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Short paper

The cryopreservation of shoot tips of Rosa multiflora

P.T. Lynch*, W.C. Harris & J.M. Chartier-Hollis Plant Biotechnology Group, Division of Biological Sciences, University of Derby, Kedleston Road, Derby DE22 1 GB, UK (‘authorfor correspondence)

Key words: cryopreservation, encapsulation/dehydration, rose, Rosa multijora, shoot tips

Abstract

Shoot tips of in vitro grown plantlets of Rosa multijlora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24 h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.

Abbreviations: RSCl = modified Murashige and Skoog medium for Rosa multifora shoot culture

Introduction

The market for roses is a large and valuable one for the horticultural industry. It has been estimated that more than 200 million rose bushes are planted annually world-wide, which represents a retail market of US $720 million [ 151. For rose breeders to maintain their market share they must be able to regularly produce novel cultivars. To achieve this they require access to extensive genetic resources. As roses do not come ‘true-to-type’ from seed, characterised germplasm must be sustained vegetatively. Plants maintained for breeding purposes in the field or glasshouse are vulnerable to loss due to pests, diseases and abiotic factors. Similarly, plant material under in vitro micro- propagation conditions may be lost due to microbial contamination and equipment failure [4] and is subject to the effects of somaclonal variation [7,14,16]. How- ever, cryogenic storage provides a stable and secure means of long-term storage while safeguarding against genetic erosion [ 1,121. This report describes the initial results of an investigation aimed at the development of a protocol for the cryopreservation of rose shoot tips using the dehydration/encapsulation method of Fabre and Dereuddre [5].

Materials and methods

Tissue culture and encapsulation

Apical shoot tips for these studies were excised from in vitro shoot cultures of Rosa multi$ora. The shoot cultures had been maintained on a semi - solid Murashige and Skoog-based medium [lo] containing 30 gl-’ sucrose, 8.0 gl-’ Difco Bacto agar (Difco Ltd., Surrey, UK) and supplemented with 1.0 mgl-’ 6-benzylaminopurine, 0.1 mgl-’ gibberellic acid and 0.004 mgl-’ naphthalene acetic acid [8], this media was designated RSCl [9]. Unless indicated other- wise, all in vitro plant material was maintained at 18 ‘C&2 “C, with a 16 h photoperiod and a light intensity of 3-7 pmol m-* s-l PAR. Shoot tips were excised under filter-sterilised anti-oxidant solution (0.2 M phosphate buffer, pH 5.7, supplemented with 50 gl-’ ascorbic acid and 15 gl-’ sodium borate). The excised shoots were encapsulated in calcium-alginate beads using a modified version of method described by Fabre and Dereuddre [5]. Excised shoot tips were suspended in liquid calcium-free RSCl containing 30 gl-’ sodium alginate (Sigma, Dorset, UK). The shoot tips were drawn up in to sterile 10 cm3 disposable pipettes (SLS, Nottingham, UK) and dripped in to

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liquid RSCl containing 14.7 gl-’ Ca Clz. 2Hz0, in order to form calcium-alginate beads around the shoot tips. Beads contained one shoot tip and were 5-6 mm in diameter.

Cryopreservation treatments

The calcium-alginate beads were surface dried on sterile filter paper (Whatman No. 1) and transferred to 5 cm Petri dishes (5 beads per dish) containing 10 cm3 of liquid RSCI containing 85.6-342.3 gl-’ (0.25 M-1.0 M) sucrose. The beads were incubated in the dark at 18 ‘C with shaking (40 rpm) for 24 h. After which the beads were surface dried on sterile filter paper and dehydrated for between l-24 h with silica gel using the method described by Hatakana et al. [6]. Prior to freezing the beads were placed in to 2 cm3 polypropylene vials (Starstedt Ltd., Leicester, UK, 2 beads/vial). Vials were transferred to aluminium canes and the encapsulated shoot tips frozen by either:- a) Rapid freezing: Samples were plunged directly in

to liquid nitrogen, or b) Two stage freezing: Samples were held at +20 ‘C

for 10 min, reduced to -35 ‘C at -1 ‘C per min. using a controlled rate freezer (Planer Kryo 10 Series; Planer Biomed, Sunbury-on-Thames, Middlesex, UK.) and then plunged into liquid nitrogen. Samples were stored in liquid nitrogen for at least

24 h. The frozen beads were removed from the vials and thawed by plunging in to sterile water at +40 ‘C. The shoot tips were, unless otherwise indicated, excised from the calcium-alginate beads and placed in 5 cm Petri dishes containing semi-solid RSCl. Samples were maintained at 18 “C in the dark for 7-10 d post-thaw, after which they were transferred to the same light conditions as described previously. A minimum of 10 replicate shoot tips were used for each treatment variable in 2 replicate experiments. The statistical significance of the experimental data were determined using analysis of variance.

Results

Growth of non-pretreated, non-frozen shoot tips

Excised non-encapsulated shoot tips of Rosa multijlora exhibited shoot elongation and development with- in 28d of culture on RSCl. Subsequently 100% of this material developed into rooted shoots. However,

encapsulated shoot tips exhibited only slight shoot elongation after a similar culture period, and took up to 90 d to grow away from the calcium-alginate beads. Excision of the shoot tips from the calcium-alginate beads prior to culture resulted in a frequency and rate of shoot tip growth comparable with non-encapsulated material. Hence in subsequent studies shoot tips were excised from the calcium-alginate beads prior to culture.

Influence of sucrose and silica gel pretreatments on the growth of non-j+ozen shoot tips

Pretreatment of encapsulated shoot tips with 0.25 M and 0.5 M sucrose did not affect their subsequent growth, as compared with untreated encapsulated con- trols. However, the number of shoot tips which grew after pretreatment with 0.75 M and 1 .O M was signifi- cantly (p = 0.005) reduced to 75f7% and 15f7% respectively. Greater callus formation was also noted at the base of shoot tips treated with the higher sucrose concentrations. Hence 0.5 M sucrose pretreatment was employed in subsequent investigations. The number of encapsulated shoots pretreated in 0.5 M sucrose which grew was not effected by drying with silica gel for up to 2 h. However, 3 h exposure to silica gel resulted in a significant (p = 0.005) decrease in the number (50f14%) of shoots that grew. No shoot growth was observed from shoot tips subject to greater than a 3 h exposure to silica gel. After 0.5 M sucrose and 2 h silica gel pretreatments encapsulated shoot tips had 15-20% fresh wt. moisture content.

Freezing of encapsulated shoot tips and post-thaw recovery

Post-thaw shoot tip growth (25f7%) was only observed after pretreatment with 0.5 M sucrose and 2 h exposure to silica gel followed by rapid freezing. No viable material was recovered after two - stage freez- ing. Post-thaw recovering shoots tended to develop callus around their base, which occasionally overgrew the shoot. No shoot regeneration was observed from callus generated from shoot tips recovered from cryo- genic storage.

Discussion

This initial study demonstrates the ability to cryop- reserve apical shoot tips of R. multij?ora. This is the

first report of the cryopreservation of shoot tips from a Rosa sp. However, shoot tips of other member of the Rosacae, including Malus sp. and Prunus sp. [3, 111 have previously been recovered from cryogenic storage. Similar encapsulatiotidehydrationprocedures to those described here were employed, but the details of the sucrose treatment and dehydration treatment differed. Of particular significance may be the use of a 2-step sucrose treatment which has been employed with other woody species [ 11,131. The development of callus on shoot tips after cryopreservation, is generally regarded as undesirable since it is an indication of cryoinjury and is associated with somaclonal varia- tion [2]. However, as the shoots of R. multij?oru that grew after cryogenic storage did not regenerate from the callus which was localised at the base of the origi- nal shoot tips the latter problem may not be particularly significant.

These results indicate that cryopreservation can potentially provide a useful long-term storage method for roses. Investigations are continuing to improve the efficiency of the existing protocol and extend its appli- cation to other rose species.

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