Báo cáo khoa học: "The response of dehydrated Douglas fir (Pseudotsuga menziesii) pollen to three in vitro viability assays and their relationship to actual fertility"

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article Original dehydrated Douglas fir The response of (Pseudotsuga menziesii) pollen to three in vitro viability assays and their relationship to actual fertility M Bonnet-Masimbert 2 JE Webber 1British Columbia Ministry of Forests, Research Branch Laboratory, 1320 Glyn Road, Victoria, BC V8W 3E7, Canada; 2 INRA, Station d’Amélioration des Arbres Forestiers, Centre de Recherche d’Orléans, Ardon, 45160 Olivet, France accepted 30 September 1992) 16 March 1992; (Received Summary — In vitro viability response of Douglas fir pollen stored for various periods (1 to several years) was related to actual seed set. Three assay types that provided useful relationships to seed set were respiration (RESP), percent leachate conductivity (%COND) and percent germination (CLASS 1 + 2). Before developing the relationship to seed set, media effects on germination, leach- ing time for conductivity and pollen hydration effects for all assays were studied. Both simple linear and non-linear regression analyses were compared to percent filled seed per cone (%FSPC) as de- termined from controlled crossing pollinations. Media type had a significant effect on germination re- sponse which, in the time of the test (48 h), appeared to be related to osmotic rather than metabolic effects. Hydrating stored dehydrated pollen for 16 h at 100% RH and 25 °C prior to the analysis had a significant effect on improving the response for conductivity and germination, but had no significant effect on respiration. Hydration effects were also apparent on the correlation coefficient (r) using sim- ple linear regression. For unhydrated and hydrated pollen, the r values for assay response and were 0.70 and 0.85 for RESP, -0.36 and -0.86 for %COND, and 0.07 and 0.83 for CLASS %FSPC 2 germination, respectively. Using non-linear regression models, the coefficient of determination 1 + (r values for assay response of unhydrated and hydrated pollen against %FSPC were 0.76 and ) 2 0.83 for RESP, and 0.24 and 0.82 for %COND, and 0.61 and 0.84 for CLASS 1 + 2 germination, re- spectively. The regression equations developed for respiration, percent conductivity and germination can be applied to Douglas fir pollen lots when used for controlled crossing pollinations but may not result in expected seed set values when the pollen lot is expected to also compete with outcross pol- len. Pseudotsuga menziesii / Douglas fir 1 pollen 1 respiration 1 germination / viability 1 fertility 1 seed-set Résumé — Réponse du pollen de sapin de Douglas (Pseudotsuga menzesii) à 3 tests de via- bilité in vitro et relation avec la fertilité réelle de ce pollen. Les valeurs du coefficient de détermi-
(r pour le pollen sec et le pollen réhydraté pour la réponse aux différents tests et %FSPC ) 2 nation respectivement (tableau V) de 0,76 et 0,83 pour RESP (respiration), à 0,24 et 0,82 pour sont %COND (pourcentage de conductivité) et 0,61 et 0,84 pour la germination (CLASS 1 + 2). Par ailleurs, à travers une expérience de dilution de pollen, il apparaît que la relation entre le pourcentage de pollen vivant et le %FSCP n’est pas linéaire (fig 5). Au-delà d’un seuil voisin de 40-50% de pollen vivant, il n’y a plus d’amélioration du %FSCP. D’un point de vue pratique, les équations de régression développées pour la respiration (fig 6), le pourcentage de conductivité (fig 7) et la germination (fig 8) peuvent être utilisées pour estimer la qualité de lots de pollen de sapin de Douglas utilisés pour des croisements contrôlés. Toutefois, ces courbes peuvent ne pas se traduire par le résultat attendu en terme de rendement en graines si un lot donné de pollen se trouve en situation de compétition avec un autre lot, ce qui n’était pas le cas de cette série d’expérimentations. Pseudotsuga menziiesii / sapin de Douglas / pollen/ respiration / germination / variabilité / fer- tilité / lot de graines INTRODUCTION least, comparable fertility potential has, at to that of seed) competing (ability to set pollen. As advanced generation Douglas fir seed Pollen management procedures for han- orchards become established, the need to dling Douglas fir pollen have been tested protect potential genetic gain becomes and, in particular, successful storage tech- more important. In the Pacific Northwest, the threat of inferior gametic infiltration into nique are now used routinely (Webber, 1987; Webber and Painter, in preparation). orchard populations is a constant concern However, methods for assessing pollen vi- and estimated levels of contamination range from 6-56% (Smith and Adams, ability in vitro and relating the results to seed set remain rudimentary. The objec- 1983; El-Kassaby and Ritland, 1986a; Wheeler and Jech, tives of this study are to optimize the re- 1986a). Asynchronous flowering (El-Kassaby and Ritland, 1986b), sponse of 3 viability assays (respiration, disproportionated fecundity among clones leachate conductivity and germination) us- ing stored Douglas fir pollen and to relate (El-Kassaby et al, 1989), and inbreeding (Woods and Heaman, 1989) can also re- these responses to actual seed set. The duce the genetic efficiency (see Adams, study also considers the effect of pollen 1983; El-Kassaby et al, 1984) of orchard hydration on in vitro assay response and seed. One approach to reducing the ef- its relationship to actual seed set. fects of contaminating pollen and improv- ing genetic efficiency is supplemental MATERIALS AND METHODS mass pollination (SMP). SMP has been successfully used to im- prove the balance of paternal contribution Selection of pollen lots (El-Kassaby and Ritland, 1986b), improve seed yields (Webber, 1987) and reduce Douglas fir (Pseudotsuga menziesii (Mirb) Fran- the negative impact of selfing and contami- co) pollen was collected over many years from nation (El-Kassaby and Ritland, 1986b; both the tree breeding and seed orchard pro- Wheeler and Jech, 1986b). However, suc- grams. All pollen lots (referred to as a family of cess of SMP is dependent on many fac- pollen grains arising from a single clone or seed- tors (see Bridgwater et al, 1991) not least ling) were stored at a pollen moisture content of of which is ensuring that the pollen applied 8% and at -20 °C in evacuated containers <
(see Webber, 1987; Webber and Painter, in absorbent paper saturated with water. taining preparation). Storage period for each pollen lot The lid of the larger Petri dish was secured and varied and ranged from 1-5 yr. germination allowed to proceed at 25 °C for 48 h. No particular precautions were taken to ei- ther exclude light or use specified photoperiods. In vitro viability assays After 48 h, germination was scored based on the percent of grains in each of 4 categories: Class 1, pollen grains elongated greater than Germination twice the original hydrated diameter of the grain; Class 2, pollen grains showed signs of elonga- Media type tion but were still less than twice their hydrated The procedure for germinating Douglas fir pollen diameter; Class 3, pollen grains showed no sign initially followed the technique described by Ho of elongation; and finally, any pollen grains from and Sziklai (1972). Their medium was adapted either of the 3 classes showing any amount of from Brewbaker and Kwack (1963) and included plasmolysis or other damage were scored as H (0.1 mg/ml), Ca(NO H (0.3 mg/ )O 32 BO 3 .4 2 Class 4 (see fig 1). The actual number of germi- ml), MgSO H (0.2 mg/ml) and KNO (0.1 .7 O 42 3 nating pollen grains counted followed the proce- mg/ml). The germination medium was a 10% di- dures suggested by Stanley and Linskens lution (10B) of the Brewbaker and Kwack (1963) (1974) for determining significant response dif- solution which also contained sucrose (5 or ferences at the 95% confidence level. For pollen 10%) and/or indole acetic acid (10 ppm). This lots germinating in the 50% range, 300 grains = 10B medium was satisfactory to germinate were observed and for lots germinating either > Douglas fir pollen, but the presence of sucrose 90% or < 10%, ≈ 100 grains were counted. All also facilitated the growth of contaminants asso- results were expressed as percent germination ciated with the pollen. Antibiotics (nystatin and of either Class 1 or Class 1 + 2 grains. chloramphenicol) have been used to reduce the growth of contaminants (Charpentier and Bon- net-Masimbert, 1983) but for the short incuba- Conductivity tion periods used (see below), they were not re- quired in the germination medium. Leaching of pollen lots followed the procedures The first media experiments compared the Ching and Ching (1976) in which 100 mg of of germination of 8 dehydrated stored Douglas fir pollen was soaked in 30 ml deionized water pollen parents in 4 aqueous solutions: deionized (specific conductance < 2 μS/cm) at 25 °C for water (H 10% sucrose (10S), 10% Brewbak- O), 2 60 min with constant shaking. Initially, the lea- er and Kwack (1963) solution (10B), and 10% chate was filtered or centrifuged to remove the polyethylene glycol (PEG molecular weight residual pollen debris. However, it was deter- 4000...10P) using the procedures described be- mined that removing the residue had little if any low. Agar (1.0% agar in 10% Brewbaker’s solu- effect on conductivity measurements and simply tion) was also considered as a solid medium, letting the pollen suspension settle for 5 min with and without added constituents, but it was prior to measurement was sufficient. The con- not used in subsequent trials because germina- ductance of the filtrate was determined using a tion was slower and scoring response was more standard conductivity meter (Orion Model 101) difficult. In a second experiment, the germina- with an immersion cell (platinum electrodes). All tion response of the same dehydrated lots were measurements were made at 25 °C. compared in 4 concentrations of PEG-4000 (10, A time of leaching experiment was also com- 20, 30, and 40%) with or without the inclusion of for the hydrated pollen lots only. In this pleted the 10B. test, all lots were weighed (100 mg), hydrated and then leached for 1, 2, 4, 6 and 24 h. After Germination procedure leaching, the conductivity of the leachate was determined and then expressed as a percent- For comparing germination media types, 3 ml of age of the total leachate (hot conductivity). After medium were added to 35-mm Petri dishes and cold (25 °C) conductivity was determined, the 10 mg of pollen sprinkled over the surface. The solution was boiled for 60 min, cooled to 25 °C, Petri dish lid was replaced and the dish was deionized water added as required to make the then placed in a larger Petri dish (90 mm) con-
full range 1V total vol 30 ml, and the conductivity recorded. strip chart recorder using as All cold conductivity (COND) results were ex- (100%). pressed on a dry weight basis of the pollen About 100 mg of pollen (dw) was added to 3 used (ie μS/cm/g dw). Where the results sample ml deionized water contained in the cuvette of expressed as percent conductivity were the water bath assembly and allowed to equili- (%COND), the ratio of cold to hot leachate con- brate at 30 °C for 3 min with constant stirring. ductivity was determined. After equilibration, stirring was stopped, the electrode was inserted making certain all air bubbles were excluded, and stirring resumed. Respiration Oxygen uptake was recorded for a minimum of 5 min using a chart speed of 1 cm/min. The rate Measurement of oxygen uptake by pollen in an of oxygen consumption was calculated using the aqueous solution followed the procedures of percent change in volume of dissolved oxygen Binder and Ballantyne (1975). Depletion of oxy- for a 5-min period and the solubility of oxygen in gen in 3 ml vol deionized water was determined air-saturated water at 1 atm pressure as 5.48 μl by a YSI oxygen probe (Model 5331 Clark type O at 30 °C (Lessler, 1969). Variation in oxy- /ml 2 gen solubility due to changes in atmospheric polarographic electrode) using a YSI standard pressure during any particular test were small water bath assembly (Model 5301) and oxygen and, therefore, ignored. Results for oxygen con- monitor (Model 5300). Uptake was measured at sumption (RESP) μl O /min/ 2 expressed a constant 30 °C and the output recorded on a were as
where g dw dry weight of pollen gdw the Non-linear regression analysis was used. Effect of diluting douglas fir pollen Pollen preconditioning filled seed per cone on All pollen lots tested were previously stored Fertility response is seldom linear to viability re- and, therefore, in 10% a dehydrated state (< sponse. To determine the effect of a range of moisture content). Hydrating Douglas fir pollen pollen viabilities on seed set, a single Douglas prior to the assay has been shown to increase fir pollen lot with a high fertility potential (collect- both the germination (Charpentier and Bonnet- ed from Cowichan Lake Research Station) was Masimbert, 1983; Jett and Frampton, 1990) and diluted with heat-killed pollen (4 h at 85 °C). Pol- conductivity (Webber and Bonnet-Masimbert, len dilutions ranged from 100% live to 100% 1989) response. The effect of preconditioning dead (13 separate dilutions). Each dilution was pollen by hydration or in vitro assay response on each of 2 trees using 2 replicates tested and the correlation between assay response (bags) per tree. Pollination technique was slight- and filled seed per cone was considered in the 2 ly different than described in In vivo fertility. In regression experiments described below. this test, syringe pollinators were used. The sy- Where pollen hydration technique used, was ringe plunger was replaced with a small glass the procedures of Charpentier and Bonnet- tube attached to a rubber bulb. When squeezed, Masimbert (1983) were followed. Pollen lots to the rubber bulb provided a slight pressure within be hydrated were first weighed and then ex- the syringe barrel and propelled pollen out of the posed to a saturated atmosphere for 16 h at syringe needle towards the receptive flower. All 25 °C. Hydrated pollen was assayed immediate- other aspects of bagging, cone collections and ly after treatment and the response compared to seed extraction were as described. Average a sample of the unhydrated pollen. All assay re- seed yield values were expressed as filled seed sponses were based on the dry weight of pollen per cone (FSPC). used. The dry weight of pollen was then calcu- lated from the known percent moisture content viability on filled seed The effect of of the pollen prior to hydration. Mellerowicz and Bonnet-Masimbert (1986) demonstrated that hy- per cone dration of pollen prior to pollination had no effect on filled seed per cone. Consequently, hydration Ninety Douglas fir pollen lots were selected from as a factor in field fertility trials was not consid- both tree breeding and seed orchard collections. ered. Pollen samples from each lot were removed from storage and placed in glass vials with tight- fitting lids. Moisture contents were determined and oxygen uptake measured according to the Pollen moisture content effects technique described. Lots were not hydrated be- on simple linear regression fore testing. All 90 lots were ranked by oxygen uptake (μl O g dw) and then arbitrarily 2 -1 -1 /min classed into 4 viability categories: poor (0-4), Ten samples of Douglas fir pollen lots were ran- low (5-12), moderate (13-21), and good (> 22). domly selected from previously stored lots. Pol- Within each category, 10 pollen lots were ran- len lots were hydrated for 16 h and then tested domly selected. using the 3 in vitro assays described. These 2 wk prior to field polli- tests were completed The selected 40 pollen lots were tested using = nations. Field fertility trials (see also section on respiration, conductivity and germination assays In vivo fertility) used the following design: 10 pol- as previously described. Each lot was tested in len lots applied in replicate (2 bags per lot) to both its hydrated and unhydrated state. Non- each of 8 seed-cone trees. Seed-cone trees linear regression procedures (see Statistical (clones) were randomly selected among those analyses for details) were used to estimate coef- trees with a sufficient crop to provide a minimum ficients of determination between RESP, COND, %COND, CLASS 1, CLASS 1 + 2 and percent of 20 pollination bags each containing 3-6 seed- cone buds. filled seed per cone (%FSPC).
beyond bud burst (Owens et al, 1981; Ow- 4d Each of the 10 pollen lots from each viability and Simpson, 1982; Webber, 1987). For class (40 lots) was field tested for fertility using ens 4 full-sibling seedlings from the Canadian Pacif- consistency, all seed-cone buds were pollinated at 2 d beyong burst using 0.2 ml pollen. Pollen ic Forests Products low elevation seed orchard = in Saanichton, BC. A total of 80 isolation bags was applied using a compressed nitrogen driven pollination device (contact senior author for de- containing either 2 or 3 seed cones per bag were placed on each of the 4 trees. Each of the tails). In the fall, mature seed cones were col- lected when the bracts began to flex and the 40 pollen lots were randomly assigned to 2 rep- started to turn brown. Seed cones were licates on each tree. For regression analyses, cones dried and hand extracted. All seed with a devel- mean values bulked by replicate and clone were oped seed coat were separated from the non- used (ie, N=4). developed ovules and counted. This represent- Pollinations were completed using the proce- ed the total potential seed per cone (PSPC). dures described in In vivo fertility. Cones were The number of filled seed per cone (FSPC) was harvested by replicates but cones were kept determined by X-ray analyses using Kodax In- separate and hand extracted individually. All po- dustrex 620 paper and a Hewlett-Packard (Faxi- tential seed per cone were extracted from each tron series Model 43855A) operating at 15 kVp cone and the filled seed per cone determined by for 2 min. The percent filled seed per cone was X-ray analyses as described below. calculated from the ratio of FSPC to PSPC and expressed as %FSPC. In vivo fertility Statistical analyses All pollen lots tested for in vitro viability were also tested for in vivo fertility using controlled crossing pollinations. Specific details for each All statistical analyses were completed using test are given for each experiment. Common to SAS PC (SAS Institute Inc, 1988). To determine all tests was the bagging and pollination tech- significant differences (a level of 0.05) between media types by germination class, &2 statistics chi; nique. were used. For the 4 media types, individual Seed-cone trees full-sibling (either grafts or pairs were compared using the output of Proc in age from 10-20 yr old) seedlings ranging Probit. The critical P value was calculated using were selected on the basis of crop intensity and the Bonferroni correction (0.05/6 0.0083). = vigour from various orchards or clone banks on Means with the same letter (see figs 2, 3A,B) Vancouver Island. In particular, the clone banks were not significantly different at the critical P at Cowichan Lake Experimental Station, Cow- value of 0.0083. For comparing the effect of hy- ichan Lake, BC and the seed orchard of Canadi- dration on assay response, a paired t-test was an Pacific Forest Products, Sannichton, BC used. were used. On each selected seed-cone tree, pollen-cone buds on each sample branch were For in vitro assays, the experimental unit was removed and seed-cone buds were isolated in as a family of pollen grains pollen lot (defined a pollination bags prior to bud burst. In all cases, arising from a single clone including 1 or several large, white pollination bags (obtained from ramets). For field fertility trials, controlled cross- DRG Packaging Ltd, Toronto, Ontario) with ing pollination technique was used and individu- plastic windows were used for initial isolation. al clones were the experimental unit and cones Smaller brown "corn-tector" bags (product No were the sampling unit. Linear and non-linear re- 402, obtained from Lawson Pollination Bags, gression, analyses were completed on the aver- Northfield, IL) were used to isolate fewer seed- age filled seed per cone per replicate (where ap- cone buds (2-3) on sample branches. Placed plicable) then averaged per clone (tree level) or within each bag was a 1-cm cube of no-pest bulked by clones (orchard level). strip (supplied by various manufacturers but all For simple linear regressions, the variables having the active ingredient of 18% Dichlorvos) RESP, COND, % COND, CLASS 1 and CLASS to prevent insect damage. 1 2 by hydration level compared against were + time to pollinate Douglas fir seed- %FSPC. For non-linear regressions, the vari- Optimal buds for maximum seed yields is within 2- ables RESP, CLASS 1 and CLASS 1 + 2 were cone
The value S was also determined from the compared with %FSPC using logistic function y-x a square root of the residual mean square error in the form of: term and represents an average estimate of er- ror about any point on the curve of predicted val- (see figs 6-8). ues For conductivity data, a hyperbola function was RESULTS used in the form of: y = ae bx For each equation, the parameters a, b and c were approximated by iterating the best fit using Germination medium Proc Nlin (non-linear) procedures. The coeffi- cient of determination (r was calculated from ) 2 the corrected (CSS) and residual sum of Figure 1 gives examples of the 4 classes squares (RSS), ie: of germinating Douglas fir pollen. Figure 2 shows the average germination response (by class) of 8 Douglas fir pollen lots in each of 4 media types: deionized water
10% sucrose (10S); 10% Brewbak- O); 2 (H higher levels of PEG. For the 10P10B and and Kwack (1963) solution (10B); and, 20P10B media, there was no significant er 10% polyethylene glycol-4000 (10P). Me- difference between the proportion of Class dia type had a strong effect on the propor- 1 grains but there was a significant de- tion of damaged pollen grains (Class 4). crease over the 30P10B and 40P10B me- The percent of Class 4 grains for the 4 me- dia. Correspondingly, the proportion of dia types were all significantly different Class 2 grains increased significantly over from one another with 10B showing the the 4 media types. Likewise, Class 1 + 2 lowest proportion (0.08%) followed by 10S grains increased significantly over the (21.6%), 10P (33.1) and H (42.4%). The 10P10B to 30P10B media but showed no O 2 proportion of pollen grains not germinating further significant increase for the 40P10B media. (Class 3) was also lowest for the medium 10B. There was no significant difference Based on these data, the media between the percentage of Class 3 grains 20P10B was selected for testing the germi- for the other media types. The proportion nation of Douglas fir pollen in vitro. Al- of germinating Class 1, 2 and 1 + 2 grains though the 30P10B and 40P10B media was significantly highest in 10B compared yielded the highest proportion of Class 1 + to the other 3 media types. 2 grains (88.1 and 89.0%, respectively), they also yielded significantly lower propor- Figure 3 contrasts the germination re- tions of Class 1 grains (28.3 and 10.5%, sponse (by class) of 4 levels of PEG-4000 respectively). There was no significant dif- concentrations alone (fig 3A) and with the ference between the proportion of Class 1 10B medium (fig 3B). With PEG alone (fig grains for the 10P10B and 20P10B media 3A), there was a steady decrease in Class but the proportion of Class 1 +2 grains 4 damaged grains with increasing concen- was significantly higher for 20P10B. tration of PEG (all significantly different from one another). The lowest concentra- tion of PEG (10P) yielded the highest pro- Conductivity analyses: leaching time portion of Class 3 (non-germinating) grains which was significantly different from the other three. As the concentration of PEG Figure 4 shows the response of percent increased, the proportion of Class 1 grains conductivity (%COND) by viability class for showed a significant increase from 10P to 40 hydrated Douglas fir pollen lots over 5 20P, no significant difference between 20P leaching times. The 4 viability classes to 30P, then a significant decrease with the were distinguished from each other by per- 40P media. For the proportion of Class 2 cent conductivity after 1 h. The poor viabili- grains, there was a significant increase ty class pollen lots had much higher over the range of 10-40% PEG. Compar- %COND values while the moderate and ing Class 1 + 2 grains with media type, good viability class pollen lots produced there was a significant increase over the the lowest %COND values and showed range of 10-30% PEG but no significant the least differences. Over a 6-h period, difference between 30-40% PEG. %COND values rose gradually for all viabil- The addition of 10% Brewbaker’s solu- ity classes and after 24 h, the values ap- proached 80% of the total leachable mate- tion to the 4 PEG concentrations complete- rial. The coefficient of determination (r ) 2 ly eliminated the Class 4 grains (fig 3B). Also, the addition of 10B to the 4 concen- values for both COND and %COND against %FSPC were calculated for each trations of PEG further lowered the propor- of the 5 times the corrected tion of Class 3 grains but only at the 3 leaching using
and residual sum of squares from the out- put of SAS non-linear regression proce- dures (see Statistical analyses). TableI shows a slight decline of r values for both 2 COND and %COND up to 6 h leaching with a large drop in r at 24 h. Based on 2 these data, a 1-h leaching time yielded %COND values which when used in the hyperbolic function described under Statis- tical analysis, will explain nearly 82% of the variability in %FSPC. Simple linear regression: the effect of pollen moisture content Table II compares the mean (± SE) assay response for respiration, conductivity, per- cent conductivity and germination (Class 1 and 1 + 2) for 10 Douglas fir pollen lots
that either considerably better if the seed-cone parent hydrated unhydrated. were or Average moisture content of the 10 dehy- trees were bulked (N = 10) than if the drated lots was 3.5%. After 16-h exposure seed-cone trees were considered as a at 100% RH and 25 °C, the average mois- separate factor (N = 80, data not shown). ture content was 25.9%. Only respiration showed no significant improvement in re- Non-linear regression analysis sponse due to hydration. Both conductivity and germination responses were signifi- cantly improved by hydration. Total lea- diluting Douglas fir pollen The effect of chate (511.9 vs 315.0 μS/cm/g dw) and percent conductivity (57.4 vs 35.0%) were Figure 5 shows the relationship between lower and germination response for Class FSPC and the percent live pollen for each 1 (10.0 vs Class 1 48.5%) and 2 (17.7 vs of 13 dilutions. Each value point repre- + when 67.4%) higher exposed to were sents the average of 2 replicates on each 100% RH for 16 h at 25 °C prior to the as- of 2 seed-cone clones. As the proportion of live pollen rose from 0 to 50%, there was a say. steady almost linear increase in FSPC. Table III shows the correlation coeffi- However, beyond ≈ 40-50% live pollen, no cient (r) derived from simple linear regres- corresponding increase in FSPC was ob- sion analyses for mean assay response served. (both hydrated and unhydrated pollen) against seed set (FSPC and %FSPC). In In terms of threshold level, this corre- a all cases, hydrating pollen lots prior to the sponded to 35-40 FSPC. For Douglas = assay improved r values. For respiration, r fir, this represents ≈ 55% PSPC based on values were less affected by hydration an average potential of 64-70 seeds per state than those for conductivity or germi- cone (Ho, 1980) arising from 32-35 ovulif- nation. As expected, the r values for mean erous scales per cone (Owens et al, 1991). assay response against seed set were Assuming all other factors equal, higher vi-
len used in controlled crossing pollinations, it appears that pollen can be diluted to = 50% before any reduction in FSPC is ob- served. With regards to pollen viability, it is not certain whether a pollen lot yielding fewer seeds per cone compared to a more fertile pollen lot has fewer live pollen grains (as- suming a pollen grain is either fertile or not) or if all the grains are just less fertile (assuming that the fertility of a pollen grain can vary). Our in vitro viability assays can- not distinguish between the 2 possibilities. However, this pollen dilution study does in- dicate the FSPC response of a pollen lot in which the pollen grain is either alive or dead. Under these conditions, a pollen lot with < 50% functional grains is associated with decreasing fertility. Presumably under these conditions any viability assay (ie ger- of pollen is associated with higher ability mination) would be a direct indication of FSPC. However, there is a limit beyond the proportion of fully functional pollen which increasing viability is not associated grains (see fig 8). with increasing FSPC. For Douglas fir pol-
The effect of (an average estimate of error about value pollen viability any point on the curve). To determine the effect of viability and the In general, the %FSPC from each of non-linear response of FSPC, 40 pollen the 4 viability classes sort out according to lots, representing 10 from each of 4 viabili- their respective rating. The good lots (good ty classes were all tested under the same and moderate classes) produced the best conditions. Figures 6-8 show scatter plots %FSPC and the poor lots (poor and low for %FSPC against respiration, percent classes) yielded the lowest %FSPC. Com- conductivity and percent germination paring the 3 viability assays with the 4 via- (Class 1 + 2), respectively for 40 pollen bility classes, there was considerable vari- lots (segregated into 4 viability classes). ation in ranking between lots within a class Also shown is the curve of predicted val- but average values for lots by class were ues derived from the non-linear regression ranked according to their respective class. equation (parameters shown), the coeffi- The rank order for respiration is expected cient of determination (r and the S ), 2 y-x since the original ranking of the 90 lots
Considering individual pollen lots by via- using respiration values from done was bility class, all of the good lots and 9 of the lots. Table IV shows the mean unhydrated moderate class lots produced a minimum for each of the 4 classes to each response of 50% PSPC (≈ 35 FSPC). The poor and of the 3 viability assays plus the FSPC and low viability class pollen lots showed a %FSPC values by viability class. The im- wide range of variability. Of the 10 pollen proved response for conductivity and ger- lots in the low viability class, 6 produced a mination with hydration is again apparent. minimum of 50% PSPC (range for all 10 The 3 assays (RESP, %COND and pollen lots was 12.5-80.4% PSPC). None CLASS 1 + 2) as well as FSPC and of the poor viability class pollen lots pro- %FSPC all rank according to their respec- duced %FSPC > 40%. For all 4 viability tive viability classes with the single excep- tion of germination CLASS 1. classes, 25 of the 40 lots produced at least
50% PSPC. This corresponded to assay tion (Class 1 + 2) are all > 0.8, suggesting ranges of 9-33 μl O dw for respira- that > 80% of the observed variation in /min/g 2 tion, 10-45% conductivity, and 40-90% %FSPC can be explained by assay re- germination (Class 1 2). + sponse. Coefficients of determination (r be- ) 2 For all 3 assays, classifying pollen lots tween each of the 3 in vitro assays (hydrat- into good, moderate, low and poor catego- ed and unhydrated) and their correspond- ries showed responses indicative of their ing %FSPC are shown table V. The class (figs 6-8). Both the good and moder- importance of hydration on improving the ate classes produced yields that would be 2 r values, especially for conductivity and considered operationally acceptable (50% germination assays, is again apparent. Co- PSPC or better). The poor and low viability efficient of determination for respiration, class pollen lots produced %FSPC that, in percent conductivity and percent germina- unacceptable. Furthermore, general, were
conductivity and germination as in vitro germination, media type had a tests. For large effect on response. In water alone, unhydrated pollen showed extensive dam- aged (Class 4) grains (see fig 2). The os- motic potential of deionized water was high relative to the pollen grain. If the osmotic potential of the media is decreased (by adding sucrose, PEG or Brewbakers solu- the low viability class pollen lots showed the proportion of Class 4 grains de- tion), creased and the proportion of Class 1 and considerable variation suggesting that the predictive values of these pollen lots from 2 grains increased (figs 2, 3). The propor- the regression equations will be highly var- tion of Class 3 grains was relatively unaf- iable. fected by changing osmotic potential (fig 2). For most conifers, the constituents of DISCUSSION germination media are relatively sim- the ple. Sugar (generally sucrose) solutions most often used which appears to act are In vitro assays a substrate for both osmoticum and as an and Linskens, respiration (see Stanley Potential fertility of Douglas fir pollen can Sucrose has also been 1974; pp 67-76). essential component of be adequately assessed using respiration, to be an reported
the in vitrogermination of Pinus roxburghii germination response (ie Class 1 + 2), the pollen (Dhawan and Malik, 1981) but in proportions of Class 1 and Class 2 grains short-term (< 48 h) tests it may not be es- can be significantly affected by various sential carbon source but rather acts concentrations of PEG (figs 2, 3). It is also as a interesting to note that the r value for ger- 2 osmoticum (Nygaard, 1977). This as an appears to be the case for Douglas fir pol- mination Class 1 grains is very poor (table len. For short-term germination tests, su- V) even with hydration. This may be attrib- crose is not an essential but an osmoticum uted to a media effect in which case, ger- is. Among the many types of non- mination response must include both Class metabolizable substrates, polyethylene gly- 1 + 2 grains. In addition to the stabilizing col can provide a wide range of water po- effects of PEG, the inorganic constituents tentials (Stenter et al, 1981) and has been of Brewbaker and Kwack (1963) medium used successfully to germinate pollen of are also important. By comparing sucrose other species (Zhang and Croes, 1982; (10S) and PEG (10P) against Brewbaker Subbaiah, 1984). Since PEG is an inert os- and Kwack’s media (10B), the inorganic moticum, it is preferred. constituents of 10B produced the lowest proportion of damaged (Class 4) pollen Calcium and boron have also been im- grains (figs 2, 3). Whether the 10B medium as important germination media plicated osmoticum, principally acts mem- as an constituents in some angiosperms (Johri brane stabilizer or some combination of and Vasil, 1961; Brewbaker and Kwack, both is not known. Regardless, the combi- 1963) and some pines (Nygaard, 1970; nation of the osmotic stablizing effects of Dhawan and Malik, 1981).Apparently cal- PEG and the inorganic constituents of cium is essential to maintain the structural Brewbaker and Kwack’s solution (20P10B) integrity of pollen membranes (Nygaard, yielded the best germination response 1970) while the role of boron in tube which when used with the logistic regres- growth is not known. sion equation accounted for over 80% of PEG-4000 has been used previously for the variation in FSPC. pollen germination and water relationship studies, primarily because it was consid- ered to be too large a molecule to be taken Pollen moisture content up by the cell. However, recent reports suggest that PEG-4000 may be taken up For most angiosperm pollen, dehydration by the cell (Jacomini et al, 1988), in which has a deterimental effect on its fertility po- the osmotic potential of the medium case tential (Shivanna and Heslop-Harrison, and pollen grain would change. How PEG 1981). While this effect can often be re- might affect germination of Douglas fir pol- versed by rehydration, some species are len, other than osmotic effects, is not more sensitive to dehydration than others. known. The importance of water relations In corn pollen, for example, dehydration in the germination of pollen suggests PEG below 20% moisture content leads to irre- effects warrant further study. versible loss of viability (Kerhoas et al, The results of these media experiments 1987). In conifers, however, dehydration of suggest that in the early stages (< 48 h) of pollen does not have such a severe effect Douglas fir pollen germination, response fertility potential. on may be more related to the physical prop- importance of pollen moisture con- The erties of cell membrane hydration and tent (hydration state) for in vitro assay re- elasticity than to metabolic activation. Al- demonstrated sponse has been though PEG has less effect on the overall by clearly
rates in Douglas fir Charpentier and Bonnet-Masimbert (1983) Although respiration Douglas fir pollen, Jett and Frampton appear to be less sensitive to hydra- for pollen (1990) for Loblolly pine pollen, and Fou- tion, both the average respiration response (table IV) for pollen lots within the 4 viabili- shee (1990) for western White pine pollen. ty classes and the correlation coefficient (r) For Douglas fir and Loblolly pine pollen, and coefficient of determination (r meas- ) 2 the benefit of a 16-h hydration period (at ured against %FSPC (tables III and V) all 100% RH) on germination response was improved. However, the effect of hydration apparent but the magnitude of the re- sponse was dependent on the pollen mois- on conductivity and germination response and their relationship to %FSPC was more ture content prior to hydration. For Doug- las fir, the assay response to hydration apparent. greater if the pollen moisture content was 7% (Charpentier and Bonnet- was < Masimbert, 1983) whereas for Loblolly Predicting potential seed yields pine, the threshold level was ≈ 15% (Jett and Frampton, 1990). Previous reports (Ching and Ching, 1976) in these ex- For the pollen lots studied have developed the relationship between periments, moisture content average ≈ 6- various viability assays and germination 8%. The effect of rehydrating pollen prior but few have actually correlated the assay to in vitro assay increased pollen moisture with field fertility. Binder and Ballantyne content of ≈ 26% and produced the great- (1975) reported a positive relationship be- est increase in assay response for conduc- tween respiration and fertility and suggest- tivity and germination. Respiration re- ed that pollen lots with respiration rates of sponse showed no significant increase 20 nmol O mg at 30 °C (equiva- /min/100 2 with hydration. These results confirm earli- lent to 5 μl O were probably ca- /min/g) 2 ≈ er observations for both conductivity and pable of producing seed. Data collected in germination response of Douglas fir pollen our experiments suggest that pollen lots hydration (Webber and Bonnet- to with respiration values of 5 μl O dw /min/g 2 Masimbert, 1989). It is now a matter of will produce seed (= 20% PSPC or 12 protocol to rehydrate all Douglas fir pollen FSPC) but the yields would be too low for lots for 16 h at 100% RH and 25 °C prior operational use. Such a low viability pollen testing. to lot could yield seed for breeding purposes using controlled crossing technique. How- Hydration effects were also apparent ever, low viability pollen could not be ex- from simple linear regression analyses pected to compete well in open pollination (see table II) with higher r values associat- where higher viability pollen also occur ed with hydrated assay response and (Webber and Yeh, 1987; Apsit et al, 1989). seed set compared to its unhydrated pair (see table II). Apparently, hydration is re- More recently, Moody and Jett (1990) 2 reported r values between germination quired to both stabilize (lower conductivity values) and activate (increased germina- and respiration rates for Loblolly pine pol- len lots and total seed to be 0.88 and 0.81, tion values) pollen membranes. Respira- respectively. Furthermore, Moody and Jett to be less sensitive tion, however, appears to membrane hydration state. Moody and (1990) were able to generate exponential response curves for both germination and (1990) reported no significant effect Jett respiration rate against percent filled seed respiration rates due to rehydration in on as a function of pollen age. Loblolly pine.
In Douglas fir, PSPC is limited by the pollen lot would be 14 μl O dw for /min/g 2 a number of developed ovules available. Be- respiration, 25% of the total leachate for cause there is a limit beyond which any in- conductivity, or 45% germination for Class crease in pollen viability is not equally 1 + 2 grains. Again, it must be emphasized matched by an increase in FSPC, correla- that these results were obtained from con- tion analyses based on simple linear re- trolled crossing technique. gression models do not adequately de- Applying these threshold values to the scribe this non-linear response. Another 40 lots, the number of pollen lots failing to problem in developing relationships be- meet the expected %FSPC were 5, 5 and tween assay response and FSPC is ensur- 4, respectively using respiration, conductiv- ing that a wide range of pollen viabilities ity and germination assays. The number of are included in the test to generate a good pollen lots that met or exceeded the relationship. threshold value for respiration, conductivity The results shown in tables II-V confirm and germination but did not produce the the beneficial effects of hydration for im- expected 50% PSPC were 3, 2 and 2, re- spectively. Conversely, the number of pol- proving assay response and reducing the non-explainable regression variation be- len lots that produced 50% PSPC but did not meet the threshold value for respira- tween assay response and fertility. The lo- gistic regression model used for respiration tion, conductivity and germination were 2, and germination responses and the hyper- 3 and 2, respectively. bola model used for conductivity response If used for controlled pollen lot is a against %FSPC seems to fit the data well. crossing, then it may be possible to lower Although it may be possible to improve the the critical assay value, especially if < 50% relationship by using other models, the yields are acceptable. Thus, for single lot equations shown in figures 6-8 allow us to application, lots with values > 10 μl O /min/ 2 explain over 80% (see table V) of the vari- g dw for respiration, < 50% leachate for ation within the data. The remaining 20% conductivity, or > 30% Class 1 + 2 for ger- of the variability is likely related to field pol- mination can be expected to produce ac- lination technique, pollination mechanism, ceptable seed yields (30% or 20 FSPC). = and male-female interactions (Apsit et al, However, if lots falling within this viability 1989). range are used in polymixes or expected to compete with outcross pollen, then one Using the appropriate equations for pre- cannot expected similar results. Fowler dicting %FSPC from the response of respi- (1987) and Cheliak et al (1987) have stud- ration, percent conductivity and percent ied both the biological and genetic implica- germination (see figs 6-8), it is possible to tions of using polymixes and each recom- predict the seed set response from con- mend keeping the number of male parents trolled crossing experiments. However, for operational pollination programs, these within the polymix as high as possible to prevent significant distorsion of male con- models may not be applicable where com- petition between lots of differing viabilities tribution. Under these conditions, it may can occur. It may be possible to mimic controlled be better to consider only 2 viability class- crossing results under open pollination es: acceptable and unacceptable. conditions but timing of pollination and ap- If 50% PSPC is established as an oper- plication technique must be stricly con- trolled. In Douglas fir, the pollination mech- ational seed production target, then the anism is such (see Owens et al, 1981) that threshold values for accepting or rejecting
pollen arriving first at the has 8% (Webber, unpublished stigmatic tip contents < the avantage of completing the subse- data). These species also respond similar- quent steps towards fertilization over pol- ly to hydration technique, although the hy- len grains arriving later (Webber and Yeh, dration periods vary somewhat. Where 1987). Pollination technique can also af- tested, the relationships between hydrated fect FSPC values. Pollen applicators that assay response and FSPC also show propel the pollen at the receptive strobili some degree of improvement over the un- using compressed gas driven devices hydrated assay response. However, con- have consistently yielded higher FSPC val- siderable field testing is still required for ues (Webber, 1991; and unpublished data) these species to develop the predictive re- sponse for seed set that was developed for compared to more passive pollinator types (ie, paintbrushes and misting pollinators). Douglas fir. It should be possible, then, to influence the proportion of applied male parents in Douglas fir using early pollination with lots ACKNOWLEDGMENTS of high viability and applied using com- pressed gas driven pollinators. The authors wish to thank R Painter for his tech- nical assistance in the field and W Bergerud for The non-linear regression models de- assistance in statistical analyses. The authors veloped for respiration, conductivity and also wish to express their gratitude to Canadian germination procedures may also be use- Pacific Forest Products Ltd and the BC Ministry ful for estimating the relative viability of of Forests, Silviculture Branch for access to their pollen lots being used in a polymix. This seed orchards. Financial support to JEW from may have particular importance when < 10 the National Research Council of Canada (Can- lots are used within the mix. As the num- ada/France Science and Technology Coopera- tion Program) and from NATO (Collaborative ber of clones within a seed orchard is re- Research Grant (0320/88) for travel support is duced to maximize genetic gain potential gratefully acknowledged. and the number of pollen parents are re- duced to capitalize on specific traits, then the differential viability among pollen par- REFERENCES ents will become very important. Adams WT (1983) Application of isozymes in plant breeding. In: Isozymes in Plant Genet- CONCLUSION ics and Breeding, Part A (Tanksley SD, Orton TJ, eds) Elsevier Sci Publ, Amsterdam, 381- 400 The procedures described here for the in vitro assay of Douglas fir pollen have also Nakamura RR, Wheeler NC (1989) Dif- Aspit VJ, ferential male reproductive success in Doug- been used for other species within British las fir. Theor Appl Genet 77, 681-684 Columbia’s tree improvement program. Binder WD, Ballantyne DJ (1975) The respira- Respiration and conductivity procedures tion and fertility of Pseudotsuga menziesii are as described, but germination media (Douglas fir) pollen. Can J Bot 53, 819-823 varies slightly for each species. Brewbaker JG, Kwack BH (1963) The essential It is now a matter of routine to store pol- role of calcium ion in pollen germination and len from White spruce (Picea glauca), pollen tube growth. Am J Bot 50, 859-865 Western hemlock (Tsuga hetrophylla), Bridgwater FE, Blush TD, Wheeler NC (1991) Lodgepole pine (Pinus contorta) and West- Supplemental mass pollination. In: Pollen ern larch (Larix occidentalis) at moisture Management Handbook, Vol II. Proc Pollen