PlantCell Reports
Plant Cell Reports (1993) 12:260-263
© Springer-Verlag 1993
Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annuus L.) Sergei Krasnyanski and Lfiszl6 Menczel Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, POB 521, H-6701 Szeged, Hungary Received October 1, 1992/Revised version received December 30, 1992 - Communicated by H. L6rz
Abstract A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified VKM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A3 and the ethylene inhibitor AgNO3. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in rive germinated seedlings. Eighty percent of grained shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to l0 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed. Key words: sunflower, protoplasts, regeneration
somatic embryogenesis, plant
Abbreviations: BAP, 6-benzylaminopurine; GA3, gibberellic acid; MES, morpholinoethanesulfonicacid; MS, Murashige and Skoog medium; NAA, naphthaleneacetic acid; V-KM, protoplast culture medium of Binding and Nehls; 2,4D, 2,4-dichlorophenoxyaceticacid Introduction Sunflower is one of the major oil crops of the world. Traditional breeding of the crop has resulted in a steady increase of yield and oil quality but some of the problems (e.g. sensitivity to the fungi Sclerotinia and Diaporte) are still unsolved. Wild species of the genus Helianthus are possible sources of desirable traits such as cytoplasmic male sterility (Schilling and Heiser 1981) or disease resistance (Georgieva-Todorova 1976). The use of wild species in sunflower breeding, however, is limited by sexual incompatibility and sterility in interspeeifie hybrids (Whelan 1978). An alternative to sexual crosses is the use of somatic cell fusion. This technique has been worked out and extensively applied in many different species combinations (for a review see Schieder Correspondence to: L. Menczel
and Kohn 1986). In higher plants the cell wall is a natural barrier to cell fusion. Therefore, in somatic hybridization experiments the use of protoplasts is essential. The same is true for genetic transformation by the uptake of purified DNA, another important technique of in vitro genetic manipulations. Moreover, in order to utilize the hybrids or transformants, the ability of regenerating fertile plants from protoplasts is a prerequisite. Data on plant regeneration from protoplasts of Helianthus annuus are rather limited. There have been several unsuccessful attempts to regenerate plants from protoplasts (Bohorova et al. 1986; Lenee and Chupeau 1986; Moyne et al. 1988; Guilley and Hahne 1989; Sehmitz and Sehnabl 1989; Chanabe et al. 1989, 1991; Dupuis et al. 1990). Recently, Burrus et al. (1991) reported on the regeneration of fertile plants from hypocotyl protoplasts of one particular genotype. However, the frequency of regeneration was very low (0.054 percent of the protoplasts plated) and only six plants produced any seeds in the greenhouse. While this study demonstrates the possibility of regeneration, the method described is obviously not efficient enough for practical use. In other recalcitrant species identification of speoial genotypes with high regeneration ability resulted in the establishment of practically useful protoplast culture systems (a pertinent example is M6rocz et al. 1990). For this reason, we have started our investigations by screening genotypes using the test system of Paterson and Everett (1985). In this paper we report on the development of a practically usable system of plant regeneration from protoplasts of the best genotype identified. Materials and methods Plant material: Seeds of commercial hybrids were obtained from the Hungarian Seed Company, Budapest. The following hybrids were used: Barbara, Blumix, Citosol-3, Citosol-4, Clubsol, Florakisz, Florom-206, Florom-328, Florom-350, Fundulea-206, GK-Napszirom, Granchaco, J550, Multibest. Seeds of inbred lines were kindly provided by Dr. l_Aszl6 Takdcs (Feed Crop Research Institute, Iregszemese, Hungary). Germination of seeds: Seeds were surface sterilized by soaking in diluted (5-fold) commercial bleach for 20 minutes. They were then washed three times in distilled water and were kept overnight in distilled water at 4°C. Next day the seed coats were removed and the seeds were sterilized again by soaking in 70 % ethanol for one minute followed by soaking in diluted (5-fold) commercial bleach for ten minutes. They were then washed three
261 times in sterile distilled water. For protoplast isolation seeds were germinated in the dark at 26°C on half-strength MS salts (Murashige and Skoog 1962) supplemented with 0.5 % sucrose and 0.6 % agar. Hypocotyls used in the screening for regeneration ability were germinated according to Paterson and Everett (1985). Testing of regeneration ability: Hypocotyl segments were cultured and tested for the presence of embryos and shoots according to the method of Paterson and Everett (1985). Isolation of protoplasts: Hypocotyls were cut into 5 to 10 mm sections and each section was cut in half longitudinally. One gram of cut hypocntyl tissue was placed in 10 ml of enzyme solution containing 2.0% cellulase "Onozuka" R10 and 0.5% macerozyme R10 (Yakult Honsha Co., Ltd., Japan). The enzymes were dissolved in the protoplast washing solution KS (18.0 g 1"t NaC1, 0.4 g 1"1 KCI, 6.13 g 11 CaCI 2 x 2H20 , 3.3 mM MES, pH 5.6). Digestion was carried out overnight (16 to 18 hours) at 26°C in the dark. Digested material was passed through nylon filters with 63 and 21 pan pore diameter, collected by centrifugation (80 g, 3 minutes) and washed twice in KS solution. Protoplast culture: Purified protoplasts were resuspended in hormone free V-KM medium (Binding and Nehls 1977; with the following modifications: glucose, amino acids and nucleic acid bases omitted, 80.0 g 1-1 sorbitol instead of mannitol, L-glutamine added at 1.0 g F 1, osmolarity 0.58 Os kg"1, pH 5.6) at a density twice as high as the desired final density in the agarose droplets. Aliquots (0.5 ml) were then mixed with equal volumes of V-KM medium containing different concentrations (see Results) of agarose (SERVA, cat. No. 11404) cooled to 40°C after autoclaving. The mixture was pipetted into 9 cm diameter plastic Petri dishes (GREINER, Germany). Solidified droplets were then covered by 10.0 ml of liquid V-KM medium supplemented with different amounts of NAA and BAP (see Results). When a low density of protoplasts (5 x 103 ml"1) was used inside the agarose droplets, the covering liquid medium also contained hypocotyl protoplasts at a density of 3 x 104 m1-1 ("nurse" cultures). The cultures were incubated in the dark at 26°C. Induction of embryogenesis: One week later the liquid was replaced by 10.0 ml of V-KM with different hormones (see Results). After another week in the dark plating efficiency was determined. The liquid was then replaced by 10.0 ml of V-KM supplemented with 0.05 mg 1-1 NAA and 0.2 mg 1-1 BAP (V-KM-3) and the cultures were incubated in the dark for another week. The agarose droplets were then dispersed by gentle pipetting in liquid V-KM-3 (10.0 ml). The resulting liquid cultures were diluted with V-KM-3 to give a density of about 100 colonies per ml and they were incubated for an additional 4 days in the dark. Embryo maturation and plant regeneration: Colonies growing in liquid VKM-3 were transferred onto regeneration medium containing the basic salts of MS, B5 or White's (Murashige and Skoog 1962; Gamborg et al. 1968; Shing and Krikorian 1981) supplemented with 100.0 mg I-t m-inositol, 0.5 mg 1"I vitamin B6, 0.5 mg 1"1 nicotinic acid, 0.1 mg 1-1 vitamin Bt, 1.0 mg 1"1 AgNO3, 0.2 mg 1-t BAP, 0.1 mg 1-t GA3, 0.01 mg 1-1 NAA, 0.5 g 1"1 enzymatic caseine hydrolyzate and 30.0 g 1"1 sucrose, pH 5.8. Media were solidified by 0.26 % Phytagel from SIGMA. The cultures were incubated in the light (2000 lux light intensity, 16 hour photoperiod). After 8 to 10 days developing somatic embryos were separated from the surrounding non-embryogenic calluses and were transferred to fresh regeneration medium. Two weeks later the embryos were transferred onto hormone free MS salts supplemented with I00.0 mg 1-t m-inositol, 0.2 mg 1"t vitamin B1, 20.0 g I"1 sucrose and 0.26 % Phytagel (pH 5.8). Transfer ofplantlets into soil: Shoots 1 to 1.5 cm in length were separated from the surrounding callus material. Shoots with rot~ts were directly placed into l0 cm pots containing sterilized commercial garden soil. The pots were covered by transparent plastic foil for a few days to prevent evaporation. Shoots without roots were cut about 5 to 8 mm below their tips and were grafted on hypocotyls of another commercial hybrid cultivar germinated in sterilized soil. Hypocotyls with the grafted shoots were for one week covered by glass tubes to prevent evaporation. Grafting was carried out as suggested by Dr. B. Pelissier (Rhone-Poulenc, Lyon, personal communication) with some modifications. All transplanted plants were incubated in an environmental chamber (CONVIRON, Canada; set to 24°12, 9000 lux light intensity, 12 hour photoperiod).
Results and discussion Testing the r e g e n e r a t i o n ability of genotypes Sunflower genotypes have been tested using the method of Paterson and Everett (1985). Regeneration ability was assessed by the number of shoots developed on the hypoeotyl segments cultured. Among 91 genotypes, only three hybrids and two inbred lines showed any response. T h e hybrid Florom-328 (from the Institute of Cereal and Industrial Plant Research, Fundulea, Romania) was the best responding genotype (0.6 shoots per hypoeotyl explant). T h e regeneration ability of the two inbred lines was significantly lower. Florom-328 was therefore chosen for further studies. Regeneration ability is apparently restricted to a small proportion of sunflower genotypes. Unfortunately, the genetic composition of the best genotype (Florom-328) is not known to us. However, it exhibits frequent branching of the stem when grown in the greenhouse. This trait which is not characteristic of cultivated sunflower may indicate a contribution of genetic material from wild Helianthus species to this genotype. Wild species seem to b e easier to regenerate in vitro (see e.g. Chanabe et al. 1991, Krasnyanski et al. 1992). Thus regeneration ability of the Florom genotype might have originated from a wild species used in the construction of this hybrid. The regenerable sunflower genotype o f B u r r u s et al. (1991) (PT024, designated in the paper as emslH.petiolarislcms HA89 baekeross) may also contain input from a wild species.
Figure 1. Development of protoplast derived colonies in agarose droplets. A, dividing protoplast after 4 days in culture, x 350, bar 50 #m; B and C, colonies after 10 and 12 days, x 75, bar 100 #m; D, colony after 15 days, x 80, bar 100 #m, E, globular embryo after 20 days, x 30, bar 200 /.tm.
Identification of factors affecting plating efficiency and shoot formation In the following diseussionthe term "shoot formation" refers to the typical way of plant regeneration (elongation of the shoot apex of somatic embryos without root development, see below). The average yield of protoplasts from one gram of hypoeetyl tissue was 2 to 3 million. T h e protoplast population was rather heterogeneous both in their size and in the amount of cytoplasm. A more homogeneous population was obtained when large, highly vaeuolated protoplasts were removed by filtration through nylon meshes. The age of hypoeotyl material (5, 9 or 11 days) was a critical factor. Somatic embryogensis was observed only in
262
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Figure 3. Development of embryogenic colonies on regeneration media. A, embryos at different stages of development, x 3, bar 5 nun; B, elongatingshoots on hormone free medium in 9 cm petri dish.
[mg/I]
Figure 2. Effect of initial NAA concentration on colony and shoot formation. dashed lines, colony formation (percent of protoplasts plated); solid lines, shoot formation (percent of viable colonies); (11), with 2,4D treatment; ([]), without 2,4D treatment.
colonies of protoplasts from 5 day old hypocotyls. Optimal protoplastdensity was determined first.Highest plating efficiency and colony formation was found at a density of 3 x 104 ml "I (calculatedusing the volume of agarose droplet only). Fig.1A shows firstdivisions of protoplasts in droplets of V - K M medium supplemented with 2.0 m g 1-1 B A and 0.5 m g I-I N A A and solidifiedby 0.5 percent agarose. Further divisions in most cases occurred inside the boundaries of the initialcell wall, thus the daughter cells became smaller than the original protoplasts and rich in cytoplasm. After one week the liquid over the droplets was replaced by fresh V - K M medium containing one-tenth of the original hormone concentrations.Further development of colonies followed different pathways. Developmental stages characteristic of somatic embryogenesis (Fig.IB to IE) could be observed in the cultures. However, the frequency of shoot formation was rather low (0.18 percent of viable colonies). In previous experiments on Helianthus protoplast culture compact colonies representing the first stage of somatic cmbryogcncsis could not bc observed in liquid cultures.It appears that a supporting matrix is essential for the development of embryos. For this reason, influence of the nature and concentration of gelling agent was investigated next. In cultures where 0.25 percent agarose was used, the frequency of shoot formation was only half of that observed in 0.5 percent agarose. Increasing agarosc concentration to 1.0 percent resulted in a further increase of this frequency. However, due to the high rigidity of 1.0 percent gels, colonies could not be removed from the droplet without damage. Alginic acid at 1.4 percent concentration was also tried, but there was no improvement as compared to agarose solidified cultures. In several dicotyledonous species including sunflower it has been shown that exposure of cultures to high concentrations of auxins for a limited period could induce subsequent development of somatic embryos (Finer 1987; Prado and Berville 1990). In our experiments N A A or 2,4D treatment also improved the embryogenie response of protoplast derived colonies. One week
Figure 4. Grafting shoot tips on in vivo grown hypocotyls. A, shoot developedon hormonefree MS medium;B, same shoot gratted on hypocotyl;C, floweringregeneratedplants. old colonies grown in V - I ~ t supplemented with 2.0 mg 1"1 BAP and 0.5 mg 1"1 NAA were treated with different auxins by replacing the liquid medium over the droplets by V-KM supplemented with 2.2 mg 1"1 of NAA, 2,4D or dieamba. This medium was leR on the droplets for one week, then it was replaced by V-KM with reduced NAA and BA contents as described above. Treatment with dicamba, NAA and 2,4D increased the frequency of shoot formation from 0.18 percent to 0.24, 0.8 and 1.8 percent, respectively. The protoplast density (3 x 104 per ml) optimal for initial division and colony formation appeared to be too high for later stages of colony development. Development of colonies slowed and they started to release a brown material (probably polyphenols) into the medium. Starting density was, therefore, reduced to 5 x 103 m1-1 inside the agarose droplet. The adverse effect of reduced density on plating efficiency was compensated by adding nursing protoplasts to the liquid phase. These
263 protoplasts were removed after one week when the liquid was refreshed. Plating efficiency and the frequency of colony formation were practically the same as those observed previously at higher protoplast densities. However, reduced density of colonies resulted in a significant increase in the frequency of shoot formation (1.2 percent v e r s u s O. 18).
Influence of initial auxin concentration Subsequent experiments were carried out using droplets solidified by 0.5 percent agarose, a protoplast density of 5 x 103 m1-1 and nursing protoplasts in the liquid phase at a density of 3 x 104 ml "l. A systematic investigation on the influence of initial auxin concentration was performed. Protoplasts were cultured in the presence of different concentrations (0.5 to 10.0 mg 1-1) of NAA together with 2.0 mg 1"1 BA. Interaction of initial auxin concentration and subsequent 2,4D treatment (2.2 mg 1-l) was studied by setting up parallel cultures with and without 2,4D treatment for all NAA concentrations used. Data are summarized in Fig.2. Plating efficiency (not shown) slightly decreased (from 34 to about 24 percent) with increasing NAA concentration. Colony formation was not significantly influenced by initial auxin levels. The frequency of shoot formation was highest at 4.0 mg 1-1 initial NAA concentration with subsequent 2,4D treatment. Under these conditions 9.6 percent of viable colonies formed shoots,
Embryo maturation and regeneration of plants After three weeks of culture developing colonies were released from the agarose droplets and were cultured in liquid V-KM for a few days. They were then transferred onto solid medium and were exposed to light to induce maturation and germination of somatic embryos. Three media differing in basic salt composition but otherwise supplemented with the same compounds (see Materials and Methods) were tried at this point. On B5 and White's media the colonies turned brown and developing shoots had very pale green colour. On MS medium calluses continued to grow vigorously and developing shoots had a dark green colour. When agar was used as gelling agent, callus formation became predominant, while media solidifed by Phytagel favoured embryo development. Within two to three weeks embryos at different developmental stages (from globular to torpedo-shaped) could be observed in the cultures. Development was slightly abnormal (Fig.3A, bottom row). Further development of both shoot and root was observed only in less than 5 percent of embryos. The typical way of development was shoot elongation accompanied by swelling or callusing at the root apex (Fig.3A, top row). Development of normal shoots (at a frequency of over 80 percent) occurred only on media supplemented with silver nitrate, an inhibitor of ethylene action. In the absence of silver nitrate vitrification (elongated stem, pale green watery leaves) was observed and normal shoots were infrequent. Similar effects have been described and attributed to ethylene action in other species (Chi and Pua 1989; Perl et al. 1988) and also in sunflower tissue cultures (Chraibi et al. 1991). It was essential to transfer the embryos onto hormone free MS medium as soon as shoot elongation had started. Shoots started to exhibit vitrification even in the presence of silver nitrate if they were left on the regeneration medium for longer periods. On hormone free medium the growth of callus stopped and normal shoot elongation continued (Fig.3B.). Plantlets with well developed roots could be transplanted into soil. However, in the ease of other shoots, transplanting would
have involved an additional period of rooting. Sunflower shoots in culture have a tendency to premature flowering (as noted e.g. by Burrus et al. 1991) which increases with the time elapsed in culture. The presence of flower buds in turn stops development of the shoots and causes seenescenee, usually without any seed production. Therefore, sunflower shoots should be transferred into soil at the earliest possible stage. An alternative to rooting is grafting of the shoot apex on hypocotyls of in vivo grown plants (Fig.4). Shoots as small as 10 mm in length could sueeessfuUy be grafted. More than 80 percent of 111 shoots grafted were established on the root stock and continued development. Although these plants did not grow to normal size, they flowered (Fig.4) and produced seeds. The number of seeds obtained from individual plants was in the range of 5 to 10. In conclusion, identificationof the special genotype Florom-328 allows regeneration of plants from protoplasts at practically usable frequencies. Experiments are in progress to utilize this sytem for the production of stably transformed sunflower plants by PEG-mediated direct DNA uptake. Acknowledgements This work was supported by a grant from the Feed Crop Research Institute, Iregszemcse, Hungary. We thank Dr. B. Pelissier (RlaonePoulene, Lyon) for valuable suggestionsconcerningshoot gratting.
References BindingH, Nehls R (1977) Z. Pflanzenphysiol.85,279-285 Bohorova NE, CockingEC, Power JB (1986) Plant Cell Rep. 5,256-258 Burrus M, ChanabeC, Alibert G, Bidney D (1991) Plant Cell Rep. 10,161-166 Chanabe C, Burrus M, Alibert G (1989)Plant Sci. 64,125-132 Chanabe C, Burrus M, Bidney D, Alibert G (1991) Plant Cell Rep. 9,635-638 Chi GL, Pua EC (1989) Plant Sci. 64,243-250 Chraibi KM, Latche A, Roustan JP, Fallot J (1991) Plant Cell Rep. 10,204-207 Dupuis JM, Pean M, ChagvardieffP (1990) Plant Cell Tissue Organ Cult. 22,183-189 Finer JJ (1987) Plant Cell Rep. 6,372-374 Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50,151-158 Georgieva-TodorovaJ (1976) lnterspecificrelations in the genus Helianthus. Press of the BulgarianAcad. Sei., Sofia GuilleyE, Hahne G (1989) Plant Cell Rep. 8,226-229 KrasnyanskiS, Polg~irZ, N~methG, Menczel L (1992) Plant Cell Rep. 11,7-10 Lenee P, ChupeauY (1986) Plant Sci. 43,69-75 M6rocz S, Donn G, N6methJ, Dudits D (1990) Theor. Appl. Genet. 80:721-726 MoyneAL, Thor V, PelissierB, BergouniouxC, FreyssinetG, GadalP (1988) Plant Cell Rep. 7,437-440 Murashige T, Skoog F (1962) Physiol. Plant. 15,473-497 Paterson KE, Everett NP (1985) Plant Sci. 42,125-132 Perl A, Aviv D, Galun E (1988) Plant Cell Rep. 7,403-406 Prado E, BervilleA (1990) Plant Sci. 67,73-82 Schieder O, Kohn H (1986)In: I.K. Vasil, ed., Cell Cultureand Somatic Cell Geneticsof Plants, Vol 3., AcademicPress, Orlando, pp 569-588 Shilito RD, PaszkowskiJ, Potrykus I (1983) Plant Cell Rep. 2,244-247 ShillingEE, Heiser CB (1981) Taxon 30,393-403 Sehmitz P, SehnablH (1989) J. Plant Physiol. 135,223-227 Shing M, Krlkodan AD (1981) Ann. Bot. 47,133-139 WhelanEDP (1978)In: SunflowerScienceand Technology,pp.339-369, Carter JF, ed., AmericanSociety of Agronomy,Madison, Wisconsin
Plant Cell Reports (1993) 12:260-263
© Springer-Verlag 1993
Somatic embryogenesis and plant regeneration from hypocotyl protoplasts of sunflower (Helianthus annuus L.) Sergei Krasnyanski and Lfiszl6 Menczel Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, POB 521, H-6701 Szeged, Hungary Received October 1, 1992/Revised version received December 30, 1992 - Communicated by H. L6rz
Abstract A sunflower genotype (Helianthus annuus L. cv. Florom-328) able to regenerate plants from in vitro cultures was identified by screening hybrids and inbred lines. Protoplasts of this genotype were isolated from dark grown hypocotyls and were cultured in droplets of agarose-solidified VKM medium covered by liquid V-KM supplemented with naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). One week later colonies were subjected to 2,4-dichlorophenoxyaceticacid for a one week period. Further culture in V-KM with reduced concentrations of NAA and BAP resulted in the appearence of somatic embryos. Maturation of embryos was achieved by culture on MS medium supplemented with NAA, BAP, gibberellic acid A3 and the ethylene inhibitor AgNO3. Embryos were then transferred onto hormone free MS medium for germination. The frequency of shoot formation in the best case was 9.6 percent of viable colonies (1.3 percent of protoplasts plated). Some of the shoots with roots could be transplanted into soil, others were grafted on hypocotyls of in rive germinated seedlings. Eighty percent of grained shoots and over 95 percent of rooted shoots survived. The plants flowered and produced 5 to l0 seeds each. Factors affecting the frequency of embryo formation and plant regeneration are discussed. Key words: sunflower, protoplasts, regeneration
somatic embryogenesis, plant
Abbreviations: BAP, 6-benzylaminopurine; GA3, gibberellic acid; MES, morpholinoethanesulfonicacid; MS, Murashige and Skoog medium; NAA, naphthaleneacetic acid; V-KM, protoplast culture medium of Binding and Nehls; 2,4D, 2,4-dichlorophenoxyaceticacid Introduction Sunflower is one of the major oil crops of the world. Traditional breeding of the crop has resulted in a steady increase of yield and oil quality but some of the problems (e.g. sensitivity to the fungi Sclerotinia and Diaporte) are still unsolved. Wild species of the genus Helianthus are possible sources of desirable traits such as cytoplasmic male sterility (Schilling and Heiser 1981) or disease resistance (Georgieva-Todorova 1976). The use of wild species in sunflower breeding, however, is limited by sexual incompatibility and sterility in interspeeifie hybrids (Whelan 1978). An alternative to sexual crosses is the use of somatic cell fusion. This technique has been worked out and extensively applied in many different species combinations (for a review see Schieder Correspondence to: L. Menczel
and Kohn 1986). In higher plants the cell wall is a natural barrier to cell fusion. Therefore, in somatic hybridization experiments the use of protoplasts is essential. The same is true for genetic transformation by the uptake of purified DNA, another important technique of in vitro genetic manipulations. Moreover, in order to utilize the hybrids or transformants, the ability of regenerating fertile plants from protoplasts is a prerequisite. Data on plant regeneration from protoplasts of Helianthus annuus are rather limited. There have been several unsuccessful attempts to regenerate plants from protoplasts (Bohorova et al. 1986; Lenee and Chupeau 1986; Moyne et al. 1988; Guilley and Hahne 1989; Sehmitz and Sehnabl 1989; Chanabe et al. 1989, 1991; Dupuis et al. 1990). Recently, Burrus et al. (1991) reported on the regeneration of fertile plants from hypocotyl protoplasts of one particular genotype. However, the frequency of regeneration was very low (0.054 percent of the protoplasts plated) and only six plants produced any seeds in the greenhouse. While this study demonstrates the possibility of regeneration, the method described is obviously not efficient enough for practical use. In other recalcitrant species identification of speoial genotypes with high regeneration ability resulted in the establishment of practically useful protoplast culture systems (a pertinent example is M6rocz et al. 1990). For this reason, we have started our investigations by screening genotypes using the test system of Paterson and Everett (1985). In this paper we report on the development of a practically usable system of plant regeneration from protoplasts of the best genotype identified. Materials and methods Plant material: Seeds of commercial hybrids were obtained from the Hungarian Seed Company, Budapest. The following hybrids were used: Barbara, Blumix, Citosol-3, Citosol-4, Clubsol, Florakisz, Florom-206, Florom-328, Florom-350, Fundulea-206, GK-Napszirom, Granchaco, J550, Multibest. Seeds of inbred lines were kindly provided by Dr. l_Aszl6 Takdcs (Feed Crop Research Institute, Iregszemese, Hungary). Germination of seeds: Seeds were surface sterilized by soaking in diluted (5-fold) commercial bleach for 20 minutes. They were then washed three times in distilled water and were kept overnight in distilled water at 4°C. Next day the seed coats were removed and the seeds were sterilized again by soaking in 70 % ethanol for one minute followed by soaking in diluted (5-fold) commercial bleach for ten minutes. They were then washed three
261 times in sterile distilled water. For protoplast isolation seeds were germinated in the dark at 26°C on half-strength MS salts (Murashige and Skoog 1962) supplemented with 0.5 % sucrose and 0.6 % agar. Hypocotyls used in the screening for regeneration ability were germinated according to Paterson and Everett (1985). Testing of regeneration ability: Hypocotyl segments were cultured and tested for the presence of embryos and shoots according to the method of Paterson and Everett (1985). Isolation of protoplasts: Hypocotyls were cut into 5 to 10 mm sections and each section was cut in half longitudinally. One gram of cut hypocntyl tissue was placed in 10 ml of enzyme solution containing 2.0% cellulase "Onozuka" R10 and 0.5% macerozyme R10 (Yakult Honsha Co., Ltd., Japan). The enzymes were dissolved in the protoplast washing solution KS (18.0 g 1"t NaC1, 0.4 g 1"1 KCI, 6.13 g 11 CaCI 2 x 2H20 , 3.3 mM MES, pH 5.6). Digestion was carried out overnight (16 to 18 hours) at 26°C in the dark. Digested material was passed through nylon filters with 63 and 21 pan pore diameter, collected by centrifugation (80 g, 3 minutes) and washed twice in KS solution. Protoplast culture: Purified protoplasts were resuspended in hormone free V-KM medium (Binding and Nehls 1977; with the following modifications: glucose, amino acids and nucleic acid bases omitted, 80.0 g 1-1 sorbitol instead of mannitol, L-glutamine added at 1.0 g F 1, osmolarity 0.58 Os kg"1, pH 5.6) at a density twice as high as the desired final density in the agarose droplets. Aliquots (0.5 ml) were then mixed with equal volumes of V-KM medium containing different concentrations (see Results) of agarose (SERVA, cat. No. 11404) cooled to 40°C after autoclaving. The mixture was pipetted into 9 cm diameter plastic Petri dishes (GREINER, Germany). Solidified droplets were then covered by 10.0 ml of liquid V-KM medium supplemented with different amounts of NAA and BAP (see Results). When a low density of protoplasts (5 x 103 ml"1) was used inside the agarose droplets, the covering liquid medium also contained hypocotyl protoplasts at a density of 3 x 104 m1-1 ("nurse" cultures). The cultures were incubated in the dark at 26°C. Induction of embryogenesis: One week later the liquid was replaced by 10.0 ml of V-KM with different hormones (see Results). After another week in the dark plating efficiency was determined. The liquid was then replaced by 10.0 ml of V-KM supplemented with 0.05 mg 1-1 NAA and 0.2 mg 1-1 BAP (V-KM-3) and the cultures were incubated in the dark for another week. The agarose droplets were then dispersed by gentle pipetting in liquid V-KM-3 (10.0 ml). The resulting liquid cultures were diluted with V-KM-3 to give a density of about 100 colonies per ml and they were incubated for an additional 4 days in the dark. Embryo maturation and plant regeneration: Colonies growing in liquid VKM-3 were transferred onto regeneration medium containing the basic salts of MS, B5 or White's (Murashige and Skoog 1962; Gamborg et al. 1968; Shing and Krikorian 1981) supplemented with 100.0 mg I-t m-inositol, 0.5 mg 1"I vitamin B6, 0.5 mg 1"1 nicotinic acid, 0.1 mg 1-1 vitamin Bt, 1.0 mg 1"1 AgNO3, 0.2 mg 1-t BAP, 0.1 mg 1-t GA3, 0.01 mg 1-1 NAA, 0.5 g 1"1 enzymatic caseine hydrolyzate and 30.0 g 1"1 sucrose, pH 5.8. Media were solidified by 0.26 % Phytagel from SIGMA. The cultures were incubated in the light (2000 lux light intensity, 16 hour photoperiod). After 8 to 10 days developing somatic embryos were separated from the surrounding non-embryogenic calluses and were transferred to fresh regeneration medium. Two weeks later the embryos were transferred onto hormone free MS salts supplemented with I00.0 mg 1-t m-inositol, 0.2 mg 1"t vitamin B1, 20.0 g I"1 sucrose and 0.26 % Phytagel (pH 5.8). Transfer ofplantlets into soil: Shoots 1 to 1.5 cm in length were separated from the surrounding callus material. Shoots with rot~ts were directly placed into l0 cm pots containing sterilized commercial garden soil. The pots were covered by transparent plastic foil for a few days to prevent evaporation. Shoots without roots were cut about 5 to 8 mm below their tips and were grafted on hypocotyls of another commercial hybrid cultivar germinated in sterilized soil. Hypocotyls with the grafted shoots were for one week covered by glass tubes to prevent evaporation. Grafting was carried out as suggested by Dr. B. Pelissier (Rhone-Poulenc, Lyon, personal communication) with some modifications. All transplanted plants were incubated in an environmental chamber (CONVIRON, Canada; set to 24°12, 9000 lux light intensity, 12 hour photoperiod).
Results and discussion Testing the r e g e n e r a t i o n ability of genotypes Sunflower genotypes have been tested using the method of Paterson and Everett (1985). Regeneration ability was assessed by the number of shoots developed on the hypoeotyl segments cultured. Among 91 genotypes, only three hybrids and two inbred lines showed any response. T h e hybrid Florom-328 (from the Institute of Cereal and Industrial Plant Research, Fundulea, Romania) was the best responding genotype (0.6 shoots per hypoeotyl explant). T h e regeneration ability of the two inbred lines was significantly lower. Florom-328 was therefore chosen for further studies. Regeneration ability is apparently restricted to a small proportion of sunflower genotypes. Unfortunately, the genetic composition of the best genotype (Florom-328) is not known to us. However, it exhibits frequent branching of the stem when grown in the greenhouse. This trait which is not characteristic of cultivated sunflower may indicate a contribution of genetic material from wild Helianthus species to this genotype. Wild species seem to b e easier to regenerate in vitro (see e.g. Chanabe et al. 1991, Krasnyanski et al. 1992). Thus regeneration ability of the Florom genotype might have originated from a wild species used in the construction of this hybrid. The regenerable sunflower genotype o f B u r r u s et al. (1991) (PT024, designated in the paper as emslH.petiolarislcms HA89 baekeross) may also contain input from a wild species.
Figure 1. Development of protoplast derived colonies in agarose droplets. A, dividing protoplast after 4 days in culture, x 350, bar 50 #m; B and C, colonies after 10 and 12 days, x 75, bar 100 #m; D, colony after 15 days, x 80, bar 100 #m, E, globular embryo after 20 days, x 30, bar 200 /.tm.
Identification of factors affecting plating efficiency and shoot formation In the following diseussionthe term "shoot formation" refers to the typical way of plant regeneration (elongation of the shoot apex of somatic embryos without root development, see below). The average yield of protoplasts from one gram of hypoeetyl tissue was 2 to 3 million. T h e protoplast population was rather heterogeneous both in their size and in the amount of cytoplasm. A more homogeneous population was obtained when large, highly vaeuolated protoplasts were removed by filtration through nylon meshes. The age of hypoeotyl material (5, 9 or 11 days) was a critical factor. Somatic embryogensis was observed only in
262
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Figure 3. Development of embryogenic colonies on regeneration media. A, embryos at different stages of development, x 3, bar 5 nun; B, elongatingshoots on hormone free medium in 9 cm petri dish.
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Figure 2. Effect of initial NAA concentration on colony and shoot formation. dashed lines, colony formation (percent of protoplasts plated); solid lines, shoot formation (percent of viable colonies); (11), with 2,4D treatment; ([]), without 2,4D treatment.
colonies of protoplasts from 5 day old hypocotyls. Optimal protoplastdensity was determined first.Highest plating efficiency and colony formation was found at a density of 3 x 104 ml "I (calculatedusing the volume of agarose droplet only). Fig.1A shows firstdivisions of protoplasts in droplets of V - K M medium supplemented with 2.0 m g 1-1 B A and 0.5 m g I-I N A A and solidifiedby 0.5 percent agarose. Further divisions in most cases occurred inside the boundaries of the initialcell wall, thus the daughter cells became smaller than the original protoplasts and rich in cytoplasm. After one week the liquid over the droplets was replaced by fresh V - K M medium containing one-tenth of the original hormone concentrations.Further development of colonies followed different pathways. Developmental stages characteristic of somatic embryogenesis (Fig.IB to IE) could be observed in the cultures. However, the frequency of shoot formation was rather low (0.18 percent of viable colonies). In previous experiments on Helianthus protoplast culture compact colonies representing the first stage of somatic cmbryogcncsis could not bc observed in liquid cultures.It appears that a supporting matrix is essential for the development of embryos. For this reason, influence of the nature and concentration of gelling agent was investigated next. In cultures where 0.25 percent agarose was used, the frequency of shoot formation was only half of that observed in 0.5 percent agarose. Increasing agarosc concentration to 1.0 percent resulted in a further increase of this frequency. However, due to the high rigidity of 1.0 percent gels, colonies could not be removed from the droplet without damage. Alginic acid at 1.4 percent concentration was also tried, but there was no improvement as compared to agarose solidified cultures. In several dicotyledonous species including sunflower it has been shown that exposure of cultures to high concentrations of auxins for a limited period could induce subsequent development of somatic embryos (Finer 1987; Prado and Berville 1990). In our experiments N A A or 2,4D treatment also improved the embryogenie response of protoplast derived colonies. One week
Figure 4. Grafting shoot tips on in vivo grown hypocotyls. A, shoot developedon hormonefree MS medium;B, same shoot gratted on hypocotyl;C, floweringregeneratedplants. old colonies grown in V - I ~ t supplemented with 2.0 mg 1"1 BAP and 0.5 mg 1"1 NAA were treated with different auxins by replacing the liquid medium over the droplets by V-KM supplemented with 2.2 mg 1"1 of NAA, 2,4D or dieamba. This medium was leR on the droplets for one week, then it was replaced by V-KM with reduced NAA and BA contents as described above. Treatment with dicamba, NAA and 2,4D increased the frequency of shoot formation from 0.18 percent to 0.24, 0.8 and 1.8 percent, respectively. The protoplast density (3 x 104 per ml) optimal for initial division and colony formation appeared to be too high for later stages of colony development. Development of colonies slowed and they started to release a brown material (probably polyphenols) into the medium. Starting density was, therefore, reduced to 5 x 103 m1-1 inside the agarose droplet. The adverse effect of reduced density on plating efficiency was compensated by adding nursing protoplasts to the liquid phase. These
263 protoplasts were removed after one week when the liquid was refreshed. Plating efficiency and the frequency of colony formation were practically the same as those observed previously at higher protoplast densities. However, reduced density of colonies resulted in a significant increase in the frequency of shoot formation (1.2 percent v e r s u s O. 18).
Influence of initial auxin concentration Subsequent experiments were carried out using droplets solidified by 0.5 percent agarose, a protoplast density of 5 x 103 m1-1 and nursing protoplasts in the liquid phase at a density of 3 x 104 ml "l. A systematic investigation on the influence of initial auxin concentration was performed. Protoplasts were cultured in the presence of different concentrations (0.5 to 10.0 mg 1-1) of NAA together with 2.0 mg 1"1 BA. Interaction of initial auxin concentration and subsequent 2,4D treatment (2.2 mg 1-l) was studied by setting up parallel cultures with and without 2,4D treatment for all NAA concentrations used. Data are summarized in Fig.2. Plating efficiency (not shown) slightly decreased (from 34 to about 24 percent) with increasing NAA concentration. Colony formation was not significantly influenced by initial auxin levels. The frequency of shoot formation was highest at 4.0 mg 1-1 initial NAA concentration with subsequent 2,4D treatment. Under these conditions 9.6 percent of viable colonies formed shoots,
Embryo maturation and regeneration of plants After three weeks of culture developing colonies were released from the agarose droplets and were cultured in liquid V-KM for a few days. They were then transferred onto solid medium and were exposed to light to induce maturation and germination of somatic embryos. Three media differing in basic salt composition but otherwise supplemented with the same compounds (see Materials and Methods) were tried at this point. On B5 and White's media the colonies turned brown and developing shoots had very pale green colour. On MS medium calluses continued to grow vigorously and developing shoots had a dark green colour. When agar was used as gelling agent, callus formation became predominant, while media solidifed by Phytagel favoured embryo development. Within two to three weeks embryos at different developmental stages (from globular to torpedo-shaped) could be observed in the cultures. Development was slightly abnormal (Fig.3A, bottom row). Further development of both shoot and root was observed only in less than 5 percent of embryos. The typical way of development was shoot elongation accompanied by swelling or callusing at the root apex (Fig.3A, top row). Development of normal shoots (at a frequency of over 80 percent) occurred only on media supplemented with silver nitrate, an inhibitor of ethylene action. In the absence of silver nitrate vitrification (elongated stem, pale green watery leaves) was observed and normal shoots were infrequent. Similar effects have been described and attributed to ethylene action in other species (Chi and Pua 1989; Perl et al. 1988) and also in sunflower tissue cultures (Chraibi et al. 1991). It was essential to transfer the embryos onto hormone free MS medium as soon as shoot elongation had started. Shoots started to exhibit vitrification even in the presence of silver nitrate if they were left on the regeneration medium for longer periods. On hormone free medium the growth of callus stopped and normal shoot elongation continued (Fig.3B.). Plantlets with well developed roots could be transplanted into soil. However, in the ease of other shoots, transplanting would
have involved an additional period of rooting. Sunflower shoots in culture have a tendency to premature flowering (as noted e.g. by Burrus et al. 1991) which increases with the time elapsed in culture. The presence of flower buds in turn stops development of the shoots and causes seenescenee, usually without any seed production. Therefore, sunflower shoots should be transferred into soil at the earliest possible stage. An alternative to rooting is grafting of the shoot apex on hypocotyls of in vivo grown plants (Fig.4). Shoots as small as 10 mm in length could sueeessfuUy be grafted. More than 80 percent of 111 shoots grafted were established on the root stock and continued development. Although these plants did not grow to normal size, they flowered (Fig.4) and produced seeds. The number of seeds obtained from individual plants was in the range of 5 to 10. In conclusion, identificationof the special genotype Florom-328 allows regeneration of plants from protoplasts at practically usable frequencies. Experiments are in progress to utilize this sytem for the production of stably transformed sunflower plants by PEG-mediated direct DNA uptake. Acknowledgements This work was supported by a grant from the Feed Crop Research Institute, Iregszemcse, Hungary. We thank Dr. B. Pelissier (RlaonePoulene, Lyon) for valuable suggestionsconcerningshoot gratting.
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