ExplAgric. (1969), 5, pp.

59-65
Printed in Great Britain

EFFECTS OF VACUUM-DRYING ON THE

VIABILITY OF OIL PALM POLLEN
BY J. J. HARDON

Oil Palm Genetics Laboratory, Layang Layang, Johore, Malaysia

AND M. D. DAVIES*

Chemara Research Station, Malaysia

{Accepted 20 July 1968)

SUMMARY
Pollen of the oil palm (Elaeis guineensis) was vacuum dried for various lengths of time and stored
under vacuum in ampoules at 280, 50 and — io°C. Little loss in viability over a period of 12
months was observed in any of the storage treatments when vacuum drying did not exceed one
hour. Progenies resulting from crosses with pollen stored three months under vacuum have
shown no anamolous seedling development.

The preservation of oil palm pollen over extended periods of time is of importance
for oil palm breeding and the exchange of breeding material. The oil palm
(Elaeis guineensis Jacq.) is monoecious, producing male and female inflorescences
in cycles of variable duration. Under environmental conditions that favour the
production of female inflorescences it is not uncommon to find that a particular
palm may fail to produce male inflorescences for periods of 6-12 months thereafter.
A unique feature of the oil palm is that seed production is based on a cross
between two types that exhibit monofactorial inheritance of a naturally occurring
gene or gene block, controlling shell thickness of the nut. The two homozygotes
are respectively thick-shelled (Dura) and virtually shell-less (Pisifera) while the
heterozygote (Tenera) is thin shelled. By virtue of the decreasing amount of shell
a relatively greater amount of oil-bearing mesocarp can be obtained in Teneras as
compared with Duras. The Pisifera is not grown on any commercial scale, because
the inflorescences have a tendency to abort at an early stage of their development.
Pisiferas are therefore commonly used as male parents in the Dura x Pisifera cross
to produce the Tenera hybrid. Female cycles in Pisiferas tend to be prolonged,
probably because of failure to produce ripe fruit bunches. The restricted production
of male inflorescences in many Pisiferas, and the limited number of such palms
that have been progeny-tested, emphasizes the desirability of efficient methods of
pollen storage.
Under natural conditions in oil palm plantings pollen remains viable for an
average of one week after an thesis of the male inflorescence (Hardon and Turner,
1967). Broekmans (1957) and Devreux and Malingraux (i960) reported that oil
palm pollen, pre-dried at 4O°C, could be stored over silica gel at ambient tempera-
ture for 2-3 months, whilst storage at 5°C could extend this period to 6-12 months.
Henry (1959) showed that high viability was maintained in pollen stored at — 5°C

* Present address: Department of Horticulture, University of Illinois, Urbana, Illinois 61801, U.S.A.
60 J. J. HARDON AND M. D. DAVIES

and in partial vacuum (1/15 atm.) over at least 6 months and probably longer.
More recently, various organizations in Malaysia and West Africa have stored
pollen in deep freeze (—10 to — 2o°C) with good results. However, when such
pollen is transported under uncontrolled conditions of temperature, there is often
rapid loss of viability.
In a large number of species successful preservation of pollen has been obtained
by freeze drying and vacuum drying (King, 1965; Whitehead, 1965; and others).
The present experiments were carried out to test the feasibility of these techniques
for oil palm pollen.

METHODS
Pollen was collected from oil palms in an estate planting in Southern Malaya.
Male inflorescences were surface-sterilized and covered by a bag a week prior to
an thesis. After harvesting, the whole male inflorescence was pre-dried in a
germinator at 4O°C for 24 hours, after which pollen was extracted, sieved,
and further dried in an incubator at 4O°C for 6 hours or for 6-12 hours in a
desiccator. Although preliminary drying is not absolutely necessary prior to
vacuum drying, pre-drying facilitates the separation of pollen from the stamens
and also extends the effective life of the desiccant (phosphorous pentoxide) used
in the vapour trap of the freeze drier. An average male inflorescence yields
20-30 grams of pollen.
Pollen viability was determined by germination tests in 15 per cent sucrose in
moist petri dishes; 400 pollen grains per sample were examined after 8-12 hours'
germination time.
The freeze drying equipment used was a Speedivac 5 PS centrifugal freeze drier
(Edwards High Vacuum Ltd.) with an interchangeable centrifuge assembly and
ampoule sealing header. In the normal procedure, ampoules containing pollen
were placed in the centrifuge and spun in vacuum for seven minutes, after which
the centrifuge motor was stopped and drying allowed to proceed by sublimation.
When the required degree of dryness was obtained, the centrifuge assembly and
glass cover were replaced by the ampoule sealing header. The necks of the
ampoules were then partially constricted and attached to the header nipples for
re-evacuation to 0-05 mm. Hg. Extended drying was then continued (if required)
and the ampoules finally sealed.
Sealed ampoules were tested for vacuum with a high frequency tester Model TI
(Edwards High Vacuum Ltd.).

RESULTS
Pollen viability
The effect of vacuum drying on viability of the pollen immediately after drying
was examined. Ampoules, each containing 1 gm of fresh (not pre-dried) pollen
were vacuum dried for various lengths of time (I to 24 hours) using 2 ampoules
per treatment. Drying was conducted in two ways, namely (a) by the standard
procedure (including centrifuging) and (b) by drying on the sealing header only.
 Viability of oil palm pollen 61

Pollen viability was determined, following a suitable period of rehydration

(Table i). After vacuum drying by the standard procedure for periods in excess of
two hours there was a marked decline in pollen viability. Viability of the pollen
following drying on the sealing header only was maintained at a high level for up
to twelve hours' continuous drying, but declined gradually with longer periods.

Pollen moisture status

The moisture status of the pollen was determined after each drying treatment
(Table 2). There was little difference in drying efficiency between the two treat-
ments, and hence the difference observed in pollen viability appears to be not
simply a matter of the moisture status of the pollen. The experiments were

Table 1. Percentage pollen viability after vacuum drying for various lengths of time
Drying time (hr)1
Initial
viability i i 1 2 4 6 8 10
Standard vacuum ( i ) 78-8 78-0 78-2 74- 0 69-4 32-1 24-3 0 0
drying ( 2 ) 84-3 — — — — — 14-9 —
Drying on sealing (0 77-4 78-8 77-4 78- 1 78-0 78-2 77-8 78-2 74-2
header only ( 2 ) 88-2 — — — — — 87-2 —
Initial
viability 12 14I 16 18 20 22 24
Standard vacuum (i) 78-8 0 0 0 0 0 0 0
-
drying . ( 2 ) 8o 3 — 3.2 — — — 7-7
Drying on sealing (0 77'4 69-8 64- 1 59'2 58-0 54-2 53-2 50-1
header only (2) 88-2 — 68-4 — — — 28-4

repeated, with different drying times, but again the results were in general
agreement with those given in Table 1, although actual values varied somewhat
from experiment to experiment, possibly due to such factors as the stage of anthesis
of the male inflorescences and the moisture content of pollen at collection.

Table 2. Percentage moisture on dry weight of pollen after vacuum drying for various
lengths of time
Drying time (Hours)

Fresh
pollen i i 1 2 4 8 16 24
Standard vacuum
drying 66-8 48-7 35-7 22-8 8-6 2-8 2-6 2-2 '•9
(*) Drying on sealing
header only 66-i 41 '3 28-5 I5-6 5-0 2-9 2-7 2-5 2- I

Consequently, the sharp reduction in percentage pollen viability following

standard vacuum drying procedures for periods over two hours remains un-
explained by the present data.

Rehydration
In agreement with observations on most other pollen, vacuum dried oil palm
pollen regained maximum germinability only after the dried pollen had been
62 J. J. HARDON AND M. D. DAVIES

exposed for a certain length of time to conditions allowing rehydration. The time
required to reach maximum germination at ambient temperature (28°C) and
60-90 per cent R.H. increased with increasing length of the drying treatments, but
did not seem to differ significantly for the two methods of drying. For clarity of
presentation, only the results of pollen germination after \, 2, 12, and 24 hours'
drying on the sealing header are summarized in Fig. 1, since they clearly establish

0 12 24 36 48 Hours
Period of rehydration
Fig. 1. Percentage pollen germinability after rehydration at ambient temperature and humidity,
following vacuum-drying for \, 2, 12 and 24 hours.

the general pattern. Pollen dried for periods of £-2 hours required 12-24 hours at
ambient temperature and humidity for maximum germination, but after 4-6 hours'
drying this period increased to 48—72 hours. Pollen dried for 8—16 hours only
reached maximum germination after 6 days' exposure, and pollen dried for
18-24 h ° u r s required up to 10 days. After 10 days a sharp drop in pollen viability
was usually observed, which is characteristic also for fresh pollen (Hardon and
Turner, 1967), and apparently represents the time oil palm pollen can maintain
its viability in an ambient environment. The addition of sodium borate to the
germination medium did not seem to affect the speed at which germinability
was required.

Pollen storage
The main investigation was concerned with a study of the behaviour of treated
pollen under storage. Fresh pollen was collected from 18 Pisifera inflorescences,
pre-dried in a hot room for 24 hours and further dried for 6 hours in an incubator.
The percentage moisture on dry weight at this stage appeared to be approximately
18 per cent (Table 3). Treatment 1 consisted of drying pollen for 15 minutes on the
sealing header only. Treatments 2-6 comprised standard vacuum drying for £,
\, 1, 2 and 4 hours respectively followed by re-evacuation on the sealing header
prior to sealing under vacuum. Treatments 7-10, begun 3 months later with a
similar sample of pollen, comprised £, \, 1 and 2 hours' drying on the sealing
header only, followed by sealing under vacuum. A control was included (Treat-
ment 11) of pollen pre-dried in the hot room and incubator and then stored in a
 Viability of oil palm pollen 63

sealed ampoule but not under vacuum. The moisture status of the pollen after the
various drying treatments is summarized in Table 3.
Six ampoules of each treatment were stored under 3 different conditions, viz.
28, 5 and — io°C. One ampoule of each treatment was opened at 3-monthly
intervals and the results after 12 months' storage are summarized in Table 4.
The ampoules of treatments 7-10, stored at ambient temperature, were acci-
dentally broken shortly after the period of 6-month storage, so no results are
available. Fortunately, however, Treatment 1 also included pollen dried on the
sealing header and hence results for this drying treatment are available for 9 and
12 months' storage at room temperature. It was decided not to test the viability

Table 3. Effect of various drying treatments on moisture content of pollen

Moisture content
(%dry-matter)
Fresh pollen 81
6 hours in incubator
(4°°C) >7"9
Drying time Normal vacuum Drying on sealing
(min.) drying (i) header (2)
15 8-9 7-0
30 7-5 6-6
60 4-9 5-2
120 3-2 4-6
180 2-6 3"'
240 2-3 2-7

of the pollen dried on the sealing header at 9 months' storage, to save ampoules for
testing after 2 years. From the results it appears that a high level of germinability
is maintained under all storage conditions if the drying treatments do not exceed
1 hour. The lower viability for Treatment 3, stored at room temperature after
12 months' storage, may have been due to incomplete vacuum in the ampoule.
After 2 hours' vacuum drying, results became variable and at 4 hours there was a
definite reduction in pollen viability. Non-vacuum dried pollen (Treatment 11),
stored at 28°C, showed a sharp drop in viability between 3 and 6 months, while
some reduction was apparent at 5°C after 6 and 12 months' storage. Stored at
— io°C, pollen germinability was maintained at a high level and appears to have
been approximately the same as for vacuum dried pollen.

CONCLUSION
Oil palm pollen, air-dried and stored in a desiccator, may retain its germinability
for 2-3 months. During this period, however, there is a gradual reduction in
viability. Where such pollen is stored in a deep freeze, good viability is maintained
for periods of one year or longer. However, results are sometimes variable, and
when such pollen is transferred to room temperature (28°C) there is usually a
rapid loss of viability.
The results obtained with vacuum-dried pollen are encouraging. Vacuum
drying for 15-30 minutes did not reduce the pollen moisture content below the
 T a b l e 4. Pollen viability (%) after various drying treatments and storage conditions
Storage conditions

Initial pollen 3 months 6 months 9 months 12 months

No. Treatment viability Amb. 5°C — io°C Amb. 5°C — io°C Amb. 5°C — io°C Amb. 5°C — io°C >
I 15 min. drying
on sealing header 89 9° 93 93 93 89 87 78 79 79 79 78 79 D
O
Standard vacuum drying (min.) 2;
2 '5 89 94 90 89 91 82 89 84 75 77 79 79 78
3 3O 89 88 88 87 85 88 80 78 76 47 76 72
60
9°
86 81 80 72
4 89 83 76 75 78 79 77 78 71
5 120 89 79 80 76 77 74 74 77 79 73 67 70 68
6 240 89 41 39 43 41 44 40 37 43 48 0 38 0
Drying on sealing header
7 "5 73 73 73 74 75 — — — * 70 80
75 72 O
8 73 72 75 74 73 73 73 — — — * 70 71
3°
60 *
9 73 68 70 73 7" 75 75 — — — 74 74
10 120 73 72 74 70 — — — * 56
74 69 55 47
11 Control 78 76 74 73 0 62 74 — — — 0 64 70
' Ampoules were broken.
 Viability of oil palm pollen 65

level obtained by drying in a desiccator for 12-24 hours (6-10 per cent moisture
on a dry weight basis). However, storage in sealed ampoules under vacuum
appears to be superior to storage in a desiccator at room temperature, as little loss
in viability was observed over 1 year. The difference in viability directly after
drying for 1 hour or more between the two drying treatments (standard vacuum
drying and sealing header drying) is rather unexpected and cannot be explained
from the present data.
The observation which prompted the present investigation was that progenies
occasionally exhibited a high frequency of seedling abnormality. These abnor-
malities could not be traced back to certain parents or parent combinations, and
indicated that relationships may exist between length of pollen storage, storage
conditions, pollen viability and abnormal seedlings. Such progenies are character-
ized by irregular seed germination, high frequency of 'blind' seedlings (i.e.
seedlings where growth ceases after the production of the initial leaf) and seedlings
with long, narrow and erect leaves. Investigations are continuing into the nature
of these abnormalities, but it appears possible that, although pollen can be
successfully air-dried and stored over extended periods of time, certain storage
conditions may induce pollen aberrations of a genetic nature.
Progenies resulting from crosses with pollen from the various treatments
presented here are being studied. So far, after three months' storage, no evidence
of seedling abnormality has been found.

Acknowledgement. Technical assistance by Mr. T. Vanialingam during these

investigations is gratefully acknowledged.

REFERENCES
BROEKMANS, A. F. M. (1957). J. West Afri. Inst.for Oil Palm Res. x, 133.
DEVREUX, M. & MALINGRAUX, C. (i960). Bull. Agr. du Congo Beige. 51, 543.
HARDON, J . J . & TURNER, P. D. (1967). Expl Agric. 3, 105.
HENRY, P. (1959). C. R. Acad. Sc, Paris, 722.
KING, J . R. (1965). Bull Torrey Bot. Club. 9a, 270.
WHITEHEAD, R. A. (1965). Econ. Bot. 19, 267.

Expl Agric. 5, 1