Protocols to Determine Pollination Requirements and ... Theme III... · Carrot Case Study Tested pollination by hand, honey bees, flies, and a combination of bees and flies, all in

Agri-Food Canada Agroalimentaire CanadaAgriculture and Agriculture et

Protocols to Determine Pollination Requirements and Optimal Pollinators

for Plant Genetic Resource Regeneration

Ken Richards & Mark WidrlechnerCanadian Genetic Resources Program, Saskatoon, SK

North Central Regional Plant Introduction Station, Ames, Iowa

The Challenge / NeedsDiversity of plant species

300,000 to 500,000 species of higher plants(250,000 described taxonomically)30,000 edible; 7,000 cultivated or collected by humans at one time or another 30 crops “feed the world” providing 95% of dietary energy (calories) or protein When considering all animal/plant products, 1/3 of total human diet depends directly or indirectly on insect-pollinated crops

The Challenge / NeedsLack of information

General patterns of pollen transfer understoodBut little known about pollination requirements for most cultivated plantsBreeding systems are under genetic control; rarely fixed or static, often fluid and responsive to selection pressureChallenge: most seed plants have a range of reproductive options – They arise intermittently through mutation

and evolve in response to selective conditions

The Challenge / NeedsLack of information

Although detailed pollination are requirements “known” for major cultivated crops, they are often based on modern, improved cultivars and may not reflect the full range of variation– Other crop species (incl. wild relatives,

underutilized or minor crops) are insufficiently understood

% Natural % Natural SelfingSelfing in in MelilotusMelilotus SpeciesSpeciesSano (1977) Ecol. Plant. 12: 383Sano (1977) Ecol. Plant. 12: 383--394394

00 5050 10010000

5050

100100

% H

and

% H

and

Selfi

ngSe

lfing

indica

eleganssulcata

messanensisitalica

neapolitana

speciosainfesta

macrocarpa

segetalis

officinalisalba

altissima

hirsutataurica

dentatasuaveolens

wolgica

suaveolens

dentata

alba

officinalis

polonica

Annual - MicromelilotusAnnual - Eumelilotus

Biennial - Eumelilotus

The Challenge / NeedsRegeneration protocols

Seed regeneration is a key part of ex situgermplasm conservationEven under optimal storage conditions, seed viability declines and leads to a loss of genetic diversityFor unique material, such losses may be irreplaceableTherefore, monitoring of viability and timely seed regeneration must be a priority

The Challenge / NeedsFactors influencing regeneration

Loss of viability (seed longevity)Small original samplesNumber of requests for a given accessionScheduled morphological characterizationNeed to separate mixtures (for inbred crops or multiple-species collections)

The Challenge / NeedsRegeneration

Ideally conducted under neutral selection conditions (minimal competition, optimal growing environment, sufficient population size)– To minimize genetic change during through

selectionBut not always possible– Certain degree of genetic change is inevitable– Especially under conditions markedly different

from those where the accession was best adapted

How to Proceed?A Basic Field Determination of

Pollination Requirements

Open pollinated

Caged with insects- Optimal pollination achieved only when appropriate pollinator is used

Pollinator excluded (control)

A More Thorough Determination of Pollination Requirements --

Detailed Greenhouse Protocol

Flowers bagged to self-pollinate (SP)Shaken to stimulate movement by windSP flowers with brush from same flowerSP flowers with brush from same racemeSP flowers with brush from same plantCross-pollinate with brush from different plantFlowers open pollinated (control)

Important Metrics

Measures of self incompatibility (Zapata & Arroya 1978)Self incompatibility = seed set from self-pollination

seed set from cross-pollination- Caution: level may vary both among populations and among individuals within populations

Estimate of cross pollination (Cruden1977)Ratio between the number of pollen grains and ovules (the P:O ratio)

Important Metrics

Rate of self-pollination (Charlesworth& Charlesworth 1987)Selfing rate = value from outcrossing – value from open pollination

value from outcrossing – value from selfing

- Caution: formula only valid when value from outcrossing > value from OP or selfing levels

Sainfoin(Onobrychis viciifolia)

Case Study I

Non-bloat forage legume for livestock productionHighly out-crossing Alfalfa leaf cutter & honey bees

Sainfoin (Alberta Data)

Flowers/raceme

Pods/raceme

%seed set

Seedyield(kg/ha)

Open 58.0a 29.7a 51.1a 1837.9aCaged & Bees

57.6a 26.7a 46.3b 1502.9a

Exclusion 46.7b 0.9b 1.9c 9.6b

Diversity of pollinators (n=4974; Alberta)

%

Megachile rotundata 55.9Apis mellifera 40.5Bombus huntii 2.7Bombus occidentalis 0.4Bombus rufocinctus 0.4Bombus fervidus 0.1

Much different array than reported in European literature

Probability of Floral Visitation

Pv = 1 – e –m, where

m = bees / area x visitation rate x pollination timeflower density

Sainfoin Pollinator Efficiency(Visitation Rate)

Bee NSeconds / flower

Flowers / minute

honey bee 595 5.66 10.61alfalfa LC 514 5.54 10.83bumble bees 55 3.55 16.91

Pollination Time

Maximum 15 hr / d(determined by photoperiod)Optimum 0800 – 2000 hr(varies by species – also midday heat in field cages)Practical 12 hr / d ?Actual ?

Activity Curves by SpeciesVary by time of day & season

Literature Values for tTereschenko (1949): 1 dayDubbs (1967): 7-12 daysRichards (unpublished): 1 day

- Generally not known or recorded

Stigma Receptivity(t = time stigma is receptive)

Alfalfa (Medicago sativa) flowers are receptive longer than a day

Flower density

Curvilinear over season

Constancy to Raceme(needs to be compared among pollinators --

indicator of outcrossing)

Apis melliferaMegachile rotundataBombus spp.

flowers visited / raceme1 2 3 4 > 553 25 13 5 456 27 12 4 160 22 10 4 4

Probability of SainfoinFlower Visitation

Predicts flower visits for studied pollinators1 Bombus is as effective as 1.5 Apis or MegachileThis approach is most valuable for planning field isolations

Probability of Visitation in Relation to Flower Density and Bee DensityBombus requires lower bee density for the same probability of visitation

Cicer Milkvetch(Astragalus cicer)Case Study II

Non-bloating forage legume for livestock productionCross – pollinatedBumblebee, alfalfa leafcutter, honey bees

Pollination Requirements for Cicer Milkvetch

open pollinated

pollinator excluded

flowers / raceme 26.3 + 0.4 27.1 + 0.5seed set % 71.1 + 8.8 1.8 + 2.6seeds / raceme 98.0 + 2.7 2.7 + 0.1seeds / pod 5.2 + 0.1 1.8 + 0.1

Rate of Flower Visitation

Sec. / fl. fl. / min.

Bombus nevadensis 2.7 + 0.1 22.2B. huntii 6.1 + 0.1 9.8B. rufocinctus 5.9 + 0.5 10.2Apis mellifera 13.7 + 0.6 4.4Megachile rotundata 6.3 + 0.2 9.5

Pollinator EffectivenessCaged Seed Yield

(seeds / pod)1979 1980 1981

Bombus nevadensis 7.92a 6.83a 7.18aB. fervidus 7.22aB. appositus 5.97b 6.10b 6.40bB. huntii 5.87b 6.05bB. rufocinctus 5.80bB. californicus 5.80bApis mellifera 3.27c 3.94c 4.19cMegachile rotundata 3.45c 3.48c 3.31c

Carrot Case Study III

(Daucus carota)(Wilson et al. 1991)

Widely cultivated root vegetableMixed mating systemMany insect visitors, but

which species are effective pollinators?

Carrot Case StudyTested pollination by hand, honey bees, flies, and a combination of bees and flies, all in field cagesThree years of data on seed weight per cage, also collected relative time measures forinsect management

Carrot Case StudyTotal Seed Weight per Cage (g)

Method 1985 1986 1988

Honeybees 384.5 a 46.9 a 80.2 ab

Flies 113.2 b 30.9 a 44.8 b

Both 67.3 a 122.1 a

Hand 26.2 b

Carrot Case Study

Combination of bees and flies worked best, without a major increase in labor investmentDaucus and other umbels vary not only in seed production by pollinator, but also can vary widely in quality (% germination)- See Pimpinella poster

Potential Managed Pollinators for Genebank Use

What pollinators are potentially available for genebank managers today?

Potential Managed Pollinators for Genebank Use

•Honey bee (Apismellifera) •Bumble bees (Bombus spp.)

Potential Managed Pollinators for Genebank Use

Mason bees (Osmia spp.)Alfalfa leafcutter bee

(Megachile rotundata)Other solitary bees

Potential Managed Pollinators for Genebank Use

Blow flies (Calliphora spp.)House fly (Muscadomestica)Syrphid flies

Avoiding Pollen Contamination through Isolation

Achieving adequate isolation depends on:- Possible pollen vectors (wind, insect, birds, bats, etc.)- Floral morphology and breeding system

Isolation mechanisms:- Spatial or temporal - Natural or artificial barriers, trap crops- Bagging with hand crossing (if needed)

Assumes knowing adequate isolation distance, how far?

- Pollen and / or pollinator movement- Determine via morphological and / or molecular markers

Pollination CagesPros and Cons

Positives

- Effective isolation barriers- Excludes certain pests, which can vector diseases or otherwise reduce seed yield / vigor- Can manipulate pollinators and control pollinator density

Pollination CagesPros and Cons

Negatives

- Expensive, laborious to construct- May shade plants / modify ambient temperature / humidity affecting plant growth, seed production, & pollinator foraging behavior- Favorable environment for certain insect pests / pathogens- May result in negative pesticide-pollinator interactions

Pollination CagesPros and Cons

Unknowns

- Pollen longevity (heat and humidity)- Pollen movement (especially with multiple crops in a cage)

Pollinator Choice and Management

Optimal crop pollination can only be achieved if an appropriate pollen vector is available to visit flowers

Challenges:• Identify primary, secondary & tertiary pollinators

– Combinations of pollinators– Efficiency, effectiveness, abundance

• Differentiate between visitors and pollinators• Develop standard methods to ID pollinators• Limited palette of managed pollinators at

present

Pollinator Choice

Determine general surface/volume relationships

Tongue length of pollinators

vs.

Floral morphology (corolla tube length)

Pollinator Choice and Management

More Challenges:Number of pollinators needed on small isolation plots

- Integrate bee behavior, plant biology, agronomy- Bee drift from small plots and other plant competition

Management techniques- Need handbook for pollinator management for genebanks

Staff training and specialized facilitiesWorking in cages with bees (sting / allergy hazard)Introducing new pollinators to regions and risks

Conclusions

Pollination requirements for major crops are known in part; minor crops inadequate

Optimal pollinator needs to be determined

Compare to available “managed” pollinators

Need to integrate agronomy, plant pollination requirements and biology, and pollinator behavior

Many Thanks to…

Sharon McClurg and Jeff Carstens for assistance with images and carrot data

Larry Deack and Beatriz Moisset for fly images from the Internet

John Virosteck, Tim Myers and Peggy Edwards for assistance with obtaining plant seed set and pollinator foraging data