A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts. Ruth Wingender, Hans-Joachim Henn, Stefan Barth, Dirk Voeste, Hassan Machlab, ...
Plant Cell Reports
Plant Cell Reports (1996) 15:742-745
9 Springer-Verlag1996
A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts Ruth Wingender, Hans-Joachim Henn, Stefan Barth, Dirk Voeste, Hassan Machlab, and Heide Schnabl Institut f/ir Landwirtschaftliche Botanik der Universit/it Bonn, Meckenheimer Allee 176, D-53115 Bonn, Germany Received 26 June 1995/Revised version received 8 September 1995 - Communicated by H. L6rz
Summary.
Hypocotyl
protoplasts
of
four
different
Helianthus annuus genotypes were cultivated for 22 - 28
days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.
sunflower species seeming to be less recalcitrant to in vitro culture (Chanabe et al. 1991; Krasnyanski et al. 1992) than the cultivated species. It has therefore been speculated that the regeneration potential of two genotypes PTO24 (Burrus et al. 1991) designated as cms /H.petiolaris/cms HA89 backcross and Florom-328 (Krasnyanski & Menczel 1993) exhibiting branching, might be contributed by the wild type genome (Krasnyanski & Menczel 1993). Moreover, the regeneration of. fertile plants from protoplasts of one inbred line out of sixteen tested genotypes lacking any wild type contribution has been reported (Fischer et al. 1992). Our objective was to develop a reproducible shoot and root regeneration protocol applicable even to H. annuus genotypes of low regeneration potential. Four cultivars differing in their in vitro culture response were selected and regenerated. Since vitrification and precocious flowering have been observed generally during in vitro cultivation of sunflower (Fischer et al. 1992; Krasnyanski and Menczel 1993), another aim of the work presented was to overcome these problems.
Abbreviations: BAP, 6-benzylaminopurine; 2,4-D, 2,4dichlorophenoxyacetic acid; FeNaEDTA, ethylenediaminetetraacetic acid ferric sodium salt; IAA, indole acetic acid; MES, morpholinoethane sulfonic acid; NAA, 1-naphtalene acetic acid
Introduction The regeneration of fertile plants from protoplasts of sunflower has been achieved by protocols differing considerably with respect to basal medium and growth regulators added. Both embryogenesis (Krasnyanski and Menczel 1993; Barth et al. 1993) as well as organogenesis (Binding et al. 1981; Burrus et al. 1991; Fischer et al. 1992) have been described resulting in shoot regeneration. In contrast to Krasnyanski and Menczel (1993) we did not succeed in shoot elongation of isolated embryos which prompted us to reinvestigate organogenesis of sunflower protoplasts derived callus. Regeneration via both pathways is apparently restricted to a small proportion of sunflower genotypes, with wild Correspondence to." R. Wingender
Materials and Methods Plant material and protoplast culture. Seeds of four cultivars ('Cerflor'
and 'Euroflor'from RS Sacon Pflanzenzucht GmbH, Hamburg,Germany; 'Florom-328' from the Institute of Cereal and Industrial Plant Research, Fundulea, Romania; Frankasol from Cargill-Saaten, Hamburg, Germany) were surface sterilized in NaOCI (4.5%, 60 rain). After three washes in sterile water., the seeds were germinated and grown for one week in the dark as described (Schmitz & Schnabl 1989). Hypocotylprotoplastswere isolated according to Lennee & Cbupeau (1986) and Schmitz & Schnabl (1989). The protoplasts were cultivated at a density of 2x104 mlqin 50 ~tl agarose droplets (Shillito et al. 1983) 20 per Petri dish coveredwith 10 ml liquid mKM medium in the dark at 26~ The inorganic compoundsof this medium are given in Table 1 while vitamins, organic acids, sugar and sugar alcohols as well as casaminoacid were added according to Kao and Michayluk (1975), excluding the high glucose concentration used for cultivation of protoplasts, L-aminoacids,nucleic acid bases and coconut water.
743 Table 1. Inorganiccompoundsof mediummKM CaC12 10mM KH2PO4 0.5 mM KNO3 7.52 mM NH4NO3 5mM MgSO4 4 mM COC12 0.11 p.M CuSO4 0.1 laM H3B03 0.05 mM KI 4.9 laM MnSO4 50 laM Na2MoO4 0.48 I.tM ZnSO4 5.1 p.M FeNaEDTA 105 ~tM The osmolaritywas adjusted to 0.6 osmol kg H2O-I with mannitol and the pH to 5.6. Concomitant with weekly changes of the medium the osmolarity was reduced to 0.3 osmol kg HzO1 in steps of 0.1 osmol kg H201. Supplementationwith plant growth regulators was as depicted in Figure 1. In the last week the cultures were shifted to a 12 h light period (50 lamol sec-l m"2 ) at 26~ which was maintained in all further cultivation steps. Optimization steps i.e. medium composition, light treatment, growth regulator regime were performed with Cerflor and Euroflor. The values given here refer to three independent experiments performed in parallel with all four genotypes.
Shoot differentiation. In the 5th week, agarose droplets (6-7 drops per Petri dish) were transferred to solid differentiation(D) medium based on MS salts (Murashige and Skoog, 1962) with the followingadditions:87.6 mM sucrose, 2.7 mM myo-inositol,3raM MES, 7.4 ~tM thiamine-HCI,2 nM nicotinic acid, 1.2 nM pyridoxine-HCl,13/.tM glycine, 5.8 ~tM silver nitrate and 4 g 11 phytagel, pH 5.7. The plant growth regulators BAP and NAA were added to a final concentration of 4.4 ~tM and 0.1~tM, respectively. Morphogenic colonies were transferred to hormone free SE medium (1/2 MS salts, 58.43 mM sucrose, 2.7 mM inositol, 7.4 l.tMthiamine-HCl, 2 nM nicotinic acid, 1.2 nM pyridoxine-HC1, 3 mM MES, 5.8 ~tM AgNO3, and 4 g I1 phytagel, pH 5.7). 20
Transfer of plants into soil Shoots were cut, dipped shortly into 5.3 M NAA solution and cultured on modified SE20 supplementedwith 2 g 1-1 casein-hydrolysate(accordingto Sato et al. 1993) and 13 laM glycine (pH 5.7). Plants with well developed roots were transferred to the greenhouse in a 50/50 sterile mixture of vermiculiteand garden soil and subjected to different hardeningstations.
dividing protoplasts were obtained in the case of Euroflor and Frankasol. The regeneration protocol is summarized in Figure 1. For the genotypes of higher regenerative potential, Florom-328 and Cerflor, the agarose droplet culture was shortened by reducing the cultivation time in the third week to 5 days and to 3 days in the fourth week. Factors found to be important for subsequent normal shoot development were: medium composition, gradual reduction of osmolarity and light treatment in the fourth week. Cultivation in V KM (Binding & Nehls 1977) was less efficient than in mKM. The gradual reduction of osmolarity was fotmd to be essential for normal shoot development during subsequent culture on solid medium otherwise only stunted shoots were obtained. Cultures that were shifted before the third week to 12 h light period showed reduced shoot formation.
Callus cultivation (5th- 7th week) The agarose droplets were transferred onto solid D medium and gently cut into pieces in order to release the small calli (size 0.1-0.3 mm). The plating efficiency (calculated from the total number of protoplasts) varied between the genotypes. The highest of ca. 5% was obtained with Florom-328 followed by Cerflor3%, Euroflor2% and Frankasol 1%. During the first week of solid phase culture the first differentiation processes became visible. Agarose droplet culture of protoplasts covered with mKM culture 1. week 2. week 3. week 4. week
dark dark dark light
growthregulators (~tM) 4 10 4 4
mosmol kg H2O-1
BAP,5 NAA 2,4 D BAP,0.5 NAA BAP,0.5 NAA
600 500 400 300
Callus cultivationon solid medium medium 5. week
D
sucrose (mM) 4
BAP,0.1 NAA
87.6
Results
Agarose droplet culture of protoplasts (1-4 week) Four cultivars were selected for this study. Florom-328 exhibiting the best regeneration potential out of 91 genotypes tested (Krasnyanski & Menczel 1993), Cerflor, Euroflor (Barth et al. 1993) and Frankasol, the latter being especially recalcitrant to in vitro culture (not shown). The avera6ge ryield of isolated protoplasts was in a range of 12x10 g" fresh weight irrespective of the genotype used. An initial plating density of 2.0x104 protoplasts ml "l agarose was found to be crucial for the further fate of the developing calli. Higher densities led to brownish callus in the third week which stopped growing in most cases. The initial division rates varied between the genotypes. In the second week about 60-70% Florom-328 and Cerflor protoplasts showed cell division while only 50-60%
Transfer ofmorphogenic callus ( shoot formation ) 8. week
SE20
hormonefree
58.4
Cutting of shoots, root formationand transferto greenhouse 11. week SE20* 13. week
hormonefree after dipping into NAA transfer to greenhouse
58.4
SE20* modified SE20medium Fig.1. Regeneration protocol of plants from hypocotyl protoplasts of sunflower cultivars Florom-328, Celflor, Euroflor and Frankasol. For media see material and methods.
744 I993) was also performed but proved to be more laborious. All plantlets obtained were stunted, often branched and flower buds were initiated soon after transfer (Fig. 2d). Depending on the number of heads 5 - 25 seeds were obtained per plant. Discussion
Fig. 2. Morphogeniccalli formed globular green structures (a) which subsequently developed small shoots (b). Shoots were rooted after dipping into 5.3 M NAA (c) and transferredto the greenhouse(d). Morphogenic calli exhibited green globular structures (Fig. 2a) which formed leaf primordia and subsequently short shoots (Fig 2b). One shoot was formed per callus. The yield of these calli was highest for Florom-328 8.5% and lowest for Frankasol 3.5% (calculated from total number of calli obtained). Higher BAP concentrations resulted in better yields i.e. in the presence of 17.7 gM BAP about 12% shoot producing calli were found in the case of Cerflor. However, concomitant vitrification became a serious problem.
Shoot differentiation (8th-I Oth week) Elongation of shoots was obtained on SE20. Since this medium is devoid of growth regulators most of the nonmorphogenic part of the transferred calli had to be removed carefully. Otherwise these regions turned brown rapidly and inhibited further development of the shoots. After about 2 - 3 weeks on SE20 the shoots reached a size of 0.5 cm. Based on the number of 10 104 protoplasts plated 35 (_+ 4) shoots were Obtained for Florom-328, 14 (_+ 3) for Cerflor, 8 (+ 3) for Euroflor and 5 (+ 2) for Frankasol. The shoots were cut, dipped in 5.3M NAA solution and subcultured on modified SE20, In about seven days, roots were formed (Fig. 2c) and the plantlets were transferred to the greenhouse one week later. About 90% of the shoots produced roots and about 1% showed precocious flowering. Without hormonal treatment the shoots produced no roots, on the other hand supplementation of the medium with NAA (4.4-0.44 gM) resulted always in calussing. Grafting of the shoots (Fischer et al. 1992; Krasnyanski & Mencze!
As reported here, we succeeded in establishing a regeneration protocol for four different genotypes of Helianthus annuus L., three of them lacking any wild type contribution (Cerflor, Euroflor, Frankasol). Seeds of the fourth, Florom-328, were heterogenous (revealed by isoenzyme analysis, not shown here) but showed a high regeneration potential probably restricted only to a part of the material. Furthermore, we present a regeneration protocol for sunflower hypocotyl protoplasts which involves a culture time of only 13 weeks up to transfer into the greenhouse. As an additional advantage of the protocol described here, we show a generation of rooted shoots after dipping the cut shoots in high auxin solution. In contrast to Burrus et al. (1991) we used NAA instead of indole acetic acid which might indicate that both compounds can be used for the induction of root formation. A good rooting ratio was obtained due to the short culture period and the healthy appearance of the shoots. In previous reports most of the shoots had to be grafted (Fischer et al. 1992; Krasnyanski & Menczel 1993). An overall efficiency of 0.4% considering the number of protoplasts plated to number of organogenic calli producing shoots was obtained in the case of Florom-328 and of 0:03% for Frankasol. These low yields are mainly due to the low plating efficiency as only few microcalli develop into growing colonies. This problem is common to sunflower tissue culture and remains to be solved. The overall yields of morphogenic calli are comparable to those from previous reports. Fischer et al. (1992) reported a frequency of shoot formation of 0.01%, Burrus et al. (1991) of 0.054% and Krasnyanski & Menczel (1993) of 1.3%. Since the latter used a culture regime optimized for Florom328, higher yields with respect to the protocol given here should be expected. Concomitant with excess cytokinin vitrified shoots were observed as previously described (Bornmann & Vogelman 1984). Normal shoots were obtained using the growth regulator concentrations reported here in combination with silver nitrate, an inhibitor of ethylene action in the differentiation medium. AgNO 3 has been tested in sunflower tissue culture; there are, however, conflicting reports on its usefulness. Less vitrification in the presence of silver nitrate was observed in two cases (Chraibi et al. 1991; Krasnyanski & Menzel 1993) whereas no effect was found by Fischer et al. (1992). Here protoplasts from four different genotypes were regenerated which all reacted well to AgNO 3. Therefore, we suppose that the time and the overall culture regime trigger the response not the addition of this compound per se. Comparing the regeneration potential of sunflower protoplasts reported here with respect to previous results (Barth et al. 1993) a wide spectrum of factors was changed. Besides the nitrogen source and Ca 2+ concentration, we
745 tested effects of phytohormones, light treatment and gradual decrease of osmolarity. The nitrogen source contained in mKM was modified compared to V Km (Binding & Nehls 1977) by reducing NO3" from 18 mM to 12,5 mM and by raising NH4+ from 3 mM to 5 raM. In previous reports glutamine (Lenne & Chupeau 1986), casamino acids ( Burrus et al. 1991) as sole nitrogen source as well as supplementation of V KM with glutamine (Krasnyanski & Menczel 1993) were used in liquid media for the cultivation of sunflower hypocotyl protoplasts. These considerable differences suggest that the nitrogen source has a strong effect on tissue culture response in sunflower however, they might also reflect genotype specific requirements. 2+ With respect to V KM, the Ca concentratton was increased twofold. At the whole plant level addition of Ca 2+ enhances the number of chloroplasts (Bowler et al. 1994) which might also affect the generation of green spots on calli. There is also a strong response of Ca 2+ on the development of tracheidal elements (Roberts & Haigler 1990) a process coupled to organization and regeneration of calli (von Keller et al. 1994). In some cases regeneration was o nl y observ e d after Ca 2+ treatment of calli (Montoro et al. 1993) or when donor plants for protoplast isolation were grown in the presence of Ca 2+ (O'Brien & Lindsay 1993). Therefore we suggest that high Ca2+-concentrations stimulate organogenesis. The rational behind the growth regulator regime chosen was to increase cell divisions by adding BAP and NAA at an 0.8/1 ratio in the first week of protoplast cultivation. Subsequently relatively high concentration of 2-4,D found to stimulate shoot formation (Krasnyanski & Menzel 1993) was applied followed by a BAP to NAA ratio of 8/1. In order to favour organogenesis rather than embryogenesis, the ratio of cytokinin to auxin was increased to 40 in the differentiation medium. Much lower ratios have been applied in previous reports (Burrus et al. 1991; ratio 4, Fischer et al. 1992; ratio 3.2, Krasnyanski & Menzcel 1993; ratio 16). Since higher ratios resulted in better yields of morphogenic calli we assume that high BAP concentrations are crucial for the organogenic response in Helianthus irrespective of the genotype in the presence of a low NAA concentration. Beside the medium composition we found that additional factors required for normal shoot development were: light treatment during the fourth week of agarose droplet culture of protoplasts and the gradual decrease of osmolarity from 600 osmol kg H20"1 in the first week to 300 osmol kg H20"1 in the fourth week. Indeed the whole spectrum of the above mentioned factors seems to induce the regeneration potential of sunflower cultivars and to result in the establishment of a regeneration protocol which might be used in general for cultivars of Helianthus annuus. These results will probably help to render other genotypes of choice amenable to tissue culture. Acknowledgements. The authors would like to thank the DFG and DARA for financial support to H.S.
References Barth S, Voeste D, Wingender R, Schnabl H (1993). Bot. Acta 106: 22022 Binding H, Nehls R (1977) Z. Pflanzenphysiol. 85:279-280 Binding H, Nehls R, Kock R., Finger J. & Mordhorst G (1981) Z. Pflanzenphysiol. 101:119-130 Bornman CH, Vogelman TC (1984) Physiol Plant. 61:505-512 Bowler C, Neuhans G, Yamagata H, Chua N-H (1994) Cell 77:73-81 Burrus M, Chanabe C, Alibert G, Bidney D (1991) Plant Cell Reports 10:161-166 Chanabe C, Burrus M, Bidney D, Alibert G (1991) Plant Cell Rep. 9: 63438 Chraibi KMB, Castelle JC, Latche A, Roustain JP, Fallot J (1991) Plant Cell Rep. 10:204-207 Fischer C, Klethi P, Hahne G (1992) Plant Cell Rep.11 : 632-636 Kao KN, Michayluk MR (1975) Planta 126:105-110 Krasnyanski S, Menczel L (1993) Plant Cell Rep. 12 : 260-263 Krasnyanski S, Polgar Z, Nemeth G, Menczel L (1992) Plant Cell Rep. 11:7-10 Lennee P, Chupeau Y (1986) Plant Science 3:69-75 Montoro P, Etienne H, Michaux N, Carton M-P (1993) Plant Cell Tissue and Organ Culture 35:279-286 Murashige T, Skoog F. (1962) Physiol Plant. 15:473-497 Roberts AW, Haigler CH (1990) Planta 180:502-509 Sato S, Hagimori M, Sumoi I (1993) Plant Cell Rep. 12:370-374 Schmitz P, Schnabl H (1989) J. Plant Physiol. 155:223-227 Shillito RD, Paszkowski J, Potrykus I (1983) Plant Cell Rep. 2: 244247 von Keller A, Frey-Koonen N, Wingender R, Schnabl H (I 994) Plant Cell Tissue and Organ Culture 37:277-285
