THIS ISSUE AT A GLANCE

Page Article

2 Tree Seed Workshop Overview

3 Conifer Reproductive BiologyEvolutionary Pathways

4 Broadleaf Reproductive Biology

5 Reproductive Biology System and itsImpact on Genetic Architecture

5 Conifer Pollination MechanismsRevisited

5 Seed Production in Lodgepole Pine

6Variation in Reproductive Capacity ofLodgepole Pine (Pinus contorta var.latifolia) in British Columbia

7 Lodgepole Pine Crop Statistics FromSelectSeed Ltd. Orchards

8Questions About the Role of Seed BugHerbivory in Production Coming FromInterior Lodgepole Pine Seed Orchards

9Lepto-Cams and Thermocouples: Whydo Insect Bags Prevent August Pli SeedLoss in the Interior of BC?

10 Seed Shortfall in Lodgepole Pine

10

Methodology to Organize OperationalLodgepole Pine Cone Harvests byUsing Observations of Seed SetDeclines

11 Testing Containers for Research andConservation Seed Storage

15 Upcoming Meetings

15 Recent Publications

CANADIAN FOREST GENETICS ASSOCIATIONASSOCIATION CANADIENNE DE GÉNÉTIQUE FORESTIÈRE

Tree Seed Working Group

NEWS BULLETIN

No. 57 August 2013

CHAIR’S ‘ARMCHAIR’ REPORT

Hello! I hope everyone is having a greatsummer. For many parts of BC it has been ourdriest July ever with no recorded precipitation inthe Vancouver area and beyond. Initialindications are that it certainly hasn’t helped ourlodgepole pine seed set issues in the northOkanagan and our falldown appears to beoccurring earlier in this hot dry summer. This isour most serious seed set issue in BC (by far)and was the subject for the majority of our TreeSeed Working Group workshop in Surrey onJuly 22nd. This dry weather may also haveimplications for reproductive bud determinationas most of our species are differentiating theirmeristematic tissues at that stage and hot, dryweather has often been a positive factor in thepromotion of reproductive bud formation.

I’d like to thank all of the speakers whopresented at our Tree Seed Working Groupworkshop at the BC Tree Seed Centre. Themajority of the content for this News Bulletinedition is the abstracts from the variouspresentations. Thank you to Lindsay Robb forher article on testing seed storage containers. Inaddition, a webpage has been set up thatprovides a pdf version of the presentations asw e l l a s t h e a b s t r a c t s(https://www.for.gov.bc.ca/hti/treeseedcentre/tsc/workshops&presentations/tswg_2013/index.htm). The workshop went smoothly (furtherdetails inside), although a sombre element waspresent with the recent passing of Tim Lee whomanaged the Vernon Seed Orchard Company.Tim was a strong tree improvement advocate,friend to many, and one who could always becounted on to do the right thing. He will surelybe missed and I send my deepest sympathies tofamily, friends and colleagues. The workshopwas dedicated to the memory of Tim Lee.

In trying to promote submitting articles for theNews Bulletin we have been having a lottery at

2

our workshop for everyone who has contributed anarticle since the last TSWG workshop. This year’sgrand prize winner was Fabienne Colas, whoreceived a copy of Claire Williams’ book “ConiferReproductive Biology”. Other prizes were awardedto Al Foley, Michele Fullarton, and Michael Stoehr.

There has not been a dedicated theme for the lastfew Newsbulletins and I thought it’s time to try andreintroduce that concept. Articles do not need to beon the indicated subject, but it does offer a cohesivetie to the other submitted articles. The theme for theDecember 2013 News Bulletin # 58 will be “FromSeed Collection to Seed Storage” and is intended toprovide a forum for processing both conifer andangiosperm tree species. It will also be anopportunity for those to sound in on storagepractices prior to propagule processing. Do youprocess a unique species? Do you have a uniqueprocessing method for our conventionalreforestation species or have specific processingproblems? News Bulletin # 58 may provide thevenue for introducing the topic to a wide audience?or it may win you a cool prize in the future. Pleaseconsider contributing an article to the TSWG NewsBulletin – Thank you.

Dave KoloteloTSWG Chair

EDITOR’S NOTES

The normal June issue was delayed in order topublish abstracts of speakers from the seedworkshop that was held at the BC Tree Seed Centrein Surrey prior to the Canadian Forest GeneticsAssociation conference. The workshop was verywell attended and all the presentations wereexcellent. Kudos go to Dave for organizing theworkshop and to his staff for setting up the venue.

Lindsay Robb has provided a very interestingarticle about testing storage containers for long-term storage of small quantities of seed. Thisshould be helpful to anyone who may want torevisit the type of containers that they are using.

I hope that you enjoy the remainder of the summerand look forward to a great fall. May the seed godsbe kind to you!

Dale SimpsonEditor

TREE SEED WORKING GROUP

ChairpersonDave Kolotelo

BC Ministry of Forests, Lands and NaturalResource Operations

Tree Seed Centre18793 - 32nd AvenueSurrey, BC V3S 0L5

Tel.: (604) 541-1683 x 2228Fax.: (604) 541-1685

E-mail: Dave.Kolotelo@gov.bc.ca

EditorDale Simpson

Natural Resources CanadaCanadian Forest Service

National Tree Seed CentrePO Box 4000

Fredericton, NB E3B 5P7Tel.: (506) 452-3530Fax.: (506) 452-3525

E-mail: Dale.Simpson@nrcan.gc.ca

Comments, suggestions, and contributions forthe News Bulletin are welcomed by theChairperson and Editor.

All issues of the News Bulletin are available at:www.for.gov.bc.ca/hti/treeseedcentre/tsc/tswg.htm

TREE SEED WORKSHOP OVERVIEW

On July 22nd, 2013 the BC Tree Seed Centrehosted the TSWG workshop with the overalltheme of “Reproductive Biology”. There were48 people in attendance and the agenda belowillustrates the speakers and their topics. The daystarted with a general review of ReproductiveBiology topics and then after our morningrefreshment break switched to focusing in onour lodgepole pine seed set problems in northOkanagan seed orchards. There was an openQ&A session in the afternoon followed by a tourof the Tree Seed Centre. Some were fortunate tothen be able to go to Whistler for the ForestGenetics 2013 meeting.

Abstracts for most talks are included in thisedition, but holidays complicated that for a fewindividuals and the Abstracts and pdf versions ofthe Powerpoint presentations will be uploaded toth is webpage for fu tu re referen cehttps://www.for.gov.bc.ca/hti/treeseedcentre/tsc/workshops&presentations/tswg_2013/index.htm.

3

Welcome Heather Rooke

Conifer Reproductive Biology evolutionary pathways Dave Kolotelo

Broadleaf Reproductive Biology Dale Simpson

Reproductive Biology System and Its Impact on Genetic Architecture Jodie Krakowski

Conifer Pollination Mechanisms Revisited Dr. Patrick von Aderkas

Seed Production in Lodgepole pine Dr. Joe Webber

Variation in reproductive traits of lodgepole pine in British Columbia Anne Berland

Lodgepole pine crop statistics from Select Seed Orchards Jack Woods

Questions About the Role of Seed Bug Herbivory in Seed Production Coming from Interior Lodgepole Pine Orchards Jim Corrigan

Lepto-cams and Thermocouples: Why do Insect Bags PreventAugust Pli Seed Loss in the Interior of BC? Dr. Ward Strong

Seed Shortfall in Lodgepole Pine Dr. Lisheng Kong

Methodology to Organize Operational Pli Cone Harvests by Using Observations of Seed Set Declines Gary Giampa

I’d like to thank everyone who contributed to theworkshop – speakers for their time and effort –you all did a great job, staff at the TSC whohelped with planning and organizing andespecially Aggie Ellis who organized most ofthe catering, and Michael Postma and DianeDouglas who took time out of their Sunday tohelp set up and then help with audiovisual andregistration logistics. Finally I’d like to thank theVernon Seed Orchard Company and SelectSeedLtd. for their financial contributions towards theworkshop.

Dave Kolotelo

CONIFER REPRODUCTIVE BIOLOGY EVOLUTIONARY PATHWAYS

The intent of this talk was to provide a baselinefor conifer reproductive biology from its earlybeginnings to the current diversity found amongnorth-temperate conifers. I consider it a work inprogress. Time estimates of significant eventsare primarily based on fossil evidence and aretherefore restricted to samples which have been‘fossilized’ under appropriate conditions andhave been found. Land plants appeared

approximately 450 million years ago (MYA)and coincided with the appearance ofmycorrhizal associations. These early landplants reproduced like ferns and were dependenton water for spore dissemination. Xylemevolution preceded seeds and this allowed plantsto reduce potential for embolism under freezingor drought conditions, in addition to significantheight increases and an increased competitiveability for sunlight.

The evolution of seeds is generally considered interms of three large-scale evolutionary changes:1) Heterospory – The differentiation of twoseparate spore types differing in size and sex.Considered to have occurred multiple times inplant evolution.2) Megaspore Reduction / Retention inSporophyte – The reduction to one femalesprore (megaspore) from many and maintenanceof the structure within the body of the parentplant (endospory).3) Integument Evolution – The evolution ofprotective tissues to protect the innermoststructures.

In conifers, a variety of pollen receptionmechanisms evolved and these finalized theconversion to the water independence of sporesfor movement and fertilization. Seeds, inaddition to mycorrhiza and xylem, areconsidered the major evolutionary factors

4

allowing conifers to dominate large land areas,especially those with stressful or sub-optimalenvironments. Seeds can be thought of as“concentrated life” as the seed contains thefuture diploid tree, the initial food reserves, andthe seed coat for protection. Seeds are thedispersal package and they also ‘allowed’ for thedevelopment of seed dormancy to synchronizegermination with the appropriate environmentalconditions to maximize survival and growth.

The two main groups of seed plants,Angiosperms (about 250,000 species) andGymnosperms (600!700 species) divergedapproximately 310 MYA. Inverted ovules,pointing towards the central axis, is acharacteristic of most conifers and evolvedapproximately 265 MYA. The inverted ovules’orientation (up or downwards pointing) isrelated to the type of pollen present (saccate ornon-saccate). Subsequent to ovule inversionthere was also a division of the Pangeaensupercontinent about 200 MYA resulting inspecies isolation and evolution of some highlyvariable reproductive biology mechanismsamong conifers. One third of all conifers areattributed to two genera: Pinus in the Northernhemisphere and Podocarpus in the Southernhemisphere.

Conifer cones are basically structures that houseovules, allow for pollen to enter, and then closeto protect the embryo. In many species this is awoody structure, but Taxus and Podacarpus areexamples of conifer genera with fleshystructures to house the seed. In general, pollencones (microsporangia arranged spirally along acentral axis) are relatively simple and eachmicrosporangium is considered to be a modifiedleaf. These pollen cones do not show a greatdeal of diversification among genera. The seedcones (bract scale complexes arranged spirallyalong a central axis) are considered to be morecomplicated as each bract-scale complex isconsidered to be a modified shoot vs. a modifiedleaf as for pollen cones. In contract, seed coneshave differentiated greatly in terms of grossmorphology, ovule orientation, and type ofovular secretion mechanism.

Dave KoloteloMinistry of Forests, Lands and Natural Resource

OperationsTree Seed CentreSurrey, BCE-mail: Dave.Kolotelo@gov.bc.ca

BROADLEAF REPRODUCTIVE BIOLOGY

Seed plants started to evolve 340 million yearsago with Angiosperms arising about 130 millionyears ago, but it was about 100 million years agothat the number of species began to rapidlyevolve. Angiosperms produce true flowerswhich can be either perfect or imperfect. Perfectflowers contain both male and femalereproductive structures whereas imperfectflowers are either sex. Flowers occur in a varietyof arrangements such as: spike, raceme, panicle,corymb, cyme, umbel, catkin, and head. Somegenera or species are monoecious where bothmales and females occur on the same tree asopposed to dioecious species where males andfemales occur on separate trees. Pollen that isdispersed by wind (anemophilous) is small andis dispersed over great distances. Pollen fromspecies that produce showy, fragrant flowers isgenerally dispersed by insects (entomophilous).This pollen is larger and its dispersal is generallylimited. Broadleaved species produce fruit thatcontains the seed. The type of fruit produced isspecies-dependent. Examples include: achene –sycamore (Platanus occidentalis); doublesamara – maple (Acer spp.); single samara – elm(Ulmus spp.), ash (Fraxinus spp.); acorn – oak(Quercus spp.); nut – hickory (Carya spp.),butternut (Juglans cinerea); legume - Kentuckycoffeetree (Gymnocladus dioicus); capsule –poplar (Populus spp.); pome – apple (Malusspp.), mountain ash (Sorbus spp.); and drupe –cherry (Prunus spp.). Fruits are shed by mostspecies in late summer and fall and are primarilydispersed by wind. Dispersal by animals isimportant for species such as mountain ash,cherries, oaks, and butternut. Seed ofbroadleaved species often exhibits dormancydue to seed coat, embryo, morphology or acombination. Seed coat dormancy can bealleviated by soaking seed in cold or hot wateror treating the seed with acid to allow moistureto enter. Moist chilling or combinations ofwarm, moist incubation and moist chillingalleviate embryo and morphological dormancy.

Dale SimpsonNatural Resources CanadaCanadian Forest ServiceNational Tree Seed CentreFredericton, NBE-mail: Dale.Simpson@nrcan.gc.ca

5

REPRODUCTIVE BIOLOGY SYSTEM AND ITS IMPACT ON GENETIC

ARCHITECTURE

Since the days of Darwin, scientists haveobserved that life history traits are associatedwith patterns of population adaptation anddiversity. Significant relationships betweengenotypic and adaptive diversity and geneticarchitecture have been demonstrated fornumerous life history traits, the most consistentbeing: mating system, taxonomy, life form,successional status, range extent and contiguity,and dispersal mechanism of seed and pollen. Innature, many of these traits are correlated andfew studies have evaluated the individualfactors. Generally, longer-lived, late-successional, outcrossing, animal-dispersedperennials that are not endemic have higherexpected and observed heterozygosity thanannual, endemic or narrowly distributed,gravity- or wind-dispersed, pioneer species. Theformer group generally have less differentiatedpopulations than the latter. However, there iswide variation among species. In addition to lifehistory traits, many studies have quantifiedequally strong ! sometimes even stronger !external influences on plant genetic architecture.Dominant influences include: refugia and post-glacial range expansion, spatial and temporaldisturbance patterns, soil and geology,hybridization, management interventions,sample breadth and size for genetic studies, andtype of genetic marker used. Adding to thecomplexity, these factors can also havesignificant interactions with life history traits.While general patterns of total heterozygosity,population differentiation, and heterozygotedeficiency are associated with life historycharacteristics, species-specific data based on acarefully designed study with clear objectivesare needed to answer questions about anyparticular species.

Jodie KrakowskiMinistry of Lands, Forests and Natural Resource

OperationsSquamish, BCEmail: Jodie.Krakowski@gov.bc.ca

CONIFER POLLINATION MECHANISMS REVISITED

Pollination mechanisms in Gymnosperms can bedivided into types, depending on the shape ofpollen and ovule micropyle and whetherpollination drops are present. Two general typesexist: pollen capture mechanisms (PCM) andextra-ovular capture and germination (ECG). Ofthese, PCM has the greatest diversity, as theseshow six different sub-types. Phylogeneticanalysis of modern and extinct gymnospermsreveals that pollination drops were fundamentalto pollination capture mechanisms of the earliestseed plants. The most derived types of PCM arefound within Pinaceae. ECG, also derived, isrestricted to a few conifer families and genera.Pollination drops provide a nectar function ingnetophytes and possibly in cycads. Thisprovides an independent origin to studyconvergent evolutionary patterns in plant-insectrelations.

Patrick von Aderkas, Alexandra Lunny,Stefan Little and Natalie PriorCentre for Forest BiologyDepartment of BiologyUniversity of VictoriaVictoria, BCE-mail: pvonader@uvic.ca

SEED PRODUCTION IN LODGEPOLEPINE

Seed yields (filled seeds per cone) from northOkanagan (NO) lodgepole pine (Pinus contortavar. latifolia) orchards have not metexpectations compared to seed yields routinelyrealized at Prince George (PG). Since the NO issubstantially hotter and drier than PG, earlyefforts focused on better irrigation systems(broad cast sprayers) and crown cooling(misting). Data collected from Kalamalka SeedOrchard 307 over the period of 2000 to 2005 didnot show any significant improvement in eithercone numbers or seed yields. However, conesprotected by insect bags had consistently moreseed.

Since most NO orchards were experiencing poorseed production, in 2006 we began to collectstandardized orchard statistics from eightorchards on four NO sites and two orchards at

6

the PG Tree Improvement site. All 10 orchardsproduce seed for three PG and three southerninterior seed planning units. We also comparedproduction from two orchards of the sameprovenance base at PG and NO.

Over the period of 2006 to 2012, the trend forhigher seed yields per cone but fewer cones atPG continued. Since the number of filled seedper cone (FSPC) was high and remained fairlyconsistent (20!25 FSPC) at PG, variation in thenumber of seed per tree principally resulted fromvariation in the number of cones per tree(100!300 cones per tree). Year-to-year variationoccurred in all orchards but those NO orchardsproducing seed for the PG area (with onenotable exception) consistently produced loweryields than those NO orchards producing seedfor the southern interior. Orchard site also wasimportant. Southern interior orchards inArmstrong, BC (Pacific RegenerationTechnology Inc.) (PRT) equalled or exceededthat from PG and for the last two years haveconsistently out-produced all other NO orchards.

Over the seven years of observation, the loss ofseed from un-bagged cones in all NO orchardsranged from about 2 to 11 FSPC. There was noseed loss from un-bagged cones at PG. Of thefour NO orchard sites, Kalamalka and Tolko hadthe greatest losses (about 10) with the two PRTand Vernon Seed Orchard Company orchardseach losing about 5 and 4 FSPC, respectively.The greatest loss of seed from un-bagged conesappeared to occur in August. Cones exposed fora two week period from April to the end of Julywere not significantly affected but conesexposed in August had significantly fewer seedper cone. Again, bagged and un-bagged conesfrom PG did not show any differences in seedyields.

There is no general agreement about the baggingaffect. One side of the argument suggests thelosses are too large to be caused by insects.However, bagged cones and insect management(spraying) all show better seed yields thanuntreated cones. Insect-protected cones canaccount for some of the losses but on average,we expect seed yields from lodgepole pine to bein the order of 20!25 FSPC. However,production from NO orchards ranges from10!15 FSPC. If we account for 5!10 FSPClosses from unprotected cones, we still mustaccount for the other 5!10 FSPC that may notbe attributed to insect predation.

Joe WebberSaltspring Island, BCE-mail: jewebber@telus.net

VARIATION IN REPRODUCTIVE CAPACITY OF LODGEPOLE PINE

(Pinus contorta var. latifolia) IN BRITISH COLUMBIA

Lodgepole pine (Pinus contorta var. latifolia) isthe most wide-ranging pine in North America.Populations in British Columbia vary widely inphenotypic and genotypic characteristics anddifferences between populations can be linked tolocal climate or to geographic predictors. Theeffect of climate on variation in reproductivecharacteristics has never been examined, yet isvital to the production of seed necessary forreforestation. This study aims to determine therelationship between climate and variation infemale cone and seed characteristics. The studymakes use of the Illingworth provenance trial,sixty common garden plots that are distributedthroughout British Columbia. Female conesfrom seven source populations were collected at22 sites during the summer of 2012. Dataprocessing to date includes measuring conelength, and determining the number of scales percone. Initial results indicate wide variationbetween sites for both variables. Further dataprocessing will include determining the numberof seeds per cone. The final product of this studywill be a response function relating climatevariables to cone morphology and seed yield.

Anne Berland and Patrick von AderkasCentre for Forest BiologyDepartment of BiologyUniversity of VictoriaVictoria, BCE-mail: anne.berland@gmail.com

7

LODGEPOLE PINE CROP STATISTICSFROM SELECTSEED LTD. ORCHARDS

SelectSeed Ltd. is wholly owned by the ForestGenetics Council of BC, a government –industry cooperative. It’s mandate is theproduction of genetically selected tree seedwithin a financial objective of long-termsustainable profit and a modest return on capitalemployed. Nine of the 14 seed orchards operatedby SelectSeed under contract are lodgepole pine(Pli) (Pinus contorta var. latifolia) (about 29 of35 thousand ramets). As a result, Pli seedproduction and sales are key to the SelectSeed’slong-term business prospects.

The original seed production and cash-flowexpectations used for SelectSeed’s business planwere based on the small amount of informationavailable in about 2000. More recentinformation clearly shows issues with low seedproduction (filled seeds per cone) from Pliorchards located in the north Okanagan valley ofsouth-central British Columbia relative toorchards located near Prince George in centralBC. Using seed production statistics from thenine SelectSeed orchards, comparisons are madebetween forecast and actual seed productionstatistics, with breakdowns for Pli derived from

naturally occurring stands from seed planningunits (SPU) located in southern BC vs. centralBC, and between north Okanagan orchards vs.those located in slightly cooler environmentsjust outside the Okanagan valley. A SPU is ageographic area where tree populations aregenetically similar enough to allow seed transferwithin the geographic and elevation boundariesof the SPU.

Overall seed production across the nineSelectSeed Pli orchards does not varysubstantially from initial forecasts (Fig. 1).Orchards with selected trees from SPU in centralBC, however, have had poorer seed productionthan those from SPU in southern BC. There is aconfounding of SPU origin and orchard locationthat makes direct comparison difficult, but thereis evidence that Pli populations from central BCSPU, planted in orchard locations cooler thanthe Okanagan valley but not cool enough to bewithin naturally occurring lodgepole pineforests, are producing more seed than the samepopulations in north Okanagan orchardlocations. Reduced seed production relative topotential is a significant financial concern to thecompany, even though actual production levelsare close to initial forecasts.

Figure 1. Age-adjusted grams of lodgepole pine seed produced per orchard position for selected treesfrom northern and southern seed planning units (SPU).

Jack WoodsForest Genetics Council of BC and SelectSeed

Ltd.Vancouver, BCE-mail: jwoods.fgc@shaw.ca

8

QUESTIONS ABOUT THE ROLE OF SEED BUG HERBIVORY IN

PRODUCTION COMING FROM INTERIOR LODGEPOLE PINE SEED

ORCHARDS

Herbivory by the western conifer seed bug(Leptoglossus occidentalis) has been assumed tobe the principal cause of substantial annualshortfalls of seed coming from lodgepole pineorchards located in the interior of BritishColumbia. The difference between the numberof filled seeds per cone (FSPC) produced incones protected from seed bug attacks (by beingenclosed inside nylon-mesh bags) and thenumbers of viable seeds coming from cones leftopen in the environment has been used as anindicator of the amount of seed bug herbivorytaking place in these orchards. This is the ‘SeedBug Model’ for losses occurring in this croppingsystem. Operational production records fromKalamalka lodgepole pine Seed Orchard 307 for2001 to 2010, taken with bagging trial resultsfrom this orchard for the same years, allow us tomake numerical estimates of whole-orchardFSPC shortcomings occurring annually duringthe past decade. These results are discussed inthe context of whether they are, or are not,consistent with a Seed Bug Model.

For over fifteen years, concerted efforts havebeen made to establish a relationship betweenthe number of seed bugs observed in Orchard307 and the FSPC losses attributed to seed bugherbivory. These efforts have not beensuccessful as no seed bug density:crop damagerelationship and no economic threshold haveever been established for this cropping system.Without an economic threshold, pesticidetreatments for seed bugs have never beenconducted in a manner consistent with themodern practice of Integrated Pest Management.Beyond this consideration, comparisons ofFSPC values derived from annual cropproduction statistics with FSPC values for conesheld in bags show that applications of thepesticide Sevin for seed bug control never haveraised operational seed yields to the levelsobtained inside the bags. In fact, seed deficits ofone-third or more of the crop occurred in each ofeight years in which pesticides were applied forseed bug control. These results were notconsistent with expectations coming from theSeed Bug Model. If FSPC losses were beingcaused by insect herbivory, why did treatmentsby an insecticide fail to increase FSPC numbersclose to those seen inside the bags?

Work done by Dr. Joe Webber and colleagues in2010 showed that major FSPC losses occurredin cones that were not enclosed inside bags

during the month of August. Being exposed tothe environment through any two-week periodfrom the beginning of May to the end of July didnot significantly reduce FSPC values. Timing ofharvest trials done in 2011 showed a similartemporal profile for FSPC declines. However,none of the cones used in the 2011 trial wereheld in bags during any point of the growingseason. Versions of both experiments have beenreplicated across multiple years at severallocations. Now, it is recognized that the majorityof the FSPC losses observed in north Okanaganlodgepole pine orchards occur in August.

By using the FSPC deficits derived frombagging trials and multiplying this by thenumber of cones harvested in any given year(derived from crop production statistics), we cancalculate the number of seeds ‘lost’ during agrowing season. For 2010, this number wasestimated to be about 5.4 million seeds. Baggingexperiments indicate that these FSPCshortcomings occurred in August. Using thisinformation, we can calculate the number ofseed bugs needed to cause the FSPC deficitsobserved for Orchard 307 in 2010. Over thecourse of 30 days, 5,374,000 seeds were lost,eaten by seed bugs with a maximum individualdaily herbivory rate of 5 seeds per day. This losswould require a minimum of 35,826 seed bugsto be actively feeding, each eating their dailymaximum number of seeds on every day of themonth.

Orchard 307 was harvested through August2010. Staff members spent a minimum of 175person-hours per week in the orchard in Augustand they physically handled every cone that waspicked. Although workers reported seeing seedbugs, we never received any reports of seed bugsbeing seen in anything approaching the numbersthat would be required to cause these FSPClosses under the Seed Bug Model.

In summary, there never has been a relationshipestablished between seed bug population sizesand the FSPC damage that they reputedly cause.Pesticide treatments have never raised FSPClevels in the operational harvests to thosepredicted (from bagging trials) to be possible ifseed bugs were causing the FSPC losses. Withthe recently acquired knowledge of when theFSPC declines occur during the growing season,we can calculate how many seed bugs must bepresent in Orchard 307 to cause the lossesattributed to them by the Seed Bug Model.Nothing even close to the number of seed bugsrequired to cause these deficits was observed in2010, nor have they been observed in any otheryear during the past decade. It is for thesereasons that I question the Seed Bug Model. I do

9

not believe that the differences in FSPCobserved between cones held inside bags andcones left exposed in the environment can besolely attributed to herbivory by seed bugs.

Currently, there are a number of investigationsthat are examining other aspects of this problem:1) seed samples taken through the declineperiod are being examined microscopically toobserve the physiological aspects of losses offilled seed,2) the relationship between seed set, area oforigin of the orchards, temperature and seeddeclines is being investigated in field trials,3) in-cone temperatures and seed bug herbivoryare being monitored on selected conesthroughout the growing season,4) harvesting schedules that incorporate thetiming of FSPC declines by clone are beingdeveloped to maximize the yields coming frominterior lodgepole pine seed orchards, and5) a method to detect seed bug herbivory onindividual lodgepole pine seeds is beingdeveloped.

It is hoped that this multi-faceted approach willhelp to address the unanswered questions thatremain about seed losses occurring in interiorlodgepole pine orchards.

Jim CorriganMinistry of Forests, Lands and Natural Resource

OperationsTree Improvement BranchVernon, BCE-mail: Jim.Corrigan@gov.bc.ca

LEPTO-CAMS AND THERMOCOUPLES: WHY DO INSECT BAGS PREVENT

AUGUST Pli SEED LOSS IN THE INTERIOR OF BC?

Lodgepole pine (Pinus contorta var. latifolia)seed orchards in the Southern Interior of BChave suffered from low seedset for many years.The causes largely remain a mystery, despiteyears of research. In 2011 and 2012, it wasdiscovered through bagging studies that much ofthe seed loss occured in August. The Augustseed loss is entirely preventable by enclosing thecones in mesh bags, while seed loss earlier in theseason was not affected by mesh bags.

Only two theories exist as to why mesh bagsprevent seed loss. One is that the bags alter themicroclimate, protecting the cones from, forexample, extreme heat events. The other is thatthe bags exclude Leptoglossus occidentalis, thewestern conifer seedbug, which might feed onseeds during August. Both theories were testedduring the summer of 2013.

The microclimate theory was tested by choosing4 cones on each of 10 lodgepole pine trees incommercial seed orchards in Vernon, BC. Coneswere close together, with similar aspect and treeheight. A one millimetre hole was drilled intothe base of each cone, and a small K-typethermocouple was inserted and held in placewith zip-ties. All four thermocouples on eachtree were connected to a 4-channel dataloggerthat recorded the internal cone temperatures at 5minute intervals. Two of the cones on each treewere enclosed in a mesh bag; the other two wereleft unbagged. Internal cone temperatures werecompared between bagged and unbagged cones,examining maximum temperatures and timesspent above various temperature thresholds.

The seedbug theory was tested by installing onetime-lapse camera in each of the 10 trees used inthe thermocouple experiment. Each camera wasaimed at a cluster of 3!4 cones and set to takephotos at 5 minute intervals. A similar conecluster on a nearby branch, with the same aspectand tree height, was enclosed in a mesh bag.Photos were examined for the presence ofseedbugs, which were categorized by activity:feeding, walking, mating, etc. At season's end,bagged and unbagged cones will be harvestedand seeds extracted to determine the extent offilled seed loss.

As of July 3, 79,206 cone temperaturemeasurements had been taken. Mean conetemperatures were always slightly higher inbagged than in unbagged cones. Bagged conesalso had more hours above any given thresholdtemperature, such as 35°C. There is morevariation in cone temperatures between trees andbetween cones within a treatment, than betweentreatments. No statistical analysis has beenconducted yet, but it appears that conetemperatures are unlikely to be the cause of theAugust seed loss.

By July 3, 81,425 images had been collected andexamined. Of these, 1,703 images showedseedbugs on or near the cones. Feeding was adominant activity. Each cone cluster was fedupon from 0 to 19.7 hours. Seedbugs were neverobserved feeding on two cone clusters. Thesesightings confirm previous evidence that there isa clonal difference in seedbug host preference. It

10

also confirms the likelihood that seedbugs areresponsible for the August seed loss.

However, these data only record events up toJuly 3. Things might change during the month ofAugust. Bags might shield cones from extremeheat events not experienced during May or June.Seedbugs may not feed upon cones duringAugust. Though this evidence is suggestive, wemust wait until our August data have beencollected before drawing conclusions.

Ward StrongMinistry of Forests, Lands and Natural ResourceOperationsVernon, BCE-mail: Ward.Strong@gov.bc.ca

SEED SHORTFALL IN LODGEPOLE PINE

Samples for histological study were collectedfrom a seed orchard located at KalamalkaResearch Station, Vernon, BC. Weekly samplecollections were completed between mid-Julyand mid-September, 2012. A big increase indamaged seed was confirmed during August.The percentage of filled seed per cone (FSPC)diminished progressively over the course of thefirst two weeks, reaching levels of under 50%FSPC, where it stayed until mid-September.Under a dissecting microscope, seed deathoccurred in two steps: death of themegagametophyte was followed by that of theembryo. Dying tissues ranged in colour fromyellowish-brown to dark brown. Bothgametophyte and sporophyte, i.e., embryo, weresoft in texture, unlike their healthy counterparts,which were not only firm, but of much lighterhue.

Histological analysis revealed tissuedegeneration in the seed. Healthy tissue wascomposed of storage cells that had abundantreserves of protein bodies, starch grains, andlipid bodies. The cells were tightly appressed toone another. Tissue degeneration wascategorized as three types: Type I began with theappearance of tiny intercellular spaces. Thesespaces increased gradually in size and then thetissue developed large holes. Fungal hyphaewere frequently observed in cells as well asintercellular spaces. Type II degeneration hadthe appearance of cell liquidization. Cell walls

were dissolved and cell contents wereamorphously coagulated. Yellow particulatestructures were frequently observed. Type IIIwas progressive loss of cell contents until onlycell walls remained. Protein body breakdownwas followed by vacuolation and nucleardisintegration. Tissue integrity failed with cellsshowing signs of cytoplasmic collapse and cellwall rupture. Types I and II occurred randomlyat multiple loci in a megagametophyte. Differentstages of Type I degeneration were sometimespresent in the same tissue. Types I and II werefound in samples from all collection dates,whereas Type III was observed in all dyingmegagametophytes that had softened tissue.

Our histological study does not support thepossibility of Leptoglossus occidentalis-relatedseed loss. However, our study did not excludethe possibility of other organisms that mightcontribute to seed loss. The presence of yellowparticles found in many of the samples suggeststhe presence of an unidentified organism or anunknown aggregation phenomenon. A majorreason for degeneration in seed was the presenceof a fungus. Hyphae were widespread in holesthat developed in the degeneratingmegagametophyte in many of the samples. Inconclusion, seed shortfall in lodgepole pineshows a progressive degeneration of the seedand there are different types of tissuedegeneration which can occur in the absence orpresence of a fungus and/or some unidentifiedparticles implying that there is more than onebiotic or abiotic factor responsible for seeddeath. High temperatures in August mayaccelerate the process of seed degeneration.

Lisheng Kong and Patrick von AderkasCentre of Forest BiologyDepartment of BiologyUniversity of VictoriaVictoria, BCE-mail: lkong@uvic.ca

METHODOLOGY TO ORGANIZEOPERATIONAL LODGEPOLE PINE

CONE HARVESTS BY USINGOBSERVATIONS OF SEED SET

DECLINES

Poor seed set in North Okanagan lodgepole pine(Pinus contorta var. latifolia) seed orchards hasbeen an issue for a number of years. Results

11

from numerous trials indicate that seed set levelsare acceptable until early in August. After thispoint seed set declines rapidly. Each lodgepolepine clone experiences this sudden loss of seedat a different point in time. We relate this periodto growing degree days.

In order to increase seed production, each clonemust be harvested at the correct time. If thecones from a particular clone are harvested tooearly seed quality is poor. If the same clone ispicked after its unique seed decline period hasstarted seed production is poor.

We are working on developing a method toidentify the best time to harvest each clone. Wecollect weekly cone samples from throughout thesummer and x-ray the seed in order to identifythe onset of sudden seed decline. Relating thisinformation to degree days will allow us toschedule our cone harvest timing so that eachclone is collected when seed yield and seedquality are at acceptable levels.

Gary GiampaMinistry of Lands, Forests and Natural ResourceOperationsTree Improvement BranchVernon, BCE-mail: Gary.Giampa@gov.bc.ca

TESTING CONTAINERS FORRESEARCH AND CONSERVATION

SEED STORAGE

Once dried, seed is best stored in hermetically-sealed containers (Manger et al. 2003). Ifcontainers are not effectively sealed, seed willgradually absorb moisture and storage life willbe reduced (Yanping et al. 1999). The seal thatcan be achieved is just as important as thecontainer material. Freire and Mumford (1986),Gómez-Campo (2002) and Manger et al. (2003)have shown that many containers do not sealeffectively.

There are advantages and disadvantages to thetypes of containers, their lids and materials suchas cost, weight, and reusability. Screw lids forrigid containers allow easy access but mayoccasionally loosen during freezing or thawing.Much more effective are lids with natural rubber

or bromobutyl seals that can be clamped orcrimped onto the container. Permanently sealedcontainers, such as metal cans or glass vials, arenot desirable when frequent access to the seedsis required. There are glass sealing jars that havebeen shown to seal effectively and are used inseed banks. However, these pose issues withweight and the necessity to match the quantity ofseeds to the size of the jar requiring differentsizes to be available, in order to minimizeoxidation and ageing. Other options used byseed banks are plastic and foil bags. The bagscan be cut to fit seed quantities and the air insideexpelled. The bags are heat sealed and can be cutopen and reused. There are also laminated typesof foil bags that help to prevent punctures andleakage.

At Alberta Tree Improvement and Seed Centre(ATISC), a range of glass and plastic containershas been used for long-term storage of seed forconservation or research purposes. Glass bottleswith 3 different types of screw lids and threetypes of plastic bottle of various sizes have beenused. These containers were not designedspecifically for cold storage and many do notclaim to seal even in normal conditions.

This trial was set up to test the seal effectivenessof a variety of containers currently being usedand being considered to store valuable andirreplaceable research and conservation seedlotsat the ATISC. This test looked at ten glass andplastic containers currently used at ATISC forstorage, two types of static shield plastic bags,and three types of foil bags, as well as the ‘zip-loc’ type bags often used for seed transportation.

Methods

The container testing method used was the samemethod employed by the Millennium Seed Bank,at the Royal Botanic Gardens, Kew and wasoriginally adapted from a technique developedby Gómez-Campo (2002).

Freshly dried self-indicating silica gel wasplaced into test containers at 1 g per 100 mlvolume. This is a small enough volume tochange colour at the earliest signs of leakage andnot just absorb any added moisture but largeenough to be easily visible (Fig 1).

12

Figure 1. Example of indicator silica gel in‘zip-loc’ type plastic bag showingbefore and after colour change fromdry orange to humid green.

Nine glass and plastic bottles already in use inthe research and conservation seed bank weretested (Fig. 2), as well as the heat sealed 5 milpolyethylene bags used in the Reforestation SeedProgram (RSP).

Figure 2. Nine types of bottles currently beingused for long-term seed storage in theconservation & research seed bank

Six other bags were also tested (Fig. 3). Allbags were filled 50!70% with inert Styrofoam tohelp ‘bulk out’ the volume. When calculating theamount of silica to add, the Styrofoam wasconsidered to be 50% air, e.g., a 200 ml bag with100 ml of Styrofoam would receive 1.5 g ofsilica. The containers were sealed in the usualway at ambient conditions of 8.6!15.9% RH and17.8!21.7°C (Fig. 4). Ten replicates were set upfor each container, with the exception of the 3Mfoil bag which had only two reps (Table 1).

Figure 3. Seven bag types tested, includingthose currently used in thereforestation seed bank (140) and the‘zip-loc’ type bags used in the seed labfor temporary storage (130).

Figure 4. Method of sealing bottles involvedtightening of lids and wrapping thejoin with either electrical tape or pipejoint tape (far left).

Once sealed, the containers were transferred tohigh humidity conditions of 96!98% RH atambient laboratory temperature. The colour ofthe silica gel was checked every two weeks forchanges. Once changes in colour from orange togreen were detected, indicating the seal hadfailed, the container was removed from the test.Before removal, verification that the containerhad been ‘sealed’ appropriately was confirmed.Opaque foil bags were not checked but were leftto the end of the test.

After 4 weeks at ambient temperatures, anyremaining containers were then moved to -18°Cand the silica gel checked periodically for

13

changes. The test was terminated after 70 days in-18°C.

Results

Most containers that failed did so while atambient air temperatures (Table 1). Of the tencontainers currently used for long-term seedstorage, three passed through to -18°C and onewith only a 90% pass.

At the end of the test, the glass bottles, twoplastic bottle types and all three foil bags hadpassed with all replicates still indicating ahermetic seal.

Conclusion

Five types of containers currently being used inthe research and conservation seed bank cannotbe hermetically sealed and of the ones thatpassed, all are rigid container types. From along-term conservation perspective, switchingfrom solid containers to bags that can be cut tofit seed quantities and therefore reduce oxidationshould lengthen seed life. Moving to a foilbagging system will also provide extra space andreduce weight for cold storage flooring andshelves, as well as for staff carrying seed trays.

There were four main differences between thethree foil bag types tested. These werelamination, size availability, gusseting, and theability to obtain stock. The foil bags obtainedfrom Flair Flexible Packaging Corporation,Calgary were gusseted and after testing withsmall seed, it was decided that the gussets madeit too difficult to remove seed from the bag.There was also a very limited size range and thelargest size available was not suitable for ourlarger pine collections. Therefore, the Flair foilbags were eliminated.

The 3M Canada foil bag was not gusseted anddid provide a larger size than Flair, althoughthere were not many sizes overall and the largebags would need to be cut to suit with moresealed edges. However, obtaining even a fewsamples from 3M for use in the test provedextremely difficult and time consuming. Thispoor customer service plus the risk of extrasealed edges eliminated the 3M bags fromconsideration.

The Baltimore foil bags proved to be the bestchoice for many reasons in the end. In additionto passing the test, the tri-laminate providesprotection for the foil from sharp seeds. The

bags are not gusseted, making seed removaleasy. There are four sizes available rangingfrom the 75 ml volume used in this test to thelargest at approximately 4 L (the largestavailable of any tested foil bags). In addition,these bags are used by other seed banks and thecompany is very knowledgeable about this typeof use. The only negative aspect is that thesebags are manufactured in Europe. Costs werecalculated including shipping for an initial largeorder to repackage all ~7000 research andconservation seed collections plus enough extrafoil bags to last for a few years of new storage.This worked out to 18.4¢ per bag and wasdeemed an acceptable and competitive cost.

Update

The bags were ordered and the repackagingproject was completed for all research andconservation seed collections in May 2013. Allnew seed collections stored in the conservation& research seed bank will be packaged in foilbags. Quality testing will be done on each neworder using the above method.

Literature Cited

Freire, M.; Mumford, P. 1986. The efficiency ofa range of containers in maintaining seedviability during storage. Seed Scienceand Technology 14:71!381.

Gómez-Campo, C. 2002. Long-term seedpreservation: the risk of selectinginadequate containers is very high.Monographs ETSIA, Univ. Politéchnicade Madrid 163:1!10

Manger, K.R.; Adams, J.; Probert, R.J. 2003.Selecting seed containers for theMillennium Seed Bank Project: atechnical review and survey, Pages637!652. In R.D. Smith, J.B. Dickie,S.H. Linington, H.W. Pritchard, and R.J.Probert, eds. Seed conservation: turningscience into practice. Royal BotanicGardens, Kew, UK.

Yanping, Y.; Rongqi, G.; Qingquan, S.; Shengfu,L. 1999. Effects of storage temperatureand container type on the vigour of Welshonion seeds with low moisture content.Australian Journal of ExperimentalAgriculture 39:1025!1028.

Container#

ContainerType

Volume(mL)

Description Comments Approx.Styrofoam

(mL)

Approx.silica gel

(mg)

Day 12% passed

Day 27% passed

Day 20% passed

Day 35% passed

Day 70% passed

1-10 bottle 500 Plastic, type 1 Current use n/a 5.00 100 100 100 100 100

11-20 bottle 250 Plastic, type 1 Current use n/a 2.50 100 90 90 90 90

21-30 bottle 125 Plastic, type 1 Current use n/a 1.25 100 10 0

31-40 bottle 125 Plastic, type 1 Current use n/a 1.25 80 0

41-50 bottle 125 glass/plastic lid Current use n/a 1.25 100 100 100 100 100

51-60 bottle 60 plastic, type 2 Current use n/a 0.60 100 100 100 100 100

61-70 bottle 60 plastic, type 1 Current use n/a 0.60 0

71-80 bottle 30 plastic, type 2 Current use n/a 0.30 100 0

81-90 bottle 25 plastic, type 3 Current use n/a 0.25 80 0

91-100 bag 75Baltimore heat sealedtrilaminate foil bag

New 50 0.50 n/a n/a n/a n/a 100

101-110 bag 753M Static Shieldplastic bag SCC1000- constant heatersealed

New - Noinstructions onsealing were

provided.50 0.50 0

161-170 bag 753M Static Shieldplastic bag SCC1000- plastic heater sealed

New - Noinstructions onsealing were

provided.

50 0.50 0

111-120 bag 800

3M Static Shieldplastic bag SCC1000– zip-lock typeclosure

New 400 6.00 0

121-130 bag 752 mil polyethylenebag – ‘zip-loc’closure

Temp. use 50 0.50 0

131-140 bag 8005 mil polyethylenebag - plastic heatersealed

Storage use forRSP.

400 6.00 0

141-150 bag 75 Flair 2oz foil bag -constant heater sealed

New 75 0.50 n/a n/a n/a n/a 100

151-152 bag 2503M Military spec foilbag - constant heatersealed

New 150 1.75 n/a n/a n/a n/a 100

Table 1. Description and results of containers tested for seal effectiveness. Days in light green were held in high humidity and ambient labtemperature. Days in light blue were held at -18°C.

15

Container Manufacturers Passing containers only. Prices were quoted 2011and do not include taxes or shipping.

Baltimore Innovations Ltd.http://www.baltimoreinnovations.co.uk/packaging-sector/barrier-foils/Email: sales@baltimoreinnovations.co.ukJackson’s Business Park, Wessex Rd, BourneEnd, BUCKS SL8 5DT, United Kingdom$64.00/1000

Flair Flexible Packaging Corp.http://www.flairpackaging.comEmail: cscanada@flairpackaging.com4100 72 Avenue SE, Calgary, AB T2C 2C1CanadaStand up, pouch style, bottom gusset, without zip-lock, gloss, 2oz$73.79/1000

3M Canada EMX Enterprises Limited (western Canadadistributor)250 Granton Drive, Richmond Hill, ON L4B 1H7CanadaEmail: info@emx.ca

Moisture Barrier Bag MILpac 131Pricing followed up over 5 months – neverreceived.

I can provide no ordering details for the solidcontainers that passed, beyond that they wereordered from Fisher Scientific. The specificationand manufacture of these types of containers havechanged frequently in the history of our seedbank, e.g. container shape, metal vs plastic lids,plastic lid inserts vs paper seals. The exactcontainers tested, although they are the mostrecent ordered and used at ATISC, are no longeravailable from Fisher.

Foil Bag Heat Sealer

Pal Distributors Inc.http://www.palgroup.ca/distributors/machines/sealers.htmlEmail: calgary@palgroup.ca3N, 5550-36 St. SE, Calgary, AB T2C 1P1,CanadaConstant Heat Foot Sealer, 12" 300CFN$560.00

Lindsay RobbEnvironment, Sustainable Resources and

DevelopmentSmoky Lake, ABE-mail: Lindsay.Robb@gov.ab.ca

UPCOMING MEETINGS

42nd Atlantic Forest Nursery Conference andSeed Orchard WorkshopOctober 1!3, 2013 Debert, NSContact: Blair Andres

Blair.Andres@Northernpulp.com

ISTA Annual MeetingJune 16–19, 2014 Edinburgh, UKwww.seedtest.org

RECENT PUBLICATIONS

Afrose, F.; O’Reilly, C. 2013. Breaking seeddormancy in European rown seeds and itsimplications for reforestation. NewForests 44(4):547!557.

Cook, S.P.; Sloniker, B.D.; Rust, M.L. 2013.Using systemically applied insecticidesfor management of Ponderosa pine conebeetle and Dioryctria coneworms in seedorchards. Western Journal of AppliedForestry 28(2):66!70.

Fry, D.; Stephens, S. 2013. Seed viability andfemale cone characteristics of matureknobcone pine trees. Western Journal ofApplied Forestry 28(1):46!48.

Gruwez, R.; Leroux, O.; De Frenne, P.; Tack,W.; Viane, R.; Verheyen, K. 2013.Critical phases in the seed development ofcommon juniper (Juniperus communis).Plant Biology (Stuttgart) 15(1): 210!219.

Li, S.; Shi, T.; Kong, F.; Ma, Q.; Mao, Y.; Yin,T. 2013. Methods for breaking thedormancy of eastern redbud (Cerciscanadensis) seeds. Seed Science andTechnology 41(1):27!35.

Liu, J.; Bai, Y.; Lamb, E.G.; Simpson, D.; Liu,G.; Wei, Y.; Wang, D.; McKenney, D.W.;Papadopol, P. 2013. Patterns of cross-continental variation in tree seed mass inthe Canadian boreal forest. PLoS One8(4), Article No. e61060.

16

McCarten, S.A.; Gosling, P.G. 2013. Guidelinesfor seed collection and stratification ofcommon juniper (Juniperus communis L.)Tree Planters’ Notes 56(1): 24!29.

Parker, W.C.; Noland, T.L.; Morneault, A.E.2013. Comparative mast seed productionin unmanaged and shelterwood white pine(Pinus strobus L.) stands in centralOntario. New Forests 44(4):613!628.

Tiansawat, P.; Dalling, J.W. 2013. Differentialseed germination responses to the ratio ofred to far-red light in temperate andtropical species. Plant Ecology214(5):751!764.

Tomlinson, P.B. 2012. Rescuing Robert Brown !The origins of angio-ovuly in seed conesof con i fers . Botanical Review78(4):310!334.

van Treuren, R.; de Groot, E.C.; van Hintum, ThJ. L. 2013. Preservation of seed viabilityduring 25 years of storage under standardgenebank conditions. Genetic Resourcesand Crop Evolution 60(4):1407!1421.

Viglas, J.N.; Brown, C.D.; Johnstone, J.F. 2013.Age and size effects on seed productivityof northern black spruce. CanadianJ o u r n a l o f F o r e s t R e s e a r c h43(6):534!543.

Westengen, O.T.; Jeppson, S.; Guarino, L. 2013.Global ex-situ crop diversity conservationand the Svalbard Global Seed Vault:Assessing the current status. PLoS One8(5), Article No. e64146.