Somatic hybridization in Petunia: A male sterile cytoplasmic hybrid

Plant Science Letters, 14 (1979) 49--55 © Elsevier/North-Holland Scientific Publishers Ltd.

49

SOMATIC HYBRIDIZATION IN P E T U N I A : A MALE STERILE CYTOPLASMIC HYBRID S. IZHAR* and J.B. POWER Division of Plant Genetics and Breeding, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel) and the A.R.C. Group, Department o f Botany, University of Nottingham, University Park, Nottingham MG7 2RD (United Kingdom)

(Received June 12th, 1978) (Accepted September 7th, 1978)

SUMMARY

Fusion was induced between leaf mesophyll protoplasts of a cytoplasmic male sterile (cms) and a fertile petunia line. The selection system was designed to allow only the growth of protoplasts possessing the genome of the fertile line. The majority of plants which were regenerated from protoplasts were phenotypically similar to the fertile line. Some of these plants were cytoplasmic hybrids (cybrids), combining the (S) cytoplasm from the male sterile line with the genome of the fertile line.

INTRODUCTION

Several successful cases of somatic hybridization in plants using protoplast fusion have been reported in recent years [ 1]. Perhaps the major significance of somatic hybridization is that it provides a means to obtaining heterozygosity of extra~hromosomal genetic elements (e.g. organelles) in the cytoplasm. The possibility of combining genetic elements from different cytoplasms with specific nuclear genomes in one cell may help resolve general questions of plasmon-genome interaction. It may also yield novel and useful nuclear-cytoplasmic combinations based in p ~ t on rearrangements of the organelle populations in the new hybrid cell. One of the aims of somatic hybridization is the manipulation of genetic elements between cells. However, present techniques have not yet shown to be effective in uptake and subsequent replication and expression of alien cytoplasAbbreviations: BAP, 6-benzylaminopurine; cms, cytoplasmic male sterile; cybrids, cytoplasmic hybrids; 2,4-D, 2,4-dichlorophenoxyacetic acid; M/S, Murashige and Skoog; NAA, naphthaleneacetic acid; PEG, polyethyleneglycol.

50

mic organelles or DNA [2 ]. In the present paper we describe the successful production of cybrids in which male sterility, as a single cytoplasmic marker, was combined with genome of a normal plasmotype, via protoplast fusion. In view of the great economic importance of cms, the methodology which enables quick replacement of the normal plasmon by (S) plasmon may be an i m p o r t ~ t contribution to plant breeding. MATERIALS AND METHODS

The following two lines were used for protoplast isolation and fusion: Petunia hybrida (Hort.) Vilm. line 2426, a cms line with medium-sized purple flowers, short round leaves, bushy growth type, from the collection in the Volcani Center, Bet Dagan, and Petunia axiUaris (Lam) B.S.P. line 2785, male fertile, medium size long white flowers, narrow leaves, erect growth type, from K.C. Sink, Michigan State University, East Lansing, MI. The following lines were used for testcrosses in the genetic analysis; Petunia hybrida (Hort.) Vilm. line 2340. A normal fertile line, which does not contain any male fertility restoration (mfr) alleles [3], and Line 2439, a cms line isonuclear with line 2340. In Nottingham, plants were grown according to Power et al. [4]. In Bet Dagan plants were grown either in an air~onditioned green-house, or in growth chambers (Percival, large walk-in type, Boone, IA, USA). Temperatures and daylength were similar to those in Nottingham. Standard protoplast isolation and fusion procedures were used throughout this study according to Power et al. [4,5]. PEG (M.W. 6000 Koch-Light Co.) was used at two concentrations; 15% in the first and the third experiments and 20% in the second experiment. The protoplast culture medium consisted of Murashige and Skoogs' components [6] (M/S) to which was added 9.0% Mannitol, 3.0% sucrose and the following hormone combination:- 2.0 mg/l naphthalenacetic acid (NAA) and 0.5 mg/l 6-benzylaminopurine (BAP). This medium permitted growth of P. axillaris and thus putative cybrids with the P. axillaris nucleus, whilst not facilitating the growth of the cms line 2426, or any nuclear hybrids of the two species. Plants of 2785 (P. axillaris) and the putative cybrid were ultimately regenerated on M/S medium containing 1.0 mg/l zeatin as the sole growth regulator. Each fusion experiment was designed as follows: 1. Viability controls -- Protoplasts of lines 2426 or 2785 plated separately. 2. Polyethyleneglycol (PEG) control -- Protoplasts of lines 2426 and 2785 treated with PEG and plated separately. 3. Mixture control -- Protoplasts of lines 2426 and 2785 treated with PEG separately and then mixed prior to plating. 4. Fusion -- Protoplasts of 2426 and 2785 mixed prior to fusion treatment and plating. In the first experiment a ratio of 4 : 1 (cms lines 2426 : line 2785) was employed and in the second experiment this was reduced to a ratio of 2 : 1.

51 TABLE I EXPERIMENTS ON PROTOPLAST FUSION BETWEEN P. HYBRIDA, CMS LINE 2426, AND P. AXILLARIS, LINE 2785. All regenerated plants were of line 2785 type. Experiment number and date of initiation

Protoplasts source and treatment

Number of plants regenerated from protoplasts

Experiment 1 28.1.1976

2426 PEG control 2785 PEG control 2426 + 2785 Fusion

none

2426 + 2785

F 2 of plants regenerated from protoplasts

60 60

Not attempted Three plants segregated cms offspring All normal offspring

52

Mixture control

Experiment 2 6.2.1976

24 26 PEG control 2785 PEG control 2426 + 2785 Fusion 2426 + 2785

none

80 80

Not attempted Three plants segregated cms offspring Not attempted

50

Mixture control Experiment 3

30.5.1977

2426 PEG control 2785 PEG control 2426 + 2785 Mixture control

none 100 150

i

All normal offspring All normal offspring

All p r o t o p l a s t s were p l a t e d at a final d e n s i t y o f 2.5 × 104/ml. RESULTS In t h e first e x p e r i m e n t (Table I) m o r e t h a n J 0 plants were regenerated f r o m t h e fusion t r e a t m e n t . All plants b u t o n e were male fertile and p h e n o t y p i c a l l y similar t o line 2 7 8 5 . T h e o n e male sterile p l a n t was similar to t h e 2 4 2 6 t y p e (P. hybrida) a n d was t h u s assumed to have leaked t h r o u g h t h e selection. Plants, r e g e n e r a t e d from t h e various c o n t r o l s were all of t h e 2 7 8 5 t y p e . In t h e second e x p e r i m e n t m o r e t h a n 80 plants were regenerated f r o m t h e fusion t r e a t m e n t ; all b u t one w e r e male fertile a n d p h e n o t y p i c a l l y similar to t h e 2 7 8 5 t y p e . This single p l a n t ( 6 . 2 - - 4 5 ) was very w e a k , male fertile, and i n t e r m e d i a t e b e t w e e n 2 7 8 5 and 2 4 2 6 for o t h e r markers. This plant, a possible somatic h y b r i d , was selfed a n d t h e analysis o f its successive generations is still u n d e r way. T h e o t h e r plants regenerated in t h e fusion t r e a t m e n t s o f the t w o experim e n t s were selfed a n d t h e F2 p o p u l a t i o n s were grown and observed for male

52

TABLE H GENETIC ANALYSIS OF THE PLANTS REGENERATED FROM PROTOPLASTS AFTER INDUCTION OF FUSION Each F 2 population are plants from seed obtained by selfing the plants regenerated from protoplasts. The same fertile F 2 plants were used for selfing and for testcrossing with line 2340. Only the data of plants segregating in F 2 are presented in this table. The figures in the table are numbers of plants or population numbers; in the testcrosses at least 100 plants were observed in each case. Date

28.1.1976

6.2.1976

Regenerated plants

F z populations from regenerated plants

F 3 populations of fertile plants from F2

F:S

F:S

plant 1

9 :4

plant 2

11 : 6

2 : 15 1 : 16 0 : 6 0 :

1 3 0 0 0

plant 3

8 :4

plant I

15 : 5

plant 2

21 : 6

I :

12 : 4

0 0 0 2 0

plant 3

7

: 8 : 10 : 5 : 7 : 8

0 : 14 0 : 7 0 : 10 8

: 5 : 3 : 4 : 11 : 6

Testcross progenies of male fertile plants from F2x 2340

Testcross progenies of male sterile plants X 2340

all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile

all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile

all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile

all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile all sterile ,

sterile plants. In a d d i t i o n , plants o f t h e m i x t u r e c o n t r o l s o f t h e first experim e n t were selfed and their F2 p o p u l a t i o n s w e r e grown and observed for male sterile plants. In a d d i t i o n , p l a n t s o f t h e m i x t u r e c o n t r o l s o f t h e first experim e n t w e r e selfed and their F2 p o p u l a t i o n s w e r e grown. In t h e third experim e n t (see Table I) only c o n t r o l t r e a t m e n t s w e r e a t t e m p t e d a n d all regenerated plants w,ere identical t o 2 7 8 5 and gave o n l y n o r m a l , 2 7 8 5 t y p e , offspring. T h e d a t a o b t a i n e d in t h e genetic analysis is p r e s e n t e d in Table II. The F2 p o p u l a t i o n s o f each o f t h e r e g e n e r a t e d plants (several t h o u s a n d plants) were grown in B e t Dagan in t h e fall o f 1976. Plants were observed f o r pollen prod u c t i o n as well as o t h e r characteristics. As i n d i c a t e d in Table I, only after fusion t r e a t m e n t s in each o f t h e t w o e x p e r i m e n t s , were t h r e e F2 p o p u l a t i o n s identified w h i c h segregated male sterile plants. T h r e e male sterile plants and F

53 samples of the fertile segregants were test-crossed with line 2340 (see Table II). The same fertile segregants were also selfed and the F3 populations were grown. Both testcross progenies of the male sterile segregants X 2340 and the male fertile segregants × 2340 yielded only male sterile plants. The use of fertile segregants from F2 or F3 populations in testcrosses, as male parents, on line 2439 showed segregation of male fertile and sterile plants. The proper testcrosses of fertile plants from non segregating F2 populations showed the presence of normal plasmon. In addition to transmission of cms, purple flower colour was observed in plants of the F2 populations. The expression of purple colour which is characteristic is line 2426 was not associated with the transmission of cms. Another phenomenon which was quite common among the F2 populations was variegation but again this was not associated with the transmission of cms. DISCUSSION

These experiments were designed to obtain cybrids between the nuclear genome of line 2785 and the (S) plasmon of line 2426 -- such cybrids were indeed obtained. Other cybrid types were selected against as a result of the choice of culture media. W e believe that the combination of unequal protoplast ratios during the P E G treatment and the selection against protoplasts of cms line 2426 contributed much towards achieving this goal in the following way. The different protoplast ratios in favour of 2426 (4 : 1 and 2 : 1) increased the probability that protoplasts of line 2785 will fuse with protoplasts of line 2426 rather than with each other (see Kao and Michayluk [6] and Izhar [7] for detailed discussion). By selecting against protoplasts of line 2426 and potential somatic hybrid protoplasts, except those possessing the 2426 plasmon and 2785 genome the total protoplast density in the dishes was reduced drastically thus increasing the chances of the desired cybrid lines surviving and developing into plants. It should be pointed 'out that the approach in the present work towards obtaining a cybrid was different from that used in other successful somatic hybridization attempts [1,9 ]. In the other studies in order to obtain somatic hybrids the selection pressure was against both parental lines.In the present selection scheme, the genome only of the originalnormal type (line2785) was favoured, and since it-was known that the (S) plasmon did not influence potential growth in the protoplast medium [10] it was assumed that the 2785 genome with (S) plasmon would respond similarly to the medium. In this respect the cybrids described here are different from those obtained by Belliard et al. [11] and Gleba [1] in that nuclear fusion products, if arisingin experiments, would not survive the selection procedure. A n unknown factor, at the time these expe~ments were initiated,was the presence of mfr allelesin line 2785. The necess.~.tyto selfwas suggested by the early appearance of plant 6.2--45, which was male fertile,and by the experiences of Frankel [12], Edwardson and Corbet [13] and Bianchi [14] with

54

graft transmission of cms in petunia. In these examples, cms was identified in the FI population only when the fertile scion was selfed. While in the graft transmission experiments Frankel [12] showed that only a portion of the FI plants were cms, this was not so in our case where all the F2 plants of segregating populations carried (S) plasmon (see Table II). Cybrids, in the present experiments, could arise in a variety of ways. Inadequate coalescence of protoplasts during fusion treatment could result in the exclusion of one of the nuclei. A somewhat similar product could be envisaged following the fusion of a normal protoplast with an enucleated protoplast (subprotoplast) or with a protoplast lacking a viable nucleus. The latter situation could be enhanced by the irradiation of the cms parental protoplasts. Heterokaryons, where nuclear fusion fails to occur or where unidirectional chromosome or whole nucleus elimination takes place, could give rise to cybrids, particularly under growth conditions not favouring hybrid survival. In some cases cytoplasmic competition may be expected since in many somatic hybrids elimination of one of the parental cytoplasms was observed [ 15]. Furthermore the presence of two cytoplasms yielded a weak and inferior cybrid. On the other hand when protoplasts of Nicotiana lines with plastome mutations were fused, a cybrid plant with one nucleus and two complementing cytoplasms was isolated [ 1]. Since good biochemical markers in petunia are not available [16] it was not possible to identify genetic elements of both (S) plasmon and normal plasmon in the cybrid. Nevertheless, the results of the genetic analysis show that in some of the original plants, regenerated from protoplasts, (S) plasmon was present but was n o t expressed because of the mfr alleles. The question should also be asked, whether mechanisms other than protoplast fusion were responsible for the formation of the cybrids. There are, up till now, no reports of effective nuclear or organelle transplantations in higher plants and particularly their expression [2]. It should be pointed out that conditions bringing about protoplasts fusion could lead to organelle transfer. It is perhaps important to point out thepractical application of these results. The male sterile offspring of line 2785 whose normal cytoplasm was quickly replaced by s male sterile cytoplasm of another species can be used directly as female lines for hybrid seed production in petunia.

ACKNOWLEDGEMENTS S.I. is grateful to Prof. E.C. Cocking in whose laboratory the first part of this work was carried out. Thanks are due to Mrs. Lily Feldman and Mrs. Yona Tabib (Bet Dagan) for technical assistance. Tb~ firstpart of this work was carried out while S.I. was on leave in Nottingham. This leave was s u p p o ~ : ~ in part by the Royal Society, London, the Israeli Academy of Sciences (Jerusalem) and the United States-Israel Binational Science Foundation (BSF), Jerusalem (Israel).

55 REFERENCES 1 Y.Y. Gleba, Non-chromosomal inheritance in higher plants as studied by somatic cell hybridization. In Plant Cell and Tissue Culture-Principles and Applications. Ohio State University Press, Columbus (in press). 2 E.C. Cocking, Int. Rev. Cytol., 58 (1977) 323. 3 S. Izhar, J. Hered., 69 (1978) 22. 4 J.B. Power, E.M. Frearson, D. George, P.K. Evans, S.F. Berry, C. Hayward and E.C. Cocking, Plant Sci. Lett., 7 (1976) 51. 5 J.B. Power, E.M. Frearson, C. Hayward, D. George, P.K. Evans, S.F. Berry and E.C. Cocking, Nature, 263 (1976) 500. 6 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. 7 K.N. Kao and M.R. Michayluk, Planta, 115 (1974) 355. 8 S. Izhar, in A.W. Alferman and E. Reinhard (Eds.), 'Production of natural compounds by cell culture methods' Gesellschaft fur Strahlenund Umweltforschung, Munich, 1978. (in press). 9 W.R. Scowcroft, Adv. Agron., 29 (1977) 39. 10 S. Izhar and J.B. Power, Plant Sci. Lett., 8 (1977) 375. 11 G. Belliard, G. Pelletier, and M. Ferault, C.R. Acad. Sci. Paris, D 284 (1977) 749. 12 R. Frankel, Science 124 (1956) 684. 13 J.R. Edwardson and M.K. Corbet, Proc. Natl. Acad. Sci. (U.S.A.) 47 (1961) 390. 14 F. Bianchi, Genen en Phaenen, 60 (1963) 807. 15 S.D. Kung, J.C. Gray, S.G. Wildman and P.S. Carlson, Science, 187 (1975) 353. 16 A.A. Gatenby and E.C. Cocking, Plant Sci. Lett., 10 (1977) 97.