Plant Physiol.
(1992) 99, 468-472
0032-0889/92/99/0468/05/$01 .00/0
Plant Growth Environment Effects on Rapeseed Microspore
Development and Culture1
A Flow Cytometric Study
Kwan-Hung Lo2 and K. Peter Pauls*
Department of Crop Science, University of Guelph, Guelph, Ontario, Canada, NlG 2W1
ABSTRACT
The influence of donor plant growth conditions on microspore
embryogenesis in rapeseed (Brassica napus) was studied for
plants grown at 23/180C (16/8 hours) under continuous light, 23/
180C (16/8 hours) with a light/dark (16/8 hours) cycle, 15/12°C
(16/8 hours) under continuous light and 15/120C (16/8 hours)
with a light/dark (16/8 hours) cycle. Significantly higher embryo
yields were obtained from microspore cultures initiated from
donor plants grown at 15/120C instead of 23/180C. Flow cyto-
metric measurements of the microspores isolated from 2.5- to
5.0-millimeter buds showed that the microspores isolated from
low-temperature-grown plants had significantly lower log 90-
degree light scatter to forward angle light scatter and log 90-
degree light scatter to time of flight ratios than those isolated
from high-temperature-grown plants, suggesting that the former
are more translucent than the latter. Thus, the effect of donor
plant growth temperature on microspore embryogenesis may be
mediated by a change in the physiology of the microspore cell,
which results in the reduction of its cytoplasmic granularity and/
or exine density.
Haploid plants can be produced by culturing anthers or
isolated microspores of rapeseed (Brassica napus) (3, 9, 12,
13). Microspore to haploid embryo conversion frequencies as
high as 10 to 50% have been reported (9, 16), but for most
genotypes <1% of the microspores that are cultured will
develop into embryos.
Previous research with several plant species has shown that
the conditions under which the donor plants were grown
affect the frequency of androgenesis in culture. Seasonal
variations in anther response were reported for a number of
species including Solanum tuberosum (4), Hordeum vulgare
(2), and Triticum aestivum (17). For B. napus, controlled
studies of the effects of growth temperature, light intensity,
and light duration on anther culture response showed that
embryo production could be significantly increased by grow-
ing the donor plants at a low (1 5C) temperature (3, 11, 12)
and high light (12, 20). There is no detailed report of the
'Supported by the Ontario Ministry of Agriculture and Food.
2 Present address: Department of Horticultural Science, University
of Saskatchewan, Saskatoon, Canada S7N OWO.
468
Received for publication September 4, 1991
Accepted December 20, 1991
effects of donor plant growth temperature and photoperiod
on embryogenesis in isolated microspore culture of B. napus.
The basis for the environmental effect on donor plant
responsiveness has not been determined, but it may be me-
diated by alterations in the developmental pattern of the
microspore because haploid embryo production is extremely
sensitive to the stage of development of the microspore at the
time of isolation. For B. napus, only microspores at the late
uninucleate stage or early binucleate stage are embryogenic
in culture (5, 13, 14, 16).
Microspores can be staged according to their cytological or
morphological characteristics (5, 13, 16). However, these
methods are laborious and time consuming. Rapid staging
methods based on bud, anther, or petal sizes are only poorly
correlated with embryo yield (16). Flow cytometry has been
found to be a useful method for characterizing microspore
development. In particular, it is possible to rapidly identify
microspores at different developmental stages in preparations
of unstained microspores on the basis of flow cytometric light
scatter measurements (6). TOF3, FALS,and L90°LS intensities
give information regarding cell size, cell shape, and cyto-
plasmic granularity (18, 19).
In this study, the effects of donor plant growth temperature
and photoperiod on microspore development were studied by
flow cytometry, and the significance of correlations between
the flow cytometric parameters and embryogenesis was tested.
MATERIALS AND METHODS
Donor plants of rapeseed (Brassica napus) breeding line
G23 1 were grown in four growth chambers that were set at
23/18'C (16/8 h) with continuous light, 23/18'C (16/8 h) in
light/dark (16/8 h), 15/12'C (16/8 h) with continuous light,
or 15/12°C (16/8 h) in light/dark (16 h/8 h). The light
intensities in each growth chamber were adjusted to 160 ± 10
,umol m-2 s-' of photosynthetically active radiation at the pot
level. The flower buds were sampled from the three uppermost
racemes on the 4th to 10th days after the first flower opened
and were separated into five different size ranges: 2.6 to 3.0,
3.1 to 3.5, 3.5 to 4.0, 4.1 to 4.5, and 4.6 to 5.0 mm. The
3 Abbreviations: TOF, time of flight; FALS, forward angle light
scatter; L90°LS, log 90' light scatter.
 TEMPERATURE EFFECTS ON MICROSPORES 469

microspores were isolated and cultured according to the pro- Table II. Embryo Yield in Microspore Cultures Initiated from
tocol described by Coventry et al. (1), with the modification Different Sized Buds of B. napus Plants Grown in Different
that the culture density was 3 x 104 cells/mL. The embryo Environments
yield was determined 4 weeks after culture initiation. Means ± SE are indicated, n = 3.
The light scatter properties of 10,000 microspores from Growth Environment Bud Size Embryo Yield
each sample were measured with an EPICS V flow cytometer
mm %
cell sorter (Coulter Electronics, Hialeah, FL) equipped with a
Coherent (Palo Alto, CA) 5-W argon-ion laser and a multiple 23/180C (16/8 h), continuous light 2.6-3.0 0.02 ± 0.01
data acquisition and display system. Microspores were passed 3.1-3.5 0.04 ± 0.01
through the EPICS V at a rate of 20 to 100 cells per s. The 3.6-4.0 0.18±0.15
4.1-4.5 0.28 ± 0.28
cells were illuminated by a laser emitting at 488 nm with a 4.6-5.0 0.09 ± 0.09
power output of 500 mW. A 76-,um flow cell tip and a sheath
pressure of 13 p.s.i. were used. FALS (light deflected 10 to 23/1 80C (16/8 h), light/dark 2.6-3.0 0.00 ± 0.00
(16/8 h) 3.1-3.5 0.20 ± 0.03
190 from the axis of the laser beam) was detected by a standard 3.6-4.0 0.68 ± 0.66
Coulter photodiode after attenuation with two NDl filters. 4.1-4.5 0.07 ± 0.06
L90LS and TOF were detected by a photomultiplier tube 4.6-5.0 0.00 ± 0.00
after deflection by a 488-nm dichroic filter and attenuation 15/1 20C (16/8 h), continuous light 2.6-3.0 0.05 ± 0.05
by a ND2 filter. The half-height pulse width of the L90°LS 3.1-3.5 1.04±0.17
peak signal was used to determine TOF values. Histograms 3.6-4.0 2.84 ± 0.94
of FALS, TOF, and L90LS were recorded for 10,000 micro- 4.1-4.5 1.89 ± 1.66
spores in each sample. 4.6-5.0 1.13 ± 1.03
15/1 20C (16/8 h), light/dark 2.6-3.0 0.03 ± 0.01
RESULTS (16/8 h) 3.1-3.5 1.52 ± 0.76
3.6-4.0 1.37±1.11
The different growth regimens had significant effects on 4.1-4.5 1.99 ± 1.95
days to flower as well as plant height and number of internodes 4.6-5.0 1.76 ± 1.76
at the time of first flowering (Table I). In general, the plants
grown at higher temperatures and/or under continuous light
had shorter growth periods (measured as the time from seed Bud size had a highly significant quadratic effect (at the
sowing to first flowering). Plants grown at 23/18°C with 0.1% level) on embryo yield in microspore culture (Table II).
continuous light were usually 5 to 10 cm shorter at the time Embryo yield increased as bud size increased until it reached
of first flowering than those grown in other environments. a peak in 3.6- to 4.5-mm buds and then declined for samples
The largest number of internodes (14-16) at the time of first isolated from larger buds. However, the optimal bud size was
flowering occurred in plants grown at 23/18°C under light/ not consistent among replications or among the growth en-
dark conditions. Conversely, plants grown at 15/12°C started vironments studied.
flowering about two to three internodes earlier than those Donor plant growth temperature had a statistically signifi-
grown at 23/1 8°C (light/dark) depending on the photoperiod. cant effect (at the 1% level) on the frequency of embryogenesis
The plants grown at 23/18C with continuous light had the in microspore cultures (Table II, Fig. 1). The average embryo
least number of internodes (nine to 1 1) at the time of first yield in microspore cultures initiated from 2.6- to 5.0-mm
flowering and had thin stems, small leaves, and light yellow buds of plants grown at 15/12°C was 8 times higher than
flowers. those initiated from plants grown at 23/18°C (1.36 compared
with 0.16%). The low-temperature enhancement of embryo-
genesis was seen for all the bud sizes. It also appeared that a
wider range of bud sizes could be used for microspore culture
Table I. Developmental Characteristics of Plants Grown under with plants grown at 15/12°C compared with 23/18°C. Em-
Different Environmental Conditions bryo yields of 1 to 2% were obtained from microspores of
Means ± SE are given, n = 3. Means in the same column followed 3.0- to 5.0-mm buds on cold-grown plants, whereas only 3.5-
by the same letter are not significantly different at the 5% level. to 4.5-mm buds on high-temperature plants gave microspore
Timeto No. of cultures that had embryo frequencies of approximately 1%
Growth Environment Plant Heighta
Flowering Internodesa (Fig. 1). The photoperiod did not significantly affect embry-
d cm ogenesis from microspores because an average embryo yield
23/180C (16/8 h), continuous 35 ± 2 a 47 ± 2 a 10.1 ± 0.3 a of 0.76% was obtained from microspores of plants grown
light under periodic or continuous light regimens. Also, there was
23/1 80C (16/8 h), light/dark 42 ± 1 b 55 ± 1 b 15.3 ± 0.4 d no statistically significant interaction between growth temper-
(1 6/8 h) ature and photoperiod.
15/120C (16/8 h), continuous 54 ± 2 c 52 ± 1 b 11.9 ± 0.3 b The values for all three light scatter measurements (FALS,
light
15/120C (16/8 h), light/dark 65 ± 2 d 53 ± 1 b 12.8 ± 0.4 c L90°LS, and TOF) made on the microspores increased with
(1 6/8 h) increasing bud size (Fig. 2). In general, the L90°LS values
a Measured at the time of first flowering.
increased by the largest amount early in the developmental
sequence, whereas the increase in FALS values was delayed
470 LO AND PAULS Plant Physiol. Vol. 99,1992

3.5 180
0
-J 3.0
170 A E
2.5 a) 150

2.0 E 140
T~~~ z
o 1.5 120 *' 4
2 C-, 110
1.0 c 100
Co 140L .....

0.5 Co 90
LLi 80
0
2.6-3.0 3.1-3.5 3.6-4.0 4.1-4.5 4.6-5.0 70
60
Bud Size (mm) 50
40
3.5
160
0
-O
3.0 170 C D
w
2.5 160
0 2.0 150
a)
.0 140 J
02. 1.5 E 130
co z 120
1.0
C 110
C
0.5 co ioo~~~~~~~~~.
0 C-) 90 U
C
2.6-3.0 3.1-3.5 3.6-4.0 4.1-4.5 4.6-5.0 co
a)
s0
70
Bud Size (mm) 60
50
Figure 1. The effect of donor plant growth temperature on the 40
embryogenic response in B. napus microspore cultures initiated from 2.6430
3.1-3.5
3.6-4.0
414.45
4.6-5.0 2.6-3.0
3.1-3.5
3.64.0
4.1-4.5
4.6-5.0
buds of various sizes. A, 23/180C (16/8 h); B, 15/120C (16/8).
Average values ± SE are shown; n = 3. Bud Size (mm) Bud Size (mm)
Figure 2. Light scatter measurements for microspores isolated from
until the buds were 3.6 to 4.0 mm. The microspore prepara- different sized buds of B. napus plants grown at: A, 23/180C (16/8
tions that gave maximum embryo yields had flow cytometric h) continuous light; B, 23/180C (16/8 h) light/dark (16/8 h); C, 15/
readings that were between channel 65 to 75 for FALS, 120C (16/8 h) continuous light; D, 15/120C (16/8 h) light/dark (16/8
h). Means ± SE of FALS (-), L90°LS (U), and TOF (A) measurements
channel 95 to 100 for TOF, and channel 130 to 155 for are shown; n = 3.
L90°LS, regardless of the environmental conditions under
which the donor plants were grown (Table II, Fig. 2).
The ratios of L90°LS/TOF and L90°LS/FALS were both
tures, in accordance with previous reports (20), this was not
significantly lower (at the 5% level) for microspores isolated the case at the higher temperatures in the present study. A
from cold-grown plants versus high-temperature plants (Table
more extensive study of the interactions between various
III). Figure 3 shows an embryogenesis response surface for growth conditions is required to resolve this issue.
microspore preparations according to their L90°LS versus The reason for the increase in embryogenic response at
TOF values. It illustrates that microspores from the high- lower growth temperatures is not known. It has previously
temperature plants were in a less responsive zone with respect been suggested that low temperatures cause changes in (a) the
to embryogenesis than those isolated from low-temperature
plants. endogenous hormone levels, (b) the nutritional status of the
sporogenous and the somatic tissue of the anthers, or (c) the
physiological status of the microspore. The results of the
DISCUSSION present study suggest that the developmental stage ofthe plant
The results of the present study concur with previous re-
ports that low donor plant growth temperatures enhance
androgenesis in B. napus (3, 10, 12). Previous results have Table MII. Comparison of L90°LS/TOF and L90°LS/FALS Ratios for
suggested that three- to 60-fold enhancements in embryogen- Microspores Isolated from B. napus Plants Grown at 23/180C and
esis from anther cultures were possible by growing donor 15/120C
plants at 15 to 10°C instead of 25 to 20°C. Similarly, a three- Average values are shown, n = 3. Means with the same letter in
to 10-fold increase in androgenesis frequencies (depending on each column are not significantly different at the 5% level.
the bud size category) was observed in microspore cultures in Growth Temperature L90°LS/TOF L90°LS/FALS
the present study when the donor plants were grown at
reduced temperatures. Although it appeared that continuous 12/180C (16/8 h) 1.58 a 2.24 a
light had a beneficial effect on androgenesis at low tempera- 15/120C (16/8 h) 1.45 b 2.07 b
 TEMPERATURE EFFECTS ON MICROSPORES 471

180 cultures, and the formation of P grains is controlled by the

X
A-..........
'\
growth conditions of the donor plants (8).
The results of the present study suggest that a proportion
160
of the microspores in the buds from low-temperature plants
(D
-
.* ,' followed a different pathway of development than those from
140
N/. X /,' high-temperature plants, which affected their physical prop-
ct'- |
/! /, erties and their ability to develop into embryos in culture.
120 l/: ,' / /,. Therefore, the results of this study support the concept that
CM at least a portion of the responsiveness of microspores is
E1 100 l ' je / ' determined before culture (8). Perhaps the donor plant growth
temperature effect on androgenesis is mediated by a change
in the physiological state of the microspores, which results in
oJ 80
a decrease in the number and/or the size of cytoplasmic
0
a) inclusions and/or a decrease in the density of their spore
n 60 walls.
The present study confirmed that flow cytometric light
40 scatter measurements give information about microspore on-
60 70 80 90 100 110 120 togeny. L90LS intensity increased rapidly during the early
TOF (mean channel #) stages of microspore development
thickening, and an increase in cellwhen exine deposition, wall
granularity occur. During
Figure 3. Eimnbryogenesis response surface based on the average the development of the microspore from the early round stage
L90°LS and TOF mean values measured for microspores isolated to late round or oval stage, there was a more gradual change
from plants cgrown at 23/18 or 15/120C. Mean TOF versus L90°LS in L90LS intensity but a more rapid change in FALS intensity
values for rriicrospores isolated from different bud size categories and TOF. The light scatter changes can probably be attributed
(increasing le,ft to right) of plants grown at 23/180C (@) and 15/1 2°C to the rapid swelling that these spores undergo during this
(U) are showin. The contour lines connect points of equal embryogenic time, which would lead to a change in cell size but little
frequency: ---, 1%; -----, 0.1%; ...., 0.01%. The predicted change in cell granularity.
maximal reslponse is 2.13%, which occurs when TOF is 91.6 and In accordance with many previous studies, the success in
L90°LS is 11 *9.2.
isolated microspore culture was strongly dependent on the
developmental stage of the microspore (14, 16), and the bud
may be a fa .ctor b te lsize measurement was related to the developmental stage of
cth
becaue .th
lowempereat plnse initiate
flowering aLt the 12Zth or 13th node, whereas those grown at
t
the
e

microspores. However, the optimal bud size was not

higher temi peratures deviated muchmoewithconsistent
stage of dev elopmentdeiatthedt~
elopment~~ imu fowerin.Thuesp
more
at th ieolwrn Thus .
their
bpreviously
th buds
and the miccrospores may have been in different physiological
states whern harvested from low- versus high-temperature
donor plan ts. The sensitivity of in vitro flower development
to the node from which the explant is taken has previously
been reportLed for tobacco (15). It seems clear, however, that
the differenice in embryogenic capacity between microspores
isolated froim low-temperature versus high-temperature donor
plants is in dependent of the developmental stage of the mi-
crospore be cause each bud size category that was tested was
affected in Ithe same way.
The flow cytometric results supported the conclusion that
microspore s were different in low- versus high-temperature-
grown plarnts. In particular, the lower 90°LS/FALS and
90°LS/TOF ratios for microspores isolated from low-temper-
-
among replications in the present study. It has
bud
been shown that there is a poor correlation between
b size and embryo yield in microspore culture (16). In
contrast, the flow cytometric analysis camed out in the pres-
ent study showed that the microspore preparations that gave
the highest embryo yields had similar FALS, TOF, and
L90LS values. Furthermore, a multiple regression test in-
cluding fixed effects (growth temperature, photoperiod, and
bud size) and flow cytometric covariances (mean values for
FALS, L90°LS, and TOF) showed that only growth tempera-
ture and TOF were significantly correlated with embryo yield
in the reduced model (analysis not shown).
Importantly, the statistical analysis indicated that bud size
was not an important parameter when flow cytometric covar-
iances were included in the model. Therefore, a flow cyto-

metric measurement appears to provide a better estimate of

ature versu. s high-temperature-grown plants suggest that mi-
crospores firom the former are more translucent than those
from the laltter. Although pollen polymorphism has not been
reported in B. napus, two types of pollen, normal gametophy-
tic pollen gVrains (N grains) and embryogenic P grains, are
found in mtany other species, including rye, peonia, barley,
oat, wheat, and tobacco (listed in ref. 7). The embryogenic P
grains in to tbacco are starch free and have a clear cytoplasm.
Similarly, tl he percentage of this type of pollen has been found
to be close ly related to embryo production frequencies in
the stage of microspore development than a measurement of
bud size. Presumably, this is because properties of the mi-
crospores are measured directly by flow cytometry. The other
advantages of using flow cytometry for staging microspore
development are that thousands of cells can be analyzed
within minutes, and no processing or staining of the micros-
pores is required.
In general, the results of this study show that flow cytometry
is a useful method for characterizing the physiological and
developmental status of microspore cells. Furthermore, we
472 LO AND PAULS
have shown that the flow cytometric parameters are useful
predictors of the embryogenic potential of these cells.
ACKNOWLEDGMENTS
We express our gratitude to Kathy Fuchs for her assistance with
the flow cytometer and to Jennifer Kingswell for typing the
manuscript.
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