length, strength etc) - Fabric quality (eg. Bayer’s work on flame retardance, improved chemical reactivity and anti-wrinkle) - Coloured cotton (so far unsuccessful) - Novel insect control products (eg. Dow’s work on toxins from Photorhabdus and Xenorhabdus symbionts from entomopathogenic nematodes) and toxins from spiders, scorpions, ant lions, parasitic wasps etc., and lectins, cyclotides, monoterpenes, peroxidases etc. New biotechnologies to facilitate cotton breeding Recent discoveries in cotton genomics have facilitated new biotech tools to help cotton breeders breed better cottons. New Marker-Aided Selection tools will help breeders select for rare traits of economic value or those left behind during the domestication of crops. Biotech breakthroughs will allow much of this previously tedious work to be conducted more efficiently by moving testing from the field to the lab. Gene chip microarrays will also allow the identification of large numbers (+ 10,000) of short sequences of DNA or RNA at one time which will allow the simultaneous tracking of many genes for complex traits such as fibre quality and stress tolerance. Conclusion
We have been growing commercial transgenic cotton crops for over a decade now and they have been grown or tested across a range of environments on all continents. These technologies have been rapidly adopted by cotton growers wherever they have been introduced and we have seen significant economic, environmental and human health and welfare benefits. The upcoming challenges of climate change, competing crops, production shifts and the continuing cost/price squeeze, will require us to continue developing and adopting new biotech solutions to help keep the world cotton industry viable.
New Technologies in Biotech Cotton Neil Forrester Director of Neil Forrester Consulting Pty. Ltd.
Current commercial Biotech Products 1995 - BXN from Calgene (now with Bayer) Herbicide tolerant (bromoxynil) – a photosystems II photosynthesis inhibitor – HRAC mode of action class C3
HRAC = Herbicide Resistance Action Committee
Current commercial Biotech Products 1996 - Bollgard 1 from Monsanto Insect tolerant (Cry 1Ac)
Current commercial Biotech Products 1997 - Roundup Ready from Monsanto Herbicide tolerant (glyphosate) – an aromatic amino acid inhibitor at EPSP synthase – HRAC mode of action class G - Vegetative tolerance only with limited spray opportunities (up to 4 true leaves)
Current commercial Biotech Products 1997 - Chinese Bt from Biocentury Insect tolerant Single gene product – ‘fused’ Cry 1Ab/Cry 1Ac 2 gene stacked product – ‘fused Cry 1Ab/c + CpTi CpTi = cowpea trypsin inhibitor
Current commercial Biotech Products 2002 - Bollgard 2 from Monsanto 2 gene stacked Insect tolerant (Cry 1Ac + Cry 2Ab) Improved efficacy and range of insects controlled Improved IRM = Insecticide Resistance Management
Current commercial Biotech Products 2004 - Liberty Link from Bayer Herbicide tolerant (glufosinate) – a glutamine synthesis inhibitor – HRAC mode of action class H
Current commercial Biotech Products 2004 - Widestrike from Dow 2 gene stacked Insect tolerant (Cry 1Ac + Cry 1F)
Current commercial Biotech Products 2006 - Roundup Ready Flex from Monsanto 2 gene Herbicide tolerant (glyphosate only) – both vegetative and reproductive tolerance Very flexible spray window
Current commercial Biotech Products 2007 - Indian Bt from JK Agrigenetics Insect tolerant (Cry 1Ac)
Near-term Biotech Products MONSANTO - 2 gene Bollgard 3 (based on hybrid Bt proteins?) - 2nd herbicide tolerance gene (dicamba) a synthetic auxin growth regulator HRAC mode of action class O - Sucking insect tolerance (eg. Lygus / mirids)
Near-term Biotech Products BAYER - 2 gene Twin Link Insect tolerance (Cry 1Ab + Cry 2Ae) - 2nd herbicide tolerance gene Glytol (glyphosate)
Near-term Biotech Products DOW - 2 gene herbicide tolerance DHT (Dow Agrosciences Herbicide Tolerance Trait) 2,4-D – a synthetic auxin growth regulator HRAC mode of action class O
+ “fop” (aryloxy phenoxy propionate) herbicides HRAC mode of action class A
Near-term Biotech Products SYNGENTA - 2 gene Insect tolerance VipCot (Cry 1Ab + Vip 3A) - Herbicide tolerance gene Touchdown (glyphosate)
Near-term Biotech Products DUPONT - 2 gene herbicide tolerance Optimum GAT (Glyphosate ALS Tolerant) Optimum = optimised gene expression through “gene shuffling” Glyphosate (HRAC mode of action class O) + Acetolactate synthase (ALS) inhibitor herbicides such as the sulfonylureas and imidazolinones HRAC mode of action class B
Near-term Biotech Products HEXIMA (Australia) - 2 gene Insect tolerance Unique proteinase inhibitors (NaPI + Pot 1) from wild tobacco (Nicotiana alata) & potato - 2 gene disease tolerance based on defensins
Near-term Biotech Products More Bt proteins India – Central Indian Cotton Research Institute, Nagpur (Cry 1Ac in both Gossypium arboreum and G. hirsutum) Pakistan – Cry 1Ac from NIBGE (National Instiute for Biotechnology & Genetic Engineering, Faisalabad) and CAMB (Centre for Applied Microbiology, Lahore)
Near-term Biotech Products Agronomic Traits MONSANTO – drought tolerance
Long-term Biotech Products Disease tolerance, especially Fusarium tolerance in Australia and CLCV tolerance in India and Pakistan
Disease Control Broad-spectrum pathogen signaling Selective natural compounds
Insect defensin A
Rs-AFPI
Long-term Biotech Products Disease tolerance, especially Fusarium tolerance in Australia and CLCV tolerance in India and Pakistan Nematode tolerance in the US Yield enhancement (including improved photosynthetic ability)
Yield Enhancement Published routes to enhance yield in plants: Over expression of: – Sucrose phosphorylase – Fructose 1,6 bisphosphate aldolase – ADP-glucose pyrophosphorylase – Brassinolide growth hormone Suppression of adenylate kinase Regulating cell cycle Siderophore production for micronutrient uptake Delaying leaf senescence Cellulose Binding Proteins, Expansins
Long-term Biotech Products Disease tolerance, especially Fusarium tolerance in Australia and CLCV tolerance in India and Pakistan Nematode tolerance in the US Yield enhancement (including improved photosynthetic ability) Improved nutrient use efficiency Tolerance to high temperatures
Long-term Biotech Products Chilling tolerance
Cold Tolerance Although cotton is a subtropical crop, modern genetics and production systems allows its growth in temperate climate However, the plants metabolism is still highly susceptible to cold Several strategies are being considered to enhance cold tolerance in cotton Lipid modification Stress response signaling Antioxidants
Long-term Biotech Products Chilling tolerance Salt tolerance Waterlogging tolerance Improved oil quality (eg. healthier high oleic cottonseed oils)
Oil Quality Improvement SATURATES 16:0 18:0
palmitic
stearic
MUFA ∆9 desaturase
18:1 oleic
Stable ↑ LDL
PUFA 18:2
∆12 linoleic desaturase
linolenic
Unstable ↓ LDL cholesterol
stable & healthy cooking oils
18:3
Oil Quality Improvement To use cottonseed oil for frying it is currently necessary to convert the polyunsaturated lipids to monounsaturated lipids – Hydrogenation can create trans (double bond) fatty acids from the normal cis double bonds. Trans fatty acids are considered less healthy than cis fatty acids.
Instead, can prevent the formation of polyunsaturated lipids by blocking the action of desaturases Stearic
18:0
CH3CH2CH2CH2CH2CH2 CH2CH2CH2CH2(CH2)7COOH
Oleic
18:1∆9
CH3CH2CH2CH2CH2CH2CH2CH2CH=CH(CH2)7COOH
Linoleic
18:2∆9,12
CH3CH2CH2CH2CH2CH=CHCH2CH=CH(CH2)7COOH
X
X
Linolenic 18:3∆9,12,15 CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2) 7COOH
Long-term Biotech Products Chilling tolerance Salt tolerance Waterlogging tolerance Improved oil quality (eg. healthier high oleic cottonseed oils) Improved fibre quality (length, strength etc)
Fiber Quality Proteins involved in cellulose biosynthesis are just now being elucidated. – Expansins – Extensins – CesA
Fiber Quality Proteins that control cellulose biosynthesis are just now being elucidated. Relationships between specific proteins and cellulose properties (strength) are being developed that hopefully will lead to improved fiber properties.
Long-term Biotech Products Chilling tolerance Salt tolerance Waterlogging tolerance Improved oil quality (eg. healthier high oleic cottonseed oils) Improved fibre quality (length, strength etc) Fabric quality (eg. Bayer’s work on flame retardance, improved chemical reactivity and antiwrinkle)
Long-term Biotech Products More drought tolerance
Drought Tolerance Cotton is the most drought-tolerant major crop when measured in dollars per mm of water (~$3 per hectare-mm). Enhancing drought tolerance of cotton would sustain farming in areas otherwise abandoned Several strategies are being considered to enhance drought tolerance in crops Osmotic adjustments Stress response signaling Heat shock proteins Stomatal regulation
Proline accumulation Trehalose accumulation Mannose accumulation Choline & Glycine betaine
Long-term Biotech Products More drought tolerance Coloured cotton (so far unsuccessful) Novel insect control products (eg. Dow’s work on toxins from Photorhabdus and Xenorhabdus symbionts from entomopathogenic nematodes) and toxins from spiders, scorpions, ant lions, parasitic wasps etc., and lectins, cyclotides, monoterpenes, peroxidases etc.
New Biotechnologies to Facilitate Cotton Breeding Two examples :Marker aided selection Gene Chip Microarrays
Marker-Aided Selection During the domestication of crops, genetic diversity within a species was limited – Selected for rare traits of economic value – Some traits left behind have value today
Bringing in genes from cotton’s wild relatives through traditional crossing is too time consuming Can facilitate movement of “left behind” traits Can speed variety development by moving some testing from the field to the lab
Gene Chips Microarrays Gene chip microarrays are tools to identify large numbers (+ 10,000) of short sequences of DNA or RNA at one time. Although the precision is not as great as PCR methods, microarrays can simultaneously track many genes allowing complex traits to be developed, such as fiber quality and stress tolerance.
SUMMARY SLIDES Herbicide Tolerance - Current BAYER – BXN (HRAC class C3) – Liberty Link (HRAC class H) MONSANTO – Roundup Ready & Roundup Ready Flex (HRAC class G)
SUMMARY SLIDES Herbicide Tolerance - Coming MONSANTO – Dicamba + RR Flex (HRAC / O + G) BAYER – Liberty Link + Glytol (HRAC / H + G) DOW – 2,4-D + “fops” (HRAC / O + A) SYT – Touchdown (HRAC / G) DUPONT – Optimum GAT (glyphosate + sulfonylureas / imidazolinones (HRAC / G + B)
SUMMARY SLIDES Insect Tolerance - Current MONSANTO – Cry 1Ac + Cry 2Ab DOW – Cry 1Ac + Cry 1F Chinese Bt – “fused” Cry 1Ab/Cry 1Ac Indian Bt – Cry 1Ac
SUMMARY SLIDES Insect Tolerance - Coming MONSANTO - Hybrid Bt proteins - Lygus (sucking pest) tolerance
HEXIMA - dual gene PIs (proteinase inhibitors) BAYER
- Cry 1Ab + Cry 2Ae
SYNGENTA
- Cry 1Ab + Vip 3A
India & Pakistan – more Bt proteins
