HELIA. 22. Nr. 30. o.p. 6l-7O. ( 1999) ' UDC 631.524.8:633.854.78

AN OPTI]MIZDD PROCEDURE FORSUNFLO1ryER PROTOPLAST (Helianthus ssp.)

CULTwATTON rN LrgUrD CULTURE

P.C. BinsfeldI'2, R. Wingenderl, H. Schnabll

! InstitutJitr Landu;irtschsliche Boto,nik der lJniDersttat Bonn, MeckenheirnerAllee. 176. D-53115 Bonn. Germanu2 Centro de Biotecnologia, IJniuer{td.ade Federal d.e Pelotas, C.P. 354, CEP960 1 O-9OO. Pelotas-RS. Brazil

Receiued: JuIg O2, 1998Accepted: September 2A, 1998

SUMMARY

Mesophyll protoplasts isolated from four sunflower species: H. o'nnuus,H. giganteus, H. maximiliani, H. nuttallii were subjected to cultivation in liq-uid media. Various factors were analyzed and their optimization resulted in ahigh and reproducible percentage ofviable and dividing cells in liquid culture.Donor plant pre-cultivation in the dark for two days, usage of 8-12 day-oldleaves, selection of undifferentiated protoplasts, a cultivation density of 6 x 1Oaprotoplasts ml-l and culûvation in day/night cycle were found to be essentialfor a high ald synchronous division rate of more TlrLan 7Oo/o dividing cells. Celldivision was obtained for all four species at different rates reflecting their culti,vation potential.

Key words: Protoplast g;rowth, Ifetianthus, cell division, cell suspension

INTRODUCTION

Protoplasts are considered as one of the most important vehicles for geneticimprovement of plants (Bekkaoui et al., 1987) and provide the start point for manytransformation and manipulating techniques. species of the Herianfhus genus pro-vide important genetic variability like stable stress tolerance or pathogen resistancethought to contribute to improvement of sunflower production (Korel ef al., 1996).On the other hand protoplast culture of these species is known to present major dif-fïculties (Fischer et aI., 1992). In the last years many efforts were made to use pro-toplast biotechnologr such as somatic hybridization (Krasnyanski and Menczel,1995: Henn et al., 1998), A. tumeJaciens transformation (Knittel et at., lg94;schonenberget aL, 1994) and direct DNA transfer (Machlab, 1996) in sunflower. Anew aspect is represented by the microprotoplast fusion technique enabling theintergeneric transfer of partial genome and alien genes from sexually incompatible

62 HELIA, 22, Nr. 30, p.p. 6l-70, ( 1999)

donor species (Ramulu et aI., 19951. A prerequisite for tfiis method is the establish-ment of s5mchronously dividing cell cultures.

This technique has so far not been applied in sunflower where protoplast culti-vation was mostly optimized for plant regeneration (Chanabe et al., l99l; Burruset aL, l99l; Polgar and Krasniansky, lg92; Fischer et al., 1992; Krasnyanski andMenczel, 1993; Wingender ef al., 1996). Since organogenic or embryogenic respon-ses were only obtained with âgarose droplet or Ca-alginate embedded protoplasts(Fischer et aI., 1996) reports on protoplast culture in liquid media are rare andwithout much success. Bohorova (1986) reported that using three different mediaand different Helianthus species no cell divisions were induced in liquid media.Lenné and Chupeau (1986) succeeded in liquid culture ofprotoplasts from varioussources but only hypocotyl protoplasts were found to divide under the conditionstested. Liquid culture of mesophyll protoplasts was first achieved by Guilley andHahne (f 989) who obtainedTOo/o dividingcells after l3 days in culture.

In this report we present an.efficient procedure for inducing cell divisions in ahigh percentage of cells in liquid culture from different Helianthus species. Thistechnique will allow to obtain synchronized cell dil'isions and thus enable genomemanipulations, like transformation, direct gene transfer, induction of micronucleior metaphase chromosome isolation from the dividing cells.

MATERIAL AND METHODS

Plant material

Plants of the cultivar Florom-32e @. annuus) (Schmitz and Schnabl, l9B9)from the Institute of Cereal and Industrial Plant Research, Fundulea, Romania andin uttro propagated (Imhoff et al., 1996) plants of three wild sunflower species (H,giganteus, H. maxîmiltani, H. nuttallti.l were cultivated for O, 12, 22, 48 and Z2hours in the dark before protoplast isolation. Leaves (l g) were placed intact in 20ml enzyme solution which was prepared as described by Henn et at. (1998) formesophyll protoplasts. The leaves were incubated at 2O'C for 14h and protoplastsliberated by subsequent rotation at 45 rpm at 27"c for 2h. All steps were performedin the dark. The enzyme-protoplast mixture was liltered through 50 and 40 pmnylon sieves and protoplasts purified as described by Henn et at. (1998).

Cultlvatlon

Prior to cultivation the protoplast suspension was enriched with undifferenti-ated protoplasts by making use of their slow sedimentation. The protoplastsobtained from 3 g material (3 ml in salt buffer) were transferred into 6 ml KMARIiquid medium (Table l)in a test tube. After O, 5, 10, 20 and 40 min the floatingprotoplasts were removed with a pipette and adjusted to densities of 2-lS x lOaprotoplasts ml-I with medium. Subsequently they were cultivated in Petri dishes

HELIA,22, Nr. 3O, p.p. 6l-70, (1999)

(0=9 cm) in lO ml liquid KMAR supplemented with different combinations of plant

gowth regulators (IAA 5.7 pM, NAA 5.4 pM, F,AP 4'4 pM and zeatine 4'6 pM) and

ascorbic acid (o, 10, 20 and 40 mg f I) ln the dark or day/night cycle [14h at 30 pmol

rn-2"-l) at 26"C. After B days the cells were transferred to fresh medium.

Table l: Composition of KMAR medium

compound Sugar Vitamine

63

KN03

QaCl22H2O

MgS0a 7H20

NH4N03

NH4H2P04

FeNa EDTA

MnC12 4H20

KI

ZnS0a

Na2Mo0a H20

CuS0a

H3803

CoC12 6H20

0rganic acid

15.0 mM Cellobiose

10.0 mM Fructose

4.0 mM Mannose

4.0 mM Rhamnose

0.5 mM Ribose

0.1 mM Sucrose

23.0 trlM Sorbitol

15.0 gM Xylose

5.0 pM Inositol

0.4 trrM Mannitol

0.06 trrM Glucose

0.05 pM

0.04irM Nucleic acid

250 mg l-1

.-1zcu mg l'250 mg l-1

,-1zbu mg r

.-1zbu mg |'250 mg l-1

250 mg l-1

100 mg l-1

2 1.0 g l-1

68.4 g l'1

250 mg l- Ascorbic acid

Pantothenic acid

Choline Chloride

Nicotinamide

Pyridoxine-HCl

Thiamine

Folic acid

Aminobenz. Acid

Vitamine B12

Biotin

Vitamine A

Vitamine D3

Amino acid

2.0 mg l'

1.0 mg l-1

1.0 mg I-1

1.0 mg l'r

1.0 mg l-r.1.0

mg l-1

0.4 mg l-1

1 0 trrg l-1

20 ltgl'11 0 irg l-1

'I 0 prg l-1

10gg l-1

Histidine

Lysrne

Proline

Tyrosine

Tryptophan

Cysteine

Glycine

Asparagi ne

Arginine

Leucine

Serine

lsoleucine

Methionine

0.1 mg l-

0.1 mg l-1

0. 1 mg l-1

0.1 mg l-1

0.1 mg l-1

0.1 mg l-1

0.1 mg l-1

0.1 mg l-1

0..1 mg l-1

0. 1 mg l-1

0.1 mg l-1

0.1 mg l-1

0.1 mg l-1

Adenine

Uracil

Xanthine

Guanrne

Thymine

Hypoxanthine

,-1u.uJ mg | ' Glutamine

0.03 mg l-1 Glutamic acid

0.03 mg l-1 Cysteine

0.03 mg l-1 Aspartic acid

0.03 mg I-1 Phenylalanine

Valine 0.1 mg l-1

Threonine 0.1 mg l-1

0.10 mg l-

1.0 mg l-

Malic acid

Fumaric acid

Citric acid

Na- Pyruvate

Other addition

40 mg l-

40 mg l-1

40 mg 1"1

40 mg l-1

0.6 mg l-1

0.2 mg l-r

0.1 mg l-1

0.1 mg I-1

Casein hydrolysate

0smolarity 600 mOsmo/kg pH

250 mg l- MES

5.7

600 mg I

Determinatlon of cell vtabtltty

One volume of cell suspension was mixed with one volume of 50 mg l-l fluores-cein diacetate which was prepared fresh by diluting a 4 g I-l stock in growthmedium. The percentage of viability was calculated as the number of cells showinggreen fluorescence under UV illumination, in relation to the total number of cells(lOOo/o), counted under white light.

Statistical analysls

All experiments were repeated three times. The analysis of variance was accord-ing to completely random design and multiple comparison test of the mean accord-ing to Duncan's test at P<0.05 (using MS-SANEST software).

64 HELIA,22, Nr. 30, p.p. 6I-70, (1999)

RESULTS AND DISCUSSION

Effects of plant growth regulators

The highest yield of 4.5 x I 06 mesophyll protoplasts per gram of fresh weightwas obtained with H. giganteus followed by H. nuttalli,i 2.4 x 106, H. annuus 2 xlOo and H. maximiliant 1.5 x 106. Incubation of the leaves for over l6h in enzymesolution increased the yield but decreased the viability of the isolated protoplasts.Preliminary tests showed that the best relation auxine/cytokinine was 1.2, inde-pendent of the type and the combination of growth regulators which is in agreementwith wingender et aI. (1996). All tested combinations of NAA, IAA, BAp and zeatinewere appropriate for induction of cell division (Table 2).

Table 2: Effect of different combinations of plant growth regulators in the medium onprotoplast division (06) after 5 days in culture

Helianthusgrowth regulator annuus giganteus maximilianiIAA/zeatine

NAA/BAP

IAA/ BAP

NM/zeatine

62.3 a

63.3 b

58.1 b

56.0 b

64.3 a

61.1 a

54.6 c

57.4 b

45.4 a

40.8 b

39.8 b

37.6 c

46.7 b

49.8 a

45.4 b

41 .3c

54.6

53.7

49.5

48.0Âoo 40.9 45.7

Values followed by the letters are significantly different in each column (p<0.05)

The overall best results were obtained with IANzeatine where also earlier (2days) onset of mitosis was observed. This combination was therefore chosen for allsubsequent experiments. Regardless of the growth regulator supplementation spe-cies speciflc differences of the division rates were obtained reflecting probablygenetical control of regeneration competence. Similar responses in sunflower geno-types were reported by chanabe et al. (1991) and wingender et al. (1996). For therapid recovery from isolation stress and a synchronous cell division it was impor-tant to use a rich and complete culture medium. In preliminary tests KMAR wascompared with mKM (Binding and Nehls, 19771 and MS (Murashige and Skoog,1962) medium and found to give the best results.

Effect of protoplast density, ascorbic acld and plant pre-cultivatlon in thedark

For most plant cells a minimum effective cell density of about I 04- 1 05 cells ml- I(Sakurai and Mori, 1996) is required in order to initiate cell division. We tested den-sities of 2-15 x loa protoplasts ml-l. only the lowest density was found to promotecell division rates of about 54o/o while with the highest density only 29o/o could beachieved (Figure l). In addition, many protoplasts became brown and died afterfour days. In order to reduce the mortality rate of protoplasts we added ascorbicacid to the culture medium and in contrast to the results reported by Xu and Jia( 1996) we observed a decreasing viability and division ability of the cells in the pres-

65

ence of more than 10 mg l-l of ascorbic acid. As shown in Figure 2, plant pre-culti-vation in the dark resulted in an increase of cell viability and division rate. The pre-cultivation for 48h in the dark was the most effective leading to a Zo/o increase in via-bility and to lolo higher division rate.

-E-I'-I*-----a

T-/-l=\T,"- ' \t

-1-"-t I

F{bjfriI . pvisidl

Ë60èR

't

g

o.*

246810 12.141ôCultivation density of protoplasts (x 1O.)

Figure 1: Elfect oJ cell densitA on dtuisionrates oJ cells deriuedJrom mesophgll pro-toplasts cultiuatedJor S dags in KMAR liq_uid medium in the presence oj 5.7 W IAAand 4.6 W zeatine, mean ualues;for the

Jo ur H e lianthus spec ies.

4a 60Plânt prê-cultivation on the dÊrk (h)

Figure 2: Elfect oJ dark pre-cultiuation on theuiabilitg and diuision rates oJ cells d.eriued.

Jrom mesophgll protoplasts oJH. gigan-teus cultiuated in liquid culture KMAR sup_plemented utith 5.7 1tM oJ IAA and 4.6 p,Mzeatlne, $ter 5 dags in culture.

12

Selection of the protoplasts with the highest growth potential

Another factor that contributed to a high protoplast division rate in liquid cul-ture was to select small protoplasts. As show in Figure 3, both of the evaluatedparameters, cell viability and division ability, increased with a cell selection. Wecould veri$r that protoplasts that quickly sedimented had low division ability, incontrast to those that floated (not shown). After lo min sedimentation, we couldselect protoplasts with higher division rates (between l4-l7o/o) in relation to controlprotoplasts' It can be speculated that this effect might be due to the differentiationstate of the cells and size of vacuole. cells with a large vacuole are supposed to sed-iment quicker than cells with small vacuoles. Xu and Jia (1996) described a poordivision ability of cells from A. sphaerocephala when the cells had a large vacuoleand only those protoplasts with a dense cytoplasm could continuously divide,accordingly Schmitz and Schnabl (19S9) obtained protoplast division after removalof the vacuole by centrifugation.

Age of the leaves for protoplast lsolation

Protoplasts isolated from g-12 days-old leaves performed superior to thosefrom older material for the analyzed parameters. After five days in culture the cells

E80-d

6__ès'"

66 HELIA.22, Nr. 30, 6r-70, ( r999)

showed more than 9Oo/o viability and over 600/o of dividing protoplasts. Cells from

older leaves showed a drastic inhibition of the viability and division ability (Figure 4).

Figure 3: Elfect oJ the sedÎmentotion time on Figure 4: Elfect oJ the IeaJ age on uiabllttgthe selection oJ H. maximilio:ni proto- and diuision abilitg. oJ H. giganteus.cells

ptasts tpith higher DicLbilitg olnd. diui,sion cultiuated at 5xloa protoplasts ml-l in-crbilita

culti)ated in KMAR tiquid KMAR liquid medium uith 5.7 p'M IAA

medium u:ith 5.7 ltM IAA and 4'6 p'M and 4'6 VM zeatine'zeatine, aJter 5 dags in culture.

Leaves older than 20 days were difficult to digest leading to a reduced yield and

to a high percentage of protoplasts that quickly sedimented. Young fully developed

leaves were easily digested and gave a higher percentage offloating undifferentiated

protoplasts, these protoplasts show a satisfactory viability and division ability'

accordingly Barth et at. (f994) reported that the viability of the protoplasts of H'

6rnnuus, H. Iaetftorus and H. po:uciflorus were age dependent. In young leaves,

most of the cells exhibited a round shape and a small vacuolar system. Our obser-

vations agree with results obtained by Ochatt and Power (1992) who reported that

growth ability of protoplasts is proportional to their dedifferentiation. Older meso-

phyll protoplasts are more differentiated and had also a large developed vacuole

resulting in a low growth abilitY.

Effect of the light

A high protoplasts division rate was observed in both (light and dark) cultiva-

tion regimes (Figure 5). Light regime stimulated the protoplast division during the

exponential phase. In this phase the division was approximately 2Oo/o higlret than in

the dark. No negative effects could be observed like protoplast browning or prema-

ture death of cells. During the exponential division phase (3 to 6 days), most cells

exhibited a round shape and a small size concomitant with a relatively small vacu-

olar system. The cells became larger during the stationary phase and at this point

the medium had to be changed. For the light regime this phase was reached after 7

oèS

Rest time of the Protoplaats (min)

HELIA,22, Nr.3O, p.p. 6|-z0, (1999)

or 8 days and in the dark after ll or 12 days. Exchange of the medium after earlystationary phase lead to an increase in dead cells.

TF-.-- LGh{J *o- park

I

67

45

30

.gseo

=EèÊ

Figure 5: Effect oJ the eultiuation regime on thediDision abilitA oJH. gtganteus cells d.uring8 dags cultiuated in KMAR liquid med.iumwtth 5.7 W IAA and 4.6 W zeatine.2

crtti'atiil time (oï 8

The high division ability of cells in liquid culture obtained with the optimizationdescribed here provides the first step towards a rapid and homogeneous growthrate of cells, especially for synchronization of the culture for micronuclei inductionor metaphasic chromosome isolation.

CONCLUSION

Five factors appear to be essential for liquid culture of Helianthus protoplasts:a rich medium with an adequate growth regulator combination supplying allrequired elements for growth induction and for the rapid stabilization of the stresscaused by the isolation' rather young not fully differentiated donor tissue, selectionof floating undifferentiated protoplasts, pre-cultivation of the donor prants in thedark and cultivation in day night cycle.

ACKNOWLEDGMENT

The authors toould like to thank the DFGJorj.narrcial support.

REFERENCES

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chanabe, c., Burrus, M., Bidney, D. and Alibert, G., 1991. Studies on plant regeneration fromprotoplasts in the genus Helianthus. Plant Cell Rep', 9: 635-638'

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Krasnyanski, S. alld Menczel, L., 1995. Production of fertile somatic hybrid plalts of srmflower"and Helianthus giganteus L. by protoplast fusion. Plant Cell Rep', I I: 7-lO'

Lenee, p. and Chapeau, Y., 1986. Isolation and culture ofsunllower protoplasts (Helianthuso"nnuus L.). Factors injluencing the viability of cell colonies derived from protoplasts.Plant Sci.. 43:69-75.

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Ochatt, S.J. and Power, J.E}., 1992. Plant regeneration from cultured protoplasts of higherplalts. In: Fowler, M.W., Warren, G.S. and Moo-Young, M. (e-ds). Plant Biotechnologr.^comprehensive Biotechnolo$/, second supplement. Pergamon Press, Odord, pp. 99-l'27.

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HELLA, 22, Nr. 30, p.p. 6I-70, ( 1999) 69

OPTIIVIIZACION DE CULTIVO DE PROTOPLASTOS DEGIRASOL (Helianth,us ssp.) EN MEDIO DD CULTMLigurDo

RESUMEN

Protoplastos de células del mesôfilo fueron aislados de cuatro especies degirasol (H. 6.nnuus, H. giganteus, H. maxtmiliani , H. nuttallit). Los cultivosde protoplastos fueron sometidos en medio de cultivo liquido. Varios factoresfueron alalizados para la optimizacion y obtencion de un alto y reproducibleporcentaje de viabilidad y division celular en medio de cultivo liquido. Densi-dad de cultivo de 6 x loa protoplastos ml'l. seleccion de protoplastos no difer-enciados, precultivo de la planta donadora en la oscuridad por dos dias,utilizacion de hojas entre 8-12 dias y ciclos de cultivos dia,/noche, fueron deter-minates para la obtencidn de una alta y sincronisada tasa de divisiôn celularmayor de TOoA de division celular. Division celular fue obtenida en las cuatroespecies en diferentes tasas, evidenciando el potencial del protocolo de cultivo.

uNE TECHNTgUE OplrMrSÉE DE CULTURE DESPROTOPLASTES AU MILTEU LTgUIDE CHDZ LETOURNESOL (Helisnthus ssp.)

RESUME

Des protoplastes du mesophylle de quatre espèces d'Helianthus (H.

annuus, H. giganteus, H. maximtliani, H. nuttallii) ont été isolés et mis en cul-ture en milieu liquide. De nombreux facteurs ont été analysés et un pourcent-age élevé et reproductible de cellules vivantes et en division a été obtenu. Pource résultat les paramètres suivants étaient importants: culture des plartes pen-dant deux jours à l'obscurité, isolement des protoplasts à partir des feuillesâgées de 8 à 12 jours, sélection des protoplasts peu differentiés, culture desprotoplasts à raison de 6 x lOa protoplasts ml-l et à un régime jour/nuit. Unpourcentage de division jusqu'à 7Ool" a été obtenue quoique Ie nombre de divi-sions étaient variable parmi les espèces selon leurs aptitude à la culture aumilieu liquide.

HELIA, 22, Nr. 30, p.p. 6I-70, ( 1999)