Annals of Botany 82 : 455–461, 1998Article No. bo980703

Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosum L.)

Clones by Vitrification

DEBABRATA SARKAR* and PRAKASH S. NAIK

Di�ision of Genetics and Plant Breeding, Central Potato Research Institute, Shimla-171 001,

Himachal Pradesh, India

Received: 27 February 1998 Returned for revision: 28 April 1998 Accepted: 30 May 1998

In �itro-grown shoot tips of five tetraploid potato (Solanum tuberosum L.) clones were cryopreserved by vitrification.Excised shoot tips (0±5–0±7 mm) were pre-cultured on filter paper discs over half strength liquid Murashige and Skoog(MS) medium supplemented with 8±7 µ GA

$and different combinations of sucrose (0±3, 0±5 and 0±7 ) plus mannitol

(0, 0±2 and 0±4 ) for 2 d under a 16 h photoperiod at 24 °C. The pre-cultured shoot tips were either successivelyloaded with 20 and 60% PVS 2 solutions or directly exposed to concentrated vitrification solution before physicalvitrification during liquid nitrogen treatment. The vitrified shoot tips were warmed rapidly and treated with dilutionmixture (MS­1±2 sucrose) for 30 min before plating on regrowth medium. Addition of mannitol to the pre-culturemedium improved survival of vitrified shoot tips. Direct dehydration of pre-cultured shoot tips with concentratedPVS 2 was detrimental to survival of vitrified shoot tips. Shoot tips pre-cultured on medium containing 0±3 sucroseplus 0±2 mannitol, and loaded with 20% PVS 2 for 30 min followed by 15 min incubation in 60% PVS 2 and 5 minincubation in 100% PVS 2 at 0 °C resulted in up to 54% survival after vitrification. About 50% of vitrified andwarmed shoot tips formed shoots directly. Post-thaw culturing of vitrified shoot tips on medium containing anelevated level of sucrose (0±2 ) under diffuse light for the first week enhanced the survival rate. Continuousculturing of vitrified shoot tips on high-sucrose medium induced multiple shoot formation.

# 1998 Annals of Botany Company

Key words : Solanum tuberosum L., potato, cryopreservation, germplasm conservation, in �itro conservation,meristems, shoot tips, tissue culture, vitrification.

INTRODUCTION

Minimal growth may be used for the conservation of potato(Solanum tuberosum L.) germplasm in �itro (Sarkar andNaik, 1998). This medium-term conservation approach isnot only time-consuming and labour-intensive, but inaddition may not even ensure good genetic stability of in�itro micropropagated plantlets (Harding, 1991). Cryogenicstorage of shoot tips in liquid nitrogen has therefore beenrecognized as a major method for the long-term storage ofpotato germplasm. Storage in liquid nitrogen ensured a highdegree of genetic (ploidy) stability in dihaploid potatoclones and wild Solanum species (Ward et al., 1993). Workon the cryopreservation of potato shoot tips started as earlyas the late 1970s, and since then different methods have beendeveloped using slow or ultrarapid cooling or other modifiedcooling approaches (Bajaj, 1977; Grout and Henshaw,1978; Towill, 1981, 1984; Benson, Harding and Smith,1989; Scha$ fer-Menuhr, Muller and Mix-Wagner, 1996).Some of these classical methods are time-consuming, requiresophisticated freezing equipment and involve specific ad-aptation of cooling rates to optimize the results. Comparableresults were not obtained in early potato cryopreservationwork because of differential responses observed betweenand within groups and}or species. Except for the droplet

* For communication. Fax ­91 177 224460, e-maild.sarkar!mailexcite.com

freezing technique (Scha$ fer-Menuhr et al., 1996), no onemethod can be routinely applied to a wide range of speciesand accessions within species. In addition, many of thesetechniques involve regeneration of cryopreserved shoot tipsthrough an intermediary callus phase. However, concernshave been raised regarding the genetic stability of plantsregenerated in this way (Henshaw, O’Hara and Stamp,1985).

In recent years, an alternate method has been developedto cryopreserve cells or small tissue segments throughvitrification. Vitrification is the phase transition of waterfrom a liquid directly into a non-crystalline amorphousphase, a glass, by an extreme elevation in viscosity duringcooling (Fahy et al., 1984). In contrast to classical techniquesbased on freezing, cell dehydration in vitrification-basedprocedures is performed prior to freezing by exposure ofsamples to concentrated cryoprotective media and}or airdesiccation followed by rapid cooling (Engelmann, 1997).In the encapsulation-vitrification technique, the explants areencapsulated in alginate beads and treated with vitrificationsolutions before freezing (Matsumoto and Sakai, 1995).Although the encapsulation-vitrification procedure was notemployed in potato cryopreservation, efforts were made tocryopreserve potato shoot tips by vitrification based on theencapsulation-dehydration method (Fabre and Dereuddre,1990;Bouafia et al., 1996).However, vitrification proceduresbased on encapsulation-vitrification and encapsulation-dehydration are lengthy, cumbersome and highly labour-

0305-7364}98}100455­07 $30.00}0 # 1998 Annals of Botany Company

456 Sarkar and Naik—Cryopreser�ation of Potato Shoot Tips by Vitrification

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F. 1. Pre-growth effect on potato shoot tip viability before and aftercryoprotective loading and}or dehydration. Interaction effects ofsucrose and mannitol on shoot tip survival after : pre-culturing for 2 dunder a 16 h photoperiod (approx. 40 µmol m−# s−" light intensity) at24 °C (A); sequential loading of pre-cultured shoot tips with 20%(30 min) and 60% (15 min) PVS 2 solutions followed by dehydrationwith concentrated PVS 2 for 5 min (B); direct dehydration of pre-cultured shoot tips with concentrated PVS 2 for 5 min (C) ; Data (%)are averaged over five genotypes. Bars with common letters are notsignificantly different at P% 0±05, according to Student-Newman-Keul

test.

intensive. In contrast, vitrification in the presence of ahighly concentrated cryoprotective solution is simpler. Inthis method, the cells or shoot tips are sufficiently dehydrated

with a highly concentrated vitrification solution beforedirect transfer to liquid nitrogen (Sakai, 1995). Thevitrification procedure has been applied to a wide range ofshoot tips (Yamada et al., 1991; Towill and Jarret, 1992;Matsumoto et al., 1997; Takagi et al., 1997).

To the best of our knowledge, no detailed work has beenpublished on the cryopreservation of potato shoot tips byvitrification employing chemical additives. The presentstudy was therefore undertaken to standardize an effectivechemical-induced vitrification procedure for cryopreser-vation of potato shoot tips. Most vitrification studiesreported so far suggest that the key to successful vitrificationof cells or small tissue segments is the effective increase inthe concentration of cellular solutes achieved by chemicalpre-treatments. Therefore, one of the main objectives of thepresent study was to develop suitable pre-culturing con-ditions for potato shoot tips before vitrification in liquidnitrogen.

MATERIALS AND METHODS

Plant material

In �itro-grown plantlets of five potato (Solanum tuberosumL.) genotypes, viz. ‘Kufri Badshah’, ‘Kufri Chandramukhi ’,‘Kufri Lalima’, ‘Kufri Lauvkar’ and ‘Kufri Sindhuri ’,were used in the present study. Disease-free stock cultures ofthese plants were multiplied and maintained over severalyears through shoot cuttings following the method describedearlier (Sarkar, Chandra and Naik, 1997). Apical shoot tipsabout 0±5–0±7 mm long were dissected from 30-d-oldplantlets. Shoot tips were pre-cultured on filter paper discsover half strength liquid MS (Murashige and Skoog, 1962)medium supplemented with 8±7 µ GA

$and different

(factorial) combinations of sucrose (0±3, 0±5 and 0±7 ) andmannitol (0, 0±2 and 0±4 ) for 2 d under a 16 h photoperiod(approx. 40 µmol m−# s−" light intensity) at 24 °C.

Vitrification procedure

The vitrification procedure involved the following steps :(a) loading of pre-cultured shoot tips with cryoprotectivesolutions; (b) dehydration of the loaded shoot tips byconcentrated PVS 2 vitrification solution (30% w}wglycerol, 15% w}w ethylene glycol, 15% w}w dimethylsulphoxide in MS medium supplemented with 0±4 sucrose ;Sakai, Kobayashi and Oiyama, 1990) ; (c) plunging theminto liquid nitrogen; (d ) rapid warming (thawing) of vitrifiedshoot tips ; (e) unloading of PVS 2 by transferring the shoottips to dilution medium (MS­1±2 sucrose) ; and ( f ) post-thaw culturing of shoot tips.

Twenty pre-cultured shoot tips were placed in a 5 ml testtube and loaded with 3 ml 20% PVS 2 for 30 min at 24 °C.Subsequently, 20% PVS 2 solution was removed using aPasteur pipette, and 3 ml of 60% PVS 2 was added. Thetubes were placed in an ice bath and incubated for 15 min.After removal of the 60% PVS 2 solution, shoot tips weretreated with 3 ml of ice cold concentrated PVS 2 for 5 min,and then transferred to 1 ml cryotubes (CRYOSTM, Sumi-tomo Bakelite Co., Japan). In the direct vitrificationprocedure, the pre-cultured shoot tips were not loaded with

Sarkar and Naik—Cryopreser�ation of Potato Shoot Tips by Vitrification 457

T 1. Analysis of �ariance for shoot tip sur�i�al and direct shoot formation during cryopreser�ation of potato shoot tipsby �itrification following direct and sequential (20% PVS 2U 60% PVS 2 ) exposure to concentrated PVS 2 solution

MS

Direct vitrification Sequential vitrification

Source d.f. % Survival% Direct

shoot formation % Survival% Direct

shoot formation

Genotype 4 578±25** 582±27** 206±72** 222±18**Sucrose 2 353±13** 54±62 3267±52** 2630±13**Genotype¬sucrose 8 11±98 23±80 6±78 8±68Mannitol 2 4501±79** 1913±54** 10274±95** 8355±60**Genotype¬mannitol 8 36±31 39±54 6±25 12±01Sucrose¬mannitol 4 1083±92** 945±89** 2075±86** 2975±92**Genotype¬sucrose¬mannitol 16 18±32 30±04 12±21 14±66Error 90 69±19 61±15 21±47 28±05

** P% 0±01.

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F. 2. Cryopreservation of potato shoot tips by direct vitrificationwith concentrated PVS 2 without being loaded with 20 and 60% PVS 2cryoprotective solutions. Pre-freeze incubation effects of sucrose andmannitol on survival of, and direct shoot formation from, vitrifiedshoot tips following direct exposure to concentrated PVS 2 solution(5 min) before vitrification in liquid nitrogen. Data (%) were recordedafter 4 weeks of post-thaw culturing of vitrified shoot tips and averagedover five genotypes. Bars with common letters are not significantly

different at P% 0±05, according to Student-Newman-Keul test.

diluted PVS 2 solution, but treated directly with concen-trated PVS 2 for 5 min. The cryotubes (each containing fiveshoot tips suspended in 0±7 ml of PVS 2) were plunged intoliquid nitrogen, and held there for at least 60 min. Cryotubeswere warmed in a water bath for 1 min at 35 °C. After rapidwarming, the contents of the cryotubes were flushed withdilution medium, expelled into a 5 ml test tube, and themixture (diluent­PVS 2) was immediately removed using aPasteur pipette. Shoot tips were subsequently maintainedin dilution medium (approx. 20 shoot tips 4 ml−" of dilutionmedium) for 30 min.

Control experiments

In control experiments, the viability of shoot tips wasassessed after : (a) pre-conditioning on different pre-culturemedia for 2 d at 24 °C; (b) sequential cryoprotection-dehydration by 20 and 60% PVS 2 solutions followed byconcentrated PVS 2; and (c) direct cryoprotection-dehy-dration by concentrated PVS 2 without being loaded withdiluted PVS 2 solutions.

Post-thaw culturing and �iability

The vitrified (cryopreserved) shoot tips were plated onsemi-solid MS medium containing 0±2 sucrose, 5±8 µ

GA$, 1±0 µ BA and 6 gl−" Nobel agar (HiMedia, India),

and cultured under 16 h diffuse light (approx. 6 µmol m−#

s−") at 24³1 °C. After 1 week, shoot tips were transferredonto standard shoot tip medium based on MS supplementedwith 0±09 sucrose, 2±9 µ GA

$and 6 gl−" agar, and

incubated under a 16 h photoperiod (approx. 40 µmolm−# s−") at 24³1 °C. Shoot tip recovery was recorded atweekly intervals. Two types of growth were observed:regrowth of the shoot apex or callus formation. Survivaland direct shoot formation were recorded as the percentageof the total number of shoot tips showing regrowth anddirect shoot formation, respectively, 4 weeks after initialplating.

458 Sarkar and Naik—Cryopreser�ation of Potato Shoot Tips by Vitrification

Data analysis and statistical procedures

For each genotype, twenty apical shoot tips were testedfor each of three replications for each treatment, and theexperiment was repeated once. The percentage data werepooled over individual experiments, and transformed intoarc sine square root before statistical analysis. The three-way analyses of variance (five genotypes¬three levels ofsucrose¬three levels of mannitol) were computed using thestandard procedure, and mean separations were carried outby Student-Newman-Keul multiple range test (Steel andTorrie, 1980).

RESULTS

In a preliminary experiment, excised apical shoot tips wereeither successively loaded with 20 and 60% PVS 2 solutionsbefore vitrification or directly vitrified (without beingloaded) with concentrated PVS 2. Little or no survival wasobserved in the vitrified and warmed shoot tips cooled to®196 °C in either of these cases. This suggested that pre-culturing (before vitrification) might be an essential step forsuccessful cryopreservation of potato shoot tips by vitri-fication.

Control experiment

The effects of pre-culture media on shoot tip viabilitywere studied before and after cryoprotective loading and}ordehydration. Variation due to genotypic differences washighly significant (P¯ 0±01) for shoot tip survival. Therewas a significant (P¯ 0±01) interaction between sucrose andmannitol for shoot tip survival in all the three controlconditions studied (see Materials and Methods), suggestingthat the effect of sucrose was not uniform over differentlevels of mannitol or �ice �ersa (Fig. 1). After 2 d of pre-culturing on medium supplemented with 0±3–0±5 sucroseor 0±3 sucrose plus 0±2 mannitol, more than 90% of the

T 2. Sur�i�al of, and direct shoot formation from, cryopreser�ed shoot tips in fi�e potato genotypes under two �itrificationconditions

Direct vitrification Sequential vitrification

Genotype % Survival% Direct

shoot formation % Survival% Direct

shoot formation

Pre-culture: 0±3 M sucrose­0±2 M mannitolKufri Badshah 40 (39±21)* 25 (29±92) 60 (50±79) 56 (48±62)Kufri Chandramukhi 32 (34±15) 20 (26±26) 55 (47±98) 55 (47±88)Kufri Lalima 35 (36±18) 18 (25±00) 57 (49±02) 52 (45±97)Kufri Lauvkar 25 (29±80) 10 (15±19) 52 (46±30) 48 (44±04)Kufri Sindhuri 20 (26±45) 8 (16±60) 47 (43±57) 40 (39±26)

Pre-culture: 0±5 M sucrose­0±2 M mannitolKufri Badshah 35 (36±24) 18 (25±19) 55 (47±88) 50 (45±11)Kufri Chandramukhi 33 (35±11) 20 (26±26) 45 (42±09) 45 (42±11)Kufri Lalima 37 (37±12) 23 (28±78) 52 (45±97) 45 (42±09)Kufri Lauvkar 25 (29±74) 10 (15±38) 48 (44±04) 40 (39±28)Kufri Sindhuri 18 (24±81) 5 (10±64) 41 (39±95) 33 (35±24)Sd- 4±80 4±51 2±67 3±06

* % Data averaged over three replications and rounded; arc sine transformed means are within parenthesis.Shoot tips were pre-cultured on media containing 0±3–0±5 sucrose plus 0±2 mannitol and cryopreserved following direct and sequential (20%

PVS 2U 60% PVS 2) exposure to concentrated PVS 2 solution before vitrification in liquid nitrogen.

shoot tips survived (Fig. 1A). A significant reduction inviability was observed when shoot tips were pre-cultured onmedium containing 0±7 sucrose or 0±5 sucrose plus 0±2

mannitol. A higher concentration of mannitol (0±4 ) in thepre-culture medium was detrimental to shoot tip viability.After cryoprotective loading with diluted PVS 2 solution,about 70–80% of shoot tips survived when they were pre-cultured on medium containing 0±3–0±7 sucrose or0±3–0±5 sucrose plus 0±2 mannitol (Fig. 1B). Sucrose atthe highest concentration (0±7 ) in combination withmannitol, or mannitol at the highest concentration (0±4 )in combination with sucrose was detrimental to shoot tipviability following cryoprotective loading. In general, areduction in viability occurred when the pre-cultured shoottips were not cryo-loaded, but dehydrated directly withconcentrated PVS 2 (Fig. 1C). Unloaded shoot tips recordedmaximum survival when pre-cultured on medium supple-mented with 0±3–0±5 sucrose and 0±2 mannitol.

Vitrification-cryopreser�ation experiment

The pre-cultured shoot tips were either directly vitrified orsuccessively loaded with 20 and 60% PVS 2 solutionsbefore vitrification with concentrated PVS 2 in liquidnitrogen. Analysis of variance for both vitrification con-ditions is presented in Table 1. The main effects of genotypeand mannitol were highly significant (P¯ 0±01) for shoot tipsurvival and direct shoot formation in both vitrificationconditions (i.e. direct and sequential) employed. Variationdue to sucrose concentration in the pre-culture media wassignificant (P¯ 0±01) for both the survival characters,except for direct shoot formation in the direct vitrificationcondition. The genotype did not show any significantinteraction with sucrose or mannitol, suggesting that theeffect of sucrose or mannitol was uniform over the genotypestested. Two-way interaction between sucrose and mannitolwas highly significant (P¯ 0±01) for both characters. This

Sarkar and Naik—Cryopreser�ation of Potato Shoot Tips by Vitrification 459

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F. 3. Cryopreservation of potato shoot tips by sequential vitrificationwith PVS 2. Pre-freeze incubation effects of sucrose and mannitol onsurvival of, and direct shoot formation from, vitrified shoot tipsfollowing sequential cryoprotective loading with 20% (30 min) and60% (15 min) PVS 2 solutions, and subsequent dehydration (5 min)-vitrification with concentrated PVS 2 in liquid nitrogen. Data (%) wererecorded after 4 weeks of post-thaw culturing of vitrified shoot tips andaveraged over five genotypes. Bars with common letters are notsignificantly different at P% 0±05, according to Student-Newman-Keul

test.

showed that the effect of sucrose was not constant overdifferent levels of mannitol or �ice �ersa. The three-waygenotype¬sucrose¬mannitol interaction was not signifi-cant, suggesting that sucrose and mannitol together do nothave a major effect on the expression of genotypic variationover and above their individual effects.

In general, shoot tips pre-cultured on medium containing0±3 sucrose gave only 6% survival when they werevitrified directly with concentrated PVS 2 without beingloaded sequentially with 20 and 60% PVS 2 solutions (Fig.2). Pooled over genotypes, up to 30% of the unloaded shoottips survived after vitrification when they were pre-culturedonmediumcontaining 0±3–0±5 sucrose plus 0±2 mannitol.Unloaded shoot tips remained green immediately aftervitrification-warming, but most of them turned necrotic 1week after plating on regrowth medium. Callus developedfrom the basal portion of necrotic shoot tips after 2 weeksof reculture. About 12–16% of vitrified shoot tips remainedgreen continuously after plating, and formed shoots directly

within 2 weeks (Fig. 2). Following direct vitrification,maximum shoot tip survival (40%) and direct shootformation (25%) were observed in ‘Kufri Badshah’ whenthe shoot tips were pre-cultured on medium containing0±3 sucrose plus 0±2 mannitol, whereas ‘Kufri Sindhuri ’was the least responsive recording only 20 and 8% shoot tipsurvival and direct shoot formation, respectively (Table 2).Intermediate responses were observed in the other cultivars.The genotypic response to direct vitrification was more orless similar when shoot tips were pre-cultured on mediumcontaining 0±5 sucrose plus 0±2 mannitol. However, inboth pre-culture treatments, a reduction in direct shootformation was recorded in all the genotypes following directvitrification (Table 2).

The survival of vitrified shoot tips increased significantlywhen the pre-cultured shoot tips were loaded successivelywith 20 and 60% PVS 2 solutions before vitrification inliquid nitrogen (Fig. 3). Shoot tips pre-cultured on mediumcontaining 0±3 sucrose recorded about 27% survivalfollowing cryoprotective loading and vitrification. Increas-ing the sucrose concentration in the pre-culture mediumimproved the survival of vitrified shoot tips (38–42%). Ingeneral, a significant (P¯ 0±05) increase in survival ofvitrified shoot tips occurred following pre-culturing onmedium containing 0±3–0±5 sucrose plus 0±2 mannitol. Ahigher concentration (0±4 ) of mannitol in the pre-culturemedium was detrimental to shoot tip viability followingvitrification. Averaged over genotypes, the highest survival(54%) was observed when shoot tips were pre-cultured onmedium supplemented with 0±3 sucrose and 0±2 mann-itol, and loaded successively with 20 and 60% PVS 2solutions before vitrification (Fig. 3). About 31% of vitrifiedshoot tips resumed normal shoot growth when pre-culturedon medium containing sucrose (0±5–0±7 ) alone. In com-parison, about 50% of vitrified shoot tips formed shootsdirectly when they were pre-cultured on medium containing0±3 sucrose and 0±2 mannitol (Fig. 3). Followingsequential vitrification, maximum shoot tip survival (60%)and direct shoot formation (56%) were observed in ‘KufriBadshah’ when the shoot tips were pre-cultured on mediumcontaining 0±3 sucrose and 0±2 mannitol (Table 2).Minimum shoot tip survival (47%) and direct shootformation (40%) were observed in ‘Kufri Sindhuri ’ underthe same pre-treatment and vitrification conditions. In allgenotypes, a general reduction in survival and direct shootformation occurred when shoot tips were pre-cultured onmedium containing 0±5 sucrose plus 0±2 mannitol andcryopreserved by sequential loading with diluted PVS 2solutions before vitrification. However, in contrast to directvitrification, a higher percentage of vitrified shoot tipsresumed direct shoot growth following sequential vitri-fication under both the pre-culture conditions (Table 2).

A single shoot developed per vitrified shoot tip using thestandard reculturing method (see Materials and Methods).However, if the vitrified shoot tips were cultured con-tinuously on semi-solid MS medium containing 0±2

sucrose, 5±8 µ GA$

and 1±0 µ BA under 16 h diffuselight for the first week, and subsequently under a 16 hphotoperiod of 40 µmol m−# s−" light intensity, multipleshoots developed. After 6 weeks of reculture on this high-

460 Sarkar and Naik—Cryopreser�ation of Potato Shoot Tips by Vitrification

F. 4. Regeneration of multiple shoots from cryopreserved potato shoot tips in ‘Kufri Badshah’. A, Multiple shoots developed from vitrifiedshoot tips 6 weeks after plating on semi-solid MS medium containing 0±2 sucrose, 5±8 µ GA

$and 1±0 µ BA; B, 2-week-old plantlets

recovered from multiple shoot cuttings growing on semi-solid MS medium supplemented with 0±09 sucrose, 0±29 µ GA$and 0±054 µ NAA.

sucrose (0±2 ) medium, about 30–50 shootlets developedfrom each vitrified shoot tip (Fig. 4A). The shootlets weremultiplied, and they resumed growth to develop intoplantlets under standard culture conditions (Sarkar et al.,1997) and were morphologically normal (Fig. 4B).

DISCUSSION

The present study showed that pre-conditioning of excisedshoot tips was essential for successful cryopreservation ofpotato shoot tips by vitrification. It is well established thatsucrose is an important pre-growth additive for theacquisition of desiccation tolerance during cryopreservationof shoot apices by vitrification. A high level of sucroseduring pre-culture was reported to be essential in achievinghigh survival rates of cryopreserved shoot tips in a numberof studies (Dumet et al., 1993; Gonzalez-Arnao, Moreiraand Urra, 1996). However, in the current study, we observedthat sucrose in combination with mannitol was more potentthan sucrose alone for attaining effective pre-conditioningof potato shoot tips for vitrification. Mannitol was alsoreported to be most effective for reducing the water contentand increasing the freeze resistance of sycamore andcapsicum suspension cells during cryopreservation byconventional approaches (Withers and Street, 1977; Pritch-ard, Grout and Short, 1986a). The effects of this type of pre-growth have not been investigated in detail, but it seemsprobable that they include solute accumulation, reductionof isotonic water content, reduction in vacuolar volumes(Pritchard, Grout and Short, 1986b ; Reed, 1989) andchanges in membrane structure (Webb and Steponkus,1990). In addition, we observed that sequential loading ofpre-cultured shoot tips with 20 and 60% PVS 2 solutionsbefore vitrification with concentrated PVS 2 greatly im-proved the survival of vitrified potato shoot tips. Thisbeneficial effect may be due to the presence of DMSO, ahighly penetrating cryoprotectant, in the loading solutions.

Direct exposure of pre-cultured shoot tips to concentratedvitrification solution was detrimental to the viability ofvitrified shoot tips. This harmful effect may be due toexcessive osmotic stress or chemical toxicity exerted byconcentratedPVS 2 (Matsumoto, Sakai andYamada, 1994).It is particularly important that cryopreserved shoot tips arecapable of producing shoots without intermediary callusformation. The optimum pre-culturing and loading con-ditions developed in the current study induced direct shootformation from vitrified potato shoot tips. Post-thawculturing of vitrified shoot tips under diffuse light and onmedium containing an elevated level of sucrose (0±2 ) forthe first week was found to be highly beneficial to shoot tipsurvival and direct shoot formation. It is well documentedthat cryopreserved shoot tips of potato require a lowlight level during the initial post-thaw culture phase(Henshaw et al., 1985). The beneficial effect of post-thawculturing of vitrified shoot tips on a high-sucrose mediumreported in this study might relate to some sort of favourableosmotic adjustment that allows the cryopreserved shoot tipsto recover from the trauma of cryo-shock. Continuousculturing of vitrified shoot tips on this high-sucrose mediuminduced multiple shoots. This is highly advantageous formultiplying clones that show low survival after cryo-preservation. However, there are potential risks of geneticor somaclonal variation in plants regenerated throughadventitious shoot development (Benson et al., 1996). Thus,this mode of multiplication should only be used withcaution until evidence is obtained of acceptable levels oftrueness-to-type among the plantlets regenerated via mul-tiple shoot formation.

ACKNOWLEDGEMENTS

We thank Dr G. S. Shekhawat, Director, Central PotatoResearch Institute and Dr P. C. Gaur, Head, Division ofGenetics and Plant Breeding for providing necessary

Sarkar and Naik—Cryopreser�ation of Potato Shoot Tips by Vitrification 461

facilities. Photographic assistance from Mr S. K. De ismuch appreciated. We also thank Professor Akira Sakai forvaluable comments and suggestions during his visit to ourlaboratory. Comments and suggestions on the manuscriptfrom the anonymous reviewers are gratefully acknowledged.

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