View of Protoplast culture and plant regeneration of different agronomically important Brassica species and varieties

Maataloustieteellinen^Aikakauskirja Vol. 63: 371—378, 1991

Protoplast culture and plant regeneration

of different

agronomically important

Brassica

species and

varieties

JANOS PAUK*,

SANDOR

FEKETE*, JUHA VILKKI** and SEPPO PULLI**

*Cereal Research Institute, POB. 391, H-6701 Szeged, Hungary

**Agricultural Research _Centre, Institute

of

Plant Breeding, SF-31600 Jokioinen, Finland

Abstract.Protoplastcultureswerepreparedfrom 6-day-old hypocotyls of six spring,sev- enwinter cultivars of Brassica napusL.and ^oneline of Brassica campeslrisL.The molarity ofenzymesolutionwasraised to0,714 Mmannitol resultinginwell manipulable, cytoplasm dense protoplasts.Inthe protoplast purification procedure densitygradient centrifugationwas used to minimize physical damage of protoplasts.

Three different protoplast culturesystems—(1) liquid, (2) 2nd dayembedded,(3) directly embedded inlowmelting agarosewerecompared.The two different protoplast embedding techniquesresultedinthesame efficiencyof cell divisionasthe liquid culture method and over this fact the colony browning wasavoided. Using protoplast agarose-embedding and culture techniques, healthycalliwereobtained for plant regeneration experiments.

Incorporationof silver nitrate into the regeneration medium improved the efficiency of plant regenerationinresponsive genotypesand the regenerationwasinduced inthreenon- responsive(without silver nitrate)genotypes,too.The supplement of silver nitratein^regener- ation mediumwas especially advantageousinplant regenerationof B.campeslris.

Outof fourteen commercial cultivars of BrassicanapusandB.campeslris, there is only onerecalcitrant genotypeinobtaining plantlets from protoplast-derived calli.

Index words: protoplastisolation,protoplastculture, silvernitrate,plant regeneration, BrassicanapusL,Brassica campeslrisL.

Introduction

Since several ^{of the most widespread} methods of introducing foreign ^genes into plant cells rely on protoplasts, the establish- ment of areproducible protoplast-plant sys-

temis ofacrucial importance in any plant spe- cies of biotechnological interest.

The first successful! protoplast isolation and plant regeneration experiments have been per- formedbyWenzel (1973). The Brassica spe- cies vary considerably in their ability to

JOURNALOF AGRICULTURAL SCIENCEIN FINLAND

regenerate plant from protoplast. While the Brassica napus and the Brassica oleracea (Sidney et al. 1983, Robertson et al. 1984) generally give good results in protoplast re- generation experiments (Kartha etal. 1974, Li et al. 1982, Glimelius 1984), in case of turnip rape (B. campestris, B. rapa) the plant regeneration from protoplast derived callire-

mained a sporadic event (Glimelius 1984, Ulrich etal. 1980).

The aim of thepresentstudywastodevelop alargelygenotypeindependent, efficient plant regeneration procedure for B. napus L. and B. campestris L. with agronomical impor-

tance.

Material and methods

To extend the protoplast-plant system to differentgenotypes of agronomic importance various spring and wintertypevarietieswere includedin the study. Springcultivarsof Bras- sica napus L. spp.

oleifera

^{cv. Hanna,Kun-}

to, Olga, Omega,Varma, Westar and winter cultivars Arabella, Belinda, Danubia, Dar- mor,Santana, Ujfertodi, Vikingweretested.

One Brassica campestris L. ssp.

oleifera

^breed-

ing line Jo. 4001 was used, ^too(Table 2). The datawere calculated from2 or 3 in- dependent experiments.

For protoplast^isolation, the seeds were sur- face sterilized with 75 ^% alcohol for I—21—2 minutes, then immersed in2^%NaO’Cl with some drops of Tween 80 and continually shaken for20 minutes followed by athorough wash in autoclaved sterile distilled water.

Sterilized seedsweregerminatedon ahormon- free MS medium (Murashige and ^Skoog

1962) for 5—6 ^days in darkness at 28°C.

The5—6cmlong hypocotylswerecutinto smallpieces of about 1.0 mm and treated for 17—18 hours (generally overnight) with 1 ^% Cellulase R-10 and Macerozyme R-10 enzymes dissolved ⁱⁿ

K 3

^{(Nagy and Maliga 1976)}

medium. The osmotic pressure of the enzyme mixturewasraised to0.714 M using mannitol asosmotic stabilizer. This treatmentresulted ina greatnumber of comparatively small pro-

toplasts with dense cytoplasm. The filtration of the isolated protoplastswasomittedtomin- imizephysical damage. The enzyme mixture together with the protoplast and the hypocotyl debris were diluted with W 5 salt solution (Menczel _etal. 1981)to 5—6 times the origi- nal volume and centrifuged for 5 minutes at

120g.Thesupernatant wasdiscarded and the protoplasts, mixed withdebris, ^{were suspend-} ed in 0,6 M ^sucrose and

W 5 was

^{layered on}

thetop, then centrifugated _at 120 rpm for 5 minutes.The floating protoplastswerecollect- ed by Pasteur pipette and washed twice with

W 5 solution.

The isolated protoplasts were cultured in 0,4 M K-75 medium (Kao and

Michayluk 1975) modified by Glimelius (1984), at28°C in dark. Protoplasts werecul- turedat a density of 6 X 10⁴in three differ-

ent culture systems: (1) liquid medium, ⁽²⁾ embedded on the 2nd day from liquid into agarose and (3) embedded into agarose direct- ly after isolation. The embedded cultureswere solidified ^by 0,8 ^% low melting agarose (Sigmatype ^VII).After about 10 days the liq- uid and agarose embedded cultures_were fed with liquid medium with reducedosmoticum (3 ^% sucrose). The cellaggregates wereready toplate from the beginning of the third week.

The liquid cultureswereplated_on agar solid- ified regeneration medium and the agarose blocks were floated in liquid medium before plating. The hormones _were decreased to quater strength in the nutrient and floating mediumasdescribed by^Vamlingand Glime-

lius(1990).

The plant regeneration wascarried outon

K 3 medium

supplemented with 0.5 mg/1BAP, 0,5mg/1 Zeatin and 0,1 mg/1 lAA (Nagy and Maliga 1976, Vamling and Glimelius 1990).

To increase the efficiency of plant regenera- tion 5 mg/1 AgNG₃ was added ^to the re- generationmedium. Regenerantsweregrown up with spaceisolationin different greenhouse chambers.

Results

In each variety the protoplasts (Fig. la)

were isolated from hypocotyl. The relatively high osmotic pressure (0,714 M) results in small, cytoplasm-dense protoplasts. Small, cytoplasm-rich (Fig. lb) protoplasts_werewell manipulable during isolation procedure and

proved to be excellent for different culture procedures.

The culture methods applied resulted in difference in initiation of cell division ^at 24- hour age (Table 1). Protoplasts cultured in liq-

Fig. I. Developmentof Brassica protoplast-callus systemusing^agaroseembedding techniquewith ’Varma’ cv.

a.Freshly-isolated protoplastscollected from the interphase of gradient centrifugation.^{Bar =}40pm.b. Cytoplasm- rich protoplast embedded inagarosesolidified medium.^{Bar =}40pm.c.Freshlydivided protoplast embedded into agarosefrom liquid mediumon the second day of culturing. Bar ⁼40 pm.d. Second cell division within4days of culturing. Bar ⁼40pm.e.Cellclusters obtained within⁴⁰daysof culturing.^{Bar =}0,1mm.f.Protoplast-derived colonies of ’Varma’ cultivarinagarose-solidifiedmedium and^agaroseblocks floatedinliquidmedium. Magnifica- tion ⁼2x.

TableI.Percentageof dividingcells using three different culture methods inprotoplast cultures of threeB.napus cv. The numbers of division (in percent)werecounted after 24-, 48-and 96hours.

Genotype Culture Percentageof dividing

method* cells after

24 48 96

hours hours hours

liquid 1,1 18 48

emb. ^2ndd. 1,0 14 46

emb. direct 0 16 47

Olga

liquid 2,3 12 51

Varma emb. 2nd d. 2,4 14 53

emb. direct 0,1 10 49

liquid 0,7 9 44

Arabella emb. 2nd d. 0,9 7 42

emb. direct 0 7 45

* Seein Materials and methods

uid mediumwere able to undergo divisions from the end of the Istdaydependingoncul- turemethods. Protoplasts embedded directly after isolation into agarose were capable to divide from the 2nd day of culturing showing visible divisions at 48th hour (Fig. 1c). The

agarosetechniqueseemstobeahindering fac- torin the starting of celldivision,but this hin- dering effect disappeared on the fourth day of culturing. There was no significant differ- ence in _percentage of dividing cells obtained from differentvarietiesusing different culture techniques _onthe fourth day of culturing(Ta- ble 1).Each variety produced about thesame percentage of divisionson the fourth day (in 96 hours) of subculturing. Second divisions (Fig. Id)wereobserved from the third day of subculturing.

Among thevarieties, ^Varmawas the most responsive in the efficiency of cell divisionat the end of the 4th day ofculture,but incom- mencementof microcolony development there was nosubstantial difference between culture methods regarding the percentage of devel- oped colonies.

Observing of cell division after 96 hours of culturingwaseasier in agarose embedded cul- turesbecause the well-spreadcells,protoplast- derived colonies in agarosewere in fixed po-

sitions while in liquid medium the cells were aggregated spontaneously and it was difficult todistinguish between division-derived micro- colonies and spontaneous cell groups.

After about tendays in cultures small calli ormicro colonies (Fig. le) had developed in both culture systems. Therewas an essential difference between liquid culture technique and agarose solidified culture. In the liquid culturesa brown precipitation startedto de- velop around microcalli from the second week. This phenomenon, observed by other authors ^as well^(Schenckand ^Hoffman 1979, Glimelius 1984),was detrimentaltothe sub- sequentculturework,inhibitingorcompletely stopping the growth. This harmful phenome- non wasnotobserved in agarose solidified cul- tures(Fig. If) indicatingmorefavourableout- comewhen using agarose-embedding in Bras- sica protoplast culture.

Exceptfor onevariety Ujfertddi—, ev- ery variety in both Brassica napus and cam- pestris formeda higher number of calli for plant regeneration experiments (Table 2). At the end of2 nd and 3rdweek,the number of formed microcalli varied, ^{depending on in-} dividual cultures and_genotypes,butno essen- tial differenceswerefound between cultivars.

In most cases white calli with green or greenish morphogenestructures(Fig. 2,^indi- cated by arrows)wereobtainedonthe regener- ation medium but the efficiency of plant regeneration remained low (Table 3). Initial- ly, only intenof fourteen Brassica genotypes plant regeneration was successful. The ^re- generants (Fig. 3) were obtained from green organogeneticcentresonthe surface of white calli, and generallyone ortwoshootswereob- tained from onecallus (Fig. 3). Consequent- ly, to test its effect onregeneration, AgNQ₃ wasaddedtothe regeneration medium. In the presence of 5 mg/I AgN0₃ the regeneration efficiencywas substantially increased includ- ing multiple shoots (Fig. 4 and 5) in the posi- tivecases. Out of the fourteen Brassica geno- types studied, thirteen gave plantlets with varying numbers of regenerants (Table 3).

This modified regeneration procedure re-

Table2.Summaryof resultsinprotoplastisolation, culture,callusinduction,plant regenerationand sourceofgeno- typesused ininvestigations.

Species, varietyand Life form Protoplast Plant regene-

source ^{~ ' ;} I ^{' ; " ~} ration

isolation culture derived calli B. napus

Olga S spring ^>

Varma SF ^{» >}

Westar CAN ^{» >}

Kunto S ^{» >}

Hanna S ^{» >}

Omega S ^{» > •>}

Viking DK winter ^>

Arabella D ^{» >}

Darmor F ^{» >}

Santana D ^{» >}

Danubia H ^{» > >}

Belinda D ^{» >}

Ujfertddi H ^{» >}

B. campestris

Jo. 4001 SF spring ^{> •>}

> without silver nitrate inregeneration medium

■> with silver nitrate inregenerationmedium

generation medium supplemented with AgNG₃ has aparticularly advantageouseffect ^on regeneration of B. campestris which has been

Table3.The effect of silvernitrate (AgNO,)onplantre- generationfrom protoplast derived calli ofB.napusand B. campestris varieties.

Name ofgenotype ^% of shoot regenerating

and life form calli

—AgNO, ⁺AgNO,

B. napus

Olga *s. 24 41

Varma s. 31 39

Westar s. 19 33

Kunto s. 11 29

Hanna s. 9 19

Omega s. 0 4

Viking **w. ⁹ 23

Arabella w. 10 17

Darmor w. 4 14

Santana w. 6 10

Danubia w. 0 8

Belinda w. 2 6

Ujfertddi w. 0 0

B. campestris

Jo.4001 s. 0 22

•s⁼spring, ^** w⁼winter Regeneration without AgNOj(—)

with AgN03(⁺)

known torespond poorly toin vitro manipu- lation.

The regenerated plantlets weretransferred

to

K 3 medium

without AgN03 which al- lowed the plantlets to develop rapidly.

Discussion

The molarity of enzyme solutionwasraised from 0,4 ^M sucrose suggested by Glimelius (1984) and^Vamlingand Glimelius (1990)to

Fig. 2.Protoplast-derivedcalli^withmorphogene struc- tureontheir surface (arrows)onplant regenerationme- dium without silver nitrate.

0,714 M mannitol. We thus used higher molarity as Kohlenbach et al. (1982) who used 0,55 M and 0,65 M mannitol for leaf and stemembryo protoplast isolation,respective- ly.^Eapen etal. (1989)obtained^goodresults using0,6 M mannitol in enzyme solution in mesophyll protoplast isolation of Brassica

juncea. The cytoplasm dense small protoplasts proved to be well manipulable during isola- tion procedure. They will be ^an essential ad- vantage in direct protoplast transformation experiments where protoplasts are manipulat- ed washing etc. several times.

The agarose embedding culture technique developed by Shillito et al. (1983) has proventobe asuccessful method in Brassica also. Our results confirmed the applicability of the embedded protoplast culture technique (directly or on the 2nd day of subculturing) which produced the same division^{results as} the liquid culture method (basic culture meth- od) published for Brassica protoplast culture (Bidney etal. 1983, Glimelius 1984, Jourdan et al. 1989, Loudon et al. 1989).This tech- nique has been already used for 2—3- week old colony embedding by ^Vamling and Gli-

melius (1990) resulting in a good culture practice, but_ourresults (early embedding af- terisolation) show other advantagesoverthis culture technique,too.The colony browning,

Fig. S. Typical rapeseedwhite callus with oneshooton regenerationmedium without silver nitrate.

Fig. 5. One shootleton surface of green callus develo- ping onAgNOi supplemented regenerationmedium.

Fig. 4.Well differentiated greenprotoplast-derivedcal- lus with multiple shoot primordia (arrows)on regenera- tion medium supplemented with 5mg/1 AgN03.

widely reported _to be a feature of Brassica protoplast culture (Xu etal. 1982,^Glimelius

1984, Loudonetal. 1989)wasprevented and healthy calli_were induced for plant regenera- tion experiments.

One of the noticeable features of this study is the wide rangeofresponses observed(Ta- ble 2). These results show,ⁱⁿaccordance^with other studies (Jourdan and Earle 1989,^Lou-

don etal. 1989) that various Brassica varie- ties ofgreateconomic importancecanbesuc- cessfully regenerated from protoplasts. There was onlyone (Ujfertodi) outof the fourteen varieties tested that failed to produce plant- lets.

Incorporation of silver nitrate (5 mg/1) into theregeneration medium improved the effi- ciency of plant regeneration. With ^{the in-} troduction of AgNOj three of the examined varieties became responsive in regeneration ex- periments (Table 3) and regeneration was moreefficient in each variety. There is only onerecalcitrant genotype Ujfertodi in

ourexperiments (Table 2 and 3). Inagreement withourresults, the positive effect of AgNQ3

on the induction of plant regeneration has been recently published by other authors as well (Lentini et al. 1988, ^{Songstad et al.}

1988) in Brassica and in other crop plants (Purnhauser et al. 1987).

Our results _support the findings of Purn-

hauser et al. (1987) and Williams et al.

(1990) that AgNQ₃ has the mostfavourable effecton plant regeneration when appliedat alaterstageof callus development (whenmor- phogenezoneshad already been formed) rath- erthan right from the beginning of callus in- duction.

Acknowledgements.The authors^arethankful to Miss OutiManninen,Mrs. Maija-LiisaPenttila,Mrs. lldiko D.Bartok,for their skilful assistance and Mr. B.Dusha andMr. B.Kdrmdninpreparingthe photographicma- terial. They also wish to thank Mr. L. Nagyfor kindly supplyingone partof seed material. This workwas sup- ported byJ. Pauk’s 6-month fellowshipinFinland.

References

Sidney, D.L., Shepard, J.F., Kaleikau, E. 1983.Re- generationof plants from mesophylle protoplasts of Brassica oleracea. Protoplasma 117: 89—92.

Eapen, S.,Abraham,V.,Gerdemann, M.,Schieder, O.

1989.Direct somatic embryogenesis, plantregenera- tion and evaluation of plants obtained from mesophyll protoplastsof Brassica juncea. Annals of Botany63, 369—372.

Glimelius,K. 1984.High growth rateand regeneration capacityofhypocotyl protoplastinsomeBrassicaceae.

Physiol.Plant. 61: 38—44.

Jourdan,P.S. Earle, E.-D. 1989. Genotypicvaria- bilityinthe frequency of plant regeneration from leaf protoplastsof four Brassica spp.and of Raphanus salivas.^{J. Amer.} Soc.Hort. Sci. 114(2): 343 —349.

Kao, K.N. ^Michayluk, M.R. 1975. Nutritionalre- quirementsfor growth of Vida hajaslana cells and protoplasts atverylow population densityinliquid media. Planta 126: 105—110.

Kartha, K.K. ^Michayluk, M.R. Kao, K.N.

Gamborg, O.L. Constable, F. 1974.Callus for- mation and plant regeneration from mesophyllpro- toplastofrapeplants(Brassicanapuscv.Zephir) Plant Sci. Lett.3: 265—271.

Kohlenbach, H.W., Wenzel,G., Hoffmann, F. 1982.

Regenerationof Brassicanapusplantletsincultures from isolated protoplasts of haploid stem embryos^as comparedwith leaf protoplasts.Z.Pflanzenphysiol.

Bd. 105. S. 131—142.

Lentini, Z. Mursell, M.A. Mutschler,M.A.

Earle,E.D. 1988.Ethylen generationand reversal of ethyleneeffects during developmentinvitro of rapid- cyclingBrassica campestrisL.Plant Sci.54: 75 —81.

Li,L.C. Kohlenbach,H.W. 1982.Somatic embryo- genesisinquiteadirectwayincultures of mesophyll protoplastsof Brassica napus^L.Plant Cell Rep. 1:

209—211.

Loudon, P.T. Nelson,R.S. Ingram, D.S. 1989.

Studiesof protoplast culture and plant regeneration from commercial and rapid-cycling Brassica species.

Plant Cell Tiss. Org. Cult. 19: 213—224.

Murashige,T. Skoog,F. 1962.Arevised medium for rapid growthand bioassays with tobacco tissue cul- tures. Physiol.Plantarum ^15:473—497.

Nagy,J. Maliga,P. 1976.Callusinduction and plant regenerationfrom mesophyll protoplasts of Nicotiana silveslris.Z.Pflanzenphysiol.78: 453—455.

Purnhauser,L. Medoyesi,L. Czak6,M. Dix,

P.J. Marton, L. 1987. Stimulation of shoot regeneration in Triticum aeslivum and Nicotiana plumbaginifoliaViv. tissue culture using the ethylen inhibitor AgNG3Plant Cell Rep. 6: I—4.

Robertson, D. Earle, E.D. Mutschler, M.A.

1984.Enhanced rates ofplant regeneration from^pro- toplastsof cultivarGreenComet Broccoli Brassica

oleracea. Plant Physiol. 75: 134.

Schenk, H.R., Hoffmann, F. 1979. Callus and root regenerationfrom mesophyll protoplasts of basic Brassica species: B. campestris,B. oleracea andB.

nigrra. Z.Pflanzenziichtg. 82: 354 —360.

Shillito, R.D., Paszkowski, J., Potrikus, I. 1983.

Agarose plating and beadtypetechniqueenable and stimulate development ofprotoplast-derivedcolonies inanumber of plant.

Songstad,D.D. Duncan, D.R. Widholm, J.M.

1988.Effect of 1-aminocyclopropane-l-carboxylic acid, silver nitrate and norbornadiene ^on plantre- generation.Plant Cell Rep.7: 262—265.

Ulrich, T.H. Chowdhury, J.B. Wildholm,J.M.

1980.Callusand root formation from mesophyllpro- toplasts of Brassicarapa. Plant Sci. Lett. 19; 347

—354.

Vamling,K.,Glimelius,K. 1990.Regenerationof plants from protoplasts of oilseed Brassica crops.In:^Bajaj (ed.); BiotechnologyinAgricultureand Forestry. Vol.

10. 385—417.Springer-Verlag, Heidelberg.

Wenzel, G. 1973. Isolation of leaf protoplasts from haploid plants of petunia,^rape and^rye. Z.Pflanzen- ziich. 69: 58—61.

Williams,J. Pink,D.A.C. Biddington,N.L. 1990.

Effect of silver nitrate ^on long-term culture and regenerationof callus from Brassica oleraceavar. gem-

mifera.Plant Cell Tissue Org. Cult. 21:61 —66.

Xu, Z.H., Davey,M.R., Cocking, E.C. 1982.Plant regenerationfrom root protoplasts of Brassica. Plant Sci. Lett. 24: 117—121.

Ms received ^June9, 1991

SELOSTUS

Maataloudelle tarkeiden Brassica-lajien protoplastien viljely ja kasvien regenerointi

JanosPauk*, Sandor_Fekete*, Juha Vilkki**

and Seppo Pulli**

•CerealResearch Institute, Pob. 391, H-6701 Szeged, Hungary

**AgriculturalResearch ^Centre, Institute ofPlant Breeding, SF-31600 Jokioinen,Finland

Tutkimuksessa eristettiin protoplasteja kolmentoista rapsilajikkeen(Brassica napus) ja yhden kevatrypsilinjan (B. campestris)6vrk:n ikaisista hypokotyyleista.

Protoplastien eristyksessa kaytettiintavanomaista kor- keampimolaarista entsyymiliuosta(0.714Mmannitoli).

Main saatiin pienia,kestavia, runsaasti solulimaa sisal- tavia protoplasteja. Protoplastien vaurioitumisen valtta- miseksi eristyksessa kaytettiin tiheysgradienttisentrifugoin- tia.

Tutkimuksessa verrattiin kolmea eri protoplastivilje- lymenetelmaa: 1) nesteviljely, 2) maljaus agaroosiin toi-

senaviljelypaivana,3)suoramaljaus agaroosiin. Agaroo- siviljelmissasolut jakautuivat yhta aktiivisesti kuin nes- teviljelyssa,lisaksi valtyttiin solukolonioiden ruskettumi- selta.

Hopeanitraatin (AgNG₃⁾kaytto regeneraatioalustassa (5 mg/1) lisasi kalluksesta muodostuvien versojen maa- raaniilla10lajikkeella, joilla havaittiin vahaista^regene- raatiota jo ilman hopeanitraattia. Kolmella lajikkeellare- generaatioonnistui ainoastaan silloin kuin regeneraatio- alustassa oli hopeanitraattia. Erityisen suotuisa vaikutus hopeanitraattilisayksellaoli kevatrypsin regeneraatioon.