Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry

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<ul><li><p>Plant Science, 87 (1992) 199-206 199 Elsevier Scientific Publishers Ireland Ltd. </p><p>Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry </p><p>Takao Ni ino a and Ak i ra Sakai b </p><p>aNationai Institute of Agrobioiogical Resouces, Shinjo, Yamagata 996 and bAsabucho 1-5-23, Kitaku, Sapporo 001 (Japan) </p><p>(Received June 10th, 1992; revision received August 26th, 1992; accepted August 28th, 1992) </p><p>Alginate-coated shoot tips from in vitro-grown apple (Malus domestica Borkh cv. Fuji) were successfully cryopreserved following dehydration. Shoot tips cold-hardened at 50C for 3 weeks, were progressively precultured on MS agar media with increasing sucrose (0.1, 0.4 and 0.7 M) daily at 50C. The precultured shoot tips trapped into alginate-coated beads containing 0.5 M sucrose were treated in a medium supplommated with 1.0 M sucrose for 16 h at 5C. Beads containing 1 shoot tip were then dehydrated up to about 33% water content (fr~h weight basis) on sterile dry silica gel at 250C before being immersed in liquid nitrogen (LN). The average rate of shoot formation after warming was about 80%. This method was successfully applied to three apple, one mulberry and three pear species or eultivars. This encapsulation-dehydration method also permitted the shoot tips to be stored at -135C for 5 months with little or no decrease in the rate of shoot formation. This modified method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of deciduous trees. </p><p>Key words: cryopreservation; shoot tips; apple; alginate coated beads; encapsulation-dehydration technique; pear and mulberry </p><p>Introduction </p><p>The development of a simple and reliable method for cryopreservation would allow much more widespread use of cryopreserved cultured cells, meristems and somatic embryos. Recently, vitrification [1-3], simplified freezing [4] and air drying methods [5,6] have been presented to simplify the complicated procedures for cryo- preservation. Potentially valuable cryogenic pro- cedures would be vitrification and drying methods for meristems and somatic embryos. In air drying methods, the induction or modification of dehydration tolerance may be the key to successful cryopreservation [5-91. </p><p>Alglnate-eoated shoot tips of in vitro-grown pear, potato and grape were successfully cryopreserved following air drying [6,10,11]. The encapsulation-dehydration technique is easy to handle and simplifies the dehydration process. In this method, resistance to dehydration and deep </p><p>Correspondence to: Niino Takao, National Institute of Agrobiological Resources, Shinjo, Yamagata 996, Japan. Abbreviations: BA, 6-benzylaminopurine; LN, liquid nitrogen. </p><p>freezing was induced by preculturing encapsulated shoot tips in medium enriched with sucrose before dehydration. Preculture in sucrose solutions may eliminate the use of other cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol [5,10,11]. </p><p>In our previous paper [12], the alglnate-coated shoot tips from winter hardy mulberry buds were treated in a medium supplemented with 1.0 M sucrose for 16 h at 5C and then dehydrated up to about 22-25% prior to a plunge into liquid nitrogen (LN). Approximately 70% shoot forma- tion after warming was achieved. However, in the meristems from in vitro-grown apple plants, the encapusulated-dehydration technique [6] produc- ed only about 30% shoot formation. </p><p>To increase the rate of development and to eliminate the use of LN for long-term storage, the present study was performed using apple, pear and mulberry shoot tips from in vitro plantlets. </p><p>Materials anti Methods </p><p>Materials Tissue-cultured apple plants Malus domestica </p><p>0168-9452/92/$05.00 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland </p></li><li><p>200 </p><p>Borkh. cv. Fuji were mainly used in the present study. Stock cultures of three other apple cultivars cv. Golden Delicious (M. domestica Borkh.); the apple rootstocks M. 9 and M. 26 (M. paradisiaca Shneid.) were also used. Three pear cultivars, cv. Beurr6 d'Amanlis and cv. winter Nelis of Pyrus communis L. and cv. Senryo of Pyrus pyrifolia Burm. were tested for cryopreservation as well as one mulberry cultivar, Kenmochi, of Morus bombysis. </p><p>Stock cultures of apple plants [13] were main- tained on Murashige and Skoog (MS) medium [141 containing 1.0 mg 1-1 BA, 2.5% (w/v) sucrose and 0.7% (w/v) Bacto-agar (Difco, Detroit, USA). The medium was adjusted to pH 5.6 prior to autoclav- ing at 121C for 15 rain. The stock cultures were subcultured every 40 days. The stock cultures of pear plants [15] were maintained on MS medium containing 2.5 mg 1-1 BA, 2.5% sucrose and 0.8% agar (Wako Chemical Co., Japan) at pH 5.6 [161. The stock cultures of mulberry [17] were maintain- ed on MS medium containing 1.0 mg 1 -I BA, 2.5% sucrose and 0.8% agar (Wako Chemical Co., Japan) at pH 5.6. </p><p>These tissue-cultured plantlets of apple, pear and mulberry were all grown on 25 ml of medium in 100-ml glass flasks under white fluorescent light (52 t~mol s -I m -2) during a 16-h/day photoperiod, at 25C. </p><p>Cold-hardening and preculture Three-week-old plantlets were cold-hardened at </p><p>5C for 3 weeks under an 8-h/day photoperiod. Shoot tips (about 2 mm long) were dissected from cold-hardened plantlets. Progressive preculture was performed at 5C by successive daily transfer of the shoot tips onto MS media supplemented with 0.1, 0.4or 0.7 M sucrose under an 8-h/day photoperiod. Direct preculture was also perform- ed by transferring shoot tips to MS medium sup- plemented with sucrose at various concentrations (0.1, 0.4 or 0.7 M) for a total of 1 or 2 days before further treatment. </p><p>Alginate beads Alginate gel beads containing shoot tips were </p><p>prepared according to the technique of Bapat et al. [18] with some alterations. Shoot tips were suspended in calcium-free culture medium sup- </p><p>plemented with 3% (w/v) Na-alginate solution and 0.5 M sucrose. The mixture was dispensed from a dispenser with a sterile tip into 50 ml of culture medium containing 100 mM calcium chloride and 0.5 M sucrose and held for 30 min at 25C. Beads of about 5 mm in diameter containing one shoot tip were treated in MS medium supplemented with 1 M sucrose for 16 h at 5C. </p><p>Dehydration After treatment with 1.0 M sucrose, the surface </p><p>solution was wiped off and the beads were sub- jected to dehydration in Petri dishes (9 cm in diameter) containing 50 g dry silica gel held at 25C for up to 24 h. After dehydration, about 10 dried beads were placed in a 2-ml cryotube and then transferred onto agar MS medium without or after cooling to -196C. Two cooling procedures were used: rapid cooling, by direct immersion of the cryotube in LN (cooling rate: about 190C/min) and two-step cooling by prefreezing at -20 or -30C for 24 h prior to a plunge into LN. </p><p>To test the feasibility of storing dehydrated, en- capsulated shoot tips at temperatures higher than that of LN, dried beads were directly transferred to -70 or -135C (using a deep-freezer, Ultra Low -135C, Sanyo Co. Japan) without prior cooling to -196C. Other beads were immersed in LN and stored there or subsequently transferred to -70 or -135C. </p><p>Viability and regrowth Samples were warmed by placing the cryotubes </p><p>in a water bath at 25C (warming rate: 140C/min). Regrowth of alginate-coated shoot tips was achieved by transfer onto the appropriate MS medium under standard culture conditions described above. The rate of shoot formation was defined as the percent of shoot tips that produced normal shoots about 40 days after plating. Water content was expressed on a fresh weight basis. Dry matter was determined after drying for 48 h at 100C. The shoot tips were tested in each of 2-4 replicates for each experiment. </p><p>Results </p><p>To increase dehydration tolerance, cold- hardened shoot tips were precultured by direct </p></li><li><p>transfer to MS agar medium containing 0.1, 0.4 or 0.7 M sucrose, or successive transfer daily to media containing 0.1, 0.4 and 0.7 M sucrose. As shown in Table I, preculture using increasing sucrose daily produced a higher level of shoot for- marion amount ing to about 80% after cooling to - 196C. </p><p>The effect of treatment with 1.0 M sucrose solu- tion before dehydration was tested with shoot tips trapped into beads with or without 0.5 M sucrose. As shown in Table II, the experiment proved that sucrose at relatively high concentrations is necessary to produce high rates of shoot formation after cooling in LN following dehydration. </p><p>Cold-hardened and precultured shoot rips of apple were subjected to dehydration without being trapped into alginate beads. The rates of shoot for- mation of dried apple shoot tips before and after exposure to LN at various water contents are shown in Fig. 1. Shoot formation increased with decreasing water content from 45 to 22%. Subse- quently, shoot formation rapidly decreased due to </p><p>201 </p><p>Table H. Effect of treatment with 1.0 M sucrose solution before dehydration on shoot formation of in vitro-grown apple shoot tips enclosed in alginate gel beads with or without 0.5 M sucrose cooled to -196C after dehydration. Material: M. domestica, cv. Fuji. Cold-hardened: 3 weeks at 5C. Progressive precuhuring: on MS agar media with increasing sucrose con- centrations (0.1 M, 0.4 M and 0.7 M) daily. Cold-hardened, precultured shoot tips were encapsulated in alginate-gel beads with or without 0.5 M sucrose and then treated with 1.0 M sucrose at 5C for 16 h or not, before dehydration. The alginate-coated shoot tips were subjected to dehydration for 7 h (water content: about 33%) at 25C and then immersed in LN. Shoot formation (%): percent of shoot tips that produced normal shoots 40 days after plating. Approximately 10 shoot tips were treated for each of 3 replicates. </p><p>Shoot formation (% 4. S.E.) </p><p>No-treatment (%) </p><p>Sucrose treatment (%) </p><p>Aiginate beads 7 4. 4 64 4. 5 (without sucrose) </p><p>Alginate beads 47 4. 4 73 4. 7 including 0.5 M sucrose </p><p>Table I. Effect of preculturing conditions on shoot formation of alginate-coated in vitro-grown apple shoot tips cooled to -196C after dehydration. Material: M. domestica cv. Fuji. Cold-hardened: 3 weeks at 5C. Cold-hardened, precultured shoot tips were encapsulated in alginate-gel beads including 0.5 M sucrose and then treated in MS medium supplemented with 1.0 M sucrose at 5C for 16 h prior to dehydration. The alginate-coated shoot tips were subjected to dehydration for 7 h (water content: about 33% fresh weight basis) at 25C prior to a plunge in LN. Shoot formation (%): percent of shoot tips that produced normal shoots 40 days after plating. Approx- imately 10 shoot tips were treated for each of 3 replicates. </p><p>Preculturing conditions: Shoot duration, sucrose formation concentration (M) (% 4. S.E.) </p><p>Non-precultured 40 10 0.1 M, 1 day 37 9 0.4 M, 1 day 33 3 04M, 2days 43 4. 3 0.7 M, 1 day 53 4. 9 0.7 M, 2 days 67 4. 3 0.1 M, 1 day &amp; 0.4 M, 1 day 404- 10 0.4 M, 1 day &amp; 0.7 M, 1 day 77 4. 3 0.1 M, 1 day, 0.4 M, 1 day &amp; 0.7 M, 1 day 80 4- 0 Non-hardened and 0.1 M, 1 day, 0.4 M, 0 4. 0 </p><p>1 day, and 0.7 M, 1 day </p><p>100 </p><p>z o I- &lt; 50 ! </p><p>0 </p><p>t- AFTER EXPOSURE ~r I </p><p>0 e 70 60 50 40 30 20 </p><p>WATER CONTENT ( ~ FW ) </p><p>Fig. 1. Shoot formation of in vitro-grown apple shoot tips before and after exposure to LN at various water contents. MaRtial: M. domestica cv. Fuji. Cold hardening: 3 weeks at 5C; Preculturing: at 5C on agar MS medium supplemented with 0.7 M sucrose for 1 day. Dehydrated shoot tips were plac- ed in a cryotube and then transferred on agar MS medium without (-LN) or after (LN) cooling to -196C. After 1 day, the cryotube was rapidly transferred into a water bath at 25C. Shoot formation (%): percent of shoot tips that produced nor- mal shoots 30 days after plating. Approximately 20 shoot tips were treated for each of 2 replicates. </p></li><li><p>202 </p><p>desiccation injury (Fig. 1). Alginate-coated shoot tips cooled to -196C following dehydration pro- duced a higher rate of shoot formation (70%) than air dried shoot tips. The maximum rate of shoot formation was obtained at 33% water content which was about 10% higher than that of air dried ones (Fig. 2). </p><p>The alginate-coated shoot tips of 37% water content yielded a very low rate of shoot formation (15%) after direct immersion in LN, while prefreezing at -20 or -30C following dehydra- tion produced about 70% shoot formation (Table III). However, the alginate coated shoot tips of 33% water content which were prefrozen at -20 or -30C for 1 day showed only a slight increase but were at a much higher level. </p><p>The modified encapsulation-dehydration tech- nique was successfully applied to shoot tips from </p><p>100 </p><p>so </p><p>2 </p><p>o </p><p>0 80 </p><p>o o </p><p>DEHYDRATION ~ / </p><p>70 60 50 40 30 20 WATER CONTENT (% FW) </p><p>Fig. 2. Shoot formation of dehydrated alginate-coated in vitro-grown apple shoot tips before or after being cooled to -196C. Material: Malus domestica cv. Fuji. Cold hardening: 3 weeks at 5C; Preculturing: at 5C on agar MS medium sup- plemented with 0.7 M sucrose for 1 day. Cold-hardened, preeultured shoot tips in alginate-gel beads including 0.5 M sucrose were treated in MS medium containing 1.0 M sucrose at 5C for 16 h. Dehydrated beads were placed in a cryotube and transferred onto agar MS medium without ( -LN) or after (LN) cooling to -196C. After 1 day, the cryotube was rapidly transferred into a water bath at 25C. Shoot formation (%): percent of shoot tips that produced normal shoots 40 days after plating. Approximately 20 shoot tips were treated for each of 2 replicates. </p><p>Table III. Effect of prefreezing at -20 or -30C for 16 h after partial dehydration on shoot formation of alginate-coated in vitro-grown apple shoot tips cooled to -196C following dehydration. Material: Malus domestica, cv. Fuji. Cold- hardened: 3 weeks at 5C. Progressive precultureing: on MS agar media with increasing sucrose concentrations (0.1 M, 0.4 M and 0.7 M) daily. Cold-hardened, precultured shoot tips were encapsulated into alginate-gel beads including 0.5 M sucrose and then treated with 1.0 M sucrose at 5C for 16 h. These beads were subjected to dehydration for 6 or 7 h (water content; about 37 or 33%) at 25C prior to a plunge into LN. Shoot formation (%): percent of shoot tips that produced nor- mal shoots 40 days after plating. Approximately 10 shoot tips were treated for each of 4 replicates. </p><p>Temperature of Water content Shoot formation pre-freezing (C) of beads (%) (% S.E.) </p><p>-20 37 61 4 -30 37 72 + 6 Non-freezing a 37 15 5 -20 33 77 4 -30 33 83 4 Non-freezing a 33 73 4 </p><p>aCooled to -196C directly from room temperature. </p><p>Table IV. Shoot formation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry cooled to -196C after dehydration. Cold hardening: 3 weeks at 5C ; Progressive preculturing: on MS agar media with increasing sucrose con- centrations (0.1 M, 0.4 M and 0.7 M) daily. Cold-hardened, precultured shoot tips were encapsulated into alginate-gel beads including 0.5 M sucrose and then treated in MS medium supplemented with 1.0 M sucrose...</p></li></ul>

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