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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 highyielding clones have been developed to produce large fruit with desirablecharacteristics. The clone, `Blue' was planted over wide areas by many growersbecause the original selection was high yielding with fruit of excellent canningquality, but the trees failed to set fruit for several seasons after the juvenile phasehad ended.

Scientia Horticulturae 82 (1999) 255±263

* Corresponding author. Tel.: +61-8-8303-7401; +61-8-8303-7249; fax: +61-8-8303-7116

E-mail address: [email protected] (M. Sedgley)

0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 6 1 - 8

Some varieties of apricot have been found to be self-sterile or have pollinationproblems (McLaren and Fraser, 1996), but it seemed that in `Blue' femalesterility may have been a possible contributor to low fruit set. Very low levels ofset were achieved even when pollen from a different cultivar was introduced tothe trees via bouquets from fertile orchards. It was also possible that male sterilitycontributed to the low fruit set. Female and male sterility have been reportedindependently in apricot (Eaton and Jamont, 1964; Nakanishi, 1983; Medeira andGuedes, 1991; Burgos and Egea, 1994) as well as in many other tree crops(Sedgley and Griffin, 1989), but have been attributed to adverse environmentalconditions.

In this study, the female and male structures of `Trevatt Blue' were observedmicroscopically and compared with those of `Trevatt Knight' which is known tobe fertile. A comparison was also made with the cultivar `Moorpark' in case therewas a problem inherent in the `Trevatt' cultivar.

2. Materials and methods

Flower samples of the low yielding `Trevatt Blue' were randomly collectedfrom a single variety apricot orchard in Griffith, New South Wales. Fertile`Trevatt Knight' flowers were obtained from a mixed planting at Loxton, SouthAustralia, and fertile `Moorpark' flowers were obtained from the Waite orchardof the University of Adelaide, South Australia.

Flowers were collected at the late balloon stage just prior to anthesis and fixedin FPA50 (90% ethanol at 50%, 5% propionic acid and 5% formaldehyde).Ovules and anthers were dissected from each flower under a microscope and thesamples dehydrated using a tertiary-butyl alcohol series and embedded in GMA(glycol methacrylate). Serial longitudinal 4.0 mm sections through each ovule (upto 150 sections per ovule) and �48 sections of 4.0 mm through each anther werecollected onto a microscope slide, stained with periodic acid-Schiffs reagent(PAS) and toluidine blue O (TBO) stain and mounted in methyl methacrylate inxylene (O'Brien and McCully, 1981). The sections were observed under a ZeissAxiophot photomicroscope with bright field illumination. Ovules and anthersfrom 36 `Trevatt Blue', 15 `Trevatt Knight' and 9 `Moorpark' flowers wereexamined. The structure of male and female reproductive structures was recordedfor 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

256 A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255±263

smaller in size than the fertile `Moorpark' and `Trevatt Knight' ovules but allsimilar 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% hadthree and 22.2% had four (Table 1).

Of the ovules examined of the `Moorpark' and `Trevatt Knight', all had an outerintegument, 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 andnucellus, 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 observedcontained eight nuclei, and 19.2% of ovules were at the megaspore mother cellstage. Other abnormalities included small, spherical ovules, ovules joinedtogether and ovules with underdeveloped or abnormal nucellus (Fig. 2(B)) ornucellus incompletely surrounded by the integuments.

The anthers of the `Moorpark' and `Trevatt Knight' flowers were bright yellowin colour and plump compared to those of the `Trevatt Blue' flowers which werered-brown and shrunken. Anthers from `Moorpark' and `Trevatt Knight' apricotflowers had an endothecium and degenerated tapetum (Fig. 3(A)). Fullydeveloped pollen grains with an exine, intine, germination pores, vegetative

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) 2 � 0 2 � 0 2.88 � 0.79

Range 2±2 2±2 1±4

Percentage ovules with

outer integument 100 100 100

inner integument 100 100 100

nucellus 100 100 100

micropyle 100 100 88.5

embryo sac 100 100 50

Embryo sac with

0 nuclei 0 0 19.2

1 nucleus 0 0 19.2

2 nuclei 0 0 8.7

4 nuclei 0 0 2.9

5±8 nuclei 100 100 0

A.M. Lillecrapp et al. / Scientia Horticulturae 82 (1999) 255±263 257

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 remainderwithout tapetal degeneration (Fig. 4(B)). No pollen grains were observed in theanthers and only degenerated lumen contents were visible (Table 2).

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.


4. Discussion

The results show that both female and male sterility contributed to low fruit setin `Trevatt Blue' apricot trees. `Trevatt Knight' was both female and male fertileand there are no other reports of sterility in the cultivar indicating a simultaneousmutation in female and male function. `Trevatt Knight' was structurally similar to

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 in E.


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 100 100 100

degenerated tapetum 100 100 72.2

degenerated lumen contents 0 0 100

pollen grains 100 100 0

intine 100 100 0

bexine 100 100 0

germination pore 100 100 0

vegetative and generative nuclei 100 100 0


`Moorpark' showing no inherent problem in the `Trevatt' cultivar. Female sterilitywas due to multiple ovules and retarded development, and the clone was malesterile due to microspore degeneration and some failure in tapetal breakdown.The findings explain the lack of success achieved using pollinator bouquets andpollen mixtures, although occasional fruit were produced. This suggests thatsome fertile embryo sacs were formed despite none being observed bymicroscopy.

Since the clone was both female and male sterile and since similar observationswere noted for all flowers, there must have been a mutation in the original

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.


`Trevatt Blue' tree from which the budwood was taken and from which the treeswere clonally propagated. Trees maintained for budwood are generally preventedfrom flowering to limit spread of pollen-borne viruses, so a mutation would nothave been recognised until the progeny flowered. The magnitude of the problemsuggests that a single mutation or mutational event affected both the female andmale fertility of the clone.

Sterile plants can show both male and female sterility as many processes duringmicrosporogenesis and megasporogenesis are under combined genetic control(Chaudhury, 1993). In particular, disruptions during meiosis result in mutationsthat 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 maleand female sterility occurring in the same variety of apricot but the phenomenonhas been reported in other crops. Abnormal embryo sacs of the femalegametophytes and failure in gametogenesis of both male and female gametophyteswas 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 whichreduces male and female fertility due to failure of cytokinesis following meiosis(Kennell and Horner, 1985). Failure in meiosis is suggested in this study by thelow number of embryo sacs with nuclei, and the lack of pollen grains.

Sterility of apricots has previously been attributed to factors other than amutation, such as adverse environmental conditions (Eaton and Jamont, 1964).Multiple ovules, as described here, have been described in apricot (Egea andBurgos, 1995), and it was suggested that there may be a relationship between thenumber of ovules per ovary and temperature (Egea and Burgos, 1998). In otherstudies, abnormalities such as degeneration of ovules which had developednormally, ovules with twin nucellus and shortened integuments were reported(Burgos and Egea, 1994; Egea and Burgos, 1994). Some authors have found thatmale sterile apricot trees have shrunken anthers with little or no pollen(Nakanishi, 1983; Medeira and Guedes, 1991), but there are no previousmicroscopy studies.

There has been an increase in apricot breeding activity around the world inrecent years, which has exposed previously unrecognised fertility problems(J. Witherspoon, personal communication 1998). Failure of female and malefertility, and self-incompatibility problems were selected against in the oldapricot cultivars which are widely planted, but there is now a need for new highyielding firm fruited selections. This breeding activity is unmasking fertilityproblems, such as the one reported here, and understanding these problemsis fundamental to successful apricot breeding in the future. `Trevatt Blue' andother genotypes showing fertility failure are located at the Loxton ResearchStation in South Australia, and further research is required to characterisethe genetic basis of these problems. In the meantime, similar failures can beavoided by ensuring the establishment of multiple budwood mother trees of a


particular cultivar or clone, so that a mutation in one will not result in widespreadloss of yield.

Acknowledgements

The authors acknowledge Peter Burn for reporting the problem, John Zirilli forsupply of the `Trevatt Blue' flowers, Jenny Witherspoon for comments on theproblem and supply of the `Trevatt Knight' flowers and Mike Harms forassistance.

References

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