MISCELLANEOUS REPORTS 85 ISSN 0253-6749

N-VITRO MICRO-PROPAGATION OF BANANA CULTIVARSFROM APICAL MERISTEM

G.J. Minas

AGRICULTURAL RESEARCH INSTITUTEMINISTRY OF AGRICULTURE, NATURAL RESOURCES

AND THE ENVIRONMENT

FEBRUARY 2002

CYPRUSNICOSIA

Editor - in Chief

Dr A.P. Mavrogenis, Agricultural Research Institute, Nicosia, Cyprus.

All responsibility for the information in this publication remains with the author(s). The use oftrade names does not imply endorsement of or discrimination against any product by theAgricultural Research Institute.2

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SUMMARYApical meristem tips from banana cultivars Dwarf Cavendish, Gerlick, Grand Naine and

Williams, 2 to 3 mm long were cultured in-vitro on a modified MS medium and microplantswere massively produced. Two media were used. The in-vitromedium BPM for establishmentof cultures, proliferation and micro-shoot growth (stages I, II and III) and the medium BRMfor rooting micro-shoots in-vitro (final phase of stage III). The BPM consisted of MS basicsalts and vitamins plus3% sucrose, 4.5 mg/l BAP, 0.009 mg/l IBA, 55.7 mg/l ascorbic acid andwas solidified with 2.5 g/l phytagel. The BRM consisted of MS basic salts and vitamins plus3% sucrose, 2.25 mg/l BAP, 0.009 mg/l NAA, 55.7 mg/l ascorbic acid and was solidified with2.5 g/l phytagel. Immediately after placing meristem tips on the media a dark spherical halowas formed. Daily transfer to fresh media during the first week eliminated the halo. A pro-gressively increasing enlargement of tissues with proliferation started at week four, andgrowth of micro-shoots were rooted in-vitro on BRM. The highest proliferation rate was 4 foldper week, which is considered adequate for mass micro-propagation. Forteen plants from eachcultivar were grown and finally 14 were planted at Zygi Experimental Station for furtherinvestigations. Five years after being planted in the field they showed no symptoms ofpathogen infection or genetic abnormalities. The plants will be subjected to true-to-type andpathogen-free tests. The use of this method for the production of superior quality true-to-typeand pathogen-free micro-plants for direct distribution to the growers, as well as the use of themethod for safe in-vitro storage of propagation material at stage II, is discussed.

ΠΕΡΙΛΗΨΗΑκραία κορυφαία µεριστώµατα των ποικιλιών µπανάνας Dwarf Cavendish, Gerlick,

Grand Naine και Williams µήκους 2 ως 3 mm καλλιεργήθηκαν in-vitro σε τροποποιηµένοMS θρεπτικό υπόστρωµα και παρήχθηκαν µικρόφυτα. Χρησιµοποιήθηκαν δύοδιαφορετικά θρεπτικά υποστρώµατα ένα για κάθε ένα από τα πιο κάτω στάδια. Tο in-vitro υπόστρωµα ΒΡΜ για εγκατάσταση των καλλιεργειών, µερισµό και ανάπτυξηµικροβλαστών (στάδια Ι, ΙΙ και ΙΙΙ) και το υπόστρωµα ΒRΜ ριζοβολίας µικροβλαστώνin-vitro (τελική φάση του σταδίου ΙΙΙ). Το ΒΡΜ αποτελείτο από τα βασικά άλατα καιβιταµίνες του MS µε προσθήκη 3% σακχαρόζη, 4.5 mg/l BAP, 0.009 mg/l IBA, 55.7 mg/lασκορβικό οξύ, στερεοποιηµένο µε 2.5 g/l phytagel. Το ΒRΜ αποτελείτο από τα βασικάάλατα και βιταµίνες του MS µε προσθήκη 3% σακχαρόζη, 2.25 mg/l BAP, 0.009 mg/lΝΑA, 55.7 mg/l ασκορβικό οξύ, στερεοποιηµένο µε 2.5 g/l phytagel. Αµέσως µετά τηντοποθέτηση τους στα υποστρώµατα όλες οι µεριστωµατικές κορυφές παρουσίασαν µιασκούρα σφαιρική άλο γύρω τους. Καθηµερινές διαδοχικές µεταφυτεύσεις κατά τηνπρώτη εβδοµάδα σε φρέσκα υποστρώµατα σταµάτησε την εµφάνιση της. Μεπροοδευτικά αυξανόµενο ρυθµό είχαµε διόγκωση των µεριστωµάτων και από τηντέταρτη βδοµάδα µερισµό και ανάπτυξη µικροβλαστών που ριζώθηκαν in-vitro στοθρεπτικό υπόστρωµα ΒRΜ. Το µέγιστο της παραγωγικότητας ήταν τετραπλασιασµόςτου υλικού στις καλλιέργιες ανά βδοµάδα, ρυθµός που κρίνεται αρκετά ικανοποιητικόςγια χρήση της µεθόδου για µαζικό µικροπολλαπλασασµό. ∆εκατέσσερα φυτά από τηνκάθε ποικιλία φυτεύτηκαν στον Πειραµατικό Σταθµό Ζυγίου για περαιτέρωπαρατηρήσεις. Τα φυτά αυτά, πέντε χρόνια µετά την εγκατάσταση τους στο χωράφι, δενπαρουσίασαν καµία ένδειξη γενετικής δυστροφίας και/ή παρουσία παθογόνων. Ταπαραχθέντα µικρόφυτα θα περάσουν από σειρά δοκιµασιών για να διαπιστωθεί, στοµέγιστο δυνατό βαθµό, η γενετική τους πιστότητα και η καθαρότητα από παθογόνα.Σχολιάζεται η χρήση της µεθόδου µικροπολλαπλασιασµoύ για παραγωγή µικροφύτωνανώτερης ποιότητας, για άµεση διάθεση στους παραγωγούς καθαρού και γενετικάπιστού πολλαπαλσιαστικού υλικού, καθώς και για οικονοµικότερη ασφαλή αποθήκευσηγενετικού υλικού in-vitro στο στάδιο ΙΙ του µικροπολλαλασιασµού.

IN-VITRO MICRO-PROPAGATION OF BANANA CULTIVARSFROM APICAL MERISTEM

G.J. Minas

INTRODUCTIONBanana is currently the most prominent

tropical crop in the Mediterranean, the mainproducers being Spain, Egypt and Israel witha total of 25 350 ha (Sauco, 1994). In Cyprus,banana (260 ha) is cultivated in the area ofKissonerga and Peyia and other villages ofPaphos producing 12000 t/year (Papandreou,personal communication).A true milestone insome Mediterranean areas is the use of plas-tic houses, a technique which began in theCanary Islands at the end of the 1970’s andwas extended to other subtropical areas(Sauco et al., 1988).Cultivation of bananas under plastic has

three important consequences: a) temperatureincrease, b) increase in leaf surface and c)reduction in water consumption, all of capitalimportance in reducing costs and increasingyields. In some areas of Canary Islands,Dawrf Cavendish yields have increased up to61.7% in bunch weight (Sauco et al., 1991).Similar prospects are expected for GrandNaine. The reduction of water consumption ofgreenhouse grown bananas is also of majorsignificance for the cultivation of bananas inCyprus.A second importand breakthrough was the

development of a specific tissue culture tech-nique which allows cultivation at greater den-sity and at only one or two cycles, a currentpractice in Israel ( Israeli and Numeri, 1988).Combined use of tissue culture and green-house inmild areas of subtropics should resultin a tremendous increase in yield and reducecosts, which may permit cultivation in someMediterranean countries to continue understricter antagonism (Sauco, 1994).The main problem detected so far with tis-

sue cultured plants has been the appearance ofsomaclonal variation (Reuveni, et al., 1990;Stover 1987, 1988) causing failure of manynew plantations (Reuveni and Israeli, 1990).Special attention has been given to the earlyidentification of rogue dwarf types, which canremain genetically stable for generations(Israeli et al., 1990; Smith and Drew, 1990)and eliminate transmission of bunchy-topvirus (Drew et al., 1989) a major constraint tothe commercial application of this technique.On the other hand, somaclonal variation isalso a useful breeding tool and is beingexplored to obtain new clones (Smith andDrew, 1990).In the past decade, the most dramatic event

affecting banana industry was the appearanceof races of Fusarium wilt pathogen thatattacks cultivars previously considered resist-ant, and of more virulent forms of the BlackSigatoka and Black Leaf Streak caused byMycosphaerella fifiensis, a fungus thatreduces yields by 30 to 90%.Apical meristem tip culture has been

recognised as one of the best means to pro-ducing plants free of systemic pathogens.Micro-propagation starting from apicalmeristem tip offers the advantages of safe,without the risk of reinfection, and rapid clon-al multiplication of pathogen free or virusindexed plants on a continuous basis. The ear-liest reports for banana plants produced by in-vitro shoot-tip culture came from Taiwan inthe early 1970’s (Ma and Shii, 1974;Ma et al.,1978). It was then considered a necessity todecapitate the banana shoot-tips cultured in-vitro in order to reduce apical dominance andencourage the growth of axillary buds. Ben-erjee et al. (1986) indicated that the decapita-tion of shoots was not necessary if an appro-priate amount of cytokinin was used in theculture medium.Since 1980, however, a wide range of

Musa species and cultivars have been foundamenable to in-vitro shoot tip culture(Cronauer and Krikorian, 1984a; 1984b; Jar-ret et.al., 1985; Muller and Sandoval, 1986;Novak et al., 1986; Sun, 1985; Vessey andRivera, 1981; Vuylsteke, 1983; Vuylsteke andDe Langhe, 1985; Wong, 1986; Zamoraet etal., 1986).Large scale field establishment of in-vitro

propagated banana plants has been reported inmany banana producing countries (Vuylsteke,1989). Such plants were establishing faster,were taller and had a shorter time of bunchemergence and harvest than conventionalplanting material (Drew and Smith, 1990).

Musa germplasm in tissue culture form hasbeen exchanged internationally since the firsthalf of 1980’s (Jarret et al., 1986; Zamora, etal., 1986). Since 1985, the International Insti-tute for Tropical Agricultural (IITA) atIbadan, Nigeria, has used meristem culturesfor regional and intercontinental exchange ofgermplasm; more than 120 Musa species andcultivars have been introduced and over 100were distributed to many parts of the world(Vuylsteke, 1989). In Cyprus, the cultivarsGerlick and Grand Naine were introduced in1990. There is also increasing interest for

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using in-vitro techniques in bananagermplasm conservation and storage. Satis-factory results have been obtained usingshoot-tip cultures stored under minimalgrowth conditions (Banerjee et al., 1986; Jar-ret et al., 1986).

MATERIALS AND METHODSApical meristem-tips 2 to 3 mm long were

excised from fruit bearing sword suckers,with the typical characteristics of each culti-var of Dwarf Cavendish, Gerlik, Grand Nineand Williams. Suckers bearing also small eti-olated suckers on them were taken from thefield and maintained for at least 4 weeksbefore excision in a 75% shaded glasshouseplanted to a 50/50% v/v peat-moss/horticul-tural grade perlite to facilitate better responseto sterilisation procedures. All plants wereuprooted at the right time, transferred to thelaboratory and used as meristem donors. Sur-face sterilisation, prior to excision was byplacing liquid household detergent and underrunning tap water for two hours, then rinsedin 95% ethanol twice for 15 and 30 sec andimmersed in soaked 25% commercial bleach

(1.3% w/v sodium hypochloride) and finalthree 15 min rinses with glass double distilledsterile water. Apical meristem tips 2 to 3 mmlong were excised under a binocular stereo-scope in a laminar flow cabinet using alcoholflame sterilized blades no11 mounted onscalpel no5 and were placed on the upperscratched surface of the medium in boron freeglass test tubes 22x150 mm, covert withtransparent plastic caps. The media were pre-pared by a stock solution (Table 1) which is aMS (Murashige and Skoog, 1962) modifica-tion by Vuylsteke (1989) plus 30 g/l sucrose,solidified with 2.5 g/l phytagel. Media weresterilized for 15 min in a pressure cooker at1.5 atm pressure. Cultures were incubated ina growth roomwith 1000, 2000 or 3000 lux ina constant 25+2 oC 16 h photoperiod provid-ed by cool white fluorescent irradiation and6000 lux illumination, the last being full phas-ma daylight provided byAgro-T-Plus sodiumilluminators. Cultures were transferred tofresh media and larger vessels according totheir needs. Beach nut, 330 and 500 ml jarscovered with transparent poly-carbonate capswere used as tissues enlarged.

Table 1. Stock solutions and media formulations use for apical meristem micro-propagation in-vitro ofbanana cultivars clones

Stock Amount Stock solution/solution Ingredient (mg) medium (ml/l)

BPM BRMA:Macro KNO3 95000

NH4NO3 82500CaCl2.2H2O 22000

B:Macro MgSO4.7H2O 18500 20 20C:Macro KH2PO4 8500 20 20D:Micro MnSO4.H2O 1690 1 1

H3BO3 620KI 83ZnSO4.4H2O 860Na2Mo4.2H2O 25CuSO4.6H2O 2.5CoCl2.6H2O 2.5

E:Fe-stock4 FeSO4.7H2O 2785 5 5Na2EDTA.2H2O 3725

F:Vitamins Glycine 200 1 1and Thiamine-HCl 40aminoacids3 Pyridoxin-HCl 50

Nicotinic acid 50G:Antioxidant5 Ascorbic acid 1000 1 1H:IBA5 IBA 8.75 1J:NAA6 NAA 9.30I:BAP7 BAP 11.25 2

RESULTS AND DISCUSSIONSoon after the placement of the apical

meristem on the medium sap was penetrateda halo started to form in an area extendingapproximately 5 mm hemispherical aroundthe initial tissue. The halo was progressivelybecoming darker and the next day the meris-tems were transferred to fresh media. Haloformation was probably considered by to bedue to penetration of phenolics from the planttissue to the medium, which with the catalystaction of pheloloxidases in the presence ofMn and Mg cations turns to tannins causingthe black colouring. After a few transfers tofresh media the penetration of phenolicsstoped and enlargement of the tissues and pro-liferation (Fig. 1) started. Growth was speed-ed up by transfers to fresh media, incorpora-tion of the tissues into the media and divisionof the tissues (approximately 5 mm wide).Growth and proliferation speed was affectedeither positively or negatively by the mediumand the air phase in the culture.Several factors affect plant micro-propa-

gation of which the most important constraintis the luck of skilled labour. Since tissues in-vitro are heterotrophic they need sucrose asenergy source; however sucrose is the growthsubstrate for many other micro-organismswhich may contaminate and destroy thewhole balance of the culture. For the tissuesto be autotrophic, the conditions whichenhance photosynthesis must be increased.Konzai (1991), among others, suggested toincrease CO2 concentration and light intensi-ty and phasma with a parallel decrease of

sucrose concentration, all leading to a benefi-cial effect by decreasing contamination at theculture phase and increasing plantlet adapt-ability to external conditions. Relative humid-ity, which can be enhanced by scratching theupper surface of the medium up to a point,increase enlargement and proliferation. How-ever at very high levels, it is considered (Ziv,1991) as a major factor for vitirificationwhich is expressed as soaky and fleshy tis-sues, a major constrain in tissue culture as itproduces plantlets which cannot survive ex-vitro.The present results are in agreement with

those of the other investigators who showedthat the removal of apical dominance byremoving the apical meristem tip can bereplaced by incorporating the appropriateamount of cytokinin in the culture medium.After 8 weeks in culture many cultures reachthe rate of 4 fold in proliferation (Fig. 1).Shoots longer than 30 mmwere transferred tothe BRM for rooting and then harvested andhardened in peatmoss/perlite 50/50% v/vmixture (Fig. 2). More than 1000 micro-plants were produced from which 40 fromeach cultivar were grown (Fig. 3) and finally14 were planted at Zygi Experimental Station(Fig. 4) for further investigation. Five yearslater they show no symptoms of pathogeninfection or genetic abnormalities. Genetic,epigenetic and non genetic variations (from 0to 38%) on micro-propagated plantlets havebeen reported by many authors (Vuylsteke,

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Figure 1. Banana cultures at in-vitro stage II ini-tiated from apical meristem tips.

Figure 2. Well established (end of stage IV)banana micro-plants produced in-vitro.

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1989) for a number of cultivars. Severalingredients in the media as well as some envi-ronmental conditions (UV selectivelyabsorbed and destroying DNA) have beenreported to change the genotype and /or the

habit of the plantlets produced and were notused in this study.Growth from micro-plants, although

appearing healthy and vigorous, must under-go phytosanitary and genetic observations.For genetic uniformity tests, molecularmethodologies, such as RAPD-PCR (Ran-dom Amplified Polimorphic DNA- Poly-merase Chain Reaction) or other convention-al methods, like isoenzyme analysis, as fasterand more accurate methodologies of to day,are sugested. Counting of chromosomes andmorphological characteristics could provideadditional valuable information.This method could be used for mass micro-

propagation of superior quality true-to-typeand pathogen-free micro-plans for direct dis-tribution to the growers, as well as for clean-ing existing germplasm from certainpathogens.

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Drew, R.A., J.AMoisander andM..K. Smith. 1989.Trasmission of banana bunchy-top virus inmicropropagated bananas. Plant Cell Tis-sue and Organ Culture 16:187-193.

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Figure 3. Grown-up banana plants from apicalmeristem tip cultured in-vitro. Cov-ered by glass jar in a small pot arebanana micro-plants at stage IV.

Figure 4. Apical meristem-produced bananas inplantation at Zygi.

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