Confirmation of cytotype stability in autotetraploid black wattle (Acacia mearnsii) trees using flow cytometry and size differences of the reproductive gametes

Abstract

Acacia mearnsii (black wattle) is grown commercially in South Africa for its timber and bark. However, the invasive nature of the species has resulted in it being considered an alien invader and for this reason research has been aimed at producing a sterile triploid variety that would be highly desirable for the South African commercial forestry industry. Tetraploids were successfully induced by soaking germinating diploid seeds in colchicine. Seed from these tetraploids was used to establish a field trial, where crossing diploid with tetraploid parent plants only produced diploid and tetraploid progeny and failed to produce any triploid progeny. Control-crossed seed set between diploids is generally low in A. mearnsii and, together with the possibility of an unstable tetraploid population, this could be reducing the chances of producing triploid seed. Thus identification and confirmation of stability within the existing advanced-generation tetraploid population (aged 10–11 years) was critical to ensure the production of sterile triploids. Flow cytometry was used to determine the stability of the ploidy of leaf vegetative tissues, whereas polyad and ovule size measurements were used to determine the stability of ploidy of the reproductive tissues. Results from the study revealed that the tetraploidy of within the leaf vegetative tissue was stable. For both the ovule and polyad size measurements, a size range was determined for diploids and tetraploids and, within the population under investigation, no overlap was apparent. This allowed for the conclusion that the advanced-generation tetraploid population was stable and that the absence of triploid progeny must be because of post-zygotic reproductive barriers within the ovary.

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Confirmation of cytotype stability in autotetraploid
black wattle (Acacia mearnsii) trees using flow
cytometry and size differences of the reproductive
gametes
Sascha L Beck-Pay a
a Institute for Commercial Forestry Research, PO Box 100281, Scottsville 3209,
Pietermaritzburg, South Africa
Published online: 04 Apr 2013.
To cite this article: Sascha L Beck-Pay (2013) Confirmation of cytotype stability in autotetraploid black wattle (Acacia
mearnsii) trees using flow cytometry and size differences of the reproductive gametes, Southern Forests: a Journal of
Forest Science, 75:1, 1-6, DOI: 10.2989/20702620.2013.743763
To link to this article: http://dx.doi.org/10.2989/20702620.2013.743763
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Southern Forests 2013, 75(1): 1–6
Printed in South Africa — All rights reserved
Copyright © NISC (Pty) Ltd
SOUTHERN FORESTS
ISSN 2070-2620 EISSN 2070-2639
http://dx.doi.org/10.2989/20702620.2013.743763
Confirmation of cytotype stability in autotetraploid black wattle (Acacia
mearnsii) trees using flow cytometry and size differences of the
reproductive gametes
Sascha L Beck-Pay
Institute for Commercial Forestry Research, PO Box 100281, Scottsville 3209, Pietermaritzburg, South Africa
E-mail: sascha.pay@icfr.ukzn.ac.za
Acacia mearnsii (black wattle) is grown commercially in South Africa for its timber and bark. However, the
invasive nature of the species has resulted in it being considered an alien invader and for this reason research
has been aimed at producing a sterile triploid variety that would be highly desirable for the South African commer-
cial forestry industry. Tetraploids were successfully induced by soaking germinating diploid seeds in colchicine.
Seed from these tetraploids was used to establish a field trial, where crossing diploid with tetraploid parent plants
only produced diploid and tetraploid progeny and failed to produce any triploid progeny. Control-crossed seed set
between diploids is generally low in A. mearnsii and, together with the possibility of an unstable tetraploid popula-
tion, this could be reducing the chances of producing triploid seed. Thus identification and confirmation of stability
within the existing advanced-generation tetraploid population (aged 10–11 years) was critical to ensure the produc-
tion of sterile triploids. Flow cytometry was used to determine the stability of the ploidy of leaf vegetative tissues,
whereas polyad and ovule size measurements were used to determine the stability of ploidy of the reproductive
tissues. Results from the study revealed that the tetraploidy of within the leaf vegetative tissue was stable. For both
the ovule and polyad size measurements, a size range was determined for diploids and tetraploids and, within the
population under investigation, no overlap was apparent. This allowed for the conclusion that the advanced-genera-
tion tetraploid population was stable and that the absence of triploid progeny must be because of post-zygotic
reproductive barriers within the ovary.
Keywords: chimera, flow cytometry, ovule, polyads, polyploids
Black wattle (Acacia mearnsii de Wild) is one of the commer-
cially grown hardwood forestry species in South Africa
contributing to c. 7.6% of the total area under commer-
cial forestry plantations (Forestry South Africa 2009). It is
grown for high-grade tannins present in the bark, which are
used to produce tanning extracts and adhesives, as well as
for high-quality pulp for the pulp and paper industry. Black
wattle was introduced into South Africa from Australia and,
as an exotic species, is a prolific seed producer. Seed
can remain dormant for many years and, unless managed
correctly, can pose an environmental threat. For this reason,
it has been classified as an alien invader species in South
Africa. Consequently, the production of a sterile variety of
black wattle would be beneficial both to the wattle industry
and to address environmental concerns.
The production of a triploid variety of black wattle is
one approach of introducing sterility into the species.
Triploids (2n  3x  39) can be produced by crossing
colchicine-induced tetraploids (2n  4x  52) with diploids
(2n  2x  26) (Beck et al. 2003). Colchicine is the most
commonly used chemical inducer of polyploidy, which
prevents spindle formation during meiosis, preventing
separation of homologous chromosomes. As it only
affects actively dividing cells, it is generally applied to the
actively growing meristematic regions of young plants
(e.g. nodes) or to germinating seeds (Moffett and Nixon
1960, Singh 1993, van Harten 1998, Blakesley et al. 2002,
Beck et al. 2003, Shao et al. 2003, Harbard et al. 2012).
Triploid production has been successful with a number
of species in the fruit industry (Geraci et al. 1982) for
the production of seedless table cultivars. In woody tree
species, limited research has been conducted on triploid
production by crossing diploids with colchicine-induced
tetraploids. Most of this work in tree species has been
conducted on poplars (Bradshaw and Stettler 1993, Li et
al. 2008) and oak (Johnsson 1946, Burda and Shchepotiev
1973, Butorina 1993, Naujoks et al. 1995, Lefort et al.
1998, Lefort and Douglas 1999, Lefort et al. 2000, Dzialuk
et al. 2005). Research into producing a sterile variety of
A. mangium Willd. is being conducted by Harbard et al.
(2012), who have successfully produced neotetraploids
that will be used for sterility breeding in the future. Garg
et al. (1996) reported successful regeneration of triploid
A. nilotica (L.) Willd. ex Delile plants using endosperm
culture. With A. mearnsii, endosperm culture has been
unsuccessful because of the endosperm being absorbed
at a developmental stage where isolation was impossible
(Bairu unpublished data). However, colchicine-induced
Introduction
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Beck-Pay2
A. mearnsii tetraploids have been successfully induced
(Moffett and Nixon 1960, Beck et al. 2003), yet despite a
number of seasons of reciprocal crossing with diploids,
only diploid and tetraploid seed and no triploid seed has
been set. There are a number of problems (meiotic aberra-
tions, physiological effects of polyploidy and genetic
factors) associated with polyploids and seed produc-
tion from crosses between diploids and tetraploids is
by and large poor (Ramsey and Schemske 1998, 2002).
Controlled-cross seed production between diploids in
A. mearnsii is generally poor (Moffett and Nixon 1974,
Beck-Pay 2012) and, in the absence of any reproductive
barriers, the stability of the tetraploid population under
investigation could further be restricting the number of
triploids being produced.
During polyploid induction, the different cell layers in
the apical meristems (LI, LII and LIII) are exposed to the
chemical mutagen. It is possible that the chemical only
penetrates some cells and cell layers and not all, resulting
in the outgrowing plant tissue containing two cell lines; the
original cell line (e.g. diploid) and the polyploidy cell line
(e.g. tetraploid) (Burge et al. 2002). This is what is termed
a chimera (Dermen 1940). Cochicine-induced polyploids
can often result in a population containing mixed ploidys
(Moffett and Nixon 1960, Harbard et al. 2012). Reversions
to diploidy have been reported for Rhododendron hybrids,
especially with mericlinal and sectorial chimeras (Vainola
2000). Lehrer et al. (2008) also noted some reversion
of tetraploids to diploids with Japanese barberry (4%
of the colchicine-induced tetraploids and 2% of the
oryzalin-induced tetraploids). Harbard et al. (2012) had
a low conversion (7%) of colchicine-induced A. mangium
tetraploids yet more than 80% of these remained stable.
Thus examination of the stability of induced tetraploids is
critical and flow cytometry (Blakesley et al. 2002, Beck et
al. 2005, Lehrer et al. 2008, Harbard et al. 2012), together
with cytological differences such as chromosome counts
(Moffett and Nixon 1960), stomatal size and frequency
(Evans 1955, Moffett and Nixon 1960, Speckmann et
al. 1965, Tan and Dunn 1973, Przywara et al.1988, Beck
et al. 2003, Harbard et al. 2012), chloroplast numbers
(Hamada and Baba 1930, Mochizuki and Sueoka 1955,
Butterfass 1960, Bingham 1968, Chaudhari and Barrow
1975, Mathura et al. 2006), chloroplast content (Joseph and
Randall 1981, Warner and Edwards 1993, Romero-Aranda
et al. 1997, Fossey et al. 2009) and gamete sizes (Moffett
and Nixon 1960, Tan and Dunn 1973, Beck-Pay 2012),
have been widely used for this purpose.
The plant material under investigation in this study was
an advanced-generation tetraploid population, which had
been established from open-pollinated seed collected
from colchicine-induced neotetraploids. These tetraploids
were induced in the 1950s and confirmed through chromo-
some counts, stomata and pollinia sizes (Moffett and Nixon
1960) shortly after induction. However, no further valida-
tion of the stability of the ploidy was conducted as the
trees matured and thus the reversion to diploids and the
presence of chimeras or mixoploids was not confirmed.
Open-pollinated seed was collected from these tetraploids
in 2001. This seed was sown under nursery conditions and
after three months flow cytometry analysis together with
stomatal length and frequency measurements confirmed
the tetraploid status of the seedlings.
Thus the aim of this study was to reconfirm the ploidy of
this mature advanced-generation tetraploid population and
to check for stability. Flow cytometry was used to quantify
the DNA content of the vegetative leaf material, and
morphological size differences of the gametes (polyads
and ovules) were used for ploidy determination and to
check for stability.
Materials and methods
Flow cytometry analysis of vegetative material
In the 2009 flowering season, leaf samples were taken
from each of the 10 tetraploid trees (31, 43, 50, 52, 53,
66, 67, 71, 74 and 77) aged 10–11 years. Ten leaf sample
points throughout each tree were selected, in order to
get representation of the tree. Flow cytometry analysis
was undertaken by the Agricultural Research Council
(ARC) Institute for Tropical and Subtropical Crops using
a flow cytometer (PA-I, Partec, Germany) equipped
with a 100 W high-pressure mercury lamp. A standard
reagent kit (Partec CyStain Uv Precise P) developed
by Partec for nuclei extraction and DNA staining was
used. Approximately 0.5 cm2 of plant material was finely
chopped with a razor blade in a petri dish containing 400
l extraction buffer and vortexed for 10 s. Thereafter,
the sample was filtered through a Partec 50 m Cell-Tric
disposable filter and 1.6 ml staining buffer (HR-B) was
added. The sample was left to incubate at room temper-
ature for 30–60 s and analysed in the blue fluorescence
channel (wavelengths between   435 and   500).
A diploid A. mearnsii control was used to run the unknown
samples against. When a tetraploid peak was obtained,
the unknown sample was run in isolation of the diploid
control at the same gain (parameter on the flow cytometer)
to check for the presence of chimeras/mixoploids
(2x and 4x peaks).
Ovule size measurements
Over the 2009 and 2010 flowering seasons, 10 previously
identified tetraploid trees were selected (2009 flowering
season: 52, 53, 66, 71 and 77; 2010 flowering season: 31,
43, 50, 67 and 74). Five diploid trees were selected in the
2009 flowering season to act as controls. Measurements
from all five trees were grouped together for data analysis.
Three isolation bags were placed over unopened inflores-
cences throughout each of the trees. When the inflores-
cences were fully opened, inflorescences were fixed
in 95% ethanol:acetic acid (3:1, v/v) for 1 h, washed in
sterile Millipore water and stored in 70% ethanol. Pistils
were isolated and softened using a modified procedure
developed by Martin (1959). The pistils were excised
from all the flowers that were available, using a dissecting
microscope and then softened in 3 N NaOH for 3 h, rinsed
in tap water for 1 h and then stored in glycerol. Pistils
were mounted on slides and squashed with a coverslip to
release the ovules. Three slides were made per isolation
bag and 50 measurements were recorded per slide. Ovules
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Southern Forests 2013, 75(1): 1–6 3
and polyads were viewed and measured with a Carl Zeiss
AxioImager M.2 microscope (Figure 1).
Polyad size measurements
The effect of storage conditions on polyad size was investi-
gated prior to the 2009 black wattle flowering season.
Flowers from A. podalyriifolia A.Cunn. ex G.Don were
collected and stored in paper bags at room temperature and
weekly polyad measurements were taken for a period of
five weeks. These measurements were compared against
controls of fresh pollen samples as well as pollen that had
been killed by incubation at 80 °C for 2 h. Four slides were
made and five measurements were recorded per slide.
In the 2009 flowering season, 10 isolation bags were
placed over unopened A. mearnsii inflorescences,
throughout each of the 10 previously identified tetraploid
trees (31, 43, 50, 52, 53, 66, 67, 71, 74 and 77). Ten bags
were placed on diploid trees in the 2009 flowering season to
act as controls. When the inflorescences were fully opened,
isolation bags were removed from the tree and inflores-
cences were dried in an oven at 80 °C for 2 h and stored in
paper bags in a silica-containing desiccator. Inflorescences
were then sieved (52 m sieve for diploid and 77 m sieve
for tetraploid) over slides containing a drop of analine blue
stain and polyads that were released were mounted and
viewed. Three slides were made per isolation bag and 50
measurements were recorded per slide.
Statistical analyses
All data was analysed using GENSTAT® version 12 (Lane
and Payne 1996). The data collected from A. podalyriifolia
to determine the effect of polyad size with storage were
analysed using a one-way ANOVA. Ovule size data from
A. mearnsii were analysed using an unbalanced ANOVA
and polyad size data were analysed using a one-way
ANOVA. Significant differences between treatments were
determined using least significant differences (LSD).
Results
Flow cytometry analysis of vegetative material
Flow cytometry analysis was used to confirm the stability
(Shao et al. 2003, Harbard et al. 2012) of all 10 previously
identified tetraploid trees. All 10 samples per tree were of
the same ploidy, confirming the stability of tetraploidy in the
vegetative tissues (Figure 2).
Ovule size measurements
Ovule size measurements were taken from three
isolation bags per tree in order to ascertain the stability
of the reproductive tissues in the tetraploid (4x) popula-
tion. Comparisons were made between isolation bags
within a tree, between trees and against the diploid (2x)
control. When examining the trees within the 4x popula-
tion, some significant differences (p  0.05) were noted
between isolation bags (Table 1). However, within both
the 2x control and 4x population there was a range in
ovule size, with the 2x ovules ranging from 96.2 to 105.7
m (average 102.3 m) and 4x ovules ranging from 118.2
to 137.3 m (average 123.9 m). These measurements
were in agreement with those previously recorded for A.
mearnsii (Beck-Pay 2012). On average measurements
taken from within the 4x isolation bags were, however,
significantly (p  0.05) larger than the 2x control. Trees 71
and 77 had significantly (p  0.05) lower ovule sizes than
the other tetraploid trees but their sizes were still signifi-
cantly (p  0.05) larger than the 2x control. Thus the results
confirmed the ploidy of the tetraploid population and stability
of this population under examination.
Polyad size measurements
Fresh A. podalyriifolia pollen (0 weeks) was collected
and sieved and an average polyad size of 53.83 m was
recorded. This was not significantly (p > 0.05) different
from that of pollen that was killed at 80 °C (Figure 3).
(a) (b)
200 Pm30 Pm
2x
4x
Figure 1: Reproductive gametes of Acacia mearnsii. Ovules (a) released from the ovary and (b) diploid (2x) and tetraploid (4x) polyads
housing 16 pollen grains
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Beck-Pay4
Polyad sizes after 1–5 weeks of storage were significantly
larger (p  0.05) (55.45 m to 58.80 m) than the control
treatment (53.83 m). The results from this preliminary
study confirmed that the integrity of the polyad structure did
not desiccate and shrink over time and thus polyads need
not be measured immediately after collection.
In order to ascertain the stability of the A. mearnsii 4x
population, polyad size measurements were taken from 10
isolation bags per tree. Comparisons were made between
isolation bags within a tree, between trees and against the
2x control. When examining the trees within the 4x popula-
tion, some significant differences (p  0.05) were noted
between isolation bags (Table 2). Within both the 2x control
and 4x population there was a range of polyad sizes, with
2x polyads ranging from 38.8 to 41.7 m (average 40.7 m)
and 4x polyads ranging from 46.4 to 50.9 m (average 48.7
m), confirming results previously reported by Beck-Pay
(2012). Even though the differences recorded between
isolation bags within the 4x trees were significant, they were
within the range recorded for 4x polyad sizes and thus do
not indicate chimeras. Only for bags 1 and 5 in Tree 77
(Table 2), was the polyad size deemed as outside of the
range for 4x polyads, yet in this study it was still significantly
(p  0.05) larger than the 2x polyad size. Interestingly, the
ovule size measurements for Tree 77 were also smaller
than the other 4x trees; there was marginal variation
between the isolation bags for this tree (Table 1). All
measurements taken from within the 4x isolation bags were
significantly (p  0.05) larger than the 2x control, confirming
the ploidy status and stability of the tetraploid population
under examination.
Discussion and conclusions
Testing the stability of induced polyploids is critical and
should be conducted at various stages as the plant material
matures (Harbard et al. 2012). The ploidy of the maternal
tetraploids, from which open-pollinated seed was collected
and used for this study, had only been confirmed using
chromosome counts, stomata and pollinia sizes shortly
after induction (Moffett and Nixon 1960). No further valida-
tion of the ploidy status of these individuals was conducted
as they matured and thus the presence of chimeras and
reversion to diploidy in this neotetraploid population was
unconfirmed. The stability of both vegetative and reproduc-
tive tissues should be tested because of the nature of the
meristematic tissues being treated, in which chimeras could
arise, resulting in incomplete conversion rates (Dermen
1940, Marcotrigiano and Bernatzky 1995, Szymkowiak
and Sussex 1996, Burge et al. 2002, Harbard et al. 2012).
Various methods have been used in the past for ploidy
determination in A. mearnsii (Moffett and Nixon 1960,
Beck et al. 2003, 2005, Mathura et al. 2006, Fossey et
al. 2009) and the results from this study have shown that
flow cytometry of the leaf vegetative material together with
ovule and polyad size measurements have been successful
Tree
Ovule size (m) within isolation bag
(LSD  2.7) Average
(LSD  1.5)
123
2x control 105.7a105.1a96.2b102.3g
31 131.0a132.8a132.8a132.2a
43 129.5a128.9a129.3a129.2b
50 132.9b129.4c137.3a133.2a
52 127.7a129.2a127.4a128.1b
53 127.6a120.8b122.4b123.6d
66 126.2a119.7b125.6a123.8d
67 126.2a125.2bc 122.4c124.6d
71 120.6a119.7a121.8a120.7e
74 126.7a125.3a126.7a126.2c
77 120.9a118.3ab 118.2b119.1f
4x average 123.9d
Table 1: Ovule size (m) differences between isolation bags
within trees. Treatments denoted by the same letters within a tree,
are not significantly different (p  0.05) and were derived using
LSD values
10
20
30
40
50
60
0dead12345
POLYAD SIZE (μm)
STORAGE TI ME (weeks)
c
a
cb
a
aa
Figure 3: The effect of storage on Acacia podalyriifolia polyad
size. Treatments denoted by the same letters within a tree are not
significantly different (p  0.05) and were derived using LSD values
(LSD  2.2)
Figure 2: Flow cytometry histogram output confirming the tetraploid
status (peak 2) of an Acacia mearnsii leaf vegetative sample
compared to the diploid control (peak 1)
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Southern Forests 2013, 75(1): 1–6 5
in confirming the tetraploid status of the population under
investigation and stability thereof.
The results from this study have thus revealed that the
reason for no triploid seed being produced from the diploid
 tetraploid cross-pollinations in A. mearnsii have not
been confounded as a result of instability in the advanced-
generation tetraploid population. Both diploid and tetraploid
seed was produced, even though in limited quantities from
the 2x  4x or 4x  2x cross-pollination operations and this
was as a result of unreduced diploid gametes (Ramsey
and Schemske 1998) and from selfing. Crosses between
diploid and tetraploids often fail to produce viable triploid
seed because of intercytotype interactions during seed
development and this has been referred to as the 'triploid
block' (Ramsey and Schemske 1998). It has been reported
that this triploid block is present in many taxa and that the
ratio of the embryo:endosperm and/or maternal tissue,
together with the ratio of the maternal:paternal ploidy of
the endosperm, are determining factors for normal seed
development (Ramsey and Schemske 1998). Provided
that there are no genetic factors within the embryo that
are contributing to a reduction in fertility (Ramsey and
Schemske 2002), embryo rescue through tissue culture
is a means of overcoming potential post-zygotic barriers
within the ovary. Beck-Pay (2012) concluded that in A.
mearnsii triploid production there was a post-zygotic
barrier within the ovary and in this regard this route is
being investigated as a means of producing a sterile
variety of A. mearnsii. If successful, plantlet regeneration
and multiplication using tissue culture techniques will be
employed to provide the basis of a triploid population of A.
mearnsii in the future.
Acknowledgements — I thank the Acacia Tree Improvement
funding body for their financial support. I would like to thank the
Centre for Electron Microscopy (University of KwaZulu-Natal,
Pietermaritzburg) for all their advice and help, and Dr Kerry Koen
for all her help in the laboratory.
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(LSD  0.2)
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2x control 40.2c39.5e41.6a41.6a41.7a41.5a40.1d38.8f40.9b40.7bc 40.7i
31 49.0e49.6bcd 50.0b50.0b49.4cde 49.7bc 49.1de 49.8bc 50.6a49.8bc 49.7a
43 48.8de 48.4e49.0cd 49.0cd 49.4bc 47.8f49.7b50.4a49.5bc 49.4bc 49.1c
50 50.3b48.8c46.7f46.7f48.0de 50.0b47.6e48.2d49.2c50.9a48.6ef
52 48.4abc 48.1c48.2bc 48.2bc 48.2bc 48.4abc 47.9c48.7ab 48.7ab 48.8a48.4f
53 47.8b46.9c47.3b47.3b47.7b47.7b48.9a48.8a49.0a48.9a48.0fg
66 50.6a50.0b49.2c49.2c49.3c48.6d48.8cd 49.2c48.6d50.6a49.4bg
67 50.0b50.5ab 49.2c49.2c47.0e48.3d50.5ab 48.1d50.7a50.2ab 49.4b
71 49.8b50.3ab 50.0ab 50.0ab 48.2c47.4d50.5a46.6e48.3c48.7c49.0c
74 47.8e48.5d49.0bcd 48.9bcd 49.4ab 49.4ab 49.1abc 49.5a48.8cd 48.6cd 48.9cd
77 43.2eND 47.7b47.7b44.6d46.4c47.9a46.7c48.4aND 46.6h
4x average 48.7de
Table 2: Polyad size (m) differences between isolation bags within trees. Treatments denoted by the same letters within a tree, are not
significantly different (p  0.05) and were derived using LSD values. ND  No data collected from bags because of wind damage
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Received 20 May 2012, accepted 23 September 2012
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