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2007 vol. 67, 5-15
DOI: 10.2478/v10032-007-0025-5
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REGENERATION AND EVALUATION OF ANDROGENETIC PLANTS
OF HEAD CABBAGE (BRASSICA OLERACEA VAR. CAPITATA L.)
Krystyna GÓRECKA, Dorota KRZYŻANOWSKA, Urszula KOWALSKA
Research Institute of Vegetable Crops,
96-100 Skierniewice, Konstytucji 3 Maja 1/3, Poland
Received: March 7, 2007; Accepted: August 20, 2007
Summary
Plant regeneration from head cabbage embryos obtained in anther culture,
the way of colchicine using for doubling number of chromosomes in haploid
plants and estimation of the double haploid plants with regard to vigour, typicality of leaves and fertility were worked out. The best medium for plant regeneration from androgenetic embryos was B5 medium, enriched with sucrose
20 g.L-1 and kinetin 20 mg.L-1. On this medium, the highest number of embryos
formed shoots, usually several from 1 embryo, particularly in subsequent passages. The most effective method of doubling the number of chromosomes was
soaking the roots of young plants, when they had 5-6 leaves in a 0.1% hydrous
solution of colchicine with 4.0% DMSO and 25 ppm GA3, for 20 hrs. The tested
cultivars of head cabbage differed considerably with regard to the vigour of the
plants obtained from anther culture. The highest number of plants characterized
by strong vigour was observed in the cultivar Langendijker. About 90% of head
cabbage plants obtained from androgenetic embryos had typical leaves and the
differences between the cultivars in this aspect were insignificant. The highest
number of androgenetic androfertile plants was found in the cultivar Sława z
Enkhuizen.
The genotype was the factor which had an impact in the processes examined: ability to shoot proliferation, rooting, the sensitivity to colchicine treatment, vigour of produced doubled haploid plants and their fertility.
key words: colchicine treatment, fertility, shoot proliferation, rooting, vigour
INTRODUCTION
The process of derivation of double haploid lines with the use of anther
culture is carried out in 3 stages:
- obtaining androgenetic embryos in anther culture,
- plants regeneration from obtained embryos,
- plants estimation, ploidy determination and doubling the number of haploid
plants chromosomes (Bajaj 1990).
Corresponding author:
e-mail: [email protected]
© Copyright by RIVC
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Androgenetic embryos development is a indispensable condition for next
breeding steps, but not the only one. It is necessary to regenerate the plants from
obtained embryos. The regeneration has always been critical – many embryos
die (Keller et al. 1982). The choice of proper medium for regeneration is very
important. The following factors play a very important role: lowering the level
of sucrose, elimination of aminoacids and also change of the hormones (Keller
& Armstrong 1977, Chiang et al. 1985, Nałęczyńska 1991).
In order to obtain homozygous lines from haploid androgenetic plants it is
essential to double the number of chromosomes. The agent used most frequently in this purpose is colchicine (Jensen 1974). However, colchicine is a
poison and the treatment with its use on haploid plants characterized by low
vigour, frequently caused their death. This is why new and harmless ways of
treatment with its use are being investigated. It has been observed that in androgenetic plants of the Brassica geneus a spontaneous chromosome doubling occurs (Dore 1986, Nałęczyńska 1991).
The aim of this work was the choice the best way of regeneration head
cabbage androgenetic plants, the most effective chromosome doubling method
with colchicine and also the best method of their description (leaves characteristic and fertility).
MATERIAL AND METHODS
Three very popular cultivars of head cabbage were used in the experiments: Sława z Enkhuizen, Kamienna Głowa and Langendijker. The anther
culture were carried out according to the procedure described in the work of
Górecka & Krzyżanowska (2004) for head cabbage and of Krzyżanowska &
Górecka (2004) for Brussels sprouts.
The embryos occurred on the surface of some anthers or near them after
about 3 weeks from the establishment of the anther culture. Then, the flasks
with embryos were transferred to continuous fluorescent light 30 μmol.m-2.sec-1
at the temperature +27ºC.
When the embryos started to be come green - after a couple of days of being kept in the room with constant lighting - they were accounted for and transferred onto a regeneration medium. Four regeneration media were tested for
cultivar Langendijker and two the best of them for cultivar Sława z Enkhuizen
(medium MS-2 and B5-2, because number of embryos on hand was limited).
1. MS (Murashige and Skoog 1962) - without aminoacids and hormones and
with 10 g.L-1 sucrose and 0.5 g.L-1 charcoal (marked as MS-1), used for regeneration of androgenetic plants of rapeseed (Keller & Armstrong 1977)
and of head cabbage and Brussels sprouts (Lelu & Bollon 1990).
2. MS (marked as MS-2), with 1 mg.L-1 BA, 0.001 mg.L-1 NAA and 20 g.L-1
sucrose recommended by Chiang et al. (1985) for rise androgenetic plants of
head cabbage.
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3. B5 (Gamborg et al. 1968), without aminoacids and hormones but with 20 g.L-1
sucrose (marked as B5-1), used by Keller & Armstrong (1977) to develope
plants from androgenetic embryos of rapeseed.
4. B5 (marked as B5-2), without aminoacids, but with 20 mg.L-1 kinetine and
20 g.L-1 sucrose, described by Nałęczyńska (1991) as a very effective in her
experiments on regeneration in rapeseed plants.
For all mentioned above media pH was set at the level of 5.8. After 3 weeks
observations were made and the cultures were transferred onto a fresh media. The
development of embryos, organogenesis and callus formation were described.
During the second passage small shoots and buds were transferred onto regeneration media. The bigger shoots were transferred onto the medium for root
formation - B5 containing 30 g.L-1 sucrose and 1 mg.L-1 IAA. After about 3 weeks,
shoots without roots were transferred onto a fresh medium for root formation,
whereas shoots with roots were planted in pots with peat. The pots with androgenetic plants were placed in greenhouse in the small tent made of double foil. After
adaptation, they were taken out of the tent and replanted into bigger pots.
The cytometric analyses and the cytological observations of chromosomes
were carried out. Cytometric analysis were conducted according to PARTEC
GmbH procedure. The solution A for nuclei isolation and the solution B for
DNA fixation were used. The leaf fragment (0.3-1cm2) was desintegrated with
rasor blade in A solution in Petri dish, next 2 ml of buffor B was added. Big
tissue fragments were eliminated by filtration with 30 μm filter. Preparred sample was placed in Partec cytometer and DNA content was mesured. Obtained
results were analysed for ploidy determination. After ploidy assessment the
doubling of chromosomes number in haploid plants was carried. The following
methods were used:
1. submerging of the rooted plants with a 0.1% water solution of colchicine
with 4% DMSO and 25 ppm GA3 in 100 mL flasks (a solid medium with
rooted plants coated with 2 cm of colchicine solution) and putting them in a
greenhouse at temperatures of between +25ºC and +35ºC, for 6 hrs.
2. roots soaking during plants transplantation into bigger pots (when the plants
had 5-6 leaves) in a 0.1% water solution of colchicine, with 4% DMSO and
25 ppm GA3 - in a greenhouse at temperatures of between +22ºC and +30ºC,
for 20 hrs.
3. cut shots soaking in a 0.05% water solution of colchicine with the addition
of 3 drops per 1 liter of Tween 20 - in an incubator at the temperature +24ºC,
for 18 hrs.
4. placing a cotton wool with 0,1% water solution of colchicine directly on
shoot apical meristem - in a greenhouse, at temperatures of between +18ºC
and +25ºC, for 24 hrs.
When the androgenetic head cabbage plants had 14-15 leaves, their vigour
was assessed using scale of 6 classes. Class 1 included the worst growing plants,
class 6 - the best growing plants (Photo. 1). Plants presenting a good level of vitality, contained in classes 3-6, and without any morphological anomalies were
subjected to vernalization at the temperature +4ºC, for 2 months.
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Photo. 1. Vigour rating of androgenetic head cabbage plants (cv. Sława z Enkhuizen).
The figures on the given labels denote degree (1-6), with 1 meaning the
weakest plants and 6 - the most vigorous ones
After that time they were put into bigger pots (20 cm in diameter) and
transferred to a greenhouse, and kept there until flowering. In the flowering
phase, some morphological features of the leaves were assessed according to
the UPOV nomenclature (Union Internationale pour la Protection des Obtentions Vegetables). On the grounds of these observations the percentage of androgenetic plants with leaves typical for head cabbage was calculated. During
the flowering phase male fertility and male sterility were observed.
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RESULTS
Some embryos on the regeneration media showed growth, increased their
cotyledons and shoots. Some others formed callus. However, many of them
died. Among all the embryos transferred onto the regeneration media 11.0%
grew and formed shots, 28.6% formed callus, 44.5% died during the first passage. More embryos producing shoots in the first passage (11.9%) were observed in the cultivar Langendijker. As for as Sława z Enkhuizen is concerned,
19.3% of the embryos produced callus. The influence of the cultivar on the capability of regeneration was not so strong as it was in the case of the process of
androgenesis. The embryos placed on the regeneration medium MS-2, containing 1 mg.L-1 of BA and 0.001 mg.L-1 of NAA, produced callus intensively (50%
of embryos of the cultivar Langendijker and 23.1% of embryos of the cultivar
Sława z Enkhuizen). The best regeneration medium proved to be the B5-2 medium containing 20 g.L-1 of sucrose and 20 mg.L-1 of kinetine. Most embryos of
both tested cultivars put on this medium produced shoots (Table 1). In the second passage the process of shoot formation was ever more intensive. Obtained
shoots formed roots very well (70% of them) on the B5 medium containing
1 mg.L-1 of IAA. During three passages it was possible to obtain enough plants
for further stages of the breeding process from every embryo that survived.
Table 1. Regeneration of the head cabbage embryos obtained in anther culture on different media in the first passage
Characteristic of embryos
number of emdeveloped developed
Cultivar
died
Medium* byos placed on
callus
shoots
(%)
the regeneration
(%)
(%)
medium
MS-2
13
23.1
7.8
23.1
Sława
B5-2
12
16.7
8.3
50.0
z Enkhuizen
MS-1
28
21.4
3.6
71.4
MS-2
28
50.0
3.6
42.8
Langendijker
B5-1
27
29.6
11.1
29.6
B5-2
68
26.5
19.1
41.1
non
developed
(%)
46.0
25.0
3.5
3.5
29.6
13.2
* MS-1 (Murashige & Skoog 1962) without aminoacids and hormones and with 10 g.L-1
sucrose and 0.5 g.L-1 charcoal
MS-2 without aminoacids with 1 mg.L-1 BA, 0.001 mg.L-1 NAA and 20 g.L-1 sucrose
B5-1 (Gamborg et al. 1968) without aminoacids and hormones but with 20 g.L-1 sucrose
B5-2 without aminoacids, but with 20 mg.L-1 kinetine and 20 g.L-1 sucrose
In the cytometric examination about 30% of obtained plants were haploid.
The phenomenon of spontaneous diploidization occurred in the rest of them
(70%). This was also confirmed by the microscopic observations of the chromosomes.
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Preliminary experiments showed that colchicine in 0.1% concentration
used for the soaking of cut shoots caused their death at a high rate. The concentration of 0.05% was definitely less harmful but equally effective. The treatment
of the cut shoots with colchicine is less effective, not only because of the lower
percentage of doubled haploids (57.1%), but also because of the fact that many
shoots died and those which survived did not form root - even up to 50% of
them, when the whole plant were treated with colchicine 71% of doubled haploids was obtained. The most effective way to doubling the number of chromosomes was the second method - treating of the young plants with 0.1% water
solution of colchicine during transferring them into bigger pots. 71.4% of doubled haploid plants using this method were obtained. The mortality of plants
was lower than in the case of treating cut shoots. After treating plants in flasks
63.7% of diploids was obtained. The least effective turned out to be the method
of placing a cotton wool with 0.1% water solution of colchicine directly on
shoot apical - 33.3% of double haploids were obtained (Table 2).
Table 2. Change of ploidy level of the androgenetic head cabbage haploid plants of
3 cultivars after treatment them with colchicine
Method of colchicine applying
plants in flasks, 0.1% colchicine,
4% DMSO, 25ppm GA3, 6 hrs
young plants during transplantation,
0.1% colchicine, 4% DMSO,
25 ppm GA3, 20 hrs
cut shoots, 0.05% colchicine, 18 hrs
shoot - tips, 0.1% colchicine, 24hrs
mean
Number Number
of treated of alive
plants
plants
*Ploidy level ( %)
1n
2n
4n
141
78
30.0
63.7
6.3
57
39
9.5
71.4
19.1
198
19
60
19
42.9
33.3
27.0
57.1
33.3
64.0
0.0
33.3
9.0
* Ploidy level was determined by cytometric analysis by PARTEC GmbH procedure
The vitality of doubled haploids obtained from androgenetic embryos of
head cabbage turned out to be differential, depending on the cultivar. The greatest vigour was observed in the cultivar Langendijker - 73.1% of plants of this
cultivar presented strong and very strong growth (class 4-6 in the bonitation
scale). Only 3.7% of the plants of this cultivar died. As for as the cultivar Sława
z Enkhuizen is concerned, 50% of the plants presented strong and very strong
growth, and 10% of them died.
The plants of the cultivar Kamienna Głowa showed strong and very strong
growth at the rate of 61.5%, the percentage of plants, which died, was 4.6%
(Table 3). Some plant with growth and coloring anomalies were also found.
During the assessment of morphological features of the leaves in the flowering phase it was found, that 88% to 92% of the examined plants had leaves
typical for head cabbage. The differences between the tested cultivars in this
respect were inconsiderable, but the differences in fertility were very noticeable.
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The percentage of androsterile plants of the cultivar Sława z Enkhuizen was
17%, whereas in the case of the Langendijker cultivar, this percentage was 70%
(Table 4).
Table 3. The vigour of head cabbage doubled haploids according to the bonitation scale*
Cultivar
Sława z Enkhuizen
Kamienna Głowa
Langendijker
The percentage of plants in particular grades (%)
1
2
3
4
5
6
10.3
8.8
30.9
30.9
14.7
4.4
4.6
7.3
27.6
27.6
22.0
11.9
3.7
6.1
17.1
22.0
32.9
18.2
* 1- the worst growing plants, 6 - the best growing plants
Table 4. The characteristic of head cabbage double haploids with regard to androfertility
and morphological features of the leaves
Cultivar
Sława z Enkhuizen
Kamienna Głowa
Langendijker
Number
of plants
42
76
48
Percentage of plants (%)
androsterile
androfertile
normal leaves
17.0
83.0
92.0
33.0
67.0
92.0
70.0
30.0
88.0
DISCUSSION
Different media for regeneration of the plants obtained from embryos in
the process of androgenesis were used in experiments of many authors. Takahata & Keller (1991) used B5 medium without hormones for the regeneration of
the plants obtained from the embryos of Brassica oleracea. Several passages
induce the formation of shoots. In experiments carried by Dore & Boulidard
(1988), embryos placed on medium without growth factors transferred in the
single complete plants, whereas embryos placed on a medium with 0.1 mg.L-1 of
BA formed shoots. Nałęczyńska (1991) obtained many shoots from embryos of
Brassica napus placed on the B5 medium with 20 mg.L-1 of kinetin. In the experiment presented here, the best medium turned out to be also the B5 medium
with 20 g.L-1 of sucrose and with 20 mg.L-1 of kinetine. Using this medium, the
highest number of shoots from embryos was obtained.
Keller et al. (1982) reported that about 50% of the androgenetic embryos
of rapeseed died on the regeneration medium. In experiments presented here
44.5% of the embryos of head cabbage died on the regeneration medium. Lelu
& Bollon (1990) claimed that the capability of regeneration of the plants of
head cabbage and Brussels sprouts obtained from androgenetic embryos depended on the genotype. This dependence was observed also in the case of
Brussels sprouts by Górecka et al. (1996). Experiments presented here proved
this kind of dependence in head cabbage.
Dore & Boulidard (1988) obtained root formation in the shoots of head
cabbage on a medium without hormones. Nałęczyńska (1991) used the medium
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with 5 mg.L-1 of IBA to obtain root formation in the shoots of rapeseed. In experiments presented here satisfactory root formation on the shoots of head cabbage on the B5 medium with 1 mg.L-1 of IAA was obtained.
According to Jensen (1974) colchicine is the most frequently used agent in
the process of doubling the number of chromosomes. An effective technique of
it application must presented a high rate of doubling, cannot cause a high loss
and damage to the plant material, must be easy in use and give effect quickly.
Nałęczyńska (1991) using colchicine to treat the lateral shoots obtained from
the haploid plants of rapeseed. Then she proceeded with the root formation in
conditions of high humidity. Mathias & Röbbelen (1991) exposed rooted shoots
of rapeseed to colchicine treatment at the stage of sterile culture, using liquid
medium B5 with the addition of colchicine. Then they removed the liquid medium and replaced it with a standard medium. A method, which has been frequently used for chromosome doubling of corn plants is the submerging them in
a solution with colchicine and with the addition of DMSO, when they were in
the phase of having three shoots and after cutting the roots to the length of 5 cm
(Adamski 1979, Thomas & Pickering 1979, Devaux 1988, Taira et al. 1991).
Wan et al. (1989) used colchicine to treat haploid callus of maize, obtained in
the anther culture. They treated the callus for 72 hrs and obtained mostly
diploids, whereas they did not obtain any diploid plants from the callus, which
was not treated with colchicine. In experiments with Brussels sprouts
(Krzyżanowska & Górecka 2004) and with head cabbage (described above),
colchicine was used to treat the apical meristem of the shoots, the whole plants
in flasks and out of flasks - during replantation to bigger pots. The technique
used by Nałęczyńska (1991) had in presented work a drawback - a high mortality of the shoots. A high mortality of the plant material in this experiments was
observed, regardless of the colchicine treatment method.
The necessity of chromosome duplication prolongs the breeding time of
DH lines. Nałęczyńska (1991) obtained over 40% of spontaneously doubled
haploids in her experiment on androgenetic plants of rapeseed and suggested
reducing the process of colchicine treatment to the cases, when the efficiency of
androgenesis for the tested genotype is low or the ratio of haploids to diploids is
high. Adamus (1998) obtained 50.8% of androgenetic plants of head cabbage
having diploid number of chromosomes. In the work presented here it is concluded that the spontaneous diploidization in head cabbage occurs at even a
higher rate, which is why the thesis was supported that the doubling of the
number of chromosomes can be reduced to the cases mentioned by Nałęczyńska
(1991) and presented above. The approach adapted by Möllers et al. (1994)
seems to be very interesting as well. In their experiments, they added colchicine
to the medium initiating androgenesis directly after the isolation of the microspores of rapeseed. They observed a beneficial influence of colchicine on the
efficiency of androgenesis and on the regeneration process. They obtained 8090% of diploid embryos.
The assessment and selection of androgenetic plants can be done in different ways. Nałęczyńska (1991) eliminated all DH lines of rapeseed, character-
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ized by cower vigour, long vegetation cycle, short and badly filled siliques and
small seeds, from any further investigations. Dore & Boulidard (1988) were
observing the vigour, vegetative development, the thickness of leaves, the size
of flowers and the vitality of pollen during the flowering phase of the androgenetic plants of head cabbage. In the work presented here, the plants of head
cabbage characterized by diminished vigour were eliminated in the seedling
phase. Some observations were made concerning morphological features of
plants in the flowering stage in order to choose DH lines with features most
characteristic for the cultivar tested. The morphology of flowers in order to
determine androsterility and androfertility was observed.
CONCLUSIONS
1. The influence of genotype on the regeneration of the plants obtained from
androgenetic embryos was proved.
2. The B5 medium containing 20 g.L-1 of sucrose and 20 mg.L-1 of kinetine
proved to be the best medium for plant regeneration of head cabbage embryos obtained in anther culture
3. The method of soaking the roots of young plants when they had 5-6 leaves
in a 0.1% hydrous solution of colchicine with the addition of 4% DMSO
and 25 ppm GA3, for 20 hrs was the most effective method of doubling the
number of chromosomes
4. The tested cultivars of head cabbage differed significantly with regard to the
vigour of the plants obtained from anther culture.The highest number of
plants characterized by strong vigour was observed in the cultivar Langendijker.
5. The higher number of androgenetic androfertile plants was found in the
cultivar Sława z Enkhuizen.
6. About 90% of the plants of head cabbage obtained from androgenetic embryos had typical leaves, and the differences between the cultivars in this
respect were inconsiderable
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REGENERACJA I OCENA ANDROGENETYCZNYCH ROŚLIN
KAPUSTY GŁOWIASTEJ (BRASSICA OLERACEA VAR. CAPITATA L.)
Streszczenie
Przedmiotem badań były: regeneracja roślin kapusty głowiastej z zarodków
otrzymanych w kulturach pylnikowych, wybór efektywnego sposobu kolchicynowania
w celu podwojenia chromosomów u roślin haploidalnych oraz ocena podwojonych
haploidów pod względem wigoru, typowości liści oraz ich płodności. Pożywka B5
zawierająca 20 g.L-1 sacharozy i 20 mg.L-1 kinetyny okazała się najlepsza do regeneracji
roślin z androgenetycznych zarodków kapusty głowiastej. Na tej pożywce największa
liczba zarodków wytwarzała pędy, w kolejnych pasażach po kilka z jednego zarodka.
Najbardziej efektywną metodą podwajania chromosomów okazało się moczenie przez
20 godzin korzeni młodych roślin w fazie 5-6 liści w 0,1% wodnym roztworze kolchicyny z dodatkiem 4,0% DMSO i 25 ppm GA3. Wystąpiły wyraźne różnice między badanymi odmianami kapusty głowiastej pod względem wigoru roślin otrzymanych w
kulturach pylnikowych. Najwięcej roślin o silnym wigorze obserwowano wśród roślin
androgenetycznych odmiany Langendijker. Około 90% roślin kapusty głowiastej uzyskanych w kulturach pylnikowych miało typowe liście, różnice między odmianami pod
tym względem były nieznaczne. Najwyższą liczbę roślin męskopłodnych stwierdzono
wśród roślin androgenetycznych odmiany Sława z Enkhuizen. Genotyp okazał się
czynnikiem wpływającym na badane procesy: zdolność zarodków androgenetycznych
do tworzenia pędów, ich ukorzenianie, wrażliwość na działanie kolchicyny, wigor i
płodność uzyskanych podwojonych haploidów.
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Download Date | 6/18/17 3:17 PM