Vol. 4: 513-518.

In vitro pollen culture and the regeneration of
Brassica campestris L plants

Yang-Dong Guo 1 and Seppo Pulli
Agricultural Research Centre ofFinland, Institute ofCrop and Soil Science, FIN-31600 Jokioinen, Finland

Brassica campestris (Brassica rapa L. ssp. oleifera) is an important oilseed crop, particularly in
Finland. Pollen culture techniques for haploid production have been developed, but B. campestris is
relatively recalcitrant in pollen culture. Twenty eight genotypes of B. campestris were included in
this study. The donor plants were grown in the greenhouse and transferred to the growth cabinet
before bolting. Buds (2-4 mm long) were selected, macerated in B 5 medium, then NLN liquid cul-
ture medium was added. The microspores were incubated in the dark at 32°C for 72 h, then at 25°C
for a further three weeks. Nineteen genotypes produced microspore-derived embryos. The highest
yield was more than 300 embryos per 100 buds. Activated charcoal (150 mg/L) promoted embryo-
genesis, pollen development was faster and the embryo yield was higher. Plants were regenerated
after transferring embryos to a solid B 5 medium. Colchicine solution was used to double the chromo-
some complements. About 100 regenerate plants have been obtained in our laboratory, and these
haploids will be useful for the oilcrop breeding.

Key words: oilcrop, genotype, bud size, activated charcoal, donor plant condition, embryogenesis,
plant regeneration
'Current address: Laboratory of Plant Physiology, Department of Biology, University of Turku,
FIN- 20500 Turku. Finland

ntroduction
Brassica campestris (Brassica rapa L. ssp.
oleifera) is grown in many countries, including
Finland, as an oilseed crop. It is mostly self-in-
compatible and thereforeselfed lines are expen-
sive and very difficult to obtain. Ahaploid breed-
ing technique for the Brassica species has been
developed during the past two decades. For B.
napus, pollen culture has become a tool for crop

breeding because the yield of microspore-
derived embryos is very high (about 900,000per
100 buds), (Swanson et al. 1987;Kott et al. 1988;
Siebel and Pauls 1989). Haploids are of consid-
erable use in plant breeding and genetic studies.
Haploid production is extremely attractive be-
cause it not only provides an opportunity to se-
lect at the haploid level in vitro for desirable
agronomic and seed quality characteristics, but
it also provides a means of producing genetically
stable homozygous lines, fixed by chromo-

© Agricultural Science in Finland
Manuscript received November 1995

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https://www.c-info.fi/en/info/?token=ubDPCRoHfCEkXAW2.RnN-Lw7tF9E_2NKkJ1t_qw.zSsknxyy0WOHeL1MVl6EsVLBmu26--XzsXXRUXCIiXuKkgGYZoBGs3vYt8Vv-oz_lL8ZMMSEn8n8VJhGgPxThrUnp3a6zzD6UWOwunMyDChl1u4Ruj3TGu-InhOs6Nxumq2IGzrnSxz0hDT7oxCvH3PC1cmWWMLPOyzrfSbgMBFeMvECs8v8G_0NFWyExXGXXS0XPjtOSGZlKaKnIHI5QvigyInVafKLmqFDGY_Hkb27f9oe0aNLfc-7bNBxHcvSXcFC8FyFTyQhCuDg7NjiWua2g-Y


Guo, Y. & Pulli, S.: In vitropollen culture and the regeneration ofBrassica campestris L. plants

some doubling (Kott and Beversdorf 1990). In
cross-pollinating species, such as B. campestris,
doubled haploids are more likely to be used as
parents in the production of single- or double-
cross hybrids (Dunwell 1985).

A microspore culture technique has been de-
veloped for B. campestris (Baillie et al. 1992,
Burnett et al. 1992) which has a number of spe-
cific advantages over anther culture (Pierik
1987). In particular, it is more efficient for hap-
loid production than anther culture (Siebel and
Pauls 1989). Androgenesis has become the most
important source of haploids produced in vitro
by far. This is extremely important; as pollen
grains are available in considerable quantities
and each has the potential to develop into a hap-
loid plant (Pierik 1987). B. campestris is re-
latively recalcitrant in pollen culture, however.
The yield of embryos is relatively low and em-
bryos can only be obtainedfrom a few genotypes.
Here we report our results on the pollen culture
of B. campestris, with the aim of establishing a
pollen culture system for breeding.

Material and methods

Donor plant and growth conditions
Twenty eight genotypes of B. campestris were
tested: fourteen cultivars and fourteen breeding
lines. The donor plants were grown under con-
trolled-environment greenhouse conditions with
16h photoperiod (approximately 300 (tmol/m2 s)

and a day/night temperature of 25/15°C. The
donor plants were grown in individual 12 cm
pots, watered daily and fertilized once a week.
Before bolting, the donorplants were transferred
to a growth cabinet with a 16 h/Bh, light/dark
regime. The temperature was 10°C during the
light period, with a light intensity of 300 pmol/
m2 s, and 5°C during the dark period. The donor
plants were watered every two days and fertilized
once a week.

Pollen culture
Flower buds (50-75) were removed from donor
plants older than 6 weeks. Bud length was 2-4
mm, only those in this range could produce em-
bryos. The optimum timing for microspore cul-
ture is during mid-late to very late uninucleate
stage, DAPI (4', 6'-diamidino-2-phenylindol)
was used to check pollen development. The buds
were placed in test tubes and surface sterilized
in 4.5 % sodium hypochlorite with one drop of
Tween-20 for 15 min, followed by three five-
minute washes with sterile, distilled water. The
buds were then macerated with a glass rod in
cold B 5 medium (Gamborg et al. 1968) without
iron salt, supplemented with 13% (w/v) sucrose
(in B 5 medium, CaCl2 -2H2 G was increased to
750 mg/L), filtered through a4B pm nylon mesh
and pelleted by centrifugation at 1000 rpm for
5 min in the same medium 3 times. The micro-
spores were resuspended in the required amount
of NLN medium (Lichter 1982) supplemented
with 13 % sucrose and 0.83 mg/L potassium io-
dide, but without potato extract and hormones.
The final density of microspores was sxlo 4 per
millilitre. Activated charcoal was added to some
of the NLN culture medium at 150 mg/L. Ten
millilitres of microspore suspension was dis-
pensed into a 100x15 mm sterile Petri dish. The
dishes were then wrapped with double layers of
parafilm. Every test was repeated at least three
times. The microspores were incubated in the
dark for an initial period of three days at 32°C,
followed by three weeks of dark culture at 25°C.

Plant regeneration
After three weeks of microspore culture, the coty-
ledonous embryos were counted and placed on
a slow shaker (50 rpm) under continuous light
at 25°C until they were green. At this time, the
embryos were transferred to a solid B 5 medium
containing 2% sucrose and 0.3% phytagel with-
out growth regulators (Gamborg et al. 1968) and
cultured at 25°C, 16h light period. Plantlets were
obtained and then labeled individually. Prior to

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Vol. 4: 513-518.

transfer of plantlets to soil, theroots of the plant-
lets were immersed in a 0.2% colchicine solu-
tion for 3 h followed by several water washes.
The plantlets were then potted and placed in a
greenhouse under plastic cups to maintain a high
humidity level.

Results and discussion
Influence of genotype

B. campestris genotype is considered to be a key
factor in obtaining microspore-derived embryos;
only a few genotypes can produce microspore-
derived embryos (Baillie et al. 1992, Burnett
et al. 1992). Sorvari (1985) indicated that
the highest embryo yield was from cross type
“dihaploid x dihaploid” and high-glucosinolate
genotypes. For B. napus, haploid production was
also genotype dependent (Chuong et al. 1988).
B. campestris is mostly self-incompatible and
open-pollinated, genotypes are mixed to various
degrees in the population. There is plant to plant
variation in microspore culture response within
genotypes (Ferric and Keller 1995). In our re-
search, from a total of 28 genotypes tested,
19 produced embryos.

Influence of bud size
Bud size plays an important role during pollen
culture; microspores had embryogenic potential
only during a short and specific period during
pollen development. A range of bud sizes was
tested: smaller than 2.0 mm, 2.0-2.9 mm, 3.0-
3.9 mm and bigger than 4.0 mm. Only the buds
measuring 2.0 mm to 3.9 mm in length were easy
to produce embryos. The optimum timing for
microspore culture is during the mid-late to very
late uninucleate stage. Optimum bud lengths
were different for different genotypes; for ex-
ample, the optimum bud length for Emma (ap-
prox. 3.2 mm) was slightly higher than for
Candle (approx. 2.8 mm).

Table 1. Response of genotypes in pollen culture. Activa-
ted charcoal was contained in the culture medium. Mean is
from at least 8 replicates.

SDEmbry os/100 budsGenotype

8c3059 326 64
8c3158 283 77
Kulta 120 34
Candle 78 29
8c3211 76 21
8c3063 54 17
8c3161 49 15
8c3230 36 9
Emma 32 9
8c3234 30 11
Kova 27 4
8c3066 23 9
8c3139 15 9
8c3131 13 7
Sisu 13 6
Tobin 11 8
Tyko 8 6
Hian Vankka 4 5
8c3134 3 2
8c3129 0 0
8c3163 0 0
8c3141 0 0
Turkel 0 0
Hanne 0 0
Nopsa 0 0
Ww1774 0 0
Palle 0 0
Valtti 0 0

SD: standard deviation.

Effect of activated charcoal
In some of our tests, we found that the activated
charcoal was beneficial for embryogenesis.
A series amount of activated charcoal was add-
ed to the liquid NLN media and 150 mg/L of
activated charcoal was found the best for the mi-
crospore culture (data not presented); pollen de-
velopment was faster and the yield of embryos
was higher in the culture medium withactivated
charcoal than those in the culture medium with-
out activated charcoal. It has been reported that
endogenously produced toxins within cultures

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Guo, Y. & Pulli, S.: In vitropollen culture and the regeneration o/Brassica campestris L. plants

Table 2. Microspore embryogenesis for various bud lengths
of genotype 8c3158. Activated charcoal was contained in
the culture medium. Mean is from at least 6 replicates.

Embryos/100 buds SDBud length

00< 2.0 mm
792032.0- mm

3.0- mm
> 4.0 mm

1877
22

SD: standard deviation.

play a negative role in the initiation and devel-
opment of embryos in B. napus (Kott et al. 1988).
High levels of phenylacetic acid and p-OH-ben-
zoic acid have been found in Daucus cultures;
2,6-OH-enzoic acid and benzoic acid have been
found in Allium cultures; benzoic acid, pelargon-
ic acid and caprylic acid have been found in
Haplopappus cultures (Fridborg et al. 1978). It
was also shown that p-OH-benzoic acid had in-
hibitory effects on the embryogenesis in Daucus
cultures (Fridborg et al. 1978), whereas the
media with activated charcoal did not. This evid-
ence suggests that activated charcoal adsorbs
such toxins. We noted that, in the absence of ac-
tivated charcoal, cell division, embryo formation
and development were inhibited in the culture.
Often there was no growth or development in
vitro ; or initial growth was followed by the abor-
tion of embryos. When activated charcoal was
added to the media, such effects were no longer
apparent, the reason is perhaps due to interfer-
ence with polyphenolic compounds produced by
the explants and partial adsorption of the exog-
enous auxin by the activated charcoal (Dumas
and Monteuuis 1995). In our work, the embryos
obtained from microspore culture with activat-
ed charcoal and microspore culture without ac-
tivated charcoal are normal developed, and both
of themhave the potential to develop to the plant-
lets. For the microspore culture of Brassica
species, media change was considered a good
method to enhance embryo yield (Baillie et al.
1992. Burnett et al. 1992, Hansen and Svinnset
1993). There is no toxin in the fresh medium, so
the pollen can develop well in the new medium.

Donor plant conditions
For pollen cultures of theBrassica species, donor
plant conditions are also an important factor
in embryo production. In our work, the donor
plants were tested with different lengths of ex-
posure to low temperatures. The plants were
grown in a controlled-environment greenhouse
(25/15°C,16 h lightperiod), before bolting (about
6 weeks), they were then transferred to the
growth cabinet (10/5 °C, 16 h lightperiod). We
established that embryo yield was directly pro-
portional to the length of the low temperature
pre-treatment, not to the age of donorplants. The
yield of embryo was similar, not significantly
different if buds were moved from 6th week to
9th or 10th week (data not presented). Baillie et
al. (1992) recommended a low temperature pre-
treatment for donor plants. The temperature in
the growth cabinet was lower and more stable
than it was in the greenhouse, and this benefited
the plants. Low temperature pre-treatment has
become usual in the haploid breeding of cereal
crops (Nitsch, 1972;Wang et al. 1974).

We also established that embryos could only
be produced from older plants. The donorplants
were transferred into a growth cabinet (10/
5°C,16 h lightperiod) after six weeks growth in
the greenhouse. The too young donor plants
failed to produce embryos (data not presented).
Burnett et al. (1992) also showed that four to
five week old plants did not produce embryos.
For B. napus, microspores isolated from the buds
of older plants had a higher embryo yield than
those of the younger ones (Takahata et al. 1991).

Table 3. Effect of activated charcoal (AC). Mean is from at
least 8 replicates.

Genotype Em / 100 buds ±SD Em/100 buds ±SD
with AC without AC

69 ±32 *

52 ±l2 *

196+65
101 ±l6

8c3158
Kulta

SD: standard deviation.
* Significantat 1% level.

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Vol. 4: 513-518.

Table 4. Effect of cold treatment. Activated charcoal was
contained in the culture medium. Mean is from at least
6 replicates.

Genotype Cold treatment week(s) Em/100 buds ±SD
8c3059 0 (control) 61 ±23 c

1 172±47 "b

2 194±35""
3 264 ±53 ■

#

Kulta 0 40±17 f

1 77 ±43 d

2 62±21 dc

3 63 ±29 dc

4 74 ±27 d

#

it Plants ceased to produce buds.
SD; standard deviation.
Means followed by the same letter are not significantly dif-
ferent according to the Duncan multiple range test at the
5% level.

The microspore culture technique for
Brassica species has existed for some years, and
currently the microspore culture technique for
B. napus is very successful as a breeding tool.
The yield of embryos from B. campestris is
relatively poor, however. To improve the micro-
spore cultures for B. campestris breeding, it is
important to enhance the yield of embryos. The
effect of several factors including optimal
media, donor plant conditions and culture
temperature will be tested in the future.

Acknowledgements. We wish to thank Mr. Veli-Matti Rok-
ka for his valuable discussions. Some seeds were kindly
provided by Mr. Juha Viikki. Thanks are also due to Dr.
Jonathan Robinson for reviewing the manuscript. The re-
search work is supported by The Ministry ofAgriculture &

Forestry of Finland and The Agricultural Research Centre
of Finland.

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SELOSTUS
Kevätrypsin mikrosporiviljely in vitro ja vihreiden

kasvien tuottaminen alkioista
Yang-Dong Guo ja Seppo Pulli

Maatalouden tutkimuskeskus

Tutkimus sisälsi 28 perimältään erilaista kevätrypsi-
linjaaja -lajiketta. Tutkimusmateriaali kasvatettiin al-
kuvaiheessa kasvihuoneessa ja siirrettiin ennen ku-
kintaa kasvatuskammioon. Mikrosporiviljelyssä par-
haan tuloksen tuottivat mikrosporit eli epäkypsät sii-
tepölyhiukkaset, joiden kehitysvaihe oli myöhäinen
1-tumavaihe, mikä edellyttää, että kukkasilmun pi-
tuus on 2-4 mm. Oikeassa kehitysvaiheessa olevat
kukkasilmut murskattiin 85-nestealustalla ja mikro-
sporit viljeltiin NLN-nestealustalla, ensin 72 h ajan

+32°C:ssa ja seuraavaksi kolme viikkoa +2s°C:ssa,
Viljellyistä mikrosporeista alkioita tuotti yhdeksän-
toista kevätrypsigenotyyppiä. Parhaalla genotyypillä
saatiin 100 mikrosporista 300 alkiota. Aktiivihiili 150
mg/1 paransi mikrosporien kehitystä ja lakionmuodos-
tusta sekä lisäsi alkioiden lukumäärää. Alkiot kasva-
tettiin vihreiksi kasveiksi kiinteällä 85-alustalla.
Tutkimuksen ensimmäinen vaihe tuotti noin 100 hap-
loidista kevätrypsikasvia.

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