Sex Plant Reprod (1990) 3:225-227 Sexual Plant Reproduction �9 Springer-Verlag 1990

Storage of pollen grains of CrotMaria retusa in oils

Ajay Jain and K.R. Shivanna Department of Botany, University of Delhi, Delhi l i0 007, India

Summary. Attempts were made to store pollen grains of C r o t a l a r i a r e t u s a L. in a mineral oil (par- affin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15-30 days, while those stored in oils main- tained some degree of germinability even after 60 days. Pollen samples stored in oils at - 2 0 ~ did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.

Key words: C r o t a l a r i a r e t u s a - Oils - Pollen stor- age - Pollen viability - Pollen vigour.

Introduction

In recent years there have been many studies on the efficacy of organic solvents in short- and long- term storage of pollen grains (Iwanami 1972; Shi- vanna and Johri 1985). The storage of pollen grains in organic solvents is cheap, simple and con- venient, and seems to be as effective as lyophiliza- tion or cryopreservation, which are more expensive and cumbersome (Iwanami 1984). Organic sol- vents, which are highly suitable to the storage of pollen, do not extract membrane phospholipids (Jain and Shivanna 1988a); they also provide an- hydrous storage conditions and limit the available oxygen required for long-term storage. As oils are immiscible with water, they are also likely to pro- vide conditions for pollen similar to those provided by organic solvents. So far there are no reports on the feasibility of using oils for storing pollen, although a few studies have been made on the use

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of mineral oil in preserving bacterial and plant cell cultures (Buell and Weston 1947; Caplin 1959). This paper presents the results of our studies on the efficacy of oils (mineral and vegetable oils) for pollen storage.

Material and methods

Fresh pollen grains of Crotalaria retusa L. collected from field- grown plants were desiccated overnight over dry silica and then used. For studying the efficacy of oils in pollen storage we used one mineral oil (paraffin oil) and two commercially avail- able vegetable oils (soybean oil and olive oil). For effective comparison of the efficacy of oils with organic solvents, pollen samples were also stored in n-hexane, one of the organic sol- vents most favourable to the storage of pollen (Jain and Shi- vanna 1988a, b). Pollen grains were dispersed in the oils or solvent (100 mg/5 ml) in 10-ml screw cap bottles and main- tained under laboratory conditions or at - 2 0 ~ C. As controls, one set of pollen samples was kept under uncontrolled laborato- ry conditions (temperature 24~ ~ C; RH 35% 75%), and an- other set was maintained under low humidity conditions (over dry silica in a desiccator; RH 0 % - 5 % ) in the laboratory or at - 20 ~ C.

The viability of the pollen grains stored in oils was tested by separating the pollen grains from the oils by means of filtra- tion through a millipore filter; any excess of oil from the pollen was blotted with Wha tman filter paper. The pollen sample was then washed four to five times with n-hexane to remove the oil from the pollen surface and allowed to dry for a few minutes before testing for viability. The washing of the pollen with n- hexane did not affect pollen viability. Pollen stored in n-hexane was separated from the solvent by filtration and then tested for viability.

In initial experiments pollen viability was assessed on the basis of the fluorochromatic reaction (FCR) test (Heslop-Harr- ison and Heslop-Harrison 1970) as well as by the in vitro germi- nation test. The present study confirmed our observations from earlier studies (Jain and Shivanna 1988a, b; 1989) in that there was a significant and positive correlation between FCR and in vitro germination tests of pollen samples stored under differ- ent temperature and humidity conditions. Consequently only the in vitro germination test was used in subsequent experi- ments.

Pollen grains were cultured in drops of Brewbaker and Kwack's (1963) medium containing H3BOa (100 pg ml 1),

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Table 1. Responses of pollen grains stored under laboratory conditions and at - 20 ~ C. The fresh pollen sample (control) showed 68 .8• germination and produced 475.7• tubes. Values are m e a n • Pollen samples stored in n-hexane and oils under the laboratory temperature showed significant differences (at P<0 .01 ) over the samples stored under low RH

Storage Laboratory conditions conditions

7 days 15 days 30 days

Germinat ion (%) Tube length Germinat ion (%) Tube length Germinat ion (%) Tube length (lain) (lam) (lam)

Uncontrolled 47.6_+4.2 a 368.2_+ 14.2 a 0.0 - 0.0 Low RH 69.2_+3.2 489.3_+20.9 78.2__+2.0 220.0 • 10.0 a 4.4_+1.2 a 108.5 • a n-Hexane 70.4• 329.8_+12.0 a 73.2• 360.6• 12.8 a 70.8• 166.3• Paraffin oil 65.2-t-1.6 471.7• 25.9 b 79.4• 361.5 • 8.7 a 83.6 • 2.0 a 371.8• a'b Soybean oil 63.3 • 3.4 588.0+31.3 b 74.8___2.6 336.6• 10.6" 71.6• 365.4• 15.3 a'b Olive oil 71.2+2.0 425.0• 16.6 b 72.8+_3.0 437.3 • 13.2 b 74.8+_2.0 333.2• ~'b

a Significant at P < 0.01 over the fresh pollen (control) b Values of pollen samples stored in oils significant at P<O.OI over the pollen stored in n-hexane

C a ( N O 3 ) z 4 H 2 0 (300 lag ml 1), MgSO4-7H20 (200 ~tg ml - I ) , KNO3 (100 lag m1-1) and sucrose (10%), and incubated at 27 ~ 2 ~ C. The cultures were scored for germination and tube length after 3 h. Pollen grains which produced tubes longer than twice the diameter of the pollen were considered to be germinated. For each pollen sample 5 replicates were raised, and from each replicate 100 pollen grains were scored for germi- nat ion; the length of the 100 tubes was measured with the ocu- lar micrometer.

In one experiment pollen grains stored in the oils or solvent under either laboratory conditions or at - 2 0 ~ C were removed as described earlier and transferred to laboratory conditions. Pollen viability was monitored at 7- or 15-day-intervals.

Results and discussion

Table 1 presents the effects of different laboratory conditions on pollen viability as assessed by the in vitro germination test. In this experiment FCR test was also used to assess viability (data not pre- sented). Although the FCR scores were con- sistently higher than the in vitro germination scores, the correlation coefficient between the two tests for pollen samples stored under different con- ditions was positive and highly significant ( P < 0.01). Under uncontrolled laboratory conditions there was a significant decline in germinability within 7 days of storage and within 15 days, pollen viability was lost completely. Under low humidity laboratory conditions pollen germinability was ex- tended for up to 15 days; it declined sharply during the next 15 days. There was no loss of germinabi- lity for up to 30 days in pollen samples stored in n-hexane as well as in different oils under laborato- ry conditions. Even at 60 days a small proportion of the pollen grains showed germination. In all of the pollen samples stored at the laboratory tem- perature, the reduction in tube growth was appar-

ent much earlier than the reduction in percentage germination; however, the reduction in tube length in samples stored in oils was significantly less than those stored at low RH or in n-hexane (Table 1).

There was no decline in percentage pollen ger- mination in the samples stored at - 2 0 ~ C (without n-hexane/oil) even after 6 months; however, there was a significant decline in pollen tube length. Pol- len samples stored in either n-hexane or oils, on the other hand, showed a significant improvement in percentage germination (over the fresh pollen) that was comparable to the FCR value of the fresh pollen. These pollen samples also did not show any reduction in tube length (Table 1). As reported in maize (Frova and Feder 1979) pollen grains of Crotalaria are likely to undergo some metabolic/ membrane changes during storage at - 2 0 ~ in oils/n-hexane, and thus show an improvement in germinability to the same degree as the FCR value (>80%).

In most of the earlier studies on pollen storage in which pollen viability was assessed on the basis of the in vitro germination test, the rate of pollen tube growth was not considered. A few recent sin- dies have shown that storage affects not only pol- len germinability but also pollen vigour in terms of the rate of tube growth (Nanda Kumar et al. 1988; Shivanna and Crest• 1989). As a decline in pollen vigour may affect the competitive ability of the pollen grains in the pistil, it is desirable to standardize storage conditions that would main- tain not only pollen germinability but also pollen tube vigour. In the present investigation all of the pollen samples stored at the laboratory tempera- ture a significant decline in pollen tube growth was observed much earlier than a decline in percentage

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from 30 days onwards for percentage germination and 15 days onwards for tube length. At - 20 ~ C the tube length was significantly higher in samples stored in n-hexane and oils over the pollen stored at low RH

--20 ~ C

45 days 60 days 180 days

Germinat ion (%) Tube length Germinat ion (%) Tube length (~tm) (~tm)

Germinat ion (%) Tube length (gm)

0.0 - 0 . 0 -

0 . 0 - 0.0 - 72.3 • 2.6 354.3 • 12.8 a 48.8 • 4.3 a 163,8 • 4.2 a 8,0 • 2.3 a 72.8 • 2.4 a 78.3 • 2.3 438.4 • 18.3 42 .8_ 1.6 a 246.7 • 8.0 a'b 18.3 • 1.3" 150.3 Jr 3,3 a,b 80.3_+ 4.1 453.4 • 18.3 54.4--+3.4 a 282.8_+6.7 a'b 12.4• 1.5 a 148.1_+4.5 a'b 82.3+2.9 a 488.4--+15.3 39.6 • 3.2 ~ 254.8 • 9.6 a,b 2.6 • 1.2 a 108.6 -+ 4.3 a,b 87.3 • 2.3 " 503.3 • 19.4

germination. This was true even in pollen samples stored at - 2 0 ~ C (at low RH) without oil. Pollen samples stored in any of the three oils at - 20 ~ C, however, did not show any decline in pollen tube growth, indicating that oils at low temperatures are effective in maintaining both pollen germina- tion and pollen tube vigour.

Pollen samples once removed from the oils/sol- vent after 15 days of storage at ambient tempera- ture or after 3 months at - 2 0 ~ showed similar trends: decreased germination and tube growth under laboratory temperatures and different R H with respect to fresh pollen (results not given). Pol- len samples transferred as such in either oils or solvent fr~)m - 2 0 ~ C to laboratory conditions re- tained viability for a longer period; the decline in viability became significant only by 60 days, and the viability was lost completely by 90 days.

Our results show that storage of pollen grains of Crotalaria in oils is as effective as storage in organic solvents. Low temperature storage in oils is not a serious problem for their transportation as the pollen samples retain viability for a few weeks even after their transfer to laboratory tem- perature. Oils may also have potential application in the storage of seeds, especially recalcitrant ones, which are usually short-lived and not normally amenable to desiccation or drying.

Acknowledgements. We are grateful to Professor N.S. Rangas- wamy for useful discussions. The award of a Senior Research Fellowship by the University Grants Commission and the CSIR to one of us (A.J.) is gratefully acknowledged.

References

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