WALNUT POLLEN STUDIES V. S. Polito, K. Pinney and V. Stallman investigations were initiated this year to develop field and laboratory viability assays for stored walnut pollen, indentify conditions suitable for in vitro pollen germination and pollen tube growth, and determine pollen tube growth rates in pistillate flowers. Results of this project will provide background information for future investigations of transmission of pollen-borne virus, factors influencing pistillate flower receptivity and ovule abortion, and to develop methods for assessing pollen viability and germination potential. Results described here are based on data collected in preliminary experiments conducted during 1981. This research will be continued over the next two years in conjunction with studies of pistillate flowers and pollen tube growth in vivo. Conclusions regarding the various aspects of walnut pollen biology under investigation will depend upon the results of this research. Pollen germination and pollen tube growth in vitro. 'Hartley', 'Serr', 'Chico', 'Ashley' and 'Franquette' pollen was examined for in vitro germination. Media components tested included: sucrose concentration (10-30%), boron concentration (0-800 ppm), calcium concentration (0.1 ~M - 10 roM). Pollen was tested on media solidified with 1% agar (wet, dry, and on inverted agar blocks), in liquid culture and in hanging drop culture. Effect of pollen density was also investigated. Germination of 'Hartley', 'Serr', 'Chico' and 'Ashley' pollen never exceeded 2% on any medium. Freshly collected 'Franquette' pollen did germinate on media solidified with 1% agar but not in liquid media or in hanging drop cultures. Optimum medium consisted of 25% sucrose supplemented 1-10 ~M CaC12. Boric acid concentrations from 0- 400 ppm had no effect on germination at this sucrose concentration and concentrations above 400 ppm inhibited germination. The most important factor was pollen density on the surface of the me2ium. Germination in regions of high density (200 + grains/rom) averaged 24.4% com~ared to 14.3% in regions of intermediate (~0-200 grains/rom) and 9.18% in regions of sparse ( 50 grains/rom) pollen density. Pollen density had little effect on germination at lower concentrations of sucrose. At 20% sucrose concentration germination was approximately II,t that of maximum germination in dense regions but there was ittle difference in germination percentages among the three egions of pollen density. At 10% sucrose concentration (optimum sucrose concentration for most other fruit tree pollens) germination was uniformly low, «2%). Pollen germinations in 20% sucrose was slightly promoted by high boron concentration and this effect was also independent of pollen density. The interaction between pollen density and sucrose concentration will be investigated in more detail this spring. -5-
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WALNUT POLLEN STUDIES
V. S. Polito, K. Pinney and V. Stallman
investigations were initiated this year to develop fieldand laboratory viability assays for stored walnut pollen, indentifyconditions suitable for in vitro pollen germination and pollen
tube growth, and determine pollen tube growth rates in pistillate
flowers. Results of this project will provide background information
for future investigations of transmission of pollen-borne virus,
factors influencing pistillate flower receptivity and ovule
abortion, and to develop methods for assessing pollen viability
and germination potential. Results described here are based on
data collected in preliminary experiments conducted during 1981.This research will be continued over the next two years in
conjunction with studies of pistillate flowers and pollen tube
growth in vivo. Conclusions regarding the various aspects ofwalnut pollen biology under investigation will depend upon theresults of this research.
Pollen germination and pollen tube growth in vitro.
'Hartley', 'Serr', 'Chico', 'Ashley' and 'Franquette'
pollen was examined for in vitro germination. Media componentstested included: sucrose concentration (10-30%), boron concentration
tested on media solidified with 1% agar (wet, dry, and on invertedagar blocks), in liquid culture and in hanging drop culture.
Effect of pollen density was also investigated. Germination of
'Hartley', 'Serr', 'Chico' and 'Ashley' pollen never exceeded 2%
on any medium. Freshly collected 'Franquette' pollen did germinate
on media solidified with 1% agar but not in liquid media or inhanging drop cultures. Optimum medium consisted of 25% sucrose
supplemented 1-10 ~M CaC12. Boric acid concentrations from 0-400 ppm had no effect on germination at this sucrose concentration
and concentrations above 400 ppm inhibited germination.
The most important factor was pollen density on the surface
of the me2ium. Germination in regions of high density (200 +grains/rom) averaged 24.4% com~ared to 14.3% in regions of
intermediate (~0-200 grains/rom) and 9.18% in regions of sparse( 50 grains/rom) pollen density. Pollen density had littleeffect on germination at lower concentrations of sucrose.
At 20% sucrose concentration germination was approximately
II,t that of maximum germination in dense regions but there was
ittle difference in germination percentages among the three
egions of pollen density. At 10% sucrose concentration (optimum
sucrose concentration for most other fruit tree pollens) germination
was uniformly low, «2%). Pollen germinations in 20% sucrosewas slightly promoted by high boron concentration and thiseffect was also independent of pollen density. The interaction
between pollen density and sucrose concentration will be investigated
in more detail this spring.
-5-
Enzymatic digestion of the cell wall material that serves
to plug the germination pores of the pollen grains typically ledto the formation of short pollen-tube-like protrusions of membrane-
bound cytoplasm through the pores hut normal pollen tube growth
did not occur. There is some possibility that this response mayprovide a basis for a viability assay adaptable for use in thefield.
Germination of 'Franquette' pollen remained high for up to
3 months in storage. After 1 months, attempts to germinatepollen were unsuccessful.
Pollen yia~~~~ assays
Three assays for pollen viability were tested: Fluorescein
(TTC). Results of each of these tests indicated 95-100% pollenviability for every sample examined. Positive tests of viabilitywere not obtained for heat-killed pollen. However, subsequenttests of pollen germination in vivo (described below) indicated
that the positive results did not correlate with the ability ofthe pollen to germinate on stigmas.
After 3 months of pollen storage pistillate flowers obtained
from severely pruned 'Chicl)' trees were hand-pollinated withpollen determined to be viable by the above tests. In no casewas pollen germination observed.
This was consistent with the failure of the 'Franquette'
pollen to germinate in culture and indicates that there may be(in this variety) a correlation between ability for pollen to
germinate in vitro and on stigmas. Two possible explanations
may account for this result: 1) The viability test ar~ unreliable
indicators of the ability of pollen to germinate and producepollen tubes or 2) the stigmas of the forced flowers were unable
to support pollen tube growth. Experiments planned for this
spring should resolve this question.
At this time it does not appear dS though any of these
assays are suitable for testing pollen germinability. Subjectiveassessments of the intensity of the FDA reaction indicated to us
that a decline in intensity may have occurred over the period of
pollen storage. Attempts will be made to quantify this reaction
to confirm these observations with fresh pollen this spring.However, tests which depend upon fluorometric or colorometricquantification will have little value in a field situation.
Therefore, we will continue to test other possible viabilitytests that may be appropriate in this regard.
Pollen tube growth in vivo
Controlled pollinations ofexpansion were made in order to
tube growth in vivo. 'Ashley',
flowers bagged prior to stigma
determine parameters of pollen
'Hartley' and 'Franquette'
-h-
flowers were pollinated at the time of stigma expansion and
samples collected at 6, 8, 10, 12 and 14 days. Our preliminary
indications are that at least 8 days were required for pollen
tubes to grow to the ovary under orchard conditions. High
variability was evident in our samples. Results of experiments
to determine growth rates of black-line-virus-infected and non-
infected pollen were inconclusive. This research will be expanded