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Effect of temperature on seed and fruit development in three mango (Mangifera indica L.) cultivars
Scientia Horticulturae 105 (2005) 467–474 www.elsevier.com/locate/scihorti
Effect of temperature on seed and fruit development in three mango (Mangifera indica L.) cultivars N. Sukhvibul a,*, A.W. Whiley b, M.K. Smith b a
Chiang Rai Horticulture Research Centre, Department of Agriculture, Muang, Chiang Rai 57000, Thailand b Maroochy Research Station, Queensland Horticulture Institute, Department of Primary Industries, P.O. Box 5083 SCMC, Nambour 4560, Australia Received 9 November 2004; received in revised form 8 February 2005; accepted 9 February 2005
Abstract The effect of diurnal maximum/minimum (20/10 or 25/15 8C) temperatures on seed and fruit development of ‘Irwin’, ‘Kensington’ and ‘Nam Dok Mai’ mangoes (Mangifera indica L.) was studied in a controlled-environment glasshouse. Exposure to low temperatures (20/10 8C day/night) 3 days after hand pollination significantly increased the percentage of stenospermocarpic fruit (nubbins), in which embryos were aborted at some stage during early fruit development. There were significant differences between cultivars in the percentage of nubbins produced out of the total fruit set following overnight exposure to 10 8C with 21% for ‘Nam Dok Mai’, 11% for ‘Kensington’ and 3% for ‘Irwin’. At 45 days after pollination, nubbin fruits were much smaller in size and weighed ca. 50% less than normal fruits. The lower percentage of nubbin fruits in ‘Irwin’ implies a greater adaptation to cool temperatures by this cultivar during fruit set and early embryo development. # 2005 Elsevier B.V. All rights reserved. Keywords: Mango; Low temperature; Stenospermocarpy; Nubbin fruit
* Corresponding author. Tel.: +66 53 714023; fax: +66 53 714024. E-mail address: [email protected] (N. Sukhvibul). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.007
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1. Introduction Stenospermocarpy is a term describing a small seedless fruit caused by embryo abortion after fertilisation (Soule, 1985). In mango, embryo abortion appeared to occur when trees were exposed to either low (Lakshminarayana and Aguilar, 1975; Whiley et al., 1988) or high (Davenport and Nu´ n˜ ez-Elisea, 1983) temperatures during pollination or early fruit set. In Australia, stenospermocarpic fruit are called ‘nubbins’ and are frequently found in mangoes grown in southeast Queensland (Whiley et al., 1988). Whiley et al. (1988) suggested that low temperatures (12 8C) during flowering or at an early stage of fruit development might interrupt fertilisation or ovule development. Lim et al. (1996) suggested that low temperatures during flowering might cause incomplete fertilisation and hence result in nubbin set in ‘Irwin’. However, all previous studies or observations have been done in the field and largely rely on anecdotal data with no investigations being carried out under controlled conditions. This study was carried out to establish the effect of low day/night temperatures on the production of nubbin fruit under controlled temperatures. Our objective was to determine whether low temperature contributed to poor fruit set on polyembryonic cultivars, commonly reported in mango orchards in subtropical climates, and whether this effect occurred after fertilisation.
2. Materials and methods 2.1. Plant materials and growth conditions This experiment was carried out in controlled-environment (CE) glasshouse at Maroochy Research Station, Nambour, Queensland (268370 S, altitude 30 m a.s.l.) between August and October 1997 and repeated during the same months in 1998. Trees were 6-yearold with two polyembryonic cv. Nam Dok Mai and Kensington, and a monoembryonic ‘Irwin’, all grafted to ‘Kensington’ seedling rootstock. Trees were approximately 1 m in canopy diameter and were grown in 50 L containers with sand, peat and soil mixture (4:3:3 (v/v/v)). Trees were maintained in the open where they were exposed to ambient winter temperatures at maximum/minimum of 21.6/9.5 8C to induce floral initiation (Schaffer et al., 1994). Fertiliser was applied with slow-release Osmocote Plus1 (N:P:K = 15:4.8:10.8, Scotts Europe B.V., Heerlen, Netherlands) at 1 kg m3 and 1 month before being transferred into the CE glasshouse. Three trees of each cultivar were placed in each of four CE glasshouse rooms when 50% of inflorescences reached the developmental stage of either 2 or 3 (1 cm in length) according to Oosthuyse (1991). Temperature profiles in each room were set to provide a day maximum of 25 8C at 1200 h and a night minimum of 15 8C at 0300 h. The temperature for each CE room was adjusted to ramp between two-hourly set-points using a computerized control system (detailed profiles are described by Sukhvibul et al. (1999)). Throughout the experiment, temperature was recorded every 2 h using a TinyTalk II temperature data logger (Gemini Data Loggers, Chichester, UK) with 0.4 8C accuracy. The photoperiod in each room was between 10 and 12 h over the duration of the study and photosynthetic photon flux
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density (PPFD) at 1200 h was ca. 1300 mmol m2 s1 as determined by a line quantum sensor (LI-COR Inc., Lincoln, NE, USA). Relative humidity in each room was maintained at a minimum of 70% with humidification when required. Trees were watered to field capacity every second day. Two weeks before hand pollination, trees were sprayed with Lorsban1 (chlorpyrifos) to prevent insects from facilitating pollination. 2.2. Treatments Hand-pollination was made on un-emasculated and un-bagged hermaphrodite flowers on ‘Irwin’, ‘Kensington’ and ‘Nam Dok Mai’ trees using ‘Sensation’ pollen collected from trees in a heated glasshouse. ‘Sensation’ pollen was used to ensure that there was no effect of self-incompatibility on nubbin set. Recently opened male flowers were collected from trees in a heated glasshouse where day temperatures did not exceed 25 2 8C or fall below 15 2 8C at night. Anther dehiscence was induced with a tungsten light (Sukhvibul et al., 2000). Pollen from freshly dehisced anthers was tested for viability by staining with fluorescein diacetate (FDA; Heslop-Harrison and Heslop-Harrison, 1970) as described by Sukhvibul and Considine (1993). Pollen samples that showed more than 50% viability were stored in open Petri dishes at 22 2 8C and were used for pollination the next morning. Between 0600 and 1100 h, a maximum of 10 hermaphrodite flowers per inflorescence and a minimum of 30 flowers per cultivar were hand-pollinated with freshly dehisced pollen (one dehisced anther per two flowers) at 25/15 8C for five successive days. The pollinated flowers were labelled with a small string paper tag indicating the date of pollination. Following hand-pollination, the temperature of each CE glasshouse room was maintained at 25/15 8C for a further 2 days to ensure that flowers pollinated on the fifth day had completed fertilisation. Thereafter, the temperature in two of the CE glasshouse rooms was dropped to 20/10 8C for 3 days while the other two rooms were kept at 25/15 8C. Following low temperature exposure (3 days at 20/10 8C), all trees were maintained at 25/ 15 8C for a further 45 days. 2.3. Fruit and seed development In 1997, there was damage to ‘Nam Dok Mai’ and ‘Kensington’ inflorescences caused by powdery mildew (Oidium mangiferae), hence data on fruit size and fruit weight after treatment are only presented for 1998. Percentage fruit set was calculated from the number of fruit set in relation to the number of pollinated flowers. A fruit was considered set when the diameter of the fruit was greater than 0.5 cm (approximately 14 days from pollination). At the end of the experiment (45 days after pollination), fruit were harvested. The length and width of normal and nubbin fruit was measured using digimetric callipers (Mitutoyo Co., Tokyo, Japan) and fruit were subsequently weighed. All fruits were cut longitudinally with a razor blade and the presence or absence of seed was recorded. 2.4. Statistical analyses In this experiment, only data on percentage nubbin fruit at the 20/10 8C temperature regime and at 3–6 days from pollination were analysed as there was negligible nubbin fruit
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set at 25/15 8C and at day 7 in either year. To cater for the variation in total number of fruit set per tree and the binomial distribution of the data, data on the percentage nubbin fruit set were analysed for the effect of cultivars, days from pollination and their interactions using the general linear model (GLM) and logit link (Dobson, 1990) in Genstat (Genstat Committee, 1993). Predicted means were obtained from the fitted models. All pair-wise differences between predicted means were tested using t-tests (Genstat Committee, 1993). The LSD at P < 0.05 was used to compare the predicted means of the percentage nubbin fruit. Mean differences in fruit size and fruit weight between normal and nubbin fruits were determined with an unpaired t-test (P < 0.05).
3. Results In both years, ‘Irwin’ had the highest fruit set with 198 (24.9%) and 258 (31.4%) fruits held on the six trees that were pollinated at 25/15 8C and then exposed to cold temperatures (20/10 8C) in 1997 and 1998, respectively. In comparison, ‘Kensington’ set 134 (8.8%) and 188 (21.1%) and ‘Nam Dok Mai’ set 146 (16.1%) and 174 (18.3%) fruits in 1997 and 1998, respectively. The percentage fruit set was calculated from the number of flowers that were hand pollinated. The development of nubbin fruit on ‘Kensington’ and ‘Nam Dok Mai’ trees became apparent when the fruit were ca. 10 mm in length. Nubbin fruit were generally misshapen and had a pronounced crease extending from the pedicel attachment down the suture line of the fruit. When the fruit was cut longitudinally, the nubbin fruit were found to have a shrivelled seed with a hollow cavity, while seed of the normal fruit was fully developed and almost filled the whole seed cavity. In both years, there were almost no nubbin fruit for the 25/15 8C treatment after pollination, with the exception of some nubbin fruit that developed on ‘Kensington’ (1.7%) and ‘Nam Dok Mai’ (2.7%) trees (data not presented). The number of days from pollination to low temperature (20/10 8C) exposure and the cultivar exposed, had a significant (P < 0.05) effect on the induction of nubbin fruit (Table 1). Over all treatments, the effects of main treatments are similar for 1997 and 1998 although there was a trend for a higher incidence of nubbin fruit in 1997. Following low temperature exposure, the greatest percentage of nubbin fruit that developed was from flowers that had been pollinated 3 days (14.6–26.5%) before the onset of low temperature, which was not significantly different with flowers that had been pollinated 4 days (8.0–16.5%) before the onset of low temperature. Meanwhile, there was no incidence of nubbin set on trees of any cultivar when trees were exposed to low temperature at 7 days from pollination. Averaged for all pollination dates, there was also a significantly greater percentage of nubbins induced on ‘Nam Dok Mai’ (17.9–24.8) and ‘Kensington’ (4.9–16.5) trees compared with ‘Irwin’ (2.7–3.6) trees. In addition, the percentage of nubbin fruit was consistently high for ‘Nam Dok Mai’ and consistently low for ‘Irwin’, but it was high for ‘Kensington’ in 1997 and low in 1998. Forty-five days after trees were returned to 25/15 8C following exposure to low temperatures at 20/10 8C for 3 days, normal fruit of all three cultivars were significantly (P < 0.05) longer, wider and heavier than nubbin fruit (Table 2). Nubbin fruit was less than 50% of the fresh weight of normal fruit. For example, the fresh weight of normal fruit of ‘Nam Dok Mai’ was 3.4 g while that of nubbin fruit was 1.3 g.
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Table 1 Effect of mango cultivars and the number of days from pollination to low temperature (20/10 8C) exposure on the percentage of nubbin fruit development in 1997 and 1998 Treatment
Percentage of nubbin fruit 1997
1998
Cultivar Nam Dok Mai Kensington Irwin
24.8a (26.5) 16.5a (14.9) 3.6b (3.5)
17.9a (18.0) 4.9a (4.7) 2.7b (2.7)
Days from pollination 3 4 5 6
26.5a (28.6) 16.5a (14.3) 2.8b (2.5) 2.6b (2.9)
14.6a (14.9) 8.0a (8.0) 2.2b (1.9) 0.7b (0.8)
After pollination with ‘Sensation’ pollen at 25/15 8C for five successive days, trees were kept at 25/15 8C for 2 days before exposure to temperatures of 20/10 8C for 3 days. All trees were held at 25/15 8C for 45 days before the number of nubbins was determined. Within each year, predicted means followed by different letters are significantly (P < 0.05) different as tested by GLM. Actual means are given in parenthesis.
Table 2 Comparison of length, width and fresh weight of normal and nubbin fruits of mango cv. Irwin, Kensington and Nam Dok Mai when grown at 25/15 8C for 45 days following exposure to temperatures of 20/10 8C for 3 days Length of fruit (mm)
Width of fruit (mm)
Weight of fruit (gfw)
Normal
Nubbin
Normal
Nubbin
Normal
Nubbin
Nam Dok Mai Kensington Irwin
28.5 (18) 19.8 (18) 16.7 (18)
18.1 (18) 14.0 (8) 11.5 (9)
16.3 (18) 15.7 (18) 14.4 (18)
11.6 (18) 11.5 (8) 10.6 (9)
3.4 (18) 2.7 (18) 2.0 (18)
1.3 (18) 1.1 (8) 0.8 (9)
Mean
21.7 3.5
14.5 1.9
15.5 0.6
11.2 0.3
2.7 0.4
1.1 0.1
Cultivar
Control fruit were kept at 25/15 8C. For all cultivars, there were significant (P < 0.05) differences in fruit dimension and fruit weight between the two fruit types as determined by unpaired t-test. The number of fruit measured is given in parentheses. Fruit type means (S.E.) are presented.
4. Discussion The study on the effect of low day/night temperatures on induction of nubbin fruit reported in this paper is the first known investigation of this phenomenon under controlled temperature conditions. The results clearly show the deleterious effect of low temperatures of 20/10 8C during early fruit set on the incidence of nubbin fruit (Table 1). Flowers were pollinated and held at 25/15 8C for 2 days to complete pollen tube growth and fertilisation (Sukhvibul et al., 2000) before exposure to low temperature. Hence, there is no doubt that nubbins developed following exposure to low temperatures and they are not a result of failure in pollination or fertilisation. Several researchers, Lakshminarayana and Aguilar (1975), Ram et al. (1976), Alexander (1987) and Whiley et al. (1988), reported a high correlation between the incidence of nubbin fruit and minimum orchard temperatures and
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suggested that nubbin set was caused by low temperatures. The results of our experiment contrast with recent work with ‘Irwin’ mango by Lim et al. (1996) who suggested that nubbin fruit was the result of incomplete fertilisation. Under the hot tropical conditions of the Northern Territory, Australia, deformed fruit without seed has sometimes been observed in ‘Irwin’ trees (Kulkarni and Hamilton, 1992). In this case it is suggested that high temperatures during pollination (32–34 8C) may have caused death of the embryo at a critical stage of development shortly after ovule fertilisation. Further investigations on the effect of high temperatures on in vivo pollen tube growth and seed development of mango under controlled temperatures are required to substantiate this hypothesis. The incidence of nubbin set had a high correlation with the number of days from pollination to low temperature exposure. The greatest percentage of nubbin fruit produced resulted from exposure to low temperatures 3 or 4 days after pollination and confirms the report of Whiley et al. (1988), who suggested that nubbins were the result of embryo abortion a few days after pollination or ovule fertilisation. Dissection of nubbin fruits revealed that most embryos were shrivelled and their growth was arrested at an early stage of fruit development. It is not known however, what the critical temperature or duration of exposure is required to cause embryo abortion in mango. Further studies on the histology of embryos at early stages of fruit development after exposure to low temperatures will improve understanding of the cause of stenospermatic fruit development thereby providing opportunities to minimise their occurrence. There was a significant difference in the number of nubbins set on the three mango cultivars after exposure to low temperatures (Table 1). ‘Nam Dok Mai’ had the highest percentage of nubbins set while ‘Irwin’ had the lowest. The genotypic differences substantiate field observations and reflects differences in the susceptibility of embryos of each mango cultivar to low temperatures during development. Under subtropical climates of Australia, the polyembryonic cv. Kensington, generally has a much higher number of nubbin fruit compared with monoembryonic cultivars such as ‘Tommy Atkins’, ‘Irwin’ and ‘Kent’ (A.W., Whiley, unpublished data, Australia). The sensitivity of embryos to low temperature during development may relate to ecotype differences between these cultivars. For instance, ‘Nam Dok Mai’ (polyembryonic) seems particularly susceptible to low temperatures during anthesis while ‘Irwin’ (monoembryonic) showed a greater adaptation to cool temperatures during early fruit development. For instance, ‘Nam Dok Mai’ (polyembryonic) seems particularly more susceptible to low temperatures during early fruit development compared with ‘Irwin’ (monoembryonic). Nubbin fruit were much smaller in size weighing ca. 50% less than normal fruit 45 days after pollination (Table 2). Similar reductions in fruit size have been reported in other fruits where embryos have aborted shortly following ovule fertilisation (grapes, Vitis vinifera, Nitsch et al. (1960); avocado, Blumenfeld and Gazit (1974)). Chacko and Singh (1969) reported an increase in the size of stenospermocarpic ‘Dashehari’ fruit following spraying inflorescences with a mixture of BAP (10 mg L1) and GA3 (250 mg L1). The authors concluded that the mango fruit requires cytokinins for cell division, and auxins and gibberellins to promote cell enlargement during early fruit development. In addition, they also suggested that seed abortion during early fruit development could interfere with endogenous plant growth substance production in the seed and subsequently reduce fruit growth or fruit size.
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5. Conclusions This study has clearly shown that nubbin fruit can be induced by low temperatures (20/ 10 8C) during fruit set with the most sensitive period for embryo development being within 3 days from pollination. ‘Nam Dok Mai’ followed by ‘Kensington’ were the two most sensitive to embryo abortion while few nubbin fruit were set on ‘Irwin’. This implies a greater adaptation to cool temperatures during early fruit development in ‘Irwin’ (monoembryonic) when compared with the two polyembryonic cv. Nam Dok Mai and Kensington, which is consistent with the evolutionary history of the ecotypes they represent (Whiley and Schaffer, 1997). The greater incidence of nubbins set on two polyembryonic cultivars, may indicate a major cause of excessive premature fruit drop from these cultivars when grown in the subtropics. This, together with reduced numbers of hermaphrodite flowers and an increased percentage of floral deformity, is likely to be a prime contributor to poor yields of polyembryonic cultivars grown in the subtropics.
Acknowledgements The authors thank Dr. V.J. Doogan for assisting with statistical analysis. Special thanks to Dr. D.W. Turner for critical reading of the manuscript. This study was supported from the Australian Centre for International Agricultural Research (ACIAR) with additional resources contributed by the Queensland Department of Primary Industries.
References Alexander, D.McE., 1987. The Mango in Australia. CSIRO, Melbourne. Blumenfeld, A., Gazit, S., 1974. Development of seeded and seedless avocado fruits. J. Am. Soc. Hort. Sci. 99, 442–448. Chacko, E.K., Singh, R.N., 1969. Induction of parthenocarpy in mango (Mangifera indica L.) using plant growth regulators. Hort. Sci. 4, 121–123. Davenport, T.L., Nu´ n˜ ez-Elisea, R., 1983. Abscission and ethylene production of mango (Mangifera indica L.) fruit cv. Tommy Atkins. Proc. Fla. State Hort. Soc. 96, 185–188. Dobson, A.J., 1990. An Introduction to Generalized Linear Models. Chapman & Hall, London. Genstat Committee, 1993. Genstat 5, Release 3, Reference Manual. Oxford University Press, Oxford. Heslop-Harrison, J., Heslop-Harrison, Y., 1970. Evaluation of pollen viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Technol. 45, 115–120. Kulkarni, V.J., Hamilton, D., 1992. Fruit disorders in mango. Technical Annual Report Department of Primary Industry and Fisheries, Northern Territory, Technical Bulletin No. 207, 51–52. Lakshminarayana, S., Aguilar, P.H., 1975. Effect of orchard heating in reducing parthenocarpic fruits in ‘Haden’ mango. Proc. Am. Soc. Hort. Sci. 88, 502–505. Lim, T.K., Luders, L., Poffley, M., 1996. Studies on fruit deformity and nubbins in Irwin mango. In: Proceedings of International Conference on Tropical Fruits. Malaysian Agricultural Research and Development Institute (MARDI), Kuala Lumpur, pp. 287–293. Nitsch, J.P., Pratt, C., Nitsch, C., Shaulos, N.J., 1960. Natural growth substances in Concord and Concord seedless grapes in relation to berry development. Am. J. Bot. 47, 566–576. Oosthuyse, S.A., 1991. Stages of development of the mango panicles. S. Afri. Mango Growers’ Assoc. Yrbk. 11, 59–61.
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Ram, S., Bist, L.D., Lakhanpal, S.C., Jamwal, I.S., 1976. Search of suitable pollinizers for mango cultivars. Acta Hort. 57, 253–263. Schaffer, B., Whiley, A.W., Crane, J.H., 1994. Mango. In: Schaffer, B., Andersen, P.C. (Eds.), Handbook of Environmental Physiology of Fruit Crops, Sub-tropical and Tropical Crops, vol. 2. CRC Press, Boca Raton, FL, pp. 165–197. Soule, J., 1985. Glossary for Horticultural Crops. Wiley, New York. Sukhvibul, N., Considine, J.A., 1993. Medium and long term storage of Angiozanthos manglesii (D. Don) pollen. N.Z. J. Crop Hort. Sci. 21, 343–347. Sukhvibul, N., Whiley, A.W., Smith, M.K., Hetherington, S.E., Vithanage, V., 1999. Effects of temperature on inflorescence development and sex expression on mono- and poly-embryonic mango (Mangifera indica L) cultivars. J. Hort. Sci. Biotechnol. 74, 64–68. Sukhvibul, N., Whiley, A.W., Vithanage, V., Smith, M.K., Doogan, V.J., Hetherington, S.E., 2000. Effect of temperature on pollen germination and tube growth of four cultivars of mango (Mangifera indica L.). J. Hort. Sci. Biotechnol. 75 (2), 214–222. Whiley, A.W., Schaffer, B., 1997. Stress physiology. In: Litz, R.E. (Ed.), The Mango: Botany Production and Uses. CAB International, Wallingford, pp. 147–173. Whiley, A.W., Saranah, J.B., Rasmussen, T.S., Winston, E.C., Wolstenholme, B.N., 1988. Effect of temperature on growth of 10 mango cultivars with relevance to production in Australia. In: Batten, D. (Ed.), Proceedings of the Fourth Australasian Conference on Tree and Nut Crops. ACOTANC, Lismore, pp. 176–185.
Effect of temperature on seed and fruit development in three mango (Mangifera indica L.) cultivars N. Sukhvibul a,*, A.W. Whiley b, M.K. Smith b a
Chiang Rai Horticulture Research Centre, Department of Agriculture, Muang, Chiang Rai 57000, Thailand b Maroochy Research Station, Queensland Horticulture Institute, Department of Primary Industries, P.O. Box 5083 SCMC, Nambour 4560, Australia Received 9 November 2004; received in revised form 8 February 2005; accepted 9 February 2005
Abstract The effect of diurnal maximum/minimum (20/10 or 25/15 8C) temperatures on seed and fruit development of ‘Irwin’, ‘Kensington’ and ‘Nam Dok Mai’ mangoes (Mangifera indica L.) was studied in a controlled-environment glasshouse. Exposure to low temperatures (20/10 8C day/night) 3 days after hand pollination significantly increased the percentage of stenospermocarpic fruit (nubbins), in which embryos were aborted at some stage during early fruit development. There were significant differences between cultivars in the percentage of nubbins produced out of the total fruit set following overnight exposure to 10 8C with 21% for ‘Nam Dok Mai’, 11% for ‘Kensington’ and 3% for ‘Irwin’. At 45 days after pollination, nubbin fruits were much smaller in size and weighed ca. 50% less than normal fruits. The lower percentage of nubbin fruits in ‘Irwin’ implies a greater adaptation to cool temperatures by this cultivar during fruit set and early embryo development. # 2005 Elsevier B.V. All rights reserved. Keywords: Mango; Low temperature; Stenospermocarpy; Nubbin fruit
* Corresponding author. Tel.: +66 53 714023; fax: +66 53 714024. E-mail address: [email protected] (N. Sukhvibul). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.007
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1. Introduction Stenospermocarpy is a term describing a small seedless fruit caused by embryo abortion after fertilisation (Soule, 1985). In mango, embryo abortion appeared to occur when trees were exposed to either low (Lakshminarayana and Aguilar, 1975; Whiley et al., 1988) or high (Davenport and Nu´ n˜ ez-Elisea, 1983) temperatures during pollination or early fruit set. In Australia, stenospermocarpic fruit are called ‘nubbins’ and are frequently found in mangoes grown in southeast Queensland (Whiley et al., 1988). Whiley et al. (1988) suggested that low temperatures (12 8C) during flowering or at an early stage of fruit development might interrupt fertilisation or ovule development. Lim et al. (1996) suggested that low temperatures during flowering might cause incomplete fertilisation and hence result in nubbin set in ‘Irwin’. However, all previous studies or observations have been done in the field and largely rely on anecdotal data with no investigations being carried out under controlled conditions. This study was carried out to establish the effect of low day/night temperatures on the production of nubbin fruit under controlled temperatures. Our objective was to determine whether low temperature contributed to poor fruit set on polyembryonic cultivars, commonly reported in mango orchards in subtropical climates, and whether this effect occurred after fertilisation.
2. Materials and methods 2.1. Plant materials and growth conditions This experiment was carried out in controlled-environment (CE) glasshouse at Maroochy Research Station, Nambour, Queensland (268370 S, altitude 30 m a.s.l.) between August and October 1997 and repeated during the same months in 1998. Trees were 6-yearold with two polyembryonic cv. Nam Dok Mai and Kensington, and a monoembryonic ‘Irwin’, all grafted to ‘Kensington’ seedling rootstock. Trees were approximately 1 m in canopy diameter and were grown in 50 L containers with sand, peat and soil mixture (4:3:3 (v/v/v)). Trees were maintained in the open where they were exposed to ambient winter temperatures at maximum/minimum of 21.6/9.5 8C to induce floral initiation (Schaffer et al., 1994). Fertiliser was applied with slow-release Osmocote Plus1 (N:P:K = 15:4.8:10.8, Scotts Europe B.V., Heerlen, Netherlands) at 1 kg m3 and 1 month before being transferred into the CE glasshouse. Three trees of each cultivar were placed in each of four CE glasshouse rooms when 50% of inflorescences reached the developmental stage of either 2 or 3 (1 cm in length) according to Oosthuyse (1991). Temperature profiles in each room were set to provide a day maximum of 25 8C at 1200 h and a night minimum of 15 8C at 0300 h. The temperature for each CE room was adjusted to ramp between two-hourly set-points using a computerized control system (detailed profiles are described by Sukhvibul et al. (1999)). Throughout the experiment, temperature was recorded every 2 h using a TinyTalk II temperature data logger (Gemini Data Loggers, Chichester, UK) with 0.4 8C accuracy. The photoperiod in each room was between 10 and 12 h over the duration of the study and photosynthetic photon flux
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density (PPFD) at 1200 h was ca. 1300 mmol m2 s1 as determined by a line quantum sensor (LI-COR Inc., Lincoln, NE, USA). Relative humidity in each room was maintained at a minimum of 70% with humidification when required. Trees were watered to field capacity every second day. Two weeks before hand pollination, trees were sprayed with Lorsban1 (chlorpyrifos) to prevent insects from facilitating pollination. 2.2. Treatments Hand-pollination was made on un-emasculated and un-bagged hermaphrodite flowers on ‘Irwin’, ‘Kensington’ and ‘Nam Dok Mai’ trees using ‘Sensation’ pollen collected from trees in a heated glasshouse. ‘Sensation’ pollen was used to ensure that there was no effect of self-incompatibility on nubbin set. Recently opened male flowers were collected from trees in a heated glasshouse where day temperatures did not exceed 25 2 8C or fall below 15 2 8C at night. Anther dehiscence was induced with a tungsten light (Sukhvibul et al., 2000). Pollen from freshly dehisced anthers was tested for viability by staining with fluorescein diacetate (FDA; Heslop-Harrison and Heslop-Harrison, 1970) as described by Sukhvibul and Considine (1993). Pollen samples that showed more than 50% viability were stored in open Petri dishes at 22 2 8C and were used for pollination the next morning. Between 0600 and 1100 h, a maximum of 10 hermaphrodite flowers per inflorescence and a minimum of 30 flowers per cultivar were hand-pollinated with freshly dehisced pollen (one dehisced anther per two flowers) at 25/15 8C for five successive days. The pollinated flowers were labelled with a small string paper tag indicating the date of pollination. Following hand-pollination, the temperature of each CE glasshouse room was maintained at 25/15 8C for a further 2 days to ensure that flowers pollinated on the fifth day had completed fertilisation. Thereafter, the temperature in two of the CE glasshouse rooms was dropped to 20/10 8C for 3 days while the other two rooms were kept at 25/15 8C. Following low temperature exposure (3 days at 20/10 8C), all trees were maintained at 25/ 15 8C for a further 45 days. 2.3. Fruit and seed development In 1997, there was damage to ‘Nam Dok Mai’ and ‘Kensington’ inflorescences caused by powdery mildew (Oidium mangiferae), hence data on fruit size and fruit weight after treatment are only presented for 1998. Percentage fruit set was calculated from the number of fruit set in relation to the number of pollinated flowers. A fruit was considered set when the diameter of the fruit was greater than 0.5 cm (approximately 14 days from pollination). At the end of the experiment (45 days after pollination), fruit were harvested. The length and width of normal and nubbin fruit was measured using digimetric callipers (Mitutoyo Co., Tokyo, Japan) and fruit were subsequently weighed. All fruits were cut longitudinally with a razor blade and the presence or absence of seed was recorded. 2.4. Statistical analyses In this experiment, only data on percentage nubbin fruit at the 20/10 8C temperature regime and at 3–6 days from pollination were analysed as there was negligible nubbin fruit
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set at 25/15 8C and at day 7 in either year. To cater for the variation in total number of fruit set per tree and the binomial distribution of the data, data on the percentage nubbin fruit set were analysed for the effect of cultivars, days from pollination and their interactions using the general linear model (GLM) and logit link (Dobson, 1990) in Genstat (Genstat Committee, 1993). Predicted means were obtained from the fitted models. All pair-wise differences between predicted means were tested using t-tests (Genstat Committee, 1993). The LSD at P < 0.05 was used to compare the predicted means of the percentage nubbin fruit. Mean differences in fruit size and fruit weight between normal and nubbin fruits were determined with an unpaired t-test (P < 0.05).
3. Results In both years, ‘Irwin’ had the highest fruit set with 198 (24.9%) and 258 (31.4%) fruits held on the six trees that were pollinated at 25/15 8C and then exposed to cold temperatures (20/10 8C) in 1997 and 1998, respectively. In comparison, ‘Kensington’ set 134 (8.8%) and 188 (21.1%) and ‘Nam Dok Mai’ set 146 (16.1%) and 174 (18.3%) fruits in 1997 and 1998, respectively. The percentage fruit set was calculated from the number of flowers that were hand pollinated. The development of nubbin fruit on ‘Kensington’ and ‘Nam Dok Mai’ trees became apparent when the fruit were ca. 10 mm in length. Nubbin fruit were generally misshapen and had a pronounced crease extending from the pedicel attachment down the suture line of the fruit. When the fruit was cut longitudinally, the nubbin fruit were found to have a shrivelled seed with a hollow cavity, while seed of the normal fruit was fully developed and almost filled the whole seed cavity. In both years, there were almost no nubbin fruit for the 25/15 8C treatment after pollination, with the exception of some nubbin fruit that developed on ‘Kensington’ (1.7%) and ‘Nam Dok Mai’ (2.7%) trees (data not presented). The number of days from pollination to low temperature (20/10 8C) exposure and the cultivar exposed, had a significant (P < 0.05) effect on the induction of nubbin fruit (Table 1). Over all treatments, the effects of main treatments are similar for 1997 and 1998 although there was a trend for a higher incidence of nubbin fruit in 1997. Following low temperature exposure, the greatest percentage of nubbin fruit that developed was from flowers that had been pollinated 3 days (14.6–26.5%) before the onset of low temperature, which was not significantly different with flowers that had been pollinated 4 days (8.0–16.5%) before the onset of low temperature. Meanwhile, there was no incidence of nubbin set on trees of any cultivar when trees were exposed to low temperature at 7 days from pollination. Averaged for all pollination dates, there was also a significantly greater percentage of nubbins induced on ‘Nam Dok Mai’ (17.9–24.8) and ‘Kensington’ (4.9–16.5) trees compared with ‘Irwin’ (2.7–3.6) trees. In addition, the percentage of nubbin fruit was consistently high for ‘Nam Dok Mai’ and consistently low for ‘Irwin’, but it was high for ‘Kensington’ in 1997 and low in 1998. Forty-five days after trees were returned to 25/15 8C following exposure to low temperatures at 20/10 8C for 3 days, normal fruit of all three cultivars were significantly (P < 0.05) longer, wider and heavier than nubbin fruit (Table 2). Nubbin fruit was less than 50% of the fresh weight of normal fruit. For example, the fresh weight of normal fruit of ‘Nam Dok Mai’ was 3.4 g while that of nubbin fruit was 1.3 g.
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Table 1 Effect of mango cultivars and the number of days from pollination to low temperature (20/10 8C) exposure on the percentage of nubbin fruit development in 1997 and 1998 Treatment
Percentage of nubbin fruit 1997
1998
Cultivar Nam Dok Mai Kensington Irwin
24.8a (26.5) 16.5a (14.9) 3.6b (3.5)
17.9a (18.0) 4.9a (4.7) 2.7b (2.7)
Days from pollination 3 4 5 6
26.5a (28.6) 16.5a (14.3) 2.8b (2.5) 2.6b (2.9)
14.6a (14.9) 8.0a (8.0) 2.2b (1.9) 0.7b (0.8)
After pollination with ‘Sensation’ pollen at 25/15 8C for five successive days, trees were kept at 25/15 8C for 2 days before exposure to temperatures of 20/10 8C for 3 days. All trees were held at 25/15 8C for 45 days before the number of nubbins was determined. Within each year, predicted means followed by different letters are significantly (P < 0.05) different as tested by GLM. Actual means are given in parenthesis.
Table 2 Comparison of length, width and fresh weight of normal and nubbin fruits of mango cv. Irwin, Kensington and Nam Dok Mai when grown at 25/15 8C for 45 days following exposure to temperatures of 20/10 8C for 3 days Length of fruit (mm)
Width of fruit (mm)
Weight of fruit (gfw)
Normal
Nubbin
Normal
Nubbin
Normal
Nubbin
Nam Dok Mai Kensington Irwin
28.5 (18) 19.8 (18) 16.7 (18)
18.1 (18) 14.0 (8) 11.5 (9)
16.3 (18) 15.7 (18) 14.4 (18)
11.6 (18) 11.5 (8) 10.6 (9)
3.4 (18) 2.7 (18) 2.0 (18)
1.3 (18) 1.1 (8) 0.8 (9)
Mean
21.7 3.5
14.5 1.9
15.5 0.6
11.2 0.3
2.7 0.4
1.1 0.1
Cultivar
Control fruit were kept at 25/15 8C. For all cultivars, there were significant (P < 0.05) differences in fruit dimension and fruit weight between the two fruit types as determined by unpaired t-test. The number of fruit measured is given in parentheses. Fruit type means (S.E.) are presented.
4. Discussion The study on the effect of low day/night temperatures on induction of nubbin fruit reported in this paper is the first known investigation of this phenomenon under controlled temperature conditions. The results clearly show the deleterious effect of low temperatures of 20/10 8C during early fruit set on the incidence of nubbin fruit (Table 1). Flowers were pollinated and held at 25/15 8C for 2 days to complete pollen tube growth and fertilisation (Sukhvibul et al., 2000) before exposure to low temperature. Hence, there is no doubt that nubbins developed following exposure to low temperatures and they are not a result of failure in pollination or fertilisation. Several researchers, Lakshminarayana and Aguilar (1975), Ram et al. (1976), Alexander (1987) and Whiley et al. (1988), reported a high correlation between the incidence of nubbin fruit and minimum orchard temperatures and
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suggested that nubbin set was caused by low temperatures. The results of our experiment contrast with recent work with ‘Irwin’ mango by Lim et al. (1996) who suggested that nubbin fruit was the result of incomplete fertilisation. Under the hot tropical conditions of the Northern Territory, Australia, deformed fruit without seed has sometimes been observed in ‘Irwin’ trees (Kulkarni and Hamilton, 1992). In this case it is suggested that high temperatures during pollination (32–34 8C) may have caused death of the embryo at a critical stage of development shortly after ovule fertilisation. Further investigations on the effect of high temperatures on in vivo pollen tube growth and seed development of mango under controlled temperatures are required to substantiate this hypothesis. The incidence of nubbin set had a high correlation with the number of days from pollination to low temperature exposure. The greatest percentage of nubbin fruit produced resulted from exposure to low temperatures 3 or 4 days after pollination and confirms the report of Whiley et al. (1988), who suggested that nubbins were the result of embryo abortion a few days after pollination or ovule fertilisation. Dissection of nubbin fruits revealed that most embryos were shrivelled and their growth was arrested at an early stage of fruit development. It is not known however, what the critical temperature or duration of exposure is required to cause embryo abortion in mango. Further studies on the histology of embryos at early stages of fruit development after exposure to low temperatures will improve understanding of the cause of stenospermatic fruit development thereby providing opportunities to minimise their occurrence. There was a significant difference in the number of nubbins set on the three mango cultivars after exposure to low temperatures (Table 1). ‘Nam Dok Mai’ had the highest percentage of nubbins set while ‘Irwin’ had the lowest. The genotypic differences substantiate field observations and reflects differences in the susceptibility of embryos of each mango cultivar to low temperatures during development. Under subtropical climates of Australia, the polyembryonic cv. Kensington, generally has a much higher number of nubbin fruit compared with monoembryonic cultivars such as ‘Tommy Atkins’, ‘Irwin’ and ‘Kent’ (A.W., Whiley, unpublished data, Australia). The sensitivity of embryos to low temperature during development may relate to ecotype differences between these cultivars. For instance, ‘Nam Dok Mai’ (polyembryonic) seems particularly susceptible to low temperatures during anthesis while ‘Irwin’ (monoembryonic) showed a greater adaptation to cool temperatures during early fruit development. For instance, ‘Nam Dok Mai’ (polyembryonic) seems particularly more susceptible to low temperatures during early fruit development compared with ‘Irwin’ (monoembryonic). Nubbin fruit were much smaller in size weighing ca. 50% less than normal fruit 45 days after pollination (Table 2). Similar reductions in fruit size have been reported in other fruits where embryos have aborted shortly following ovule fertilisation (grapes, Vitis vinifera, Nitsch et al. (1960); avocado, Blumenfeld and Gazit (1974)). Chacko and Singh (1969) reported an increase in the size of stenospermocarpic ‘Dashehari’ fruit following spraying inflorescences with a mixture of BAP (10 mg L1) and GA3 (250 mg L1). The authors concluded that the mango fruit requires cytokinins for cell division, and auxins and gibberellins to promote cell enlargement during early fruit development. In addition, they also suggested that seed abortion during early fruit development could interfere with endogenous plant growth substance production in the seed and subsequently reduce fruit growth or fruit size.
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5. Conclusions This study has clearly shown that nubbin fruit can be induced by low temperatures (20/ 10 8C) during fruit set with the most sensitive period for embryo development being within 3 days from pollination. ‘Nam Dok Mai’ followed by ‘Kensington’ were the two most sensitive to embryo abortion while few nubbin fruit were set on ‘Irwin’. This implies a greater adaptation to cool temperatures during early fruit development in ‘Irwin’ (monoembryonic) when compared with the two polyembryonic cv. Nam Dok Mai and Kensington, which is consistent with the evolutionary history of the ecotypes they represent (Whiley and Schaffer, 1997). The greater incidence of nubbins set on two polyembryonic cultivars, may indicate a major cause of excessive premature fruit drop from these cultivars when grown in the subtropics. This, together with reduced numbers of hermaphrodite flowers and an increased percentage of floral deformity, is likely to be a prime contributor to poor yields of polyembryonic cultivars grown in the subtropics.
Acknowledgements The authors thank Dr. V.J. Doogan for assisting with statistical analysis. Special thanks to Dr. D.W. Turner for critical reading of the manuscript. This study was supported from the Australian Centre for International Agricultural Research (ACIAR) with additional resources contributed by the Queensland Department of Primary Industries.
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