Female and male sterility cause low fruit set in a clone of the `Trevatt' variety of apricot (Prunus armeniaca)

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1 Scientia Horticulturae 82 (1999) 255±263 Female and male sterility cause low fruit set in a clone of the `Trevatt' variety of apricot (Prunus armeniaca) A.M. Lillecrapp, M.A. Wallwork, M. Sedgley * Department of Horticulture, Viticulture and Oenology, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, S.A. 5064, Australia Accepted 9 April 1999 Abstract This study investigated ovule and anther structure of the `Trevatt Blue' variety of apricot (Prunus armeniaca) using bright field microscopy following reports of low fruit set. Ovules and anthers from fertile `Moorpark' and `Trevatt Knight' flowers were compared with those of the `Trevatt Blue'. In the `Moorpark' and `Trevatt Knight' ovules, all reproductive structures were present including embryo sacs with a complete set of eight nuclei and the anthers contained mature pollen grains. Multiple ovules which were small and retarded in development were present in the `Trevatt Blue' apricot flowers and the anthers contained degenerated microspores, with some failure in tapetal breakdown. This is the first report of a simultaneous mutation in both female and male function in apricot. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Apricot; Fertility; Prunus; Microscopy; Ovule; Embryo sac; Anther; Pollen grain 1. Introduction `Trevatt' is an old self-fertile cultivar of apricot (Prunus armeniaca) and high yielding clones have been developed to produce large fruit with desirable characteristics. The clone, `Blue' was planted over wide areas by many growers because the original selection was high yielding with fruit of excellent canning quality, but the trees failed to set fruit for several seasons after the juvenile phase had ended. * Corresponding author. Tel.: ; ; fax: address: msedgley@waite.adelaide.edu.au (M. Sedgley) /99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S (99)

2 256 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255±263 Some varieties of apricot have been found to be self-sterile or have pollination problems (McLaren and Fraser, 1996), but it seemed that in `Blue' female sterility may have been a possible contributor to low fruit set. Very low levels of set were achieved even when pollen from a different cultivar was introduced to the trees via bouquets from fertile orchards. It was also possible that male sterility contributed to the low fruit set. Female and male sterility have been reported independently in apricot (Eaton and Jamont, 1964; Nakanishi, 1983; Medeira and Guedes, 1991; Burgos and Egea, 1994) as well as in many other tree crops (Sedgley and Griffin, 1989), but have been attributed to adverse environmental conditions. In this study, the female and male structures of `Trevatt Blue' were observed microscopically and compared with those of `Trevatt Knight' which is known to be fertile. A comparison was also made with the cultivar `Moorpark' in case there was a problem inherent in the `Trevatt' cultivar. 2. Materials and methods Flower samples of the low yielding `Trevatt Blue' were randomly collected from a single variety apricot orchard in Griffith, New South Wales. Fertile `Trevatt Knight' flowers were obtained from a mixed planting at Loxton, South Australia, and fertile `Moorpark' flowers were obtained from the Waite orchard of the University of Adelaide, South Australia. Flowers were collected at the late balloon stage just prior to anthesis and fixed in FPA50 (90% ethanol at 50%, 5% propionic acid and 5% formaldehyde). Ovules and anthers were dissected from each flower under a microscope and the samples dehydrated using a tertiary-butyl alcohol series and embedded in GMA (glycol methacrylate). Serial longitudinal 4.0 mm sections through each ovule (up to 150 sections per ovule) and 48 sections of 4.0 mm through each anther were collected onto a microscope slide, stained with periodic acid-schiffs reagent (PAS) and toluidine blue O (TBO) stain and mounted in methyl methacrylate in xylene (O'Brien and McCully, 1981). The sections were observed under a Zeiss Axiophot photomicroscope with bright field illumination. Ovules and anthers from 36 `Trevatt Blue', 15 `Trevatt Knight' and 9 `Moorpark' flowers were examined. The structure of male and female reproductive structures was recorded for each flower. 3. Results Two ovules were present in each `Moorpark' and `Trevatt Knight' flower. Between one and four ovules were present in each `Trevatt Blue' flower, most

3 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255± Table 1 Ovule structure for fertile `Moorpark' and `Trevatt Knight', and sterile `Trevatt Blue' apricot flowers at anthesis `Moorpark' `Trevatt Knight' `Trevatt Blue' Mean number ovules (SD) Range 2±2 2±2 1±4 Percentage ovules with outer integument inner integument nucellus micropyle embryo sac Embryo sac with 0 nuclei nucleus nuclei nuclei ±8 nuclei smaller in size than the fertile `Moorpark' and `Trevatt Knight' ovules but all similar in size to each other. One hundred and four ovules were obtained from 36 `Trevatt Blue' flowers; 2.8% of flowers had one ovule, 27.8% had two, 47.2% had three and 22.2% had four (Table 1). Of the ovules examined of the `Moorpark' and `Trevatt Knight', all had an outer integument, inner integument, nucellus, micropyle and embryo sac (Fig. 1(A)) with an egg cell (Fig. 1(B)), two synergids (Fig. 1(C)), two polar nuclei (Fig. 1(D)) and three antipodals (Fig. 1(E)), which had degenerated in some ovules. All `Trevatt Blue' ovules had an outer integument, inner integument and nucellus, and 88.5% had a micropyle (Fig. 2(A)). Some had mis-shapen nucellus (Fig. 2(B)), and embryo sacs were degenerated or not present in 50% of ovules (Table 1). The other 50% contained embryo sacs with 0, 1 (Fig. 2(C)), 2 (Fig. 2(D)) or 4 (Fig. 2(E)) nuclei. No `Trevatt Blue' apricot ovules observed contained eight nuclei, and 19.2% of ovules were at the megaspore mother cell stage. Other abnormalities included small, spherical ovules, ovules joined together and ovules with underdeveloped or abnormal nucellus (Fig. 2(B)) or nucellus incompletely surrounded by the integuments. The anthers of the `Moorpark' and `Trevatt Knight' flowers were bright yellow in colour and plump compared to those of the `Trevatt Blue' flowers which were red-brown and shrunken. Anthers from `Moorpark' and `Trevatt Knight' apricot flowers had an endothecium and degenerated tapetum (Fig. 3(A)). Fully developed pollen grains with an exine, intine, germination pores, vegetative

4 258 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255±263 Fig. 1. (A±E) Longitudinal sections of `Moorpark' (figures A, B, D and E) and `Trevatt Knight' (figure C) apricot ovules stained with periodic acid-schiff's reagent (PAS) and toluidine blue O (TBO) and photographed using bright field optics. (A) Ovule showing embryo sac (es), nucellus (n), outer integument (oi), inner integument (ii) and micropyle (m). (B) Embryo sac with egg cell (e). A polar nucleus is also visible. (C) Embryo sac showing two synergids (s). (D) Embryo sac showing two polar nuclei (pn). An egg cell and antipodal are also visible. (E) Embryo sac showing three antipodals (a). A polar nucleus and a synergid are also visible. Bar represents 200 mm in A, 700 mm in B, 750 mm in C and 600 mm in D and E. and generative nuclei were present in anthers from all flowers (Fig. 3(B) and (C)). `Trevatt Blue' anthers were shrunken (Fig. 4(A)) with an endothecium (Fig. 4(B)), 72.2% with degenerated tapetum (Fig. 4(C)) but the remainder without tapetal degeneration (Fig. 4(B)). No pollen grains were observed in the anthers and only degenerated lumen contents were visible (Table 2).

5 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255± Fig. 2. (A±E) Longitudinal sections of `Trevatt Blue' apricot ovules stained with PAS and TBO and photographed using bright field optics. (A) Ovule showing nucellus (n), outer integument (oi), inner integument (ii) and micropyle (m). (B) Ovule showing an abnormal and underdeveloped nucellus (n). (C) Embryo sac with one nucleus. (D) Embryo sac with two nuclei. (E) Embryo sac with four nuclei. Bar represents 200 mm in A, 100 mm in B, 450 mm in C, 600 mm in D and 650 mm ine. 4. Discussion The results show that both female and male sterility contributed to low fruit set in `Trevatt Blue' apricot trees. `Trevatt Knight' was both female and male fertile and there are no other reports of sterility in the cultivar indicating a simultaneous mutation in female and male function. `Trevatt Knight' was structurally similar to

6 260 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255±263 Fig. 3. (A±C) Transverse sections of `Moorpark' (figures A and B) and `Trevatt Knight' (figure C) apricot anthers stained with PAS and TBO and photographed using bright field optics. (A) Anther lobe showing endothecium (en), degenerated tapetum (dt) and pollen grains (pg). (B) Pollen grain showing intine (i), exine (ex) and nuclei (nc). (C) Pollen grain showing germinaton pores (gp). Bar represents 200 mm in A and 750 mm in B and C. Table 2 Anther structure for fertile `Moorpark' and `Trevatt Knight', and sterile `Trevatt Blue' apricot flowers at anthesis `Moorpark' `Trevatt Knight' `Trevatt Blue' Percentage anthers with endothecium degenerated tapetum degenerated lumen contents pollen grains intine bexine germination pore vegetative and generative nuclei

7 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255± Fig. 4. (A±C) Transverse sections of `Trevatt Blue' apricot anthers stained with PAS and TBO and photographed using bright field optics. (A) Anther showing four shrunken lobes (arrows). (B) Anther lobes showing endothecium (en) and non-degenerated tapetum (t) with degenerated lumen contents (dc). (C) Anther lobes showing degenerated tapetum (dt) and degenerated content (dc). Bar represents 100 mm in A and 200 mm in B and C. `Moorpark' showing no inherent problem in the `Trevatt' cultivar. Female sterility was due to multiple ovules and retarded development, and the clone was male sterile due to microspore degeneration and some failure in tapetal breakdown. The findings explain the lack of success achieved using pollinator bouquets and pollen mixtures, although occasional fruit were produced. This suggests that some fertile embryo sacs were formed despite none being observed by microscopy. Since the clone was both female and male sterile and since similar observations were noted for all flowers, there must have been a mutation in the original

8 262 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255±263 `Trevatt Blue' tree from which the budwood was taken and from which the trees were clonally propagated. Trees maintained for budwood are generally prevented from flowering to limit spread of pollen-borne viruses, so a mutation would not have been recognised until the progeny flowered. The magnitude of the problem suggests that a single mutation or mutational event affected both the female and male fertility of the clone. Sterile plants can show both male and female sterility as many processes during microsporogenesis and megasporogenesis are under combined genetic control (Chaudhury, 1993). In particular, disruptions during meiosis result in mutations that can cause sterility of both male and female gametophytes (Reiser and Fischer, 1993). This research into the `Trevatt Blue' apricot is the only report of both male and female sterility occurring in the same variety of apricot but the phenomenon has been reported in other crops. Abnormal embryo sacs of the female gametophytes and failure in gametogenesis of both male and female gametophytes was the cause of unfruitfulness in the `Swan Hill' variety of olive (Rallo et al., 1981), and a gene has been identified in the male gametophyte of soybean which reduces male and female fertility due to failure of cytokinesis following meiosis (Kennell and Horner, 1985). Failure in meiosis is suggested in this study by the low number of embryo sacs with nuclei, and the lack of pollen grains. Sterility of apricots has previously been attributed to factors other than a mutation, such as adverse environmental conditions (Eaton and Jamont, 1964). Multiple ovules, as described here, have been described in apricot (Egea and Burgos, 1995), and it was suggested that there may be a relationship between the number of ovules per ovary and temperature (Egea and Burgos, 1998). In other studies, abnormalities such as degeneration of ovules which had developed normally, ovules with twin nucellus and shortened integuments were reported (Burgos and Egea, 1994; Egea and Burgos, 1994). Some authors have found that male sterile apricot trees have shrunken anthers with little or no pollen (Nakanishi, 1983; Medeira and Guedes, 1991), but there are no previous microscopy studies. There has been an increase in apricot breeding activity around the world in recent years, which has exposed previously unrecognised fertility problems (J. Witherspoon, personal communication 1998). Failure of female and male fertility, and self-incompatibility problems were selected against in the old apricot cultivars which are widely planted, but there is now a need for new high yielding firm fruited selections. This breeding activity is unmasking fertility problems, such as the one reported here, and understanding these problems is fundamental to successful apricot breeding in the future. `Trevatt Blue' and other genotypes showing fertility failure are located at the Loxton Research Station in South Australia, and further research is required to characterise the genetic basis of these problems. In the meantime, similar failures can be avoided by ensuring the establishment of multiple budwood mother trees of a

9 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255± particular cultivar or clone, so that a mutation in one will not result in widespread loss of yield. Acknowledgements The authors acknowledge Peter Burn for reporting the problem, John Zirilli for supply of the `Trevatt Blue' flowers, Jenny Witherspoon for comments on the problem and supply of the `Trevatt Knight' flowers and Mike Harms for assistance. References Burgos, L., Egea, J., Apricot embryo-sac development in relation to fruit set. J. Hortic. Sci. 68, 203±208. Chaudhury, A.M., Nuclear genes controlling male fertility. The Plant Cell 5, 1277±1283. Eaton, G.W., Jamont, A.M., Embryo sac development in the apricot, Prunus armeniaca L. cv Constant. J. Amer. Soc. Hortic. Sci. 86, 95±101. Egea, J., Burgos, L., Year-to-year variation in the developmental stage of the embryo sac at anthesis in flowers of apricot (Prunus armeniaca L.). J. Hortic. Sci. 69, 315±318. Egea, J., Burgos, L., Supernumerary ovules in flowers of apricot. Acta Hortic. 384, 373±377. Egea, J., Burgos, L., Fructification problems in continental apricot cultivars growing under Mediterranean climate. Ovule development at anthesis in two climatic areas. J. Hortic. Sci. and Biotech. 73, 107±110. Kennell, J.C., Horner, H.T., Influence of the soybean male-sterile gene (ms1) on the development of the female gametophyte. Can. J. Genet. Cyt. 27, 200±209. McLaren, G.F., Fraser, J.A., Pollination compatibility of `Sundrop' apricot and its progeny in the `Clutha' series. N.Z. J. Crop Hortic. Sci. 24, 47±53. Medeira, M.C., Guedes, M.E., Flower bud abscission and male sterility in apricot. Acta Hortic. 293, 311±318. Nakanishi, T., Morphological and ultraviolet absorption differences between fertile and sterile anthers of Japanese apricot cultivars in relation to their pollination stimuli. Sci. Hortic. 18, 57±63. O'Brien, T.P., McCully, M.A., The study of plant structure: principles and selected methods. Termacarphi, Melbourne. Rallo, L., Martin, G.C., Lavee, S., Relationship between abnormal embryo sac development and fruitfulness in olive. J. Amer. Soc. Hortic. Sci. 106, 813±817. Reiser, L., Fischer, R.L., The ovule and the embryo sac. The Plant Cell 5, 1291±1301. Sedgley, M., Griffin, A.R., Sexual reproduction of tree crops. Academic Press, London.