New Forests

, Volume 40, Issue 2, pp 131–142

Improving controlled pollination methodology for breeding Acacia mangium Willd.

Authors

    • School of Plant ScienceUniversity of Tasmania
  • Tran Duc Vuong
    • School of Plant ScienceUniversity of Tasmania
    • Forest Science Institute of Vietnam
  • J. L. Harbard
    • School of Plant ScienceUniversity of Tasmania
  • C. Y. Wong
    • Riau Andelan Pulp & Paper
  • C. Brooker
    • School of Plant ScienceUniversity of Tasmania
  • R. E. Vaillancourt
    • School of Plant ScienceUniversity of Tasmania
Article

DOI: 10.1007/s11056-010-9188-x

Cite this article as:
Griffin, A.R., Vuong, T.D., Harbard, J.L. et al. New Forests (2010) 40: 131. doi:10.1007/s11056-010-9188-x

Abstract

Acacia mangium is a major plantation species for the pulp and paper industry in south-east Asia and there are a number of active breeding programs. The species is predominantly outcrossing, but with a demonstrated capacity to set selfed seed where outcross pollen is limited, with consequent inbreeding depression in the progeny. Current controlled pollination methods therefore include a time-consuming emasculation step. We used microsatellite genotyping of seedlings to determine the consequences of outcross pollination with and without emasculation. Only 1 of 3 mother trees set a small amount (5%) of selfed seed. Using whole inflorescences from the male parent as the pollen applicator rather than sieved pollen reduced outcross contamination rates from 19.1 to 8.7% and substantially increased worker productivity. Application of sugar solution to the female flowers immediately prior to pollination increased yield of sound seeds per spike. Additional improvements to the pollination protocols are discussed.

Keywords

Tree breedingPreferential outcrossingSeed production

Introduction

Acacia mangium Willd. occurs naturally in the coastal, tropical lowlands forest of northern Queensland, the Western Province of Papua New Guinea, Irian Jaya and Maluku (Midgley and Turnbull 2003). It was first planted in Sabah, Malaysia, in the 1960s and has proved well suited to short rotation silviculture throughout south–east Asia (Midgley and Turnbull 2003). By the early 2000s approximately one million hectares of plantation of this species had been established, primarily for pulp production (Harwood et al. 2004). A number of organisations in Indonesia, Malaysia, Vietnam and Australia now have active breeding programs.

Efficient controlled pollination (CP) methods are needed for any intensive plant breeding program. This is technically very difficult with Acacia species as the individual flowers are very small and difficult to emasculate. Also the percentage of flowers which develop into pods is typically less than 5% even if pollen is not limiting (Sedgley and Harbard 1993; Moncur et al. 1991). CP seed is therefore very expensive to produce although technically possible (Sedgley et al. 1992a). In 2005 the Indonesian company Riau Andelan Pulp and Paper (RAPP) embarked on a fast-track improvement program for A. mangium which requires the production of moderate quantities of CP seed from proven full-sib families for use as mother plants in a Clonal Family Forestry (CFF) plantation program. Family Forestry via vegetative multiplication of seedlings or CFF is well suited to tree species such as A. mangium where it is difficult to propagate tested individual clones because of maturation problems (White et al. 2007). In such an ambitious program, methodological efficiencies are obviously extremely critical. RAPP therefore started a research program exploring ways in which all aspects of the CP process can be improved, worker productivity increased and unit costs of seed reduced, within acceptable bounds of contamination from either non-target outcrossing or self pollination. This paper reports a genetic evaluation of the consequences of one option for streamlining the CP protocols—the use of whole spikes from the male parent as a pollen applicator to un-emasculated flowers of the female parent, in lieu of the conventional use of extracted pollen on emasculated flowers within spikes (Sedgley et al. 1992a; Sedgley and Harbard 1993). We will refer to this method as “inflorescence pollination”. CP of unemasculated Acacia flowers was first reported by Philp and Sherry (1949) working with A. mearnsii De Wild. A cluster of flowers from the male parent was used to dust on pollen (K. Nixon personal communication). A. mearnsii has globose inflorescences where emasculation of individual flowers is impracticable. However the flowers are more accessible in the spicate clusters of A. mangium and it has been assumed that, since high potential self fertility has been demonstrated (Butcher et al. 2004), with associated inbreeding depression (Harwood et al. 2004), emasculation would improve yield of the target outcrossed CP seed. This conclusion was supported by Sedgley et al. (1992b) who tested inflorescence pollination for production of CP seed of hybrid A. mangium × auriculiformis but retained emasculation as a preferred method because it yielded fewer self seedlings. Acceptable levels of either self or outcross contamination will vary with the aims of the cross. For genetic experiments all seeds ought to be of the target genotype, but for operational CFF it is our view that up to 10% contamination will have little effect on productivity of the tree crop. Selfed seedlings may be less vigorous in the nursery and rejected as mother plants, while outcross contaminants will probably produce an acceptable tree.

The paper reports a study of effects of modifications to the routine CP protocol in terms of outcross and self pollen contamination and on number of pods harvested per pollinated spike and on full seeds per ripe pod.

Floral biology and breeding system of A. mangium

The inflorescences of A. mangium are loosely arranged spikes 5–12 cm long. Individual flowers are pentamerous, the calyx 0.6–0.8 mm long, with short obtuse lobes and the corolla is twice as long as the calyx (Maslin et al. 2001). Mean number of flowers per spike varied from 158 to 213 in a set of A. mangium trees studied by Sedgley et al. (1992a). Flowers may be perfect or male and the proportion of male flowers per spike varied from 3 to 88% in the Sedgley et al. (1992a) study. Other studies of Acacia species reviewed by Kenrick (2003) suggest that andromonoecy is a rather plastic trait which may vary over time in an individual plant according to environmental conditions and heaviness of the flower crop. In general, flowers of Acacia tend to be protogynous (Kenrick 2003) although the period between stylar extension and that of the filaments is only a few hours in A. mangium (Sedgley et al. 1992a; Sornsathapornkul and Owens 1998) and since flowers open sequentially along the spike, an entire spike is generally not in a distinctively female or male stage. Flowering of neighbouring spikes is not highly synchronized so there must be a high probability of geitonogamous selfing by the various species of bee which appear to be the main pollinators (Sedgley et al. 1992c; Stone et al. 2003).

The microspores of Acacia develop in polyads which are composites of 8, 12, or 16 grains (Knox and McConchie 1986). Commonly a stigma is pollinated by only a single polyad (Kenrick 2003; Moran et al. 1989). A. mangium has 16 grain polyads (Sedgley et al. 1992a) but not all are viable and/or germinate. Sedgley et al. (1992b) used UV microscopy to examine styles following controlled intra-specific pollination of A. mangium. For that subset of flowers with a germinated polyad, the average number of tubes per style was 4.6. Parallel in vitro assays showed 1.5 tubes per germinated polyad for fresh pollen (range 1–4) (data derived from Tables 3 and 6 of Sedgley et al. 1992b).

The production of selfed seed in this and other Acacia species is variable and determined by both genetic and environmental factors. Butcher et al. (2004) used molecular genotyping to determine that the proportion of selfs in the open pollinated seed harvested from six seed orchards in Vietnam varied between 0 and hence little opportunity for outcrossing. Under manipulated conditions, self pollen certainly germinates on the stigma and can penetrate the ovary (Sedgley et al. 1992a), however in common with other species of the genus, pod set and seed yields are reduced compared with manipulated outcrosses. For some species of Acacia, authors report arrest of self pollen tubes within the nucellus or embryo sac (Kendrick et al. 1986; Tandon et al. 2001; Moncur et al. 1991). While such indicators of self incompatibility (SI) have been reported for many species (reviewed by Kenrick 2003) the most detailed genetic study, on A. myrtifolia (Sm.) Willd., found variation patterns which did not fit with expectations for either gametophytic or sporophytic SI (Kendrick 1994). The author suggested that the data provide support for the hypothesis that the self infertility could be the result of post-zygotic lethal genes. This is also the major genetic system underlying the mixed mating system exhibited by Eucalyptus, the dominant genus of woody perennials in Australia (Byrne 2008).

We assume that CP of unemasculated A. mangium flowers must effect some transfer of self pollen, but the outcome of crosses where some flowers within an inflorescence are selfed and some outcrossed, is unpredictable. Perhaps resource allocation issues (Wesselingh 2007) may come into play, favouring the preferential survival of pods containing more vigorous outcrossed seeds (Butcher et al. 2004).

Materials and methods

The RAPP CP methodology development program was conducted on 10-year-old trees in a clonal seed orchard of Acacia mangium located at Kerinci, Riau Province, Sumatra, Indonesia (0°18 N 101°27 E). The set of crosses reported in this paper compared four CP methods (emasculation and non-emasculation with and without a pre-pollination sugar treatment to the styles). Three mother trees were used and four males (Table 1). All parents were unrelated selections forming part of the RAPP Breeding Population.
Table 1

Parentage and pollination method for the set of seedlings genotyped

Seedlot

Pollination method

Female parent

Male parent

No. genotyped per pollination method

3145

1

66

70

17

1069

1

68

96

15

Total

   

32

3140

2

1

70

14

3127

2

66

70

6

4180

2

66

1

13

Total

   

33

3134

3

1

70

12

3138

3

66

24

12

3120

3

68

96

12

Total

   

36

3135

4

1

70

12

3137

4

66

70

12

3163

4

68

96

12

Total

   

36

Grand total

   

137

CP methods

Method 1 used the standard controlled pollination technique established for species of tropical Acacia with inflorescences in a spike (Sedgley et al. 1992b). Spikes with unopened buds changing from green to yellow were labelled and bagged an estimated 1 day prior to anthesis using polyester bags from Duraweld Plant Breeding Supplies, UK. To reduce heat damage the window of the bag was faced towards the ground. Bags were tied to branches with cable ties and the opening plugged with cotton wool to protect the branches and prevent insects entering. At anthesis the bags were removed and spikes with no open flowers trimmed away. Unopened flowers were removed on remaining spikes and open flowers thinned to approximately 30 per spike. These were emasculated by removing the stamens with fine forceps. Pollen was prepared (see below) and transferred to the stigma of emasculated flowers using a fine brush. Spikes were then re-bagged for 3 days.

Method 2 Spikes were labelled and bagged as for Method 1. The next day any spikes without any opened flowers were removed. There was no further thinning or emasculation of flowers on the remaining spikes which were then pollinated using a spike from the male parent as an applicator.

Method 3 was the same as Method 1 with one added step. Immediately prior to pollination a 20% sugar solution was applied to all stigmas in the spike with a small wooden stick. The use of sugar to assist polyad adhesion and/or germination is an initiative attributable to staff of the Forest Research Institute of Malaysia (Mohd. Zaki Abdullah personal communication)

Method 4 was the same as Method 2 but with the addition of the sugar solution as in Method 3.

In all cases pollinated spikes were rebagged for 3 days. Bags were then removed and spikes monitored for pod set and maturation until harvest.

Pollen Preparation: Spikes from the designated male parents were also bagged 24 h prior to anthesis, harvested the same morning as pollination and air dried in the field. For drying, the harvested spikes were laid out on paper under dappled shade until the anthers dehisced. The pollen is usually ready for use after 2 h. For Methods 1 and 3 which required extracted pollen, spikes were then rubbed in a 63 μm mesh sieve and pollen collected onto a smooth black plastic surface against which the pollen could easily be seen. For Methods 2 and 4 a whole dried spike was used to transfer pollen by rubbing together with those of the female parent.

Experimental families and seedlings

Over several years RAPP conducted a semi-operational pollination program in which seed yields from these and other treatments were compared under a variety of environmental and worker deployment conditions. As routine practice, numbers of flowers and inflorescences pollinated and the pods and seeds subsequently harvested were recorded on a bag-by-bag basis. Seeds from all the pods within a bag were bulked and then sorted into viable and non-viable classes, based on prior experience. Relative seed size and whether full or flat were diagnostic criteria. In 2007 a new collaboration with University of Tasmania provided an opportunity to also evaluate treatments in terms of the paternity of the harvested seed.

For the study reported in this paper the seedlots were selected from the RAPP store rather than produced from a single designed field experiment. They were chosen in Australia without prior knowledge of the effects of pollination method on pod or seed set. The seed register was sorted to exclude seedlots with less than 30 full seed and a sub-set of the remainder then chosen as the best available balance of pollination methods across a common set of mothers and pollen parents. It was not possible to find a completely balanced set of crosses with adequate seed quantities. The set of 11 crosses selected (Table 1) included three female parents, two of which were common to all methods, and 4 pollen parents. Pollination methods 2, 3 and 4 were represented by three crosses with 2 for method 1.

The design of the study necessitated an assumption that any environmental effects due to flowering season or operator were random with respect to treatments and therefore contributed only to error variance rather than influencing the main effects which we evaluated. While not an ideal experimental approach, it is our judgment that this assumption is sufficiently robust for the conclusions to be valid.

All seed from the nominated crosses were sent to University of Tasmania for genotyping. 30 full seed per family were nicked with a sharp blade at the opposite end to the aril attachment, placed in a petri dish on three layers of moist filter paper, and germinated in the dark at 23°C ±2 for 4 days. Germinated seed were grown on in a glasshouse in individual labelled pots under 26°C day and 20°C night conditions. After 3 months, when the phase change from bi-pinnate to phyllodinous foliage had occurred, an average of 12 seedlings of normal phenotype per family was selected at random for genotyping. Across CP Methods the numbers were approximately balanced [32–36 seedlings per Method (Table 1)]. The age of sampling was determined after preliminary studies which showed that it was easier to extract quality DNA once phase change had occurred. It is also appropriate from the practical point of view as this is the age at which nursery stock are progressed to CFF mother plant beds or the plantation.

DNA extraction

After three months growth in the glasshouse, the young phyllodes from 137 healthy seedlings from 11 seedlots were harvested for DNA extraction. Phyllodes were also collected from the three mother and seven father trees in Indonesia, air dried, and placed over silica gel for transportation. DNA was extracted from all parents and progenies using the protocol for the DNeasy™ 96 Plant Kit (Qiagen). Approximately 200 mg of leaf tissue from each sample was weighed, frozen in liquid nitrogen and homogenised using mortar and pestle before putting through the Qiagen protocol. We used an incubation temperature of 55°C. DNA concentration and purity were estimated by electrophoretic separation on 1% agarose gels.

Microsatellite methods

Seven of the microsatellites developed by Butcher et al. (2000) were selected for this study (Am 018, 173, 341, 429, 436, 465, and 503) based on successful amplifications. Primers were synthesized by Sigma Proligo and the forward primers of each pair labelled with a fluorescent dye. Each PCR reaction contained 1 × PCR buffer (67 mM Tris–HCL (pH 8.8), 16.6 mM (NH4)2SO4, 0.5% Triton X-100 and 0.2 mg/μl gelatin), 0.2 μM of each primer, 0.1 μM of each dNTPs, 2.5 mM of MgCl2, 0.1 mg/ml of BSA and approximate 2.5 units Taq Polymerase in a 25 μl reaction volume. Optimum annealing temperatures varied between primers (Table 2). Amplification cycles were: 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, annealing temperature in Table 2 for 30 s and 72°C for 1 min; with a final 72°C for 10 min (Butcher et al. 2000). PCR products from the seven primers were co-loaded by mixing 1 μl of each primer together and loading 1 μl of the mixture into SLS mix (made up from 200 μl of SLS and 1.25 μl 400 bp size standards per row of 8 wells and aliquot 25 μl into each well). Microsatellites were run on a Beckman sequencer. The seven loci were divided into two groups for running on the Beckman sequencer. Group one comprised: Am341, Am465, Am503 and group two: Am429, Am018, Am173, and Am436. This division was based on the size of allele and colour of primers.
Table 2

Optimum annealing temperature of seven Acacia primers described by Butcher et al. (2000)

Primers

Annealing temperature (°C)

Number of alleles

Allele size range (bp)

Am018

55

5

125–145

Am436

55

3

247–251

Am173

57

5

95–103

Am429

60

4

163–173

Am341

60

3

122–126

Am465

57

6

147–173

Am503

65

4

165–171

Statistical analysis

The difference in proportion of contaminants between emasculated (Methods 1+3) and non-emasculated (Methods 2+4) treatments was evaluated by chi-squared test. The estimates of seeds/pod and pods/spike harvested per seedlot were subject to analysis of variance using a two way factorial model with emasculation level and sugar level as the main effects.

Results

Genotype determinations

The seven microsatellites assayed exhibited on average 4.3 alleles per locus across the set of parent genotypes, ranging from 3 for Am436 and Am341, to 6 for Am465 (Table 2). This was enough variation to uniquely distinguish all the male and female parents and permit classification of each progeny seedling as target outcross, self, or contaminant outcross, based on its multi-locus genotype (Table 3). All 137 progeny showed expected maternal alleles indicating that the seed had been harvested and processed without error.
Table 3

Progeny genotype classification by parents and methods

Pollination method

Cross

Number of seedlings of each category

No. assayed

True to expectation

Self

Outcrossed contaminant

1

66 × 70

17

13

0

4

68 × 96

15

13

0

2

Total

32

26

0

6

%

100

81

0

18.7

2

1 × 70

14

11

0

3

66 × 70

6

5

1

0

66 × 1

13

13

0

0

Total

33

29

1

3

%

100

88

3

9.1

3

1 × 70

12

9

0

3

66 × 24

12

8

1

3

68 × 96

12

11

0

1

Total

36

28

1

7

%

100

77.8

2.8

19.4

4

1 × 70

12

10

0

2

66 × 70

12

11

1

0

68 × 96

12

11

0

1

Total

36

32

1

3

%

100

88.9

2.8

8.3

Female parent

Pooled crosses

    

1

 

38

30

0

8

66

 

60

50

3

7

68

 

39

35

0

4

Total

 

137

115

3

19

%

 

100

83.9

2.2

13.9

The levels of contamination from non-target outcross pollen varied significantly with pollination method. With emasculation (Methods 1+3), 13/68 (19.1%) of the seedlings were contaminants. With inflorescence pollination (Methods 2+4), this reduced to 6/69 (8.7%). This difference was significant at <0.001 level by χ2 test. The presence or absence of the sugar treatment at pollination had no effect on contamination rates.

Only one of the three female parents (Clone 66) produced any selfed seedlings—1 from each of pollination Methods 2, 3 and 4, or 5% of the 60 seedlings assayed from that mother. No selfs were recovered from the 38 seedlings from Clone 1 or the 39 seedlings from Clone 60. We did not carry out controlled self pollinations to demonstrate that these mothers were in fact self fertile, but the simplest inference is that 66 is self fertile to some extent, but the others either are completely self infertile, or sufficiently so that selfed embryos are unable to develop in competition with outcrosses on the same spikes; seed failed to germinate; or seedlings were so weak at 3 months that they were not selected for assay.

Pod and seed yields under different CP methods

Although not a primary aim of the study, we did observe some important effects of CP method on pod and seed yields. Since the crosses for genotyping were selected from store, the productivity estimates in Table 4 are only relevant to that subset of all controlled crosses which produced at least 1 viable seed.
Table 4

Pod set and seed yield from each pollination method over all parents

Method

Spikes pollinated

Spikes harvested

Pods harvested

Pods per spike pollinated

Pods per spike harvested

Total seeds

Sound seeds

Sound seeds per pod

Sound seed per spike pollinated

1

5

4

27

5.4

6.8

163

65

2.4

13.0

2

12

6

30

2.5

5.0

158

80

2.7

6.7

3

4

4

36

9.0

9.0

165

89

2.5

22.3

4

6

4

50

8.3

12.5

318

249

5.0

41.5

Total

27

18

143

  

804

483

  

Mean

   

6.3

8.3

  

3.1

20.9

Emasculation effect

 Emasculated (1 + 3)

9

8

63

7.0

7.9

328

154

2.4

17.1

 Not emasculated (2 + 4)

18

10

80

4.4

8.0

476

329

4.1

18.3

Sugar effect

 No sugar (1 + 2)

17

10

57

3.4

5.7

321

145

2.5

8.5

 Plus sugar (3 + 4)

10

8

86

8.6

10.8

483

338

3.9

33.8

The effects of emasculation and sugar application where judged through analysis of the variable number of pods/spike harvested and number of sound seed/pod. For each variable, individual cross data were subject to least squares analysis of variance using JMP 7.0 (SAS Inst. Inc., Cary, NC) (Table 5).
Table 5

Summary analysis of variance for pod and seed yield variables

Source

DF

Sum of squares

F ratio

Prob > F

Effect tests–sound seed/pod

Emasculation level

1

4.98

23.3

0.0019

Sugar level

1

3.84

18.0

0.0038

Emasc level × Sugar level

1

3.84

18.0

0.0038

Effect tests–pods/spike harvested

Emasculation level

1

0.987

0.173

0.6895

Sugar level

1

92.04

16.18

0.0050

Emas level × Sugar level

1

22.30

3.92

0.0882

Inflorescence pollination without emasculation (Methods 2+4) had no effect on the average number of pods per harvested spike but it did significantly increase the sound seeds per pod from an average of 2.4–4.1 (Table 4), while the addition of sugar prior to pollination (Methods 3+4) had a significant positive effect on number of pods per spike harvested. The interaction of emasculation and sugar was also significant with Method 4 (no emasculation plus sugar) yielding the highest number of seed/pod (5.0) and also a threefold increase in the number of sound seed per spike pollinated compared with the conventional treatment Method 1.

Discussion

The distribution of mating types following inflorescence pollination (Table 3) indicates that outcross pollination of some flowers within a spike is sufficient to reduce the proportion of selfed progeny to very low or zero levels even without emasculation. This is consistent with the estimates of the breeding system in seed orchards of A. mangium where Butcher et al. (2004) reported that, as long as there was good general flowering, the open pollinated seed was fully outcrossed. Other studies in Acacia orchards or plantations have reached the same conclusion (Millar et al. 2008). Our data do not directly estimate the production of viable self and outcross seed since selection against selfs may have occurred between germination and the 3 month assay term but they do indicate the practical consequences at the nursery gate.

One of the main reasons to favour inflorescence pollination is that it clearly increases the number of spikes which can be processed by a fixed workforce. Sedgley et al. (1992b) reported that it took about twice as long to pollinate an emasculated spike with sieved pollen as to use the inflorescence pollination method. We did not specifically measure worker productivity in this study but records over a period of 250 worker days show an even greater advantage from inflorescence pollination, which averaged 260 spikes per worker day compared to 45 with emasculation (Wong C. Y. unpublished data).

The genetic and logistical results lead to the conclusion that inflorescence pollination incorporating the sucrose application is a cost efficient CP option where a level of contamination/selfing is an acceptable outcome—for example in the production of CFF mother plants for operational use, or for hybrid breeding where it is easy to weed out selfs from the maternal parent in the nursery. It is still prudent to determine the self fertility of each potential mother as some genetic variation in this trait is expected, and highly self fertile individuals should be used as male rather than female parents. For genetic experiments, unless there is very strong a priori evidence that the maternal genotypes are self infertile, then emasculation is still recommended. An additional and unexpected advantage of inflorescence pollination was that the average level of outcross contamination was only 8.7% (Table 3) compared with 19.1% where sieved pollen was used. It seems that the more that pollen and flowers are handled the more opportunities there are for contamination of sieves, brushes, forceps and workers’ fingers. The experimental design did not allow us to distinguish between contamination during preparation of sieved pollen or during emasculation—the former is more likely. It should be possible to tighten the protocols for producing and handling sieved pollen but the simpler process of collecting and using male parent spikes directly is currently more robust. The observed levels of contamination are rather high but by no means unusual in tree breeding (see e.g. Keil and Griffin 1994; Patterson et al. 2004). It is unrealistic to expect to completely eliminate the problem but it can certainly be managed at a low level if the pollination protocol includes rigorous attention to hygiene and record keeping and the workers are well trained, supervised and monitored.

Our paper focuses on empirical outcomes of varying the CP methodology. However ability to manage a biological system is always greater if the underlying processes are understood. More work is needed to explore why more sound seeds/pod were obtained from inflorescence pollination than following emasculation and thinning to around 30 flowers per spike, as will the more general question of why so few sound seeds per pod are produced when there are enough ovules in each ovary to accommodated fertilisation by each of the potential 16 pollen tubes per polyad. In the study by Sedgley et al. (1992b) the average number of pollen tubes per style was only 4.6, so investigation into pollen quality and handling protocols is indicated, including detailed investigation of the polyad/stigma interface. The pistil of the closely related A. mangium × auriculiformis hybrid is receptive for only about 1 day (Sornsathapornkul and Owens 1998) so it is possible that the timing of the pollination also has an effect on the probability of full fertilisation. With inflorescence pollination more flowers per spike are pollinated and therefore a larger number might be at the optimal stage of development and the pods thus able to mature a higher number of full seeds. The fact that across CP methods each harvested spike matured an average of only eight pods (Table 4) is good evidence that there must be competitive abortion of many pollinated flowers. Assessment of larger numbers of mature open pollinated pod clusters within the orchard yielded a mean number of pods per cluster (spike) of 3.1 with a range of 1–12 and 45% of clusters having only 1 pod (Wong C. Y. unpublished data).

The positive sugar effect on pods per spike and seed per pod (Table 4) appears real (Table 5) but again understanding will require microscopic examination. Perhaps it increases the chance that a polyad will adhere firmly to the stigma, and/or speeds up pollen germination so increasing fertilisation rates.

Finally it must be emphasised that the seed and pod yields reported in this paper are based on a sub-set of crosses which were successful and therefore had adequate quantities of seed in store. While this is appropriate for the purposes of this study, in planning an operational program with specific seed yield targets, it is the yields per spike pollinated which is more important; i.e., the many spikes which yield 0 pods should also be taken into account.

Conclusion

The reproductive biology of Acacia makes it difficult and expensive to produce controlled pollinated seed for operational deployment or even for breeding. After pollination of unemasculated flowers with an inflorescence from the male parent, DNA analysis of resulting seed showed that levels of self and outcross contamination were acceptably low for production of operational Family Forestry seed, though not for genetic experiments. Worker productivity was greatly increased and therefore seed cost reduced. In summary the recommended operational protocol is now:
  • omit the emasculation stage

  • use dried inflorescence spikes from male parents as the pollen applicator rather than brushing with sieved pollen

  • apply sugar solution prior to pollination to enhance seed yield

Irrespective of the CP protocol, rigorous attention to hygiene is essential if contamination is to be restricted to a low level.

Acknowledgments

We would like to thank G. D. Golani, R&D Director, RAPP for permission to use data from the company crossing program; Wiwik Endah Rahayu and her team of technicians for producing the seeds; and James Marthick, Beck Jones, Adam Smolenski and Greg Jordan from the School of Plant Science, University of Tasmania for assistance with lab work, interpretation of molecular results, and statistical advice. We also acknowledge financial support from the John Allwright Fellowship Scheme for TDV and from ACIAR Project FST2003/002 for JH.

Copyright information

© Springer Science+Business Media B.V. 2010