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TOM XXXI
ROŚLINY OLEISTE – OILSEED CROPS
2010
Katarzyna Sosnowska, Laurencja Szała, Anna Olejnik, Teresa Cegielska-Taras
Plant Breeding and Acclimatization Institute, National Research Institute — Poznań
Instytut Hodowli i Aklimatyzacji Roślin – PIB, Oddział w Poznaniu
Preliminary study on resynthesis
of winter oilseed rape (Brassica napus L.)
Wstępne badania nad resyntezą rzepaku ozimego (Brassica napus L.)
Key words:
Brassica napus L., resynthesis, RAPD markers, interspecific hybridization, in
vitro culture, genetic distance
One of the most widely used methods for resynthesis of rapeseed is crossing the selected
subspecies of Brassica rapa L. and Brassica oleracea L. and obtaining plants through in vitro culture
of isolated embryos in the early stage of their development.
In this study resynthesized rapeseed (RS) was obtained as a result of crosses between Brassica
rapa ssp. chinensis var. chinensis (Chinese cabbage – pak choy), and Brassica oleracea ssp. acephala
var. sabellica (curly kale). Morphological and nuclear DNA cytometric analyses of obtained plants
has confirmed their hybrid phenotype and genotype. Molecular analysis carried out using 20 RAPD
primers showed that resynthesized plants are distinct from Brassica napus DH lines and from
cultivars of winter oilseed rape which are bred and cultivated nowadays.
Słowa kluczowe:
Brassica napus L., resynteza, markery RAPD, krzyżowanie międzygatunkowe,
kultury in vitro, dystans genetyczny
Jedną z najczęściej wykorzystywanych metod resyntezy rzepaku jest krzyżowanie wybranych
podgatunków Brassica rapa L. i Brassica oleracea L.oraz uzyskanie roślin poprzez kultury in vitro
izolowanych zarodków we wczesnym stadium ich rozwoju.
Celem pracy było uzyskanie resyntetyzowanego rzepaku ozimego wykazującego odrębność
genetyczną od linii i odmian rzepaku obecnie hodowanego i uprawianego.
W prezentowanych badaniach resyntetyzowany rzepak (RS) otrzymano w wyniku krzyżowania
Brassica rapa ssp. chinensis var. chinensis (kapusta chińska – pak choy) i Brassica oleracea ssp.
acephala var. sabellica (jarmuż). W wyniku krzyżowania międzygatunkowego uzyskano 294 zarodki
we wczesnym stadium rozwoju i wyłożono je na pożywkę MS. Otrzymane rośliny jaryzowano.
Wydajność uzyskania roślin resyntetyzowanego rzepaku w stosunku do wyłożonych zarodków
wynosiła 10%. Zawartość jądrowego DNA roślin RS analizowano cytometrycznie w celu potwierdzenia ich mieszańcowego charakteru. Wszystkie analizowane mieszańce okazały się allodiploidami
(2n). Ocena morfologiczna i cytometryczna jądrowego DNA otrzymanych roślin potwierdziła ich
mieszańcowy fenotyp i genotyp. Analiza molekularna przeprowadzona przy wykorzystaniu 20 starterów RAPD wykazała odrębność resyntetyzowanych roślin Brassica napus od linii DH i odmian
rzepaku ozimego podwójnie ulepszonego, obecnie hodowanego i uprawianego. Resyntetyzowny
gatunek Brassica napus zapewne zwiększy obszar genetycznej zmienności. Biorąc pod uwagę
zmienność gatunków podstawowych (Brassica rapa i Brassica oleracea) można się spodziewać, że
linie RS Brassica napus przyczynią się do oczekiwanego postępu w hodowli rzepaku.
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Introduction
Oilseed rape (Brassica napus L.) with the AACC genome (2n = 38) is a natural
amfidiploid which is the result of spontaneous hybridization between diploid
species Brassica rapa (2n = 20, AA genome) and Brassica oleracea (2n = 18, CC
genome). Geographical restriction of oilseed rape cultivation combined with
intensive quality breeding (double-low varieties, 00), caused significant limits in
the genetic pool of this species. However, both progenitor species: Brassica rapa
and Brassica oleracea exhibit high polymorphic features, and therefore can be
a source of genetic variation and may be used for further improvements in oilseed
rape by distant crosses (resynthesis). In order to obtain the resynthetic oilseed rape
(RS) selected subspecies of Brassica rapa and Brassica oleracea are crossed
between each other, and hybrid plants are obtained by in vitro culture of isolated
embryos. An alternative way to obtain interspecific hybrids is a fusion of protoplasts
(Prakash and Raut 1983).
Many intergeneric and interspecific hybridizations within seed crops fail due
to the inability of pollen germination on another stigma or too short pollen tubes do
not reach the germ or cracking of pollen tubes takes place. To overcome these
barriers the technique of in vitro culture of ovary and embryos at an early stage of
development has been developed (Zenkteler and Guzowska 1967).
In addition, in cross-breeding of genetically distant plants there are prezygetic
(no pollen germination, short pollen tubes not reaching the germ) and postzygotic
(death of embryos) barriers. Olsson, Ellerström (1980) and Wojciechowski (1985)
shown that the most common cause of death of embryos within the Brassica is
incomplete development of the endosperm. Wojciechowski (1985) also showed
that the reason for the decline of embryos developed from the crosses of Brassica
rapa and Brassica oleracea was excessive overgrowth of somatic tissue, which
resulted in the inhibition of nutrient inputs to the developing embryos. In such
cases in vitro culture of ovary or embryo in its early stages appeared to be useful to
obtain interspecific hybrids.
Progeny generations are obtained more easily when species with a similar
gene pool are crossed, but if species are genetically distant the percentage of
obtained hybrid plants is generally low. Olsson (1960), Hoffmann and Peters
(1958) thought that crosses within the genus Brassica sp. occur more frequently
when the maternal parent contains more chromosomes than a paternal.
Resynthesis of Brassica napus, using a wide gene pool of the parents, provides
a potential opportunity to widen the genetic variability of this species. It also
allows the transfer of various desirable traits, such as resistance to diseases, pests,
or male sterility (Snowdon et al. 2007). Currently resynthesis of oilseed rape is
widely used in yellow seeded oilseed rape breeding, because normally this feature
does not occur in Brassica napus (Liu et al. 1991). Therefore it seems possible that
Preliminary study on resynthesis of winter oilseed rape ...
259
crossing between progenitor species with yellow or brown seeds will cause
introgression of this trait (Zaman 1988). Yellow seeded oilseed rape lines are
characterized by a thinner seed coats, which is a source of fiber in the seeds, and
tend to have higher fat and protein content than brown or black seeds (Ochodzki
and Piotrowska 2002). The crossing of yellow seeded Brassica rapa and bright
seeded Brassica oleracea is one of the ways to obtain yellow seeded hybrid of
Brassica napus. So far, studies have led to only a few stable forms of yellow
seeded oilseed rape (Chen et al. 1988, Liu et al. 1991, Wen et al. 2008). Resynthesis
of oilseed rape by crossing the parent forms with the desired characteristics has
also enabled the introduction of the trait of earliness of this species (Prakash
and Raut 1983, Akbar 1987), so that oilseed rape can develop without delay in
flowering in the subtropical zone.
There are also more and more studies on the changes in the hybrid (resynthesized)
genome at the molecular level (Li et al. 2007). As indicated by Gaeta et al. (2007),
the exchange between homologous chromosomes is the most important factor in
creating new combinations of alleles and phenotypic variation in resynthesized
poliploids of Brassica napus.
In addition, the resynthesis of hybrids complements the information about the
evolution of species and verifies a taxonomic divisions (Malepszy et al. 1989).
The aim of this research was to obtain resynthesized oilseed rape which is
genetically distinct from lines and cultivars of Brassica napus bred and cultivated
nowadays.
Materials and methods
For the resynthesis of oilseed rape genetically distant species Brassica rapa
ssp chinensis var. chinensis (Chinese cabbage – pak choy), and Brassica oleracea
ssp. acephala var. sabellica (curly kale) have been selected. Crossing B. rapa ×
B. oleracea was performed by hand. After that the 3-week embryos were transferred
on MS medium (Murashige and Skoog 1962) with 3% sucrose and were carried out
in in vitro culture. After the explants had turned green, they were transferred onto
MS medium with kinetin (10-4M) and with 2% sucrose. Regenerated shoots were
rooted on MS medium supplemented with 2% sucrose and 10 mg/l IBA (Cegielska-Taras and Szała 1997). After the transfer to soil young hybrid plants were put
through the nuclear DNA content analysis with the use of flow cytometer. The
plants at 4–6 leaf stadium were vernalized in short day conditions in 4oC for
7 weeks. After that time the number of chromosomes of hybrid plants was doubled
by immersing of the roots in colchicines solution (0.05%) for 24 hours. After
careful rinsing of roots the plants were transferred back into soil. Further
vegetation took place in the greenhouse. Cellophane insolators were imposed on
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Katarzyna Sosnowska ...
inflorescences for self pollination of plants (allotetraploids). Fatty acid content and
glucosinolates composition were analyzed in the collected seeds.
From the leaves of the first five resynthetized, parental plants and genetically
diverse DH lines and two cultivars of winter oilseed rape, DNA was isolated
applying the modified method of Doyle and Doyle (1990). In the research two
cultivars of double low oilseed rape: Monolit and Cabriolet and seven DH lines
with different levels of erucic acid in the oil and glucosinolates in meal were used.
The analysis of polymorphism of examined genotypes was performed using
20 selected RAPD molecular primers from Operon Technologies. DNA amplification
products were separated in 1.8% agarose gel in TBE buffer, at a current of 80 mA
and then stained with ethidium bromide. Primers were selected on the basis of
previously performed DNA analysis of genetically diverse lines and cultivars
of winter oilseed rape. To prove the genetic diversity of resynthetic oilseed rape
plants only those RAPD primers were selected which generated a small number
(2–6) of polymorphic amplification products in PCR.
The value of genetic distance was calculated using the GENDIST program,
according to the formula developed by Nei (Nei 1972).
Results
For the resynthesis of winter oilseed rape genetically distant species Brassica
rapa ssp. chinensis var. chinensis (Chinese cabbage – pak choy), and Brassica
oleracea ssp. acephala var. sabellica (curly kale) have been selected. Chinese
cabbage originating from north China is not a headed form and exhibits high
tolerance to low temperatures (up to 4°C), and curly kale is characterized by high
winter hardiness, it is resistant to several diseases which occur within the genus
Brassica and has low soil requirements.
As a result of interspecific crosses 294 embryos were obtained, and at an early
stage of development were cultured on MS solid medium. After several weeks,
the explants were transferred to MS medium with 2% sucrose, initially with the
addition of kinetin to ensure the proper development of shoots, and then with the
addition of indolilobutyric acid (IBA) for the proper development of the roots.
Regenerated plants were vernalized.
The efficiency of obtaining of resynthetic oilseed rape plants calculated in
relation to cultured embryos was 10% (Table 1). RS plants were analyzed cytometrically
for nuclear DNA content to confirm their hybrid nature. All analyzed hybrids
turned out to be allodiploids (2n), (Fig. 1), therefore the number of chromosomes
of resynthetized oilseed rape plants was doubled using colchicine. Further
development of the plants took place in a greenhouse.
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Preliminary study on resynthesis of winter oilseed rape ...
Table 1
Results of hybridization among B. rapa × B. oleracea species by means of embryos rescue
in vitro culture — Wynik krzyżowania międzygatunkowego B. rapa × B. oleracea przy
wykorzystaniu kultur in vitro izolowanych zarodków we wczesnym stadium rozwoju
Series — Serie
I
II
III
IV
V
VI
Total
Razem
No of embryos — Liczba zarodków
34
25
135
53
17
30
294
No of hybrid plants
Liczba roślin mieszańcowych
4
6
8
5
2
4
29
Efficiency — Wydajność [%]
≈10
Brassica rapa
Brassica oleracea
B. rapa × B. oleracea
Fig. 1. Cytometric analysis of nuclear DNA in parental forms Brassica rapa i Brassica
oleracea and hybrid B. rapa × B. oleracea (before doubling of chromosones) — Analiza
cytometryczna jądrowego DNA form rodzicielskich: Brassica rapa i Brassica oleracea oraz
mieszańca B. rapa × B. oleracea (przed kolchicynowaniem)
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Katarzyna Sosnowska ...
Pollination of hybrid plants by its own pollen was hand-assisted. The
collected seeds of RS oilseed rape were analyzed on fatty acids composition and
glucosinolate content. The average content of erucic acid in total fatty acid content
of oil was 46.8%, and total glucosinolates in the seeds — 66.3 µmol/g d.m.
The evaluation of morphological features and cytometrical analysis of nuclear
DNA of plants obtained from interspecific crosses between Brassica rapa ssp.
chinensis var. chinensis × Brassica oleracea ssp. acephala var. sabellica confirmed
their hybrid phenotype and genotype.
Molecular analysis carried out using 20 RAPD primers clearly demonstrated
that resynthetized Brassica napus plants derived from crosses between Chinese
cabbage and curly kale are distinct from the DH lines and varieties of oilseed rape
cultivated nowadays. Sample image separation of DNA amplification products
using primer OPG-11 in agarose gel electrophoresis is shown in Figure 2. Genetic
distance value was calculated using the GENDIST program with a formula of Nei
(1972). The greatest genetic distance was found between B.chiniensis and
B.oleracea — 1.2669 and the lowest among interspecies hybrids RS-1 and RS-4 —
0.0358. Graphic relationship tree was drawn using the Ward method in
STATISTICA program. Created dendrogram based on 284 RAPD markers divided
tested genotypes into two main groups (Fig. 3). The first group is the Chinese
cabbage and curly kale and hybrids obtained from them, and the other — the
variety of "00" type and DH lines of winter oilseed rape.
1
2
3
1 — marker
12 — Monolit
4
5
6
7
8
2 — B. rapa
13 — Cabriolet
9
10 11 12 13 14
15 16 17
3 — B. oleracea
14–20 — linie DH EG
18
19
20 21
4–11 — linie RS
21 — marker
Fig. 2. DNA amplification products using primer OPG-11 for B.rapa parent, B.oleracea
parent, hybrid plants Brassica napus cultivars and breeding lines — Produkty amplifikacji
DNA przy użyciu startera OPG-11 form rodzicielskich (B. rapa, B. oleracea), roślin
mieszańcowych RS, odmian i linii hodowlanych B. napus
Preliminary study on resynthesis of winter oilseed rape ...
263
Discussion
The most commonly used subspecies for interspecific croning of the species
Brassica rapa L. ssp. oleifera are pekinensis, Chinensis, rapifera; and of the species
Brassica oleracea L: ssp capitata, acephala, italica, alboglabra (Seyis et al. 2005,
Zhang and Zhou 2006). In the present study an objective was to produce the RS
winter genotypes, therefore species characterized by tolerance to low temperatures
have been selected. In many publications, the methodology of obtaining interspecific
hybrids is similar. The main difference concerns the stage of development at which
the structure formed after pollination ovaries or embryos at early stage of development
are transferred to regeneration medium (Wojciechowski 1985, Seyis et al. 2005,
Wen et al. 2008). In the presented study the embryos created 21 days after
pollination were carried out in in vitro cultures. The medium in the first phase
of embryo growth was not supplemented with casein hydrolysates, or coconut milk
as proposed by Inomata (1978).
Fig. 3. Dendrogram genetic distance among B. rapa parent, B. oleracea parent, hybrid
plants (RS) Brassica napus and cultivars Monolit, Cabriolet and DH lines using 284 RAPD
markers — Produkty amplifikacji DNA przy użyciu startera OPG-11 form rodzicielskich
(B. rapa, B. oleracea), roślin mieszańcowych RS, odmian i linii hodowlanych B. napus.
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Katarzyna Sosnowska ...
For regeneration of shoots and roots previously developed method for obtaining
androgenic plants of Brassica napus was used. Oilseed rape is characterized by good
reaction to a high concentration of kinetin in the course of shoots regeneration
(Cegielska-Taras and Szała 1997). Efficiency of plants obtained in relation to the
cultured embryos was comparable with other reports. It is believed that the fewest
hybrids are created when the mother is Brassica oleracea (Stewart 2004).
However, Takeshita et al. (1980) showed that obtaining interspecific hybrids
is more likely to succeed in the culture of embryos when Brassica oleracea
is mother.
As expected, the RS lines obtained in the presented studies were characterized
by a high content of erucic acid in oil and high glucosinolate content in seed
because the phenotype of a hybrid depends on the quality of parental forms. Seyis
et al.’s research (2005) confirms that the use of species characterized by a low
content of erucic acid to the resynthesis results in almost zero erucic acid RS
genotypes of Brassic napus.
Resynthesized Brassica napus extends the range of genetic variability of this
species. Considering the variation in parental species (Brassica rapa and Brassica
oleracea) we can expect that resynthesized Brassic napus will meet some demands
of the advanced oilseed rape breeding.
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