Effects of cell type used for fusion on chromosome elimination and chloroplast segregation in Brassica oleracea (+) Brassica napus hybrids

Plant Science. 78 (1991) 89-98

89

Elsevier Scientific Publishers Ireland Ltd.

Effects of cell type used for fusion on chromosome elimination and chloroplast segregation in Brassica oleracea (+) Brassica napus hybrids E. Sundberg, U. Lagercrantz and K. Glimelius Department o f Plant Breeding, Swedish University of Agricultural Sciences, Uppsala (Sweden)

(Received March 8th, 1991; revision received April 30th, 1991; accepted April 30th, 1991) To determine how the developmental and physiological characteristics of parental cells can influence the fate of chromosomes and chloroplasts in somatic hybrids, fusions were made between Brassica napus and B. oleracea using hypocotyl and/or mesophyll protoplasts in four different combinations. Chromosome number varied greatly among the hybrids obtained. Most hybrids (58%) had more chromosomes than the parental sum, 32% had a DNA-content corresponding to the sum, and only 10% had fewer chromosomes than the sum. The frequencies of symmetric hybrids and chromosome eliminating hybrids obtained in fusions performed by combining mesophyll and hypocotyl protoplasts did not differ significantly from those obtained by combining protoplasts from the same cell type. However, the frequency of hybrids with a DNA-content corresponding to one genome from one of the parental species and two from the other and the frequency of hybrids having the reciprocal combination of three genomes differed significantly between fusion categories. Of 128 hybrids examined, 36 (28%) had B. oleroe,', ,-hloroptastsand 92(72~,0 had Br napus chloroplasts. The pattern of chloroplast segregation did not differ significantly between fusion categories. Key words: Brassica; somatic hybridization; proplastids and chloroplasts; cell cycle asynchrony; differentiation; mesophyll and

hypocotyl protoplasts

Introduction S o m a t i c h y b r i d i z a t i o n is a v a l u a b l e c o m p l e m e n t to c o n v e n t i o n a l p l a n t breeding, a l l o w i n g new genetic m a t e r i a l to be i n t r o d u c e d into cultivated species. H o w e v e r , large v a r i a t i o n s in c h r o m o s o m e n u m b e r and in the o u t c o m e o f o r g a n e l l e segregation have been f o u n d a m o n g interspecific s o m a t i c h y b r i d s (reviewed by Glimelius, Ref. 1). T h e degree o f genetic divergence between the h y b r i d ized species a p p e a r s to affect the rate o f c h r o m o s o m e e l i m i n a t i o n [2] as well as the segregation o f organelles [2-4]. O t h e r factors t h a t also might affect the genetic c o n s t i t u t i o n o f the h y b r i d s include tissue-specific differences related to to: E. Sundberg, Department of Plant Breeding, Swedish University of Agricultural Sciences, Uppsala, Sweden. Correspondence

physiological c o n d i t i o n s o r the degree o f differentiation o f cell types used for fusion. P r o t o p l a s t s f r o m different tissues are often used as m o r p h o l o g i c a l m a r k e r s , a l l o w i n g the fusion p r o d u c t s to be identified (reviewed by G l e b a a n d S h l u m u k o v , Ref. 5). Besides being m o r p h o l o g i c a l ly different, cell p o p u l a t i o n s derived f r o m different tissues usually differ in their frequency o f actively dividing cells a n d in the p r o p o r t i o n o f cells that arrest in G I o r G 2 o f the cell cycle [6]. These differences m a y result in cell cycle a s y n c h r o n y between the two c o m b i n e d genomes, which in t u r n can lead to the c h r o m o s o m e s o f one o f the genomes being e l i m i n a t e d [7,8]. F u r t h e r m o r e , the n u m b e r o f organelles per p r o t o p l a s t , the m e a n n u m b e r o f g e n o m e s in each organelle at the time o f fusion, a n d the d e v e l o p m e n t a l stage o f the organelles differ between tissues [9] a n d m a y consequently influence organelle segregation.

0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

90 To determine how the developmental and physiological characteristics of parental ceils can influence the fate of chromosomes and chloroplasts, fusions were made between Brassica napus and B. oleracea using hypocotyl and mesophyll protoplasts in the following four combinations: hypocotyl (+) hypocotyl, hypocotyl (+) mesophyll, mesophyll (+) hypocotyi and mesophyll (+) mesophyll. B. napus and B. oleracea were chosen as parental material to avoid incompatibility reactions that could hide the effects of the cell types used. These two species can be combined by sexual crossings [101. Furthermore, in B. napus, which contains the genomes of both B. oleracea and B. campestris [11], some varieties have B. oleracea organelles while others have B. campestris organelles. Thus, it should be possible to discern the effects of the cell type used for fusion on chromosome elimination and chloroplast segregation in somatic hybrids produced between these two species. Materials and Methods

Plant material Brassica napus L. ssp oleifera (2n = 38) cv. Hanna (spring rapeseed) and B. oleracea L. var italica (2n = 18) cv. Green Mountain (Broccoli) were used as fusion partners. Seeds were kindly provided by W. Weibull AB, Landskrona, Sweden. Protoplast isolation Protoplasts were isolated from either chlorophyll-containing 5-day-old hypocotyis, etiolated 5-day-old hypocotyls or leaves of 4-weekold plants cultured in vitro. Plant material was cultured and protoplasts were isolated according to Glimelius [12] and Sj6din and Glimelius [131. Staining of protoplasts prior to fusion Hypocotyl (hyp) and mesophyll (mes) protoplasts were fused in four different combinations (Table I). Chlorophyll autofluorescence and 5(6)carboxyfluorescein diacetate (CFDA, Molecular Probes) were employed as markers for selection of fusion products using flow cytometry and cell sorting in the hyp (+) hyp, hyp (+) mes and mes (+) hyp fusion experiments. Prior to fusion, the white

Table I. Protoplasts isolated from either hypocotyl or

mesophyll tissues, were combined in the followingways. B. oleracea

(+)

Cell type Fluorescent marker Hypa Hyp Mesb Mes

CFDAc CFDA chl.d CFDA

B. napus Cell type Fluorescent marker

(+) (+) (+) (+)

hyp rues hyp rues

chl. chl. CFDA scopoletin

aHyp, hypocotyl protoplasts. bMes, mesophyll protoplasts. CCFDA, 5(6)-carboxyfluoresceindiacetate. dchlorophyll autofluorescence.

hypocotyl protoplasts were stained with CFDA for 10 min according to Sj6din and Glimelius [14]. In the mes (+) mes fusion experiments, one of the fusion partners was stained with CFDA and the other with scopoletin (Sigma) according to Kanchanapoom et al. [15] with some modifications. After mesophyll tissue had been treated with cell wall degrading enzymes for 16 h, the protoplasts were filtered through a 100-/~m nylon mesh and stained with scopoletin while still in enzyme solution. A stock solution, containing 1 mg scopoletin/ml W5 was heated to about 90°C, to dissolve the dye, and thereafter cooled to 45°C. One milliliter of stock solution was added to 10 ml enzyme solution containing about 5 x 10 6 mesophyll protoplasts. The protoplasts were then incubated at 30°C for 30 min in darkness and thereafter washed according to the ordinary protoplast isolation procedure. Fusion and selection of hybrid cells Protoplasts were fused using polyethylene glycol as described by Sundberg and Glimelius [16], but with one modification i. e. the protoplasts were suspended in W5 [171 prior to fusion. In the mes (+) mes and hyp (+) hyp fusion experiments protoplasts from the two fusion partners were mixed in equal proportions (I:1) while hypocotyl and mesophyll protoplasts in the hyp (+) mes and mes (+) hyp fusion experiments were mixed in a 2:1 proportion prior to fusion. To test whether the proportion of cells from the two fusion partners

91 affects the ploidy-level distribution of the hybrid plants the mes (+) hyp fusion combination was also made using equal proportions (1:1) of hypocotyl and mesophyll cells. After fusion and washing, a solution with a modified [18] 8p culture medium [19] with 0.4 M mannitol and W5 (1:1 v/v) was added, and the cultures were stored at +8°C in darkness for at least 2 h. Before selection of the heterokaryons using flow cytometry and cell sorting, the protoplasts were detached from the Petri dish, resuspended in W5 and centrifuged at 75 x g for 5 min. The protoplast pellet was suspended in the 8p medium containing 0.4 M mannitol and diluted to a concentration of about 1 x 10 6 protoplasts/ml. Fusion products were selected with flow sorting according to Glimelius et al. [18] with some modifications. To detect C F D A emissions an argon laser was adjusted to emit 500 mW at the 488 nm line. Fluorescence emission was measured through a 530 nm band pass filter. To detect scopoletin emission from the second fluorescent dye used in the mes (+) mes fusion experiments, a second argon laser was adjusted to 100 mW at the combined 355 and 364 nm lines. Fluorescence emission was measured through a 425 nm band pass filter.

Culture of hybrid cells Fusion products were sorted into separate wells of a multivial dish (Costar R 48 wells) containing 100 /A of the modified 8p medium with 4.5 t~M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 /~M l-naphtylacetic acid (NAA) and 2.2 tzM 6-benzylaminopurine (BAP). After about 4 - 7 days or immediately after the first divisions were obtained, the cells were diluted with fresh culture medium, containing 0.55% Sea Plaque agarose (FMC R) without hormones, to 4 times the original volume. The protoplasts consequently became embedded in the medium which contained 1.1 /~M 2,4-D, 0.55 /~M BAP and 0.13 /zM NAA. After gelling, beads with the embedded protoplasts were placed in 2.5 cm Petri dishes with 3 ml of liquid 8p medium containing the same hormone concentrations as the beads. At weekly intervals the medium was discarded and replaced with fresh medium. After 2 - 4 weeks, small calli (1-2 mm in diameter) were plated on K3 medium [20] containing 0.1 M

sucrose, 0.13% agarose and the same hormone concentration as the beads. Two weeks later, the calli were transferred to K3 medium with 0.015 M sucrose, 0.4"/,, agarose, 0.6/~M indole-3-acetic acid (IAA), 2.2/~M BAP and 2.3 I~M zeatin to induce differentiation. Calli were transferred to fresh differentiation medium after 3 weeks. Light and temperature conditions and procedures used to culture the regenerated shoots were identical to those described by Sj6din and Glimelius [13].

Confirmation of hybrid character by isoenzyme and RFLP analyses The hybrid nature of the regenerated plants was determined by isoenzyme and restriction fragment length polymorphism (RFLP) analyses. Isoenzyme analyses were performed according to Sundberg and Glimelius [16]. The enzymes examined were leucine aminopeptidase (LAP), phosphoglucose isomerase (PGI), 6-phosphogluconate dehydrogenase (6-P) and phosphoglucomutase (PGM). The only one of the four enzymes to differ distinctly in its isozyme pattern between B. napus and B. oleracea was LAP. For some genotypes PG! also differed distinctly. Two probes were used for RFLP-analyses: a 570-bp Hindlll-Xhol fragment o f a napin gene [21] and a 1800-bp Pvull fragment of a cruciferin gene [22]. Total-DNA was isolated from the hybrids according to a modified [23] protocol of Bernatzky and Tanksley [24]. The Southern blot analyses were made according to Sambrook et al. [25]. Analysis of nuclear DNA-content Measurements of nuclear DNA-content using flow cytometry is highly correlated (r = 0.9) with chromosome number in somatic hybrids within Brassicaceae [26] and was used in the present investigation as an estimate of ploidy level. The nuclear DNA-content of the hybrids was determined in a flow cytometer as described by Fahleson et al. [26]. Leaf protoplasts were isolated from each hybrid plant as described by Glimelius [12], and the cell nuclei of the protoplasts were prepared and stained with propidium iodide according to VindeiCv et al. [27]. To standardize the DNA-axis two reference standards, human lymphocytes and protoplasts of B. campestris, were added to each

92 Table II. Summaryof results from the fusion experiments performed.

B oleracea

(+)

B. napus Proportion No. of of protofusion plasts experiments

Hyp (+) Hyp Hyp (+) Mes Mes (+) Hyp Mes (+) Hyp Mes (+) Mes Sum of all fusion categories

1:1 2:1 1:2 I:I l:l

1 3 1 4 6 15

No. of calli

No. of Frequency calli that of calli that regenerated regenerated shoots shoots (%)

No. of No. of Hybrid plants nuclear frequency obtained markers t%)

187 902 226 nd a 1097 -

34 70 30 nd 104 -

23 41 26 40 70 200

18 8 13 nd 9 -

4 3 3 I l -

91 95 88 90 96 93

and, not determined.

preparation. Aliquots of the lysate, corresponding to 104-105 nuclei, were analysed with flow cytometry as described by Fahleson et ai. [26] except that another instrument, a FACS Star Plus, was used. The DNA-content was determined as pg DNA/nucleus [26].

about 0.5-1 day was noted in fusions combining mesophyll protoplasts of both parents. The frequency ofcalli that regenerated shoots varied from 8 to 18% between the different fusion categories (Table II). In total, 200 plants were transferred to the greenhouse.

Analysis o f chloroplast genotype For chloroplast-DNA analysis, total-DNA was isolated from the hybrids as described above, digested with BamHI and resolved by electrophoresis. Chloroplast-DNA restriction fragments could be distinguished from the nuclear smear on total-DNA gels stained with ethidium bromide. Purified samples of chioroplast-DNA of the parental species, isolated according to Sundberg et al. [28], were used as standards.

Confirmation o f hybrid character O f the 200 plants obtained, 186 showed a hybrid pattern for at least one of the nuclear markers used (Figs. la,b). O f the remaining 14 plants, 12 showed a B. napus pattern for all markers and two showed a B. oleracea pattern. No significant difference (X 2 = 2.6, d.f. = 4, P = 0.6) in hybrid frequency was revealed between the different fusion categories (Table II). O f 67 hybrid plants analysed using three nuclear markers, 54 (80%) showed a hybrid pattern for all markers, 12 (18%) had lost one or two of the markers from B. oleracea and one (2%) had lost one marker from B. napus.

Seed set Seed set for each hybrid was recorded after selfpollination and backcrossing to B. napus. For each hybrid, 100 flowers were self-pollinated, and 50 flowers were fertilized with pollen from B. napus. Fertility was expressed as the number of seeds obtained per pollinated flower. Results

Cell culture and shoot regeneration Within 5 - 6 days after fusion and sorting, more than 50% of the hybrid protoplasts from the hyp (+) hyp, hyp (+) mes and mes (+) hyp fusions had divided. A further delay in the first divisions of

Ploidy level The DNA-content analyses of the hybrid plants revealed considerable variation in ploidy level between the plants in each fusion category (Fig. 2). Hybrids were sorted into different classes having a DNA-content corresponding to 56 (sum of the parental chromosomes), 74 (sum of two B. oleracea genomes and one B. napus genome) and 94 (sum of one B. oleracea genome and two B. napus genomes) chromosomes, respectively (Table III). A hybrid was included in a given class if it had a D N A content within -4-6.9% of that class (cor-

93

a

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2

3

4

5

6

7

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2

3

4

5

6

kb 21

-5.1 -4.3

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b 1

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Fig. I. (a) TotaI-DNA from five putative hybrids and their parents was isolated, digested with EcoRV and hybridized to a DNA probe coding for napin. Lanes 1-4, hybrids; lane 5, recovered plant with B. napus pattern; lane 6, B. napus; lane 7, B. oleracea. Bands unique for B. napus are marked with circles and bands unique for B. oleracea are marked with squares. The molecular weight standard lambda-DNA cut with EcoRI/HindIIl is indicated (kbp). (b) Isozyme pattern for leucine aminopeptidase (LAP). Lane I, a hybrid in which the parental-specific bands are of equal intensity; lanes 2 and 5, hybrids in which the intensity of the B. oleracea-specific bands is twice that of the B. napus-specific bands; lane 3, B. oleracea; lane 4, B. napus. (c) Restriction pattern of totaI-DNA and chloroplast-DNA digested with BamHI. Lane I, total-DNA of B. napus; lane 2, total-DNA of B. oleracea; lanes 3-4, totaI-DNA of two somatic hybrids; lane 5, chloroplast DNA of B. napus; lane 6, chloroplast DNA of B. oleracea. The chloroplast DNA in lanes 5 and 6 was run on a separate gel. Bands unique for B. napus are marked with circles and bands unique for B oleracea are marked with squares. The molecular weight standard lambda-DNA cut with EcoRl/Hindlll is indicated (kbp).

r e s p o n d i n g to a 9 5 % c o n f i d e n c e i n t e r v a l , a s s u m i n g a s t a n d a r d d e v i a t i o n o f 3.5%). P l a n t s n o t f a l l i n g i n t o a n y o f t h e s e classes a n e u p l o i d s . V e r y few p l a n t s that had fewer chromosomes p a r e n t a l c h r o m o s o m e s (Fig.

w e r e c o n s i d e r e d as (10%) w e r e o b t a i n e d t h a n t h e s u m o f the 2a). O f t h e h y b r i d s

w i t h initially o n e g e n o m e f r o m e a c h p a r e n t the proportion o f h y b r i d s w i t h f e w e r t h a n 56 c h r o m o s o m e s r a n g e d b e t w e e n 16 a n d 33% (Figs. 2 b - 2 e ) w i t h n o statistically s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e f u s i o n c a t e g o r i e s (X 2 = 1.5, d.f. = 4, P = 0.8). T h i r t y h y b r i d s w e r e o b t a i n e d w i t h 56

94

~2.

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.

All fusion categories

~

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D N A content (pg)

Table

chromosomes and 40 had 74 chromosomes, whereas only 12 plants had 94 chromosomes (Fig 2a). The frequency distribution of ploidy classes (56, 74 and 94 chromosomes and aneuploids, Table IV) differed significantly between the fusion categories (X 2 = 22.3 d.f. = 12, P = 0.03). However, no major category-related differences were found in the proportion of symmetric hybrids (X 2 = 3.7, d.f. = 4, P = 0.4). Thus, the statistically significant relation between fusion categories and D N A content was due primarily to differences in the relative proportions of hybrids with 74 chromosomes, hybrids with 94 chromosomes and aneuptoid hybrids (X 2 = 17.8, d.f. = 8, P = 0.02) or mainly to differences in the relative proportions of hybrids with 74 chromosomes and those with 94 chromosomes (X2 = 16.1, d.f. = 4, P = 0.003). O f 14 hybrids with a DNA content corresponding to 74

Ul.

Correspondence between DNA-content and genome number in B, oleracea (+) B. napus hybrids and their parents.

Hybrids/parents

Symmetric Polyploid Polyploid B. oleracea B.napus

Fig. 2. Frequency distribution of ploidy classes for each of the four hybrid types. Hybrids were assigned to different ploidy classes with a DNA-content corresponding to 56, 74 or 94 chromosomes, respectively. A hybrid was included in a given class if it had a DNA-content within ±6.9% of that class (corresponding to a 95% confidence interval). The mean DNAvalue for each class is marked with an arrow. Hybrids not falling into any of these classes were considered aneuploid. Ploidy class 56, black bars; ploidy class 74, striped bars: ploidy class 94, grey bars. (a) Hybrids from all fusion categories. (b) Hybrids from hyp (+) hyp fusions, (c) Hybrids from hyp (+) mes fusions, (d) Hybrids from rues (+) hyp fusions performed after mixing mesophyll and hypocotyl protoplasts 1:2. (e) Hybrids from mes (+) hyp fusions performed after mixing mesophyll and hypocotyl protoplasts 1:1. (f) Hybrids from mes (+) mes fusions.

DNA-content pg

3.30 4.45 5,45 1.15 2.15

Chromosome no.

56 74 94 18 38

No. of genomes from B. oleracea

B. napus

1 2 1 1

1 1 2 1

95 Table IV.

Frequency distribution of ploidy classes for each of the four hybrid types.

B. oleracea

(+)

Hyp Hyp Mes Mes Mes The sum of all

B. napus

(+) Hyp (+) Mes (+) Hyp (+) Hyp (+) Mes fusion categories

Proportions of protoplasts mixed

1:1 2:1 1:2 I:1 1:1

21 35 19 30 52 157

chromosomes, the isozyme pattern of nine was characterized by B. oleracea-specific bands that were twice the intensity of the B. napus-specific bands, indicating that these plants had two genomes from B. oleracea and one from B. napus (Fig. lb). Fertility and morphology Self fertility in the hybrids ranged from 0-21 seeds/pollinated flower. Self fertility in B. napus (cultured in greenhouse as the hybrids) averaged 8 seeds/pollinated flower. The highest fertility was found in hybrids with a DNA-content corresponding to 56 chromosomes or less. Irrespective of the chromosome number of the hybrids, an increase (average 38%) in seed set was generally obtained when the hybrids were fertilized with pollen from B. napus. The morphology of the hybrids varied depending on their ploidy level. The group of hybrids having approximately 56 chromosomes contained the tallest plants (average 101 cm, range 50-140 cm). With increasing chromosome number, plant height at maturity decreased

Table V. B. oleracea

No. of hybrid plants

Frequency (%) of hybrids with DNA-content corresponding to chromosome number 56

74

94

Aneuploid

43 26 21 40 31 32

14 48 16 27 33 31

19 3 26 10 4 10

24 23 37 23 32 28

(average 90 cm, range 50-120 for hybrids with 74 chromosomes and 77 cm, range 40-90 for hybrids with 94 chromosomes). In addition, the plants with nodular leaves and plants with large flowers increased in frequency with increasing chromosome number. The flower and leaf morphologies of the hybrids with a DNA content corresponding to 56 chromosomes were intermediate between those of B. napus and B. oleracea. Chloroplast segregation The chloroplast type in the hybrids was determined by R F L P (Fig. lc). Of the 128 hybrids examined, 36 (28°/,,) had B. oleracea chloroplasts and 92 (72°/,,) had B. napus chloroplasts. No significant differences (X2 = 0.3, d.f. = 3, P = 0.96) in the pattern of chloroplast segregation were found between fusion categories (Table V). Discussion

A large variation in chromosome number was found among the B. oleracea (+) B. napus hybrids

Chloroplast segregation pattern in the different fusion combinations. (+)

B. napus

No. of hybrids

Frequency C/,,) of hybrids with chloroplasts from B. oleracea

Hyp Hyp Mes Mes

(+) (+) (+) (+)

Hyp Mes Hyp Mes

16 39 25 48

31 28 24 29

B. napus

"

69 72 76 71

96 produced in this investigation. Most of the hybrids (58%) had more chromosomes than the sum of the parental chromosomes; 32% had a DNA-content corresponding to the sum, and only 10% had fewer chromosomes than the sum. Of the hybrids that initially contained only one genome from each parent about 20% had eliminated chromosomes. However, the frequency of chromosome eliminating hybrids was similar regardless of whether fusions were performed by combining mesophyll and hypocotyl protoplasts or by combining protoplasts from the same type of cell source. Thus, differences in the frequency of dividing cells and the cell cycle phase in mesophyll and hypocotyl tissues did not seem to affect the sorting out and elimination of chromosomes in the fusion products. Ashmore and Gould [29] found that fusion preferentially occurred between protoplasts in the same cell cycle phase even though fusions were observed in all possible combinations of cells in various cell cycle phases. Furthermore, they only observed nuclear fusions between G1 nuclei. If their results are generally applicable they could explain why the elimination of chromosomes occurred independently of the cell types used for fusion. Even though production of symmetric and partial hybrids was not significantly influenced by the fusion combinations, the frequencies of plants with DNA contents corresponding to 74 and 94 chromosomes, respectively, differed significantly between fusion categories. Since these chromosome numbers represent the numbers obtained from a combination of one genome from one of the parental species and two of the other, these hybrids could have resulted either from the fusion of three protoplasts or from endoreduplication [30] of one of the parental genomes, before or after cell fusion. It is well known that the number of protoplasts in each fusion event is impossible to regulate in a mass fusion system and that multiple fusions occur [31,32]. Endoreduplications are also common in cell culture [33]. Since the first division of mesophyll protoplasts usually starts after the hypocotyl protoplasts have begun dividing [12], this might cause an endoreduplication of the hypocotyl partner after fusion. Species-specific differences in cell cycle time might also cause a

delay in the division of one of the fusion partners, thus causing an endoreduplication of the other. The possibilities of endoreduplications being induced by the tissue culture system cannot be excluded either. Up to 22% of the plants regenerated from B. napus hypocotyl protoplasts had twice their normal chromosome number (Glimelius, unpublished results), indicating that the culture procedure itself may induce the development of polyploid hybrids. Evaluations of the different fusion categories did not reveal any consistent relation between polyploid hybrid development and cell type or species. However, the two mes (+) hyp combinations made, mixing hypocotyl and mesophyll protoplasts in the proportions 1:2 and l:l, respectively, resulted in reversed proportions of plants with 74 and 94 chromosomes. Even though this difference is not statistically significant (X2 = 4.5, d.f. = 3, P = 0.21), it indicates that plants resulting from fusions of more than two protoplasts are quite common. It also indicates that the obtained differences between fusion categories in frequency of plants with DNA contents corresponding to 74 and 94 chromosomes, respectively, is related to differences in proportion of protoplasts mixed from the two parental lines prior to fusion rather than to differences in cell type used. Differences in plastid development are known to exist between the etiolated hypocotyl tissue and the mesophyll tissue used in the present study. In etiolated hypocotyls, the plastids are all proplastids or etioplasts, whereas in young leaf tissues, the plastids are already fully developed chloroplasts [34]. Furthermore, mature leaf cells contain about ten times the number of plastids present in meristematic cells [34,35]. Nevertheless, these differences in physiological conditions between mesophyll and hypocotyl cells did not influence the segregation of chloroplasts in the present investigation. The relative proportions of hybrids containing B. napus and hybrids containing B. oleracea chloroplasts did not differ between fusion combinations. The biased segregation favouring B. napus chloroplasts (69-76% of the hybrids), characterizing all fusion combinations, was probably not due to incompatibility reactions between the B. oleracea chloroplasts and the B.

97

napus nuclei, since plants of B. napus with B. oleracea chloroplasts can be produced without any

negative features [36]. Furthermore, when we fused B. oleracea and B. campestris, in order to resynthesise B. napus, the chloroplasts segregated randomly [2,28]. The biased segregation probably reflects ploidy differences between B. napus and B. oleracea rather than differences in physiological condition between the cell types fused. Nuclear DNA-content and cell size both influence plastid number and chloroplast-DNA content [37]. An increase in the amount of nuclear-DNA results in an increase in the number of plastids. Thus, the biased segregation favouring B. napus chloroplasts might have been due to an unequal input of organelles resulting from the fusion between the allotetraploid B. napus and the diploid B. oleracea or to other genetic differences between the species. In conclusion, we did not find any significant relation between the type of tissue used for isolating protoplasts and (1) the frequency of symmetric plants, (2) the elimination of chromosomes or (3) the segregation of chloroplasts. Thus when producing somatic hybrids it is not possible to control their organellar composition or the frequencies of symmetric hybrids by isolating protoplasts from a specific tissue. In practice, this means that the tissue most suitable for the experimental setup can be used without influencing the frequency of symmetric hybrids produced or organelle segregation.

Acknowledgements We thank G. Sv/ird and G. R6nnkvist for valuable help with the in vitro material and I. Eriksson for help with the RFLP analyses. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research and the Nilsson-Ehle Foundation, Sweden.

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K. Glimelius, Potentials of protoplast fusion in plant breeding programmes, in: K.J. Puite, J.J.M. Dons, H.J. Huizing, A.J. Kool, M. Koornneefand F.A. Krens (Eds.), Progress in Plant Protoplast Research, Kluwer Academic Publishers, 1988, pp. 159-168. E. Sundberg and K. Glimelius, Effects of parental ploidy level and genetic divergence on chromosome elimination

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