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SOMATIC EMBRYONESIS AND SUSPENSION CULTURES

FROM MATURE ZYGGTIC EMBRYOS OF DATE PALM

(Phoenix dactyllfera L.)

H.saka

INRAA, laboratoire de Physiologie Végétale, CRP mahdl Boualem, BP37, Baraki, Alger.

Abstract : Excised embryos ofDeglet Nour, Tagaza and Takerboucht showed a poten-tial of embryogenesis . The picloram, dicamba and 2,4-D were able to induce embryogénie caiii. Genotypic différences in embryogénie responses is observed. Friable calliwas used to initiate the cell suspension. Ali the concentrations of the various growthregulators tested aliowed an increase in ceil fresh weight as well as the cell suspension growth. Howeven régénération from cell suspension cultures was very low.

Key words ; date paim, picloram, dicamba, 2,4-D, callus, embryogenesis, cell suspension, régénération.

Résumé: Les embryons excisés de la Deglet Nour, Takerboucht et Tagaza ont initiés ducals embryogénes et ce quelque soit les substances de croissance utilisées. Des différences génotypiques sont cependant notées. Le cal globulaire et friable est utilisé pourobtenir des suspensions cellulaires. Les différentes substances de croissance testées ontpermis l'établissement et la prolifération des cultures cellulaires . Le taux de germination obtenu qui devait démontrer la totipotencie des cellules s'est avéré faible.

Mots clé ; Palmier dattier, picloram, dicamba, 2,4-d, cals embryogenèse. Suspensioncellulaire, régénération.

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INTRODUCTIONDate palm (Phoenix dactylifera L.) is hundredby a disease syndrome known as Bayoud , pri-marily caused by Fusahum oxysporum f.sp.albedinis which bas aiready killed 3 millionsof trees (DJERBI ,1990) . The most succesfullstrategy for Fusarium wilt control in othercrops bas been tbe development of résistantsvarieties (FROMM et al,1985 ; DAN etSTEPHEN,1991).Tbe use of somatic ce!! gene-tic techniques, sucb as protoplast régénération, ce!! bybridization and ce!! sélectionexbibiting résistance to applied stress,basbeen widely recognized as a tool to Improvespecies (KARP et al ,1990). To explore the abi-iity to grow plant cells in isolation an efficient régénération system is necessary togenetically transform species . Althoughmocotyledonous species bave been shown tobe récalcitrant in in vitro culture, plants canbe regenerated from embryogénie cel! suspensions, and protoplasts from number of

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SCARNEC,1992), ' "^^^^^1,1979 ;Tbe efficiency of tissus cultumve cell line sélection k h.l . competiti-of cell totipotency in a fpIThe cell prolifération ^hort time .the maintainance of ceïîTdnessentiel step for cGlIm/r^- ^°^'Po^ency is antigated the establishmenrôV'emh^^suspension cultures ofthr 2 ^n^bryogenictypes . We describe ^ 9^"°"ceil culture calli and maintnerabie embryog^nfc XreTT"embryos of date palm zygotic

material and methods

experlmentTL^glTnoTarjraglt'^e,^sensitive variét és to F r. = ^a résistant cultiver ^ Takerboucht,

Seeds were surface sterilized by immersion ina fongicide solution (BENLATE ,1g/l) followedby three rinses in stérile distilled water. Seedswere aiso acid-scarified in 15% H2So4, dip-ped for a 30 minutes in 1,5% sodium hypo-chlorite solution and rinsed again .

Callus initiation and maintainanceExcised embryos were piaced on a modifiedMS médium (REYNOLDS et MURA-5HIGUE,1978) to induce embryogénie calli.This médium Is supplemented with 2,4-D,picloram and dicamba plant growth regula-tors at various concentrations ;2,5mg/l, 12.5mg/l, 25mg/!, and 100mg/l. The pH is ajustedto 5.6 before sterilizatlon. Five embryos eachwere cuitured in PetrI dishes at 27+- 2°C.Tewnty replications is done for each treat-ment. Twenty embryos are piaced on germination médium as control to assesss the viabi-

iityQftheemi)rj/o;ej<^,s^d.Atter 18 to 24 weeks of culture, seeds produ-cing compact or friable callus were transferredin a 250 mi flasks in the same basa! médiumwith lower growth regulators were tested tosustain further growth.

Initiation of cell suspensionSuspension cultures were derived by transferof small portions of friable calli, 1 to 0,5 g/1,to a liquid médium. The cultures were grownon the prolifération média ,in dark at 120rpm. Cell suspension cultures and cell aggre-gates were subcultured every 2 weeks at anInoculation rate of 20 to 25%(V/V). At eachtransfer, blomass is weighed and residual calliremoved until a suspension is obtained.Différent concentations of growth regulators( 12,5 and 2,5 mg/l) were tested to monitorprolifération .

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Régénération

To control the totipotency of the cells ,nodules transferred to llquid basai médiumfor two or four cycles are plated on Pétridishes containing the same médium butsolid, devoid of growth reguiators and undera 16 hour photoperiod.The suspension is plated and the germination of the embryosnoted.

non embryogénie calli which develop rootsonly. In ail three génotypes formation ofnodular and friable callus is obtained. No

apparent différences in the callusing fre-quency were observed between génotypes,and induction média except for the médiumcontaining 100 mg/l of 2,4-D. With thismédium no callus was formed and ail the

embryos germinated .

Results - Discussion

Callus initiation

At the end of 6 to 9 month culture, only thecalli containing smooth globular structureswere recorded as embryogénie callus .Thosehaving a nodular appearence correspond to

Tâbl@ Effecf of qrowfil reguiators on embryogénie calli of three date palm génotypesThe data are the means of 10 repiicates

ON TAK TAG

Growth Regulator(mg/L)

(1) (2) (1) (2) (1) (2)

15

Pic 2,5

12,5

32

58

32

58

25

21

25

21

32

28

32

28

OOfs

c

3

Die 2,5

12,5

28

52

28

52

50

42

50

42

25

28

25

28

CD

Z

Si

5

2,4-D 2.5

12,5

25

100

32

21

31

0

32

21

31

25

12

28

0

25

12

28

32

25

25

0

32

25

25

ÊQj

O□>0)

1-number of embryos showing calli after 6 to 9 months culture.2-frequency of embryogénie calli (%) with glodular aspect

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Table II: Embryogenesis and régénération of 6 to 9 -month old primary calli from embryosof three date palm génotypes

Frequency of calli producting embryos %

Growth Regulator(mg/1) DN TAK TAG

Pic 2,5

12,5

32

24

15

19

25

28

Die 2,5

12,5

15

12

22

36

25

27

2,4-D 2,5

12,5

25

100

22

23

25

0

12

32

28

0

21

24

28

0

Newiy initiated calli consisted of a mixture ofmorphological distinct tissue as observed inothers species (CAI et BUTLER, 1990).this mixture showed a hard ,opaque white callus, asoft watery brownish callus and a nodularhard callus. In concordance with différents

16 reports (TISSERAI, 1978 ; REYNOLDS, 1978 ;SAKA et al, 1997), embryogénie calli have a

0 compact or friable texture, globular shapeç and opaque white color. Cultures of Tak pro-^ duce large amounts of phenolic compounds2 that darkened the médium and the culture1 underwent necrosis. Decreasing the time bet-s ween initiation and subculture increased the

I rate of survival. Genotypic différences in^ embryogénie responses is observed . TheI three cultivars tested ranged from 12 to 32%

as shown in tablel. DN genotype reacted bet-ter whith picloram while Tak has higher callus percentage on dicamba médium and Tagreacted invariably to ail the growth regula-tors. However, the concentrations of the

various growth regulators tested did nothave an effect except for 100 mg/l of 2,4-D.The data suggest that optimal embryo induction médium with regard to auxin type andconcentration has to be determined for each

genotype

Genotypic effects have been previousiyreported in various species ( MAL-MERG ,1979 ; LAZZERI et al,1987) . Since ittakes at least 4 months to obtain plants wefollowed until germination (table II).The germination rate is identical for ailtypes of growth regulators and testedconcentrations.

Cell suspension culturesAttempt is made to adapt the régénérationcapability for suspension culture of onegenotype DN . Friable calli was used to initia-te the cell suspension. From the 5 day in astirred médium, embryogénie masses startedto detach from mother callus tissue . The dia-meter of the particles varied from 1 to 2 mm.For each initial callus ranging from 1 to0,5 g/1 ,a final weight is obtained. Theweight increased 4 times after 4 weeks. Ailthe concentrations of the various growthregulators tested allowed an increase in cellfresh weight (Fig 1.). The best results areobtained with 5 mg/l of picloram , This auxinlike has been USed with success to maintain

callus prolifération and totipotency in sugarcane and date palm (FITCH et MOORE ,1990;

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OMAR et NOVAK,1990). Concentrations of5mg/l and 12,5mg/l of 2,4-D induced cellgrowth of date palm . This findings is in desa-greement with TOUCHET et al (1991) whoobserved that concentrations of 25 and 50mg/l of 2,-4D were insufficient to sustain cellgrowth of oil palm .The auxine - like dicam-ba showed a cell growth identical to 2,4-D.From 4 to 16 subcultures, the cells sustainedgrowth. Beyond 16 subcultures which correspond to 32 weeks, the cell growth started todécliné for ail the growth regulators tested

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Subcultures (weeks)

Figure n°1: Weight Increase of cellsafter 28 subcultures for the various

growth regulators tested with genotypeD.N

Régénération from cell suspensionThe cells were plated at 6 to 22 subcultureson germination medim devoid of growthregulators. The germination occured after 12weeks and not on ail the plates and only atotal of 10% of the cells plated showed germination (data not shown). This long timeand low germination could be due to theprolonged stay in média with growth regulators and the remaining effect of the growthsubstances as stated in TOUCHET et al (1991).The cell suspensions contained single cells,cell aggregates, and heavily vitrified structures that could lead to inconsistent regene

ration. Régénération in many monocotyledo-nous species has been shown to occur fromcompact callus that can be maintained inlong-term cultures and friability results innon-regenerable cultures ( MORRISH et al,1987 ; HUTCHINSON et al, 1997).

CONCLUSIONExcised embryos showed a potential ofembryogenesis as in various other species.The picloram and dicamba as well as 2,4-Dwere able to induce embryogénie calli.Genotypic différences in embryogénie res-ponses is observed which is not spécifie todate palm. Friable calli was used to initiatethe cell suspension. Ail the concentrations ofthe various growth regulators tested allowedan increase in cell fresh weight. Dicamba aswell picloram auxins-like allowed the initiation of embryogénie calli as well as the cellsuspension growth. However, régénérationfrom cell suspension cultures was very lowand could be due to the remaining effect ofthe growth regulators tested or the hetero-geneity of the calli. Histological studiesshould be performed to assess the totipoten-cy of the cells and improve the method. Thispaper reported for the first time long-termcell suspension cultures of date palm. it isimportant to master the cell culture of datepalm since information on cultural conditions favoring high frequency maintainanceand régénération through cell cultures is aprerequisite to accelerate application of nonconventionnal techniques to date palmimprovment.

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•CAI T. et BUTLER L. (1990), Plant régénération from embryogénie callus initiated fromimmature inflorescences of several high- tannin sorghums.Plant Cell .TIssue and OrganCulture, 20 :101-110

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