Cryo-Letters 13, 165-174 (1992).

Published by Cryo-Letters, 7,Wootton Way, Cambridge CB3 9LX, U.K.

CRYOPRESERVATION OF IN VITRO SHOOT TIPS OF CHICORY(CICHORIL~ INTYBUS L.)

1* 2 1M.A.C. DEMEULEMEESTER , B.J. PANIS, M.P. DE PROFT·

1 Laboratory of Plant Culture, Catholic University of Leuven,De Croylaan 42, B-3001 Heverlee, Belgium

2 ~aboratory of Tropical Crop Husbandry, Catholic Universityof Leuven, Kardinaal Mercierlaan 92, B-3001 Heverlee,Belgium

SUMMARYShoot tips of about 2 mm in length, isolated from in vitrocultured root fragments of chicory (Cichorium intybus L.)variety Flash, were subjected to several cryopreservationprotocols. Cooling rates were varied between 0,1 °C/min and0,7°C/min and the influence of DMSO (during preculture andcryoprotection treatments) on the survival was examined. Thebest resul t (regrowth of 83 %) was obtained after 2 daysprecul ture on medium without DMSO, cryoprotection with 15 %

DMSO and cooling to -40°C at ë;l rate of 0,5 °C/min prior toimmersion in liquid nitrogen.

KEY WORDScichorium, chicory, cryopreservation, cryoprotection, shoot tip

ABBREVIATION LISTDMSO, dimethylsulphoxidei M&S, Murashige and Skoog mediumiBA, benzyladeninei lAA, indolyl acetic acid

I INTRODUCTION

Genebanks conserving weIl characterized plant tissues arebecoming more and more important. Existing plant species haveto be stored without loss of their specific properties to usethem in improvement programs in the future (1).Plant germplasm can be stored in different ways : fieldgenebanks (2), seed storage (3, 4, 5) and in vitro collectionsunder normal or limited growth conditions, or cryopreservation(5, 6). Each of these methods has its own limitations. Pollenand also DNA storage are recently developed methods, requiring·

165

further investigation.

cryopreservation can be put forward as the most promisingtechnique for long-term preservation of in vitro germplasmcollections. Under conditions of storage in liquid nitrogen(-196·C), there is a complete cessation of all chemicalreactions in the cello Consequently, no cell division takesplace and the cell degeneration or genetic changes that cantake place with time in culture are suspended (7, 8, 9).

Nowadays, chicory is cultivated more and more in hydroponicculture. F1 hybrids produced by the great seed houses are usedbecause they give a more homogeneous product. Therefore,ancient varieties that are the result of years of selection byindividualchicory growers are threatened with extinction. Itis of the greatest economical importance that a wide range ofthis genetically valuable plant material is preserved forfurther improvement. Up to the present, chicóry species havebeen preserved by storing seeds. However, because we neverobtain 100 % pure lines in chicory, seed storage can not befully recommended.

The present research was carried out to study the potential ofthe cryopreservation of in vitro shoot tips of chicory.

I MATERIALS ANO METHOOS

Shoot tip culture

The Cichorium intybus L. variety Flash was used in ourexperiments. Washed and peeled roots were surface sterilizedin 95 % (v/v) ethanol for 20' s, and then in 4 % sodiumhypochlorite for 15 min, followed by three rinses (10, 5,5 min) with sterilized distilled water. Excised root explantsof 5 mm3, consisting of xylem, phloem and cambium, were placedin glass jars of 380 mI on a solidified (6 9 agar/l), adaptedM&S medium (10, 11, 12, 13). This medium contains no NH4+ ,

Zn2+, cu2+ and a reduced Na6.EOTA concentration and issupplemented with 10-5 MBA, 10- M lAA, standard M&S vitaminsand 10 gil sucrose. The pH was adjusted to 6 beforeautoclaving using 0,1 N NaOH or 0,1 N Hel.

The cultures were placed at 23·C under a 16h photoperiod(ca. 20 ~E/m2 s). After 14 days, the newly formed shoot tipswere isolated (2 mm in length) under a binocular.When placedon the adapted M&S medium described above, the regrowth was100 %.

cryopreservation procedure

PregrowthOissected shoot tips were placed in a plastic petri dish on afilter paper soaked with liquid hormone-free M&S medium(2,5 mI) supplemented with OMSO (Janssen chimica ; 99,9%

166