TREATMENT OF THE STIGMA WITH AN EXTRACT OF A COMPATIBLE PISTIL OVERCOMES SELF-INCOMPATIBILITY IN PETUNIA

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1 New Phytol. (1986) 12, TREATMENT OF THE STIGMA WITH AN EXTRACT OF A COMPATIBLE PISTIL OVERCOMES SELF-INCOMPATIBILITY IN PETUNIA BY N E E L A M SHARMA AND K. R. S H I V A N N A Department of Botany, University of Delhi, Delhi 117, India {Accepted 7 November 1985) SUMMARY Treatment of the stigma with an extract of a compatible pistil, just before pollination, was very effective in overcoming self-incompatibility in Petunia hybrida Vilm. Progressive delay either in the application of the extract, or in pollination markedly reduced its efficacy in overcoming self-incompatibility. The treatment seems to mask the self-incompatibility recognition molecules of the pistil. Key words: Petunia hybrida, pollen recognition, self-incompatibility. INTRODUCTION A range of techniques have been used to overcome self-incompatibility in different taxa (see de Nettancourt, 1977; Shivanna, 1982; Shivanna & Johri, 1985). However, the degree of success is highly variable between different techniques. In recent years, the use of mentor pollen (compatible pollen made ineffective to effect fertilization) along with incompatible pollen has been shown to be effective in many systems {Brassica - Roggen, 1975; Cosmos - Howlett et al., 1975; Petunia - Sastri & Shivanna, 1976; Nicotiana-Fandty, 1977; Sree Ramulu et al., 1979; Pyrus- Visser & Oost 1982; and Lilium - van Tuyl, Marucci & Visser, 1982). Indeed, it has been suggested that mentor pollen may provide recognition or regulatory substances to incompatible pollen, thus affecting self-incompatibility recognition or subsequent inhibition (see Stettler & Ager, 1984). Another possible approach, which has not yet been tested, is to block or decrease the ability of the pistil to recognize and/or inhibit incompatible pollen by treating an incompatible stigma with the extract obtained from a compatible pistil. We have used this approach to overcome self-incompatibility in Petunia hybrida Vilm. The only other similar investigations are those of Pandey (1963) and Shivanna & Rangaswamy (1969) in which smearing of the stigma-exudate from mature flowers on to the stigma ofthe bud increased the efficacy of bud pollination. However, no correlation with incompatibility was reported as the exudate from both compatible and incompatible stigmas was equally effective. MATERIALS AND METHODS Field-grown plants of Petunia hybrida Vilm. were used throughout this investigation. The stigma together with the style from unpollinated pistils (on the day of anthesis) were homogenized in chilled - mol \~^ phosphate buffer, ph 5-9 using acid-washed sand as an abrasive. The homogenate was then centrifuged at 1 g at 4 C for 1 min, and the supernatant used as pistil extract. A drop of either X/86/ $3./ 1986 The New Phytologist

2 444 NEELAM SHARMA AND K. R. SHIVANNA incompatible (obtained from the same plant from which pistils were used for pollination) or compatible (obtained from another plant which is cross-compatible with the plant used as the pistil source) pistil extract (2-5 pistils m\~^ containing about 2 /ig protein) was placed on unpollinated stigmas (on the day of anthesis) using a syringe and left for 1 to 12 min to allow absorption. The stigma was then washed with a few drops of the buffer and left to dry for about five min before pollinating the pistils with either self- or cross-pollen. The stigma treated with buffer alone for min before pollination served as the control. In the second experiment, untreated pistils were first pollinated with self-pollen and then the stigmas were treated with compatible pistil extract 3, 6, 9 and h after pollination. In the third experiment, pistils were first treated with a compatible-pistil extract and then self-pollinated 6, 9 and h later. In another treatment, pollen grains were incubated in a compatible pistil extract (prepared in phosphate buffer containing 2 % sucrose and 2 jug ml"! boric acid to prevent bursting of pollen) for 3 min, then separated by passing the suspension through a MiUipore filter (pore size 45 /im), and used for pollination. Pollinated pistils were bagged and left on the plant until fruit maturity/abscission. Mature fruits were harvested individually and the seed number in each fruit was counted. RESULTS The results presented in Table 1 show the effects of the treatment of stigmas with pistil extract on fruit and seed set. The plants used in these experiments were strictly self-incompatible. Treatment of stigma with buffer alone had no effect on selt-incompatibihty and only marginally reduced seed set in cross-pollinated pistils biniilarly, treatment of stigmas with incompatible pistil extract had no effect on self-polhnated pistils, but further reduced seed set in cross-pollinated pistils. 1 reatment of the stigma with a compatible pistil extract was very effective in overcoming self-incompatibility but had no effect on cross-pollinated pistils. Progressive delay in the treatment of the stigma with compatible pistil extract markedly reduced its efficacy in overcoming self-incompatibility (Table 2); none of the selfed pistils developed fruits when the treatment was delayed for h. Similarly, delay in self-pollination of the pistils treated with compatible extract also reduced seed set (Table 3). The incubation of pollen in compatible pistil ex- JZ IAZ P "^"^'^ " ^^^ " t ^e^y effective in overcoming self-incompatibility and yielded only an average of seed per fruit of 68-4 after self-pollination. DISCUSSION Recent studies have indicated that in Petunia hybrida self-incompatibility recognition molecules in the pistil are present on the surface of the stigma as well as in the transmitting tissues of the stigma and upper 2-3 mm of the style (Herrero & Dickmson, 1981, Shivanna & Sharma, unpublished). Recognition of self-pollen occurs soon after pollination (van der Donk, 1975). Treatment of the stigma with a compatible pistil extract seems to mask the recognition molecules of the pistil. Drastic reduction in the efficacy of the treatment when application of the extract was delayed (by which time pollen grains would have been recognized) is m agreement with this suggestion. However, the treatment does not seem to mask the recognition molecules of the pollen as treatment of the pollen with a compatible

3 Overcoming self-incompatibility in Petunia 445 Table 1. Effect of pistil extract on fruit and seed set in Petunia hybrida Pollination treatments pollinations fruits % fruit set Average number of seeds/fruit Average number of seeds/ pollination Control Buffer Compatible extract Incompatible extract Table 2. Effect of delay in the application of a compatible pistil extract on fruit and seed set in self-pollinated pistils of Petunia hybrida Delay (h) in application of extract after self-pollination pollinations fruits % fruit set Average number of seeds/fruits Average number of seeds/pollination Table 3. Effect of delay in self-pollination of the pistils treated with compatible extract on fruit and seed set in Petunia hybrida. Period of delay in self-pollination (h) No. of pollinations No. of fruits % fruit set No. of seeds/fruit No. of seeds/ pollination pistil extract before pollination induced very limited seed set. The considerable reduction observed in the efficacy of the treatment when the pollination is delayed may be due to the continued synthesis of the recognition molecules in the pistil as has been shown in Brassica (Roberts et al., 1983). Many techniques are available which overcome self-incompatibility in Petunia

4 44^ NEELAM SHARMA AND K. R. SHIVANNA as well as in other self-incompatible taxa (see de Nettancourt, 1977; Shivanna & Johri, 1985). As judged by fruit and seed set, the most effective technique so far used is the treatment of pollen, before pollination, with sugars. In one instance over 3 seeds/pollination (about 4 % of those obtained through cross-pollination) have been realized (Sharma, Bajaj & Shivanna, 1985). In the present investigation over seeds/pollination (about 75 % of those obtained in cross-pollination) were obtained in self-pollinated flowers treated with a compatible pistil extract. Application of a compatible pistil extract is one of the simplest and least expensive methods so far used to overcome self-incompatibility and in Petunia, it is also the most effective method. The technique is now being extended to other systems, especially to sporophytic systems and to interspecific crosses. As the crude pistil extract may contain non-specific inhibitors (Hodgkin & Lyon, 1984), stigma leachates or pistil fractions free from inhibitors, may be more effective than the crude extract in other systems. ACKNOWLEDGEMENT N. S. is grateful to the Council of Scientific and Industrial Research for the award of a Senior Research Fellowship. REFERENCES DE NETTANCOURT, D. (1977). Incompatibility in Angiosperms. Springer-Verlag, Heidelberg. HERRERO, M. & DICKINSON, H. G. (1981). Pollen tube development in Petunia hybrida following compatible and incompatible intraspecific matings. Journal of Cell Science, 47, HODGKIN, J. & LYON, G. D. (1984). Pollen germination inbibitors in extracts of Brassica oleracea L. stigma. New Phytologist, %, HOWLETT, B. J., KNOX, R. B., PAXTON, S. H. & HESLOP-HARRISON, J. (1975). Pollen-wall proteins: pbysiocbemical cbaracterization and role in self-incompatibility in Cosmos bipinnatus. Proceedings of Royal Society of London Series B188, PANDEY, K. K. (1963). Stigmatic secretion and bud-pollinations in self- and cross-incompatible plants. Naturwissenschaften,, PANDEY, K. K. (1977). Mentor pollen: possible role of wall-beld pollen growth promoting substances in overcoming inter- and intraspecific incompatibility. Genetics, Al, ROBERTS, I. N., GAUDE, T. C, HARROD, G. & DICKINSON, H. G. (1983). Pollen-stigma interactions in Brassica oleracea; a new pollen germination medium and its use in elucidating the mechanism of self-incompatibility. Theoretical and Applied Genetics, 65, ROGGEN, H. P. J. R. (1975). Stigma application of an extract from rape pollen {Brassica napus L.) affects self-mcompatibility in Brussels Sprouts {Brassica oleracea L. var. gemmifera DC). Incompatibility Newsletter, 6, «v y. SASTRI, D. C. & SHIVANNA, K. R. (1976). Recognition pollen alters incompatibility in Petunia. Incompatibilitv Newsletter, 7, K. A-. SHARMA, NEELAM, BAJAJ, MADHU & SHIVANNA, K. R. (1985). Overcoming self-incompatibility through the use of lectins and sugars in Petunia and Eruca. Annals of Botany, 55, SHIVANNA, K. R. (1982). Pollen-pistil interaction and control of fertilization. In: Experimental Embryology of Vascular Plants (Ed. by B. M. Johri), pp Springer-Verlag, Heidelberg. SHIVANNA, K. R. & JOHRI, B. M. (1985) The Angiosperm Pollen Structure and Function. Wiley Eastern, New Delhi. SHIVANNA, K. R. & RANGASWAMY, N. S. (1969). Overcoming self-incompatibil.ty in Petunia axillaris (Lam.)?7o ion *^'^y^^ pollination, pollination with stored pollen and bud pollination. Phytomorphology, 19, SREE RAMULU, K., BREDEMEIJER, G. M. M., DIJKHUIS, E., DE NETTANCOURT, D. & SCHIBILLA, H. (1979). Mentor pollen effects on gametophytic incompatibility in Nicotiana, Oenothera and Lycopersicon. 1 heoretical and Applied Genetics, 54, STETTLER, R. E. & AGER, A. A. (1984). Mentor effects in pollen interactions. In: Cellular Interactions. hncyclopaedia of Plant Physiology, new series, vol. 17 (Ed. by H. E. Linskens & J. Heslop-Harrison), pp Springer-Verlag, Heidelberg.

5 Overcoming self-incompatibility in Petunia 447 VAN DER DONK, J. A. W. M. (1975). Recognition and gene expression during the incompatibility reaction in Petunia hybrida L. Molecular General Genetics, 141, VAN TuYL, M. M., MARCUCCI, M. C. & VissER, T. (1982). Pollen and pollination experiments. VII. The effect of pollen treatment and application method on incompatibility and incongruity in Lilium Euphytica, 31, VissER T. & OosT, E. H. (1982). Pollen and pollination experiments. V. An empirical basis for a mentor pollen effect obseryed on the growth of incompatible pollen tubes in pear. Euphytica, 31,

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