Jowml of Oil Palm Research Vol. 10 No. 2, December 1998, pp. l-9

STAGES IN THEVEGETATIVE

‘PROPAGATION OFOIL PALM, Elaeisguineensis Jacq.

THROUGH TISSUECULTURE

Keywords: E/a.& guineensis Jacq.. stages.vegetative propagation, tissue culture. NAA

medium, normal inflorescences

‘SOGEKE, A K

* Nigerian ln~fitute for Oil Palm Research (NIFOR).P.M.6 1030, Benin City, Nigeria.

0 il palm (Elaeis guineensis Jacq.)

was vegetatively propagated by tis-

sue culture in NIFOR on a NAA

medium which provided a very short callus

stage. The following stages were identified:

callus (initiation and maintenance), embryoid,

plantlet (ran&i, prenursery (sand culture),

nursery, and field planting. Normal male and

female inflorescences which led to normal fresh

fruit bunches (FFB), wereproduced by the tissue

culture palms two and a half years after

planting the plantlets in the field.

INTRODUCTION

V egetative propagation of the oil palm(Elaeis quineensis Jacq.) by tissue culture

has four significant advantages over conven-tional breeding. Firstly, it enables rapid mul-tiplication of uniform planting materials withthe desired attributes, thereby providing themaximum return for investment and the req-uisite materials for meaningful agronomicexperiments. Secondly, it offers a new oppor-tunity in oil palm breeding. By obtaininghaploid palms from pollen/anther culture, theprocess of hybridization to produce homozygousdiploids is considerably reduced. Thirdly, itopens new avenues of research for oil palmbiotechnology.

However, the expectations of clonal palmshave been dampened by the occurence of fruitand flower abnormalities (Corley et al., 1986;Hartley, 1988; Paranjothy, 1989). The initia-tion of callus from leaf or root tissue is gen-erally on a medium containing a high concen-tration of 2,&Dichlorophenoxy acetic acid (2,4-D) (Duval et al., 1988; Blake, 1983; Rabechaultand Martin, 1976; Smith and Thomas, 1973).However, at high concentrations, 2,4-D and2,4,5-Trichlorophenoxy acetic acid (2,4,5-T)are phytotoxic to broad leaf plants and used asherbicides (George and Sherrington, 1984; Vasiland Vasil, 1972).

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JOURNAL OF OIL PALM RESEARCH lO(2)

Furthermore, cytological examination ofcallus from roots of oil palm grown on 2,4-Dmedium contained a high proportion ofpolyploid and aneuploid cells (Smith and

Thomas, 1973). All researchers who havesuccessfully carried out in vitro vegetativepropagation of oil palm have used somaticembryogenesis on calluses obtained on 2,4-Dmedia (Jopes, 1974; Rabechault and Martin,1976; Paranjothy and Othman, 1982; Nwankwoand Krikorian, 1983; Blake, 1983; Duval et al.,1988; and Paranjothy, 1989). Hence the abnor-malities in clonal propagated oil palm might bedue to the high concentration of 2,4, -D rou-tinely used for its culture as 2,4-D is reportedto cause mitotic spindle abnormality (Bayliss,1973).

Corley et al. (1986) however reported thattwo of their clones which flowered abnormallyhad not been exposed to 2,4-D at all. This maybe due to the prolonged callus stage which isnot advantageous to cloning oil palm as ex-plained by Sogeke (1996a). Duval et al. (1988)also associated flowering abnormality with fastgrowth of the callus. However, Butcher et al.(1975) showed that there was a greater ten-dency for fast growing cultures of Helianthusannuus a non-polysomatic species, to becomepolyploid. Hence, fast growth of callus per semay not necessarily be advantageous to cloningoil palm. Above all, Shamina (1966) and Sun-derland (1973) working with Haplopappusgracilis observed that cultures maintained onmedia containing 2,4-D had cells with a greaterrange of ploidy levels than cultures on mediawith NAA as the auxin. They both independ-ently concluded that as 2, 4-D cultures hadincreased growth rates, 2,4-D may favour thegrowth of tetraploid cell lines.

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In several laboratories, therefore, inves-tigation into the hormonal status in abnormalpalms are now pursued (Paranjothy, 1989;Besse et al., 1992; 1994; Jones et al., 1995).Furthermore, Corley et al., 1986 and Hartley,1988 suggested the time of culture and themedia be adjusted to overcome the abnormality.1-Naphthyl-Acetic acid (NAA), which is muchmilder than 2,4-D, was instead used in thisstudy. The aim was to obtain callus on a non-2,4-d medium with a short callus stage, to

determine the other stages in the in vitrovegetative propagation of oil palm, and toobserve any abnormality associated with thepalms.

MATERIALS AND METHODS

Callus initiation from immature leaf explantsof young oil palms (Elaeis guineensis Jacq.) wason modified Eeuwens (1976) inorganic mediumsupplemented with sucrose 3Og/l, inositollOOmg/l, aneurine-HCl 0.5mg/l, n i a c i n0.5mg/l, pyridoxine-HCl 0.5mg/l, casaminoacid 500mg/l, and 1-naphthyl acetic acid (NAA)15mg/l. Agar at 7g/l was added and the pHkept at 5.7. This was the initiation mediumY3(3). All culture up to the plantlet stagewere incubated under continuous flourescentl ight (200 lux) at 28”Cfl”C and al l theoperations done under aseptic conditions. Fulldetails of the methods involved have beenpublished by Sogeke (1996a). Plantlets at thepre-nursery stage were grown at room tempera-ture (28”C-30°C) under fluorescent light (200lux) with the flasks uncovered while the nurs-ery and field planting were under naturalconditions.

RESULTS AND DISCUSSION

Callus Stage (initiation and maintenance)

Callus formation (CF) with pneumathodes(Sogeke 1996a; Hartley, 1988; Blake, 1983)was obtained on Y3(3) 42258 days afterinoculation of the medium with surface steri-lized leaf explants (Figures 1 and 2). Thepneumathodes were similar to those observedin this study on roots of sprouted seeds growninside white polybags under wet conditions.They were therefore exgrowths of roots causedby the moist condition of the media as wasalso reported by Hartley (1988) for sproutedseeds growth under wet conditions. Mainte-nance of the calli was on Y3(3) containing lessNAA (5mg/l)- Maintenance Medium Y3(3A)-to obtain fast growing calli one monthafter CF.

STAGES IN THE VEGETATIVE PROPAGATION OF OIL PALM, Elaeis guineensis Jacq. THROUGH TISSUE CULTURE

stage

\

Cal lus s tage ~(initiation andmaintenance) ~

Embryoidstage

Plantletsstages

Pre-nursefystage (sand

culture)

Nurserystage

Fieldstage

CultureLeaf explant

iHigh NAA

(Initiation medium)Y3 (3)

Callus rormation(CV

LW”N’NAA(Maintenance medium)

Y3 (3A)

I

Fast grazing callus

Very fast low NAA and cytokinin(Differentiation medium (DM)

IEmbryoid’formation

D M

I

ModifiedM&S basic (MSR)

6Few plantlets

wMany ilantlets Many plantlets

solut ion -~~ inorganic

,.A _-’..~.~__

‘--.,/

--:A Top soil ~II/(sterilized)

I4

NPKMgI12:12:17:2(lkgl per palm)

Time

42-56 days

1 monthafter CF

C 2 monthafter CF

C 6 monthafter CF

C 1 yearafter CF

C 1.5 yearafter CF

C 2.5 yearafter CF

Figure 1. Schematic representation of the stages in vegetative propagation of oil palm by tissueculture

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trichlorophenoxy)-propionic acid. They there-fore suggested that flower and bunch abnor-malities in oil palms might be due to hormonalfactors whereby increase in the concentrationof hormones in the palms could cause biochemi-cal and physiological disorders within the palmleading to the expression of abnormality. Theyconcluded that since this abnormality could beinduced by both natural and artificial meansthere &is a possibility of these palms havingsimilar hormonal systems which triggers offthe formation of these abnormalities.

While tissues of several plants species canbe cultured in the absence of an exogenouscytokinin, very few can be grown without anauxin in the medium (Sogeke, 1996b). Thenotable exceptions are the habituated culturesand plant crown gall tumours (Butcher et al.,1975; Sogeke and Butcher, 1976, which cansynthesize both hormones in culture (Einsetand Skoog, 1973; Miller, 1974, 1975; Petersonand Miller, 1976; Scott et al., 1980; Sogeke andButcher, 1991). Sogeke (1996a1, however, re-ported cases of embryogenesis of oil palm calluson a medium with only NAA as the exogenoushormone. Furthermore, he also observed directroot organogenesis from oil palm leaf explanton a NAA medium with no cytokinin. Aboveall, he was also able to obtain plantlets fromcallus which had never been cultured on amedium with an exogenous cytokinin.

This report, which is the first detailedpublication of vegetatively propagated oil palmby tissue culture on a non-2,4-D medium clearlydemonstrates the possibility of cloning oil palmon a medium with NAA as the only exogenousphytohormone. It creates the need to criticallyasses the role of 2,4-D in clonal oil palmabnormality and necessitates further inves-tigations of the use of NAA and other milderauxins in modified basic media for plant tissueculture.

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