Temperature effects on male fertility and flower and fruit development in Capsicum annuum L.

Scientia Horticulturae, 25 (1985) 117--127 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

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T E M P E R A T U R E E F F E C T S ON MALE F E R T I L I T Y A N D F L O W E R A N D F R U I T D E V E L O P M E N T I N C A P S I C U M A N N U U M L.

P.L. POLOWICK and V.K. SAWHNEY Department o f Biology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OWO (Canada) (Accepted for publication 26 September 1984)

ABSTRACT Polowick, P.L. and Sawhney, V.K., 1985. Temperature effects on male fertility and flower and fruit development in Capsicum annuum L. Scientia Hortic., 25: 117--127. Temperature conditions strongly influenced the development of flowers and fruits of pepper (Capsicum annuum L.) plants. Low temperatures (LTR; 18°C day/15°C night) had much more effect on flowers and fruits than intermediate (ITR; 23°C day/18°C night) or high (HTR; 28°C day/23°C night) temperatures. LTR caused the formation of abnormal petals, stamens and gynoecium in the flowers. Stamens produced were deformed, in some cases partly carpel-like, produced abnormal non-viable pollen, and were thus functionally male-sterile. In the gynoecium, the ovary size of LTR-grown flowers was larger than that of ITR and HTR flowers, but the style elongation was inhibited. Fruits produced under HTR were larger than ITR and were seeded under both temperature regimes. Under LTR, small seedless fruits were produced, but normal seeded fruits were formed if flowers were pollinated with pollen from ITR- or HTR-grown flowers. Keywords: Capsicum annuum; flower morphogenesis; fruit development; male sterility; pepper; temperature. ABBREVIATIONS GA3 = gibberellic acid HTR = high temperature regime (28°C day/23°C night) ITR = intermediate temperature regime (23°C day/18°C night) LTR = low temperature regime (18°C day/15°C night)

INTRODUCTION T e m p e r a t u r e c o n d i t i o n s are k n o w n t o i n f l u e n c e the sex expression o f m a n y species o f flowering plants. O b s e r v a t i o n s as early as 1 8 1 9 had s h o w n t h a t w a t e r m e l o n plants g r o w n u n d e r high t e m p e r a t u r e s p r o d u c e d o n l y male flowers a n d t h a t c u c u m b e r plants g r o w n in c o o l e r t e m p e r a t u r e s f o r m e d female flowers (see reviews b y Heslop-Harrison, 1 9 7 2 ; F r a n k e l and Galun, 1977). A l t h o u g h in m o s t species investigated high t e m p e r a t u r e s e n h a n c e d maleness and low t e m p e r a t u r e s p r o m o t e d femaleness, variations f r o m this

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118 pattern are known to occur. For example, in the male-sterile, single gene "stamenless-2" mutant of tomato, low temperatures p r o m o t e d the formation of normal stamens whereas high temperatures induced the formation of carpels in place of stamens (Sawhney, 1983a). In contrast, in normal t o m a t o plants, low temperatures enhanced the growth of b o t h the stamens and the gynoecium, and the effect was more pronounced on the latter than on the former. Further, different cultivars of tomato showed differences in the degree of response (Sawhney, 1983b). Variations in the sex expression of different genetic lines of cucumber in response to temperature conditions have also been reported (Saito and Ito, 1964). In many developmental processes, the temperature effects are often similar to those of applied growth substances. In the development of floral organs also, temperature and growth substances have been shown to produce similar responses (Atsmon, 1968; Blake, 1969; Heide, 1969; Garrod and Harris, 1974; Sawhney, 1983b). In pepper, gibberellic acid (GA3) induces abnormalities in the development of petals and stamens, including the production of non-viable pollen (Sawhney, 1981; Kohli et al., 1981). The present study was conducted to determine the effects of different temperature regimes on the development of pepper flowers and fruits. MATERIALS AND METHODS Seeds of Capsicum annuum cultivar 'Vinedale' (Stokes Seeds Ltd., St. Catharines, Ontario) were germinated in peat pellets ("Jiffy-7", Jiffy Products Ltd.). Seedlings with 4--5 true leaves were transplanted into 15-cm diameter plastic pots containing a loam:sand:peat moss (2:1:1) mixture. Plants were transferred to each o f three growth chambers programmed at the following day/night temperature regimes: high (HTR, 28°C/23°C), intermediate (ITR, 23°C/18°C); low (LTR, 18°C/15°C). Temperature fluctuations were generally not more than 1°C. Illumination was provided by Gro-lux wide.spectrum fluorescent tubes at an intensity of 180 pE s-1 m-2 for 16 h/day. Plants were watered daffy and fertilized weekly with a commercial fertilizer 20: 20: 20 (Plant Products Co., Ltd.). Flowers were collected from plants grown under each temperature regime. For each flower, the number of petals and stamens, the lengths of stamens and styles, ovary diameter and the number of locules per ovary were recorded, along with any abnormalities in the petals or stamens. Flowers and floral organs were photographed through a Nikon Micro flex HFM camera fitted to a Nikon SMZ-10 stereomicroscope. Some of the flowers in each temperature regime were left to self-pollinate and the resultant fruits were collected at maturity. Measurements of the fruit length, weight, equatorial diameter (the average of two measurements at right angles) and locule number were recorded. Representative samples of fruits were photographed with a Nikon F2 camera. For the study of pollen, a sample of stamens from each temperature

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regime was fixed in Farmer's Fluid (Berlyn and Miksche, 1976) and stored at -4°C. Anther squash preparations were stained with acetocarmine and photographed through a Nikon Microflex camera attached to a Nikon Optiphot microscope. RESULTS

Different temperature regimes had no apparent effect on the development of sepals, but the development of petals, stamens and the gynoecium of pepper flowers was altered in various ways. However, no significant differences were observed in the number of petals, stamens and locules produced under the three temperature regimes (Table I). development. - - In ITR and HTR, the petals were fully opened at maturity and no significant abnormalities were observed (Fig. 1, Table I). Petals produced under LTR, however, were curled and did not expand at maturity (Fig. 2). Also, all the petals in flowers formed under LTR were abnormal (Table I) and t h e y remained in that state after dehiscence.

Petal

development. - - Stamens with some carpel-like features, i.e. the occurrence of naked ovules or a basal ovary-like region (Fig. 3), were produced in flowers grown under LTR. The occurrence of such "carpelloid stamens" was greater under LTR than in ITR, whereas they did not occur under HTR (Table I). Stamen length was also inhibited in flowers produced under LTR and again no differences were observed in the stamen lengths of ITR and HTR flowers (Table I). Further, the stamens of ITR and HTR flowers produced abundant pollen (Fig. 4), but no pollen grains were apparent on the surface o f LTR-grown anthers (Fig. 5). Squash preparations of fixed anthers showed that microsporogenesis was affected in LTR-grown flowers. Most of the pollen grains produced under low temperature were either devoid o f contents or were abnormal in shave (Fig. 6) in comparison to the normal pollen produced under high or intermediate temperatures (Fig. 7).

Stamen

development. - - Different parts of the gynoecium were affected differently by temperature conditions. In flowers produced under LTR, the ovary size was larger than in ITR flowers, which in turn was larger than the HTR flowers (Table I). Conversely, the length o f the style was greatest in HTR and was shortest in LTR flowers (Table I). The increase in ovary diameter in LTR flowers was n o t related to an increase in the n u m b e r of locules (Table I). Gynoecium

development. - - Pepper fruits developed under the three temperature regimes showed striking differences (Figs. 8--13). Fruits p r o d u c e d under HTR had the greatest fresh weight, whereas those produced under LTR had

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121 the least weight (Table II). The difference between the H T R and I T R fruits, although significant, was small. The HTR-grown fruits (Figs. 8 and 9) were longer and larger in diameter than the I T R fruits (Figs. 10 and 11), which in t u rn were larger t h a n the L T R fruits (Figs. 12 and 13, see also Table II). As in the case o f ovary, the increase in fruit size of HTR-grown plants was n o t related to an increase in the locule n u m b e r (Table II}.

TABLE II Effects of three different temperature regimes -- high (HTR, 28°C day/23°C night), intermediate (23°C day/18°C night), low (LTR, 18°C day/15°C n i g h t ) - on the developmen~ of pepper (Capsicum a n n u u m 'Vinedale') fruits. Values represent the mean ± a confidence level of 95%. Sample size (n) = 50 Temperature Fresh regime weight

(g) HTR ITR LTR

Fruit length

Length of attached style

Fruit I diameter

(cm)

(cm)

(cm)

30.56±3.08 6.72+0.41 0.0 24.54±2.78 5.92±0.29 0.0 6.67±0.61 3.04±0.16 0.49±0.06

No. of locules/fruit

4.09±0.20 2.96±0.29 3.72±0.16 2.84±0.18 2.73±0.10 2.84±0.20

1Average o f two measurements perpendicular to each other.

The L T R fruits had some additional features which are n o t e w o r t h y . First, the fruits which were left to self-pollinate were always seedless (cf. Fig. 13 with Figs. 9 and 11), p r o b a b l y due to abnormal pollen (see Fig. 6). However, if the L T R flowers were hand-pollinated with pollen f r o m ITR or H T R flowers t h e y p r o d u c e d seeded fruits, and such fruits were larger than the unseeded fruits p r o d u c e d unde r L T R . Second, the non-pollinated ITR- and HTR-grown flowers abscised, but in L T R flowers the grow t h o f the ovary c o n tin u ed af ter anthesis. Third, the carpelloid stamens p r o d u c e d in L T R flowers also showed some post-anthesis growth and developed into small fruits encircling the main fruit (Fig. 12).

DISCUSSION Results presented here show t hat t e m p e r a t u r e conditions strongly influence th e d ev e l opm ent o f flowers and fruits o f p e p p e r plants. The most p r o n o u n c e d effects were p r o d u c e d u n d e r L T R , although H T R also had some e ff ect on f lo wer a nd fruit development. L o w t e m p e r a t u r e s induced various abnormalities in petal, stamen and g y n o e c i u m development, but the development of sepals was n o t affected. In t o m a t o also, low temperatures were shown t o alter the development of

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Fig. 6. Squash preparation of an anther grown under LTR, showing the presence of abnormal pollen (X 450). Fig. 7. Squash preparation o f an anther grown under ITR, showing the presence o f norreal pollen (X 450).

floral organs other than the sepals (Sawhney, 1983b). There were striking differences, however, in the responses induced in the t o m a t o and pepper flowers by the same low-temperature regime. For example, in pepper flowers, L T R inhibited the enlargement o f petals and stamens in comparison to HTR, but in t o m a t o these structures were larger in LTR than in HTR (Sawhney, 1983b). In pepper flowers, there was no difference in the n u m b e r of petals, stamens and locules produced in the three temperature regimes, but in t o m a t o the n u m b e r o f these organs was greater under LTR than under HTR. Further, whereas in pepper flowers LTR caused the production of "carpelloid stamens" and non-viable pollen, i.e. male-sterile flowers, no such abnormalities were induced in t o m a t o flowers by the same temperature regime. Finally, in both pepper (Table I) and t o m a t o (Sawhney, 1983b) flowers, the ovary size of LTR-grown flowers was larger than that o f ITR or HTR flowers; however, in t o m a t o this was associated with an increase in locule n u m b e r (Sawhney, 1983b), but this was not so in pepper (Table II). Thus, identical temperature conditions induce some similar and some different responses in different species of the same family. Furthermore, different cultivars o f the same species axe k n o w n to exhibit variations in the Fig. 1. Normal pepper flower at anthesis, grown under HTR (X 4). Fig. 2. Abnormal pepper flower grown under LTR. Note the unrolled petals (x 6). Fig. 3. Carpelloid stamen from a flower grown under LTR. Note the presence o f both an ovary and an anther (X 12). Fig. 4. Normal anther plus a portion o f the filament from a flower grown under H TR

(x 20). Fig. 5. Anther plus a portion of the filament from a flower grown under LTR. Note the absence of pollen in comparison to Fig. 4 (x 20).

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125 intensity o f response to the same temperature (Sawhney, 1983b). These observations point to the non-generality of morphogenetic patterns in plants in response to similar environmental conditions. The influence of temperature conditions on the morphogenetic patterns in flowers m a y be explained through changes in endogenous hormones. The basis of this suggestion lies in the fact that often temperatures and applied growth substances induce similar effects on a developmental process, e.g. seed germination, bud dormancy and flowering (Villiers, 1972; Wareing and Phillips, 1981; Bernier et al., 1981). In the case o f flower development also, low temperatures and exogenous gibberellins are k n o w n to induce similar responses (Garrod and Harris, 1974; Sawhney, 1983b). Further, temperature effects have also been correlated with changes in endogenous hormones (Aung and DeHertogh, 1968; Reid et al., 1974; Atherton and Harris, 1980). The low-temperature effects on pepper flowers reported here are similar to the effects of gibberellic acid reported earlier (Sawhney, 1981). Although both L T R and exogenous GA3 induce some different responses in t o m a t o and pepper flowers, the effects p r o d u c e d by both these factors were similar in the same species (see results above and Sawhney, 1981, 1983b). These observations support the contention that low-temperature effects on flowers m a y be related to changes in endogenous gibberellins. The induction of male sterility in pepper flowers by low temperatures is of interest from the point of view of hybrid seed production, L o w temperatures are k n o w n to cause pollen infertility in many species, e.g. egg plant (Nothmann and Koller, 1973), onion (Van der Meer and Van Bennekom, 1969) and sorghum (Brooking, 1979). Examples of high-temperatureinduced pollen-sterility are also k n o w n (Meyer, 1966; Edwardson, 1970). In pepper flowers, GA3 also causes male sterility (Sawhney, 1981; Kohli et al., 1981) and Kohli et al. (1981) suggested the possibility o f using GA3 as a gametocide. Observations presented here show that low temperature can be another effective m e t h o d of inducing male sterility in pepper. Temperature conditions also affected the fruit development of pepper plants. Large seeded fruits were produced under H T R which were only slightly greater in size and fresh weight than ITR-grown fruits. In contrast, LTR fruits were smaller than the ITR- and HTR-grown fruits and were seedless. The production o f parthenocarpic fruits by low temperatures is known for several species (Nitsch et al., 1952; Osborne and Went, 1953; Rylski, 1979) including pepper (Cochran, 1936; Rylski, 1973). However, as Fig. 8. Pepper fruit from HTR (× 0.6). Fig. 9. Cross-section of a fruit from HTR. Fig. 10. Pepper fruit from ITR (× 0.6). Fig. 11. Cross-section of a fruit from ITR. Fig. 12. Pepper fruit from LTR (× 0.6). Note extra carpels. Fig. 13. Cross-section of fruits from LTR. Note the absence of seeds.

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shown here, the development of seedless fruit under LTR is not related to the infertility of the ovules, since normal pollen from ITR or HTR flowers induces the development of normal seeded fruits. Nevertheless, it is interesting that in pepper, while on the one hand low temperature induces pollen infertility, on the other it stimulates ovary growth in the absence of pollination. ACKNOWLEDGEMENT

This study was supported by funds from the Natural Sciences and Engineering Research Council of Canada to V.K.S. REFERENCES Atherton, J.G. and Harris, G.P., 1980. Effects of chilling on the formation of secondary growing-centres in flowers of the glasshouse carnation. Scientia Hortic., 13: 371--376. Atsmon, D., 1968. The interaction of genetic, environmental , and hormonal factors in stem elongation and floral development o f cucumber plants. Ann. Bot., 32: 877--882. Aung, L.H. and DeHertogh, A.A., 1968. Gibberellin-like substances in non-cold and cold treated tulip bulbs (Tulipa sp.). In: F. Wightman and G. Setterfield (Editors), Biochemistry and Physiology of Plant G r o w t h Substances. Runge Press, Ottawa, pp. 943--956. Berlyn, G.P. and Miksche, J.P., 1976. Botanical microtechnique and cytochemistry. The Iowa State University Press, Ames, IA, 326 pp. Bernier, G., Kinet, J.M. and Sachs, R.M., 1981. The Physiology of Flowering. Vol. II. Transition to Reproductive Growth. CRC Press, Boca Raton, FL, 231 pp. Blake, J., 1969. The effect of environmental and nutritional factors on the development of flower apices cultured in vitro. J. Exp. Bot., 20: 113--123. Brooking, I.R., 1979. Male sterility in Sorghum bicolor (L.) Moench induced by l o w temperature. II. Genotypic differences in sensitivity. Aust. J. Plant Physiol., 6: 143-147. Cochran, H.L., 1936. Some factors influencing growth and fruit setting in the pepper (Capsicum annuum L.). Mem. Cornell Agric. Exp. Stn., 190: 1--39. Edwardson, J.R., 1970. Cytoplasmic male sterility, Bot. Rev., 36: 341--420. Frankel, R. and Galun, E., 1977. Pollination Mechanisms, Reproduction and Plant Breeding. Springer-Verlag, New York, pp. 132--134. Garrod, J.F. and Harris, G-P., 1974. Studies on the glasshouse carnation: Effects o f temperature and growth substances on petal number. Ann, Bot., 38: 1025--1031. Heide, O~/I., 1969. Environmental control of sex expression in Begonia. Z. Pflanzenphysiol., 61: 279--285. Heslop-Harrison, J., 1972. Sexuality of angiosperms. In: F.C. Steward (Editor), Plant Physiology. Vol. VI (c). Academic Press, New York, pp. 133--289. Kohli, U.K., Dua, I.S. and Saini, S.S., 1981. Gibberellic acid as an androecide for bell pepper. Scientia Hortic., 15: 17--22. Meyer, V.G., 1966. Flower abnormalities. Bot. Rev., 32: 165--218. Nitsch, J.P., Kurtz, E.B., Livermann, J.L. and Went, F.W., 1952. The development o f sex expression in cucurbit flowers. Am. J. Bot., 39: 32--43. Nothmann, J. and KoUer, D., 1973. Morphogenetic effects o f low temperature stress on flowers o f egg plant (Solanum melongena). Israel J. Bot., 22: 231--235. Osborne, D.P. and Went, F.W., 1953. Climatic factors influencing parthenocarpy and normal fruit set in tomatoes. Bot. Gaz., 114: 312--322.

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