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In vitro manipulations and strategies for the improvement of sugarcane
cultivars in South Africa
Sandy Snyman South African Sugarcane Research Institute
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SA Sugarcane Research Institute
• 500 employees in 5 Resource centres • 6 research farms • 50 scientists • 15 postgraduate students + 20 interns
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Sugar industry in SA
• ranked in top 15 producers globally • 423 000 ha sugarcane planted • 35 000 growers, 80% small-scale
account for 11% of the crop • sugarcane crush: 18.9 million tons • sugar production: 2.2 million tons • total average industry income: R 8
billion p.a • job opportunities: 77 000
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In vitro applications in:
1. Micropropagation 2. Virus elimination 3. Genetic engineering 4. Mutagenic breeding 5. Germplasm conservation
Reference: Snyman et al. (2011) In Vitro Cell Dev Biol-Plant 47:234-249
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Routes of sugarcane regeneration
Conventional vegetative propagation using setts
Indirect morphogenesis
Indirect shoot formation
Indirect somatic embryogenesis
Direct morphogenesis
Direct shoot formation
Direct somatic embryogenesis
• •
• • • •
•
• • •
• • •
Shoot meristem culture
Immature leaf roll
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1. Micropropagation
Limitations of conventional vegetative propagation make in vitro propagation an attractive alternative
Conventional vegetative propagation is routine in sugarcane Problems associated with this: • Spread of disease • Slow rate of propagation • Affects adoption rate of new cultivars
At SASRI two different routes of morphogenesis have been used: somatic embryogenesis and meristem culture
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Micropropagation: somatic embryogenesis
Collection of tops from field Immature leaf roll cultured
RITA® vessel
Embryo production
Embryo germination
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Meristem excision
Micropropagation: meristem culture
Shoot multiplication
Bud germination or field collection of stalk apices
Shoot establishment
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1. Micropropagation NovaCane®: • disease-free tissue culture plants generated in laboratory and planted in field for seedcane
• registered as a Trademark by SASRI
• The technology has been tested on several commercial cultivars N12, N16, N19, N25, N31, N32, N36, N40, N41, N46, N49, N50
• The multiplication rate after 5 months varies between genotype: 800-3,500 plants /meristem 500-10,000 plants/30 leaf discs (somatic embryogenesis)
• Two commercial labs, DuRoi (Letsitele) and Dube AgriLab (Durban) are using NovaCane® to supply plants to sugarcane growers for seedcane purposes
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2. In vitro culture and virus elimination
• Thermotherapy and meristem culture can shorten this by half and can clean up germplasm infected with viruses such as SCYLV and SCMV
• Traditional screening process involves molecular disease-indexing and vegetative growth and takes 2-3 years prior to release of germplasm
• SASRI Quarantine – facilitates import of foreign germplasm and disease-indexes to ensure free of causal agents
References: Ramgareeb et al. (2010) Plant Cell Tiss Org Cult 100:175-181 Snyman et al. (2012) Functional Plant Science and Biotechnology 6(2):12-18
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2. In vitro culture and virus elimination
Meristem size (mm)
After 14 weeks in vitro
After 9 months (glasshouse)
SCMV SCYLV SCMV SCYLV 0.5 -1 - - - - 1 - 2 - - - -
2 - 10 + + + +
Table showing incidence of viruses in plants tested by molecular diagnosis after bud thermotherapy, meristem excision and culture (n=10)
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3. Genetic engineering
References: Snyman et al. (2006) Plant Cell Reports 25:1016-1023 Snyman (2004) in Transgenic Crops of the World Ed: IS Curtis
• selection and generation of target material • process of regeneration • response to selection agents • genotypic response • novel application of suspension cultures for evaluation of transgene expression
In vitro aspects critical for genetic transformation:
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• Complex polyploid and aneuploid genome • No fertile pollen under natural conditions in SA • Limited gene pool for desirable trait exploitation • Unpredictability of obtaining elite progeny • Certain traits are negatively associated e.g. smut and eldana
3. Genetic engineering
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3. Genetic engineering
GM proof of concept
mosaic virus
NUE Insect resistance (Bt) Herbicide tolerance
Reference: Meyer and Snyman (2013) Acta Hort 974:43-50
Drought tolerance Sucrose metabolism
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3. Genetic engineering
GM proof of concept
mosaic virus
NUE Insect resistance (Bt) Herbicide tolerance
‘get it to the field’ R&D – proof of concept
The ‘whole deal’ Commercialisation vs
Reference: Meyer and Snyman (2013) Acta Hort 974:43-50
Drought tolerance Sucrose metabolism
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3. Genetic engineering Trait (gene) Stage of assessment
Herbicide tolerance - Glyphosate (Agrobacterium CP4 gene)
- Glufosinate ammonium (Streptomyces viridochromogenes pat gene)
Field trials: commercial cultivars transformed and tested over several ratoons
Reference: Leibbrandt and Snyman (2003) Crop Science 43:672-677
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3. Genetic engineering Trait (gene) Stage of assessment
Eldana resistance (Bacillus thuringiensis Cry1Ac gene)
Greenhouse pot trials
Comparison of eldana larvae per stalk in transgenic and wildtype lines
0
5
10
15
20
25
30
03TG041 03TG017 03TG030 N21 N27
Aver
age
no. l
arva
e/st
alk
Line number
R
S
Reference: Meyer and Snyman (2013) Acta Hort 974:43-50
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3. Genetic engineering Trait (gene) Stage of assessment
Sugarcane mosaic virus resistance (antisense SCMV potyvirus coat protein)
Field trials over 2 ratoons
Reference: Meyer and Snyman (2013) Acta Hort 974:43-50
Line designation and variety
Mosaic resistance rating
Plant cane (2007)
Ratoon (2008)
Transgenic NCo310 lines
TG76 5 6 TG77 2 4 TG78 5 5 TG79 4 4 TG86 4 8 TG87 4 5 TG88 3 4 TG89 4 5 TG91 6 5 TG92 4 4 TG93 4 4
Wild type NCo310 9 9 NCo376 (HS) 9 7
N19 (S) 9 9 N12 (I) 4 4 N16 (I) 5 4 N36 (I) 4 4 N21 (R) 3 4 N27 (R) 2 2
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3. Genetic engineering Trait (gene) Stage of assessment
*Sucrose metabolism and modified cell wall composition for ethanol production (collaboration with IPB, University of Stellenbosch) [neutral invertase, pyrophosphate:fructose 6-phosphate 1- phosphotransferase (PFP), sucrose synthase, UDP glucose dehydrogenase]
Glasshouse and selected lines in field trials
Drought tolerance (DREB transcription factors)
Laboratory
N use efficiency (alanine aminotransferase – Arcadia Biosciences)
Laboratory and glasshouse pot trials underway
*Reference: Watt et al. (2010) Sugar Tech 12:85-90
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3. Genetic engineering
Reference: Snyman and Meyer (2012) Proc S Afr Sug Technol Ass 85: 96-101
• Implement decision-making process on GM trait to be commercialised
• Enter commercial agreements with IP owners of desired trait
• Allocate financial resources for royalties and biosafety dossiers
• Establish agricultural and environmental stewardship programmes
• Overcome logistic and infrastructural challenges associated with sugar production from GM and non-GM sugarcane
The role of the sugar Industry in progressing GM sugarcane
• Extremely high licencing costs
• Relatively small size SA sugar industry compared with e.g. Brazil
Major restriction is access to intellectual property (IP) i.e. gene constructs
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International GM sugarcane landscape No commercial GM internationally Following countries involved in GM sugarcane research MAP
USA – GM sugarbeet partially deregulated i.e. sugar derived from GM plant on world market soon; GM sugarcane field trials Indonesia – drought tolerant sugarcane passed through biosafety committee - for local market only Brazil – Monsanto bought private breeding company and microprop company; stated will release GM sugarcane in +/-5 years Australia – GM sugarcane field trials; teaming with multinational companies for commercial release China, Japan, Argentina – research (from literature and pers comms) SA – GM field trials
Brazil
Argentina
USA
India
Australia
Japan
• GM sugarbeet deregulated i.e. sugar derived from GM plants soon on world market
• GM sugarcane field trials and regulatory dossier (mosaic virus and herbicide tolerance)
• Monsanto and Syngenta – conventional breeding, transgenesis and micropropagation
• CTC collaborating with BASF, Bayer and Dow Agroscience
• Field trials – 2nd generation ethanol, weed and insect control • GM sugarcane field trials
• Collaborating with DuPont, Syngenta for commercial release – weed control and ‘Sugarbooster’ (isomaltulose)
research
research
research
Indonesia* • Drought tolerant
sugarcane passed through biosafety committee
• For local market only
South Africa • GM sugarcane field trials • License from Arcadia
Biosciences, USA – improved N use efficiency
China research
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4. In vitro mutagenesis Somaclonal variation • auxin, UV, chemical mutagens • potential for commercial application
Herbicide tolerance: to imazapyr (Arsenal), conferred by
single point mutation in enzyme acetolactate synthase (ALS)
Eldana + Fusarium spp. tolerance: (a) Plant tolerance to Fusarium (b) ‘biological’ control of eldana
• Approach involves exposure of embryogenic callus to the chemical mutagens ethyl methanesulfonate (EMS) and/or 5-Azacytidine
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4. In vitro mutagenesis - herbicide Leaf appearance 6 weeks after herbicide application
Mut1 Mut2 Mut3 Mut4 Mut5 Mut6 Mut7 N12
Plot B imazapyr applied at 312 g a.i. ha-1
Plot C imazapyr applied at 625 g a.i. ha-1
Plot A unsprayed
References: Koch et al. (2012) In Vitro Cell Dev Biol-Plant 48:417-427 Munsamy et al. (2013) Plant Biotechnol Rep 7:489-501 Rutherford et al. (2014) J Hort Sci and Biotech 89:1-16
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4. In vitro mutagenesis - herbicide
Germination (as a % of the N12 control) 3 weeks after planting of 3-budded setts in untreated and treated (imazapyr) plots.
Untreated soil
0
20
40
60
80
100%
Ger
min
atio
n
Genotypes
Treated soil (1254 g a.i. ha-1 )
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4. In vitro mutagenesis - eldana
References: Mahlanza et al. (2013) Plant Cell Rep 32:249-262 Mahlanza et al. (2014) African Entomology (in press)
Putative tolerant plants: 2 months after acclimation
Lesion severity ratings: 2 months after inoculation by stabbing stems with F. sacchari PNG40-colonised toothpicks
Re-isolation of F. sacchari PNG40 on Fusarium semi-selective medium
Confirming identity of isolates using Inter-Simple Sequence Repeat (ISSR) analyses
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5. Germplasm conservation
Slow growth = restricting growth in vitro by • reduction in temperature and nutrients • addition of osmotically-active agents • absence of growth regulators
Advantages: • international germplasm exchange • germplasm conservation • management of in vitro material e.g. transgenic samples
Cryopreservation and Minimal Growth
Aim: to hold material in vitro at three stages embryos shoot germinated meristems plantlets
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5. Minimal growth
18 °C
24 °C
8 months 12 months
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5. Minimal growth 18°C
Reference: Watt et al. (2009) Plant Cell Tissue and Organ Culture 96:263-271
MS, 20g/l sucrose
½MS, 10g/l sucrose
MS, 20g/l sucrose, 1mg/l abscisic acid
½MS, 10g/l sucrose, 1mg/l abscisic acid
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Collaborative projects 1. International Consortium for Sugarcane Biotechnology (ICSB)
Member countries (Argentina, Australia, Brazil, Colombia, Ecuador, France/Reunion,
Guatemala, India, Mauritius, Philippines, South Africa, USA – Florida, Louisiana, Texas, Hawaii, West Indies)
Range of projects – mapping, marker assisted selection, chloroplast transformation,
germplasm introgression 2. Sugarcane genome sequencing initiative (SUGESI) Consortium (Australia, Brazil, France, South Africa, USA)
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Concluding remarks • In vitro culture integral to several applications for sugarcane
improvement • Focus – to manipulate conditions to decrease labour + time input
Sett germination in vitro Holding plantlets in water for up to 2 weeks prior to planting out in glasshouse
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Acknowledgements