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

(0-800 ppm), calcium concentration (0.1 ~M - 10 roM). Pollen was

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.

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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

diacetate (FDA), Evan's blue, triphenyl tetrazolium chloride

(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'

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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

this spring.

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