POLYPLOIDY AND AN IN VITRO-MEDIATED PROPAGATION PROTOCOL FOR POTENTIALLY INDUCING CHROMOSOME DOUBLING IN MACADAMIA Karin Hannweg and Mark Penter Plant Improvement Division Agricultural Research Council Institute for Tropical and Subtropical Crops
LAYOUT 1. Background to chromosome doubling/polyploidy what is it? 2. What does chromosome doubling/polyploidy mean for plant improvement/cultivar development? 3. How do we induce chromosome doubling/polyploidy? 4. Case studies 5. Why chromosome doubling in macs? 6. Developing methods for inducing chromosome doubling in macs 7. The future?
BACKGROUND TO CHROMOSOME DOUBLING (i.e. POLYPLOIDY) WHAT IS IT? chromosome doubling = polyploidy = tetraploidy
BACKGROUND TO CHROMOSOME DOUBLING (i.e. POLYPLOIDY) WHAT IS IT?
Chromosomes and DNA in the nucleus
BACKGROUND TO CHROMOSOME DOUBLING (i.e. POLYPLOIDY) WHAT IS IT? Tetraploid/Polyploid cells contain double the number of chromosomes diploid chromosomes in the nucleus tetraploid/polyploid
Chromosome doubling and plant improvement/ cultivar development?
Many crops which are commonly used by man are natural polyploids:
Many crops which are commonly used by man are natural polyploids: The improved size and vigour of polyploid crops has led plant breeders to the artificial induction of polyploids (their chromosomes have been doubled) since the late 1930s.
What characteristics can be improved upon? bigger tuber, rhizome or root size bigger flower & fruit size and/or colour intensity better photosynthesis bigger and/or thicker leaves higher biomass dwarfing better nutritional value better resistance to pests & diseases
How do we induce chromosome doubling?
How do we induce chromosome doubling? cell division: mitosis.
How do we induce chromosome doubling? apply inhibitor
How do we induce chromosome doubling? Tetraploid cells apply inhibitor
Testing for polyploidy? How do we know the chromosomes have been doubled? The ARC-ITSC has a dedicated plant analysis flow cytometer
Testing for polyploidy? How do we know the chromosomes have been doubled? The fluorescent stain binds to the DNA (chromosomes) and fluoresces under UV light the more DNA there is, the higher the fluorescence. A tetraploid sample will therefore fluoresce twice as much as a diploid since it has twice as much DNA (i.e. twice as many chromosomes). The ARC-ITSC has a dedicated plant analysis flow cytometer
How do we know the chromosomes have been doubled? The ARC-ITSC has a dedicated plant analysis flow cytometer
How do we know the chromosomes have been doubled? Diploid control Chromosomes doubled! The ARC-ITSC has a dedicated plant analysis flow cytometer
Practically: How do we do chromosome doubling? GROWTH POINT TREATED WITH THE INHIBITOR COLCHICINE HORTICULTURAL EVALUATION VERIFICATION
CASE STUDIES
Overview of the ARC-ITSC s chromosome doubling research: CITRUS Production of high quality seedless varieties for the export market Development of tetraploid breeding parents for use in seedless cultivar development Development of tetraploid rootstocks dwarfing, stress resistance etc.
Overview of the ARC-ITSC s chromosome doubling research: Embryo rescue Confirm/verify seedlessness Evaluate new seedless variety
Overview of the ARC-ITSC s chromosome doubling research: GINGER Growers required an improved variety on their existing selection that had higher yield and larger rhizomes together with exceptional quality
Overview of the ARC-ITSC s chromosome doubling research: Mean shoot length 24% Mean leaf area 87% Mean stem mass 143% Rhizome mass 188% Number of knobs 15% Mean knob mass 162% control tetraploid Mean flower mass 316% Mean flower length 58%
Overview of the ARC-ITSC s chromosome doubling research: PINEAPPLE altered fruit size and shape altered fruit quality characteristics altered cycle time altered yield (planting density) altered pest/disease resistance
Overview of the ARC-ITSC s chromosome doubling research: Characteristic Leaf Thickness Chlorophyll Content Cell Size and Volume Trend 2n < 4n < 8n 2n < 4n < 8n 2n < 4n < 8n Leaf Length Plant Height Stomatal Density 2n > 4n > 8n 2n > 4n > 8n 2n > 4n > 8n
Overview of the ARC-ITSC s chromosome doubling research: 1. Plants appeared to be vigorous 2. Flowering and fruiting comparable with diploid counterparts 3. Fruit shape not suitable for canning industry (pear-shaped) 4. Fruit size extremely acceptable 5. Fruit colour extremely acceptable 6. Fruit texture crisp and fibrous 7. Very low incidence of black heart disease 8. Relatively high level of translucency
Overview of the ARC-ITSC s chromosome doubling research: Indigenous plant species Plectranthus esculentus 1cm Increased starch content Nematode resistance
Overview of the ARC-ITSC s chromosome doubling research: Indigenous plant species Crocosmia aurea Fewer, larger flowers Longer flower stems
Overview of the ARC-ITSC s chromosome doubling research: Indigenous plant species Ginger Bush, Tetradenia riparia Increase essential oil content & bioactivity against PH fungi diploid tetraploid
Chromosome doubling and plant improvement NB: BUT the effect/s of chromosome doubling on a particular species are not predictable!!!
What characteristics can be improved upon? bigger tuber, rhizome or root size bigger flower & fruit size and/or colour intensity better photosynthesis bigger and/or thicker leaves higher biomass dwarfing better nutritional value better resistance to pests & diseases
WHY CHROMOSOME DOUBLING IN MACS? Bigger fruit (small-fruited cultivars): effect on kernel size? Changes in fruit characteristics: effect on shell thickness? crackout? pest complexes? Changes in quality: effect on oil profile/ TACs & TPCs / storage behaviour? Phenology: nut maturation time/market windows? Drought/Water-logging tolerance? (rootstock?) Disease tolerance? (rootstock?)
METHODS FOR PROPAGATION AND INDUCING CHROMOSOME DOUBLING? Polyploid induction (i.e. chromosome doubling) is usually more efficient in vitro (tissue culture) Macadamia is notoriously difficult to propagate in vitro from field-grown material - contamination/phenolics (Gitonga et al., 2008 had limited success) First investigated various in-field methods of inducing polyploidy
METHODS OF APPLICATION AND INDUCING 1. In-field parafilm cups : liquid colchicine applied to axillary buds, 814 (0.1 g/l; 1.0 g/l & 10.0 g/l) CHROMOSOME DOUBLING?
METHODS OF APPLICATION AND INDUCING CHROMOSOME DOUBLING? 2. In-field: colchicine dissolved in aqueous lanolin applied to axillary buds, 814 0.1 g/l; 1.0 g/l & 10.0 g/l
METHODS OF APPLICATION AND INDUCING CHROMOSOME DOUBLING? 3. Budsticks ( 814 ): overnight dip: 0.1 g/l; 1.0 g/l & 10.0 g/l : grafted onto clonal Beaumont (695) rootstocks 4. Budsticks ( 814 ): 5 day dip: 0.1 g/l : grafted onto clonal Beaumont (695) rootstocks
METHODS OF APPLICATION AND INDUCING CHROMOSOME DOUBLING? still to evaluate 5-day dipped budsticks BUT all in-field & budstick treatments resulted in either no change or mixoploidy (unstable and of no use ) when we verified chromosome doubling using flow cytometry what about trying an in vitro method?
METHODS OF APPLICATION AND INDUCING CHROMOSOME DOUBLING? 5. In vitro (tissue culture) TDZ-mediated propagation using kernel tissues TDZ (thidiazuron) = super-cytokinin in vitro used with much success on several tissue culture-recalcitrant species including various tree species In macadamia: Mulwa and Bhalla, 2006 used immature kernel
METHODS OF APPLICATION AND INDUCING CHROMOSOME DOUBLING? 5. In vitro TDZ-mediated propagation using various tissues developing a method. Incubated kernel, integument and leaf sections on TDZ-containing agar medium following a modified method described by Mulwa and Bhalla, 2006
METHODS OF APPLICATION AND INDUCING CHROMOSOME DOUBLING? 5. In vitro TDZ-mediated propagation using various tissues Incubated kernel, integument and leaf sections on TDZ-containing agar medium following a modified method described by Mulwa and Bhalla, 2006 Embryo-like structures developed on the kernel sections developing a method.
METHODS OF APPLICATION AND INDUCING 5. In vitro TDZ-mediated propagation using various tissues Incubated kernel, integument and leaf sections on TDZ-containing agar medium following a modified method described by Mulwa and Bhalla, 2006 Embryo-like structures developed on the kernel sections The structures formed shoots CHROMOSOME DOUBLING? developing a method.
developing a method. 5. In vitro TDZ-mediated propagation using various tissues Incubation of kernel, integument and leaf sections in a solution of mitotic inhibitor (colchicine): 0.1g/l; 1.0 g/l & 10.0g/l, overnight & 3 days Transfer to culture medium containing TDZ Transfer to a culture medium which encourages shoot elongation Verify whether shoots are polyploid once they are large enough for testing!
WHY CHROMOSOME DOUBLING IN MACS? HORTICULTURAL EVALUATION Long-term but we need to start looking now at approaches to address various challenges facing the industry. Bigger fruit (small-fruited cultivars): effect on kernel size? Changes in fruit characteristics: effect on shell thickness? crackout? pest complexes? Changes in quality: effect on oil profile/ TACs & TPCs / storage behaviour? Phenology: nut maturation time/market windows? Drought/Water-logging tolerance? (rootstock?) Disease tolerance? (rootstock?)
Acknowledgements ARC-Institute for Tropical and Subtropical Crops NRF-THRIP Programme