Cryopreservation of sweetpotato (Ipomoea batatas) and its pathogen eradication by cryotherapy

Biotechnology Advances 29 (2011) 84–93

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Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Cryopreservation of sweetpotato (Ipomoea batatas) and its pathogen eradication by cryotherapy Chaohong Feng 1, Zhenfang Yin 1, Yanli Ma, Zhibo Zhang, Long Chen, Biao Wang, Baiquan Li, Yushen Huang, Qiaochun Wang ⁎ Plant Cryobiology Laboratory, College of Horticulture, Agricultural & Forest University, Yangling 712100, Shaanxi, PR China

a r t i c l e

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Article history: Received 19 June 2010 Accepted 2 September 2010 Available online 16 September 2010 Keywords: Cryopreservation Cryotherapy Embryogenesis Ipomoea batatas Pathogen eradication Shoot regeneration Shoot tips Sweetpotato Phytoplasma Virus

a b s t r a c t Sweetpotato (Ipomoea batatas) ranks as the seventh most important staple crop in the world and the fifth in developing countries after rice, wheat, maize and cassava. Sweetpotato is mainly grown in developing countries, which account for more than 95% of total production of the whole world. Genetic resources, including cultivated varieties and wild species, are a prerequisite for novel sweetpotato breeding in both conventional and genetic engineering programs. Various cryopreservation protocols have been developed for shoot tips and embryogenic tissues. The former explants are preferred for long-term conservation of sweetpotato genetic resources, while the latter are valuable for sweetpotato genetic improvement. This review provides update comprehensive information on cryopreservation of sweetpotato shoot tips and embryogenic tissues. Plant pathogens such as viruses and phytoplasma severely hamper high yield and high quality production of sweetpotato. Thus, usage of pathogen-free planting materials is pivotal for sustainable sweetpotato production. Cryotherapy of shoot tips can efficiently eradicate sweetpotato pathogens such as viruses and phytoplasma. The mechanism behind pathogen eradication by cryotherapy of shoot tips has been elucidated. Pathogen eradication by cryotherapy provides an alternative, efficient strategy for production of pathogenfree plants. In addition, cryopreserved tissues may also be considered to be safer for exchange of germplasm between countries and regions. © 2010 Elsevier Inc. All rights reserved.

Contents 1. 2.

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Significance of sweetpotato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryopreservation of sweetpotato shoot tips . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Needs for long-term conservation of sweetpotato genetic resources . . . . . . . . . . . . . 2.2. Cryogenic techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Droplet-vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Encapsulation–dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Encapsulation–vitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryopreservation of sweetpotato embryogenic tissues. . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of somatic embryogenesis in sweetpotato biotechnology . . . . . . . . . . . . . . . 3.2. Problems involved in establishment and maintenance of somatic embryogenic cultures . . . . . 3.3. Cryogenic techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Encapsulation–dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Pregrowth–desiccation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Pregrowth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryotherapy of shoot tips for pathogen eradication . . . . . . . . . . . . . . . . . . . . . . . 4.1. Yield loss caused by pathogens to sweetpotato and needs for production of pathogen-free plants 4.2. Cryotherapy techniques for sweetpotato pathogen eradication . . . . . . . . . . . . . . . 4.2.1. Virus eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. E-mail address: [email protected] (Q. Wang). 1 These two authors contributed equally to the present study. 0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2010.09.002

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4.2.2. Phytoplasma eradication . . . . . . . . . . 4.3. Mechanism for pathogen eradication by cryotherapy of 5. Conclusion and prospects for further studies . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Significance of sweetpotato Sweetpotato (Ipomoea batatas (L.) Lam) ranks as the seventh most important staple crop in the whole world and the fifth in developing countries after rice, wheat, maize and cassava (Loebenstein, 2009). The annual total growing area and yield were estimated at 9 million ha and 140 million tons worldwide, respectively. About 80% of sweetpotato is grown in Asia, 15% in Africa and only 5% in other countries (Loebenstein, 2009). China is the largest sweetpotato producing country in the world. At present, the total growing area and yield reach about 6.6 million ha and 106 million tons (Zhang et al., 2009), accounting for about 73% and 75% of the total area and yield of the world, respectively. Sweetpotato is widely adapted to various types of climate and soil, and produces tubers, even without fertilizers and with poor irrigation (Loebenstein, 2009; Zhang et al., 2009). Biomass yield and nutrient contents, such as vitamins A and C, iron, potassium, and fiber, of sweetpotato are the highest among the food crops in the world (Loebenstein, 2009). Sweetpotato serves as food, vegetables, feed and industrial materials including starch, organic acids and ethanol (Zhang et al., 2009). In Africa and Asia, thousands of people are still depending on sweetpotato for food security (Loebenstein, 2009; Zhang et al., 2009). 2. Cryopreservation of sweetpotato shoot tips 2.1. Needs for long-term conservation of sweetpotato genetic resources Sweetpotato genetic resources, including cultivated varieties and wild species, are a prerequisite for novel cultivar breeding in both conventional and genetic engineering programs. Although both in situ and in vitro preservation methods have been introduced for sweetpotato germplasm (Gaba and Singer, 2009), cryopreservation, i.e. storage of living materials at an ultra-low temperature, usually that of liquid nitrogen (LN, −196 °C), has been considered an ideal means for longterm conservation of plant germplasm (Engelmann, 1997; Wang and Perl, 2006; Benson, 2008; Reed, 2008). Under cryostorage conditions, all cellular divisions and metabolic processes cease. Theoretically, plant materials can thus be stored without any change for an indefinite period of time (Engelmann, 1997; Wang and Perl, 2006; Benson, 2008). Moreover, such storage requires a small volume, which largely avoids contamination and demands very limited maintenance. To date, cryopreservation techniques have been successfully applied to a large number of plant species including woody and herbaceous plant species ranging from temperate to tropical regions, using shoot tips, cell cultures, embryos and seeds (Engelmann, 1997; Engelmann and Takagi, 2000; Towill and Bajaj, 2002; Wang and Perl, 2006; Reed, 2008). Shoot tips are preferred for long-term conversation of genetic resources, because they are genetically more stable than cells and callus cultures (Bajaj, 1991; Engelmann, 1997; Wang and Perl, 2006). 2.2. Cryogenic techniques 2.2.1. Vitrification Vitrification refers to a physical transition process in cells from an aqueous solution to an amorphous or glassy solid without crystallization at an applied cooling rate (Fahy et al., 1984; Sakai et al., 2008). Therefore, vitrification is an effective freeze-avoidance mechanism in both intra-

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and extra-cellular tissues. In order for them to be able to vitrify upon rapid cooling in LN, cells have to be sufficiently dehydrated with the vitrification solution without chemical damage and excessive dehydration. Thus, the limiting factor for successful cryopreservation by this technique is to ensure acquisition of the samples' tolerance high enough to the vitrification solution (Sakai and Engelmann, 2007; Sakai et al., 2008). The technique employs a highly concentrated solution to treat the explants for some periods of time ranging from 15 min to 2 h, depending on plant species, prior to a direct immersion in LN (Panis and Lambardi, 2006; Sakai and Engelmann, 2007; Benson, 2008; Sakai et al., 2008). The detailed steps involved in the vitrification procedure can be found in several recently published articles (Wang and Perl, 2006; Sakai and Engelmann, 2007; Sakai et al., 2008). Schnabel-Preikstas et al. (1992) were the first who introduced vitrification for cryopreservation of sweetpotato. Shoot tips (1 mm in size) containing the apical dome (AD) with 1–2 leaf primordia (LP) were precultured with 0.4 M sucrose, followed by dehydration with Steponkus's vitrification solution (Steponkus et al., 1992), prior to direct immersion in LN. Steponkus's vitrification solution contains 50% (w/v) ethylene glycol (EG), 15% (w/v) sorbitol and 6% (w/v) bovine serum albumin (BSA) in Murashige and Skoog medium (MS, Murashige and Skoog, 1962). Following thawing in MS medium containing 1.5 M sorbitol, cryopreserved shoot tips were post-cultured on solid MS medium containing IAA and kinetin for recovery. With this protocol, although 71% of the shoot tips survived in LN, most of the surviving shoot tips formed callus without shoot regrowth, and only 23% of the surviving shoot tips regenerated shoots. Nevertheless, this protocol set up a basic vitrification line for cryopreservation of sweetpotato shoot tips. Based on these results, Schnabel-Preikstas et al. (1992) suggested that further studies should be conducted on improvement of shoot regeneration of cryopreserved shoot tips with special concentration on post-culture medium. By modifying the postculture medium, Plessis and Steponkus (1996) obtained 76% and 47% of survival and shoot regrowth, respectively, when cryopreserved shoot tips were post-cultured on MS medium supplemented with kinetin and GA3 for 2 weeks and then transferred to a hormone-free medium for shoot regrowth. However, shoot regrowth was still excessively slow. Using a modified post-culture medium composed of MS containing 2 mg/l calcium panthotenate, 100 mg/l arginine, 200 mg/l ascorbic acid, 20 mg/l putrescine, 20 mg/l GA3, 5 ml/ l coconut milk and 0.09 M sucrose, Golmirzaie et al. (2000) obtained high shoot regrowth. Results previously reported indicate that high survival rates can be obtained in shoot tips cryopreserved by vitrification. However, shoot regrowth is a key factor limiting success of this technique. The vitrification method is time-saving, usually gives highly reproducible results and has been widely applied to a wide range of plant species ranging from temperate and tropical regions (Panis and Lambardi, 2006; Wang and Perl, 2006; Sakai and Engelmann, 2007; Sakai et al., 2008; Benson, 2008). However, two main disadvantages are involved in this technique: manipulation of small, non-encapsulated shoot tips is troublesome; dehydration with vitrification solution requires very precise and short time period, which is hard to control. These two weak points make this technique difficult to simultaneously treat a large number of samples. Up to date, only a few studies have been published on successful cryopreservation of sweetpotato shoot tips by this technique (Table 1).

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Table 1 A list of studies on cryopreservation of sweetpotato shoot tips. Cryogenic procedure

Cultivar or genotype

Size of shoot tips used

S (%)a

SR (%)b

Ref.

Vitrification

Georgia Red Georgia Red PI 508515 PI 290657 PI 290657 PI 290657 PI 573324 PI 508515 W-235 Jewel Maria Angola Jonathan Morada Inta Chugoku TIB 10 PI 290657 Beni-azuma Chikou-1gou Kogane-sengan 199004.2 Lushu 8 Lizixiang Xushu 18 Jishu 15 Beijing 553

1 mm with 1–2 LP NSc 0.5–0.7 mm with 3–4 LP 0.5–0.7 mm with 3–4 LP 0.5–1.0 mm with 2–3 LP 0.5–1.0 mm with 2–3 LP 0.5–1.0 mm with 2–3 LP 0.5–1.0 mm with 2–3 LP 0.5–1.0 mm with 2–3 LP 0.5–0.7 mm 0.5–0.7 mm 0.5–0.7 mm 0.5–0.7 mm 0.5–0.7 mm 0.5–0.7 mm 0.5–1.0 mm with 2–3 LP 1.0 mm with 3–4 LP 1.0 mm with 3–4 LP 1.0 mm with 3–4 LP 1.0–1.5 mm with 3–4 LP 1.0–1.5 mm with 3–4 LP 1.0–1.5 mm with 3–4 LP 1.0–1.5 mm with 3–4 LP 1.0–1.5 mm with 3–4 LP 1.0–1.5 mm with 3–4 LP

71 76 91 NDd 66 ND ND ND ND ND ND ND ND 0 0 ND ND ND ND 88 85 68 60 58 43

23 47 64 26 66 93 83 62 63 26 24 17 8 – – 67 82 94 81 85 80 90 75 62 68

Schnabel-Preikstas et al. (1992) Plessis and Steponkus (1996) Towill and Jarret (1992)

Droplet-vitrification

Encapsulation–dehydration Encapsulation–vitrification

a b c d

Pennycooke and Towill (2000) Pennycooke and Towill (2001)

Golmirzaie et al. (2000)

Pennycooke and Towill (2001) Hirai and Sakai (2003)

Wang and Valkonen (2008a,b) Q.C. Wang et al. (unpublished)

S = survival. SR = shoot regrowth. NS = not specified. ND = not determined.

2.2.2. Droplet-vitrification Droplet-vitrification is based on the droplet procedure described by Kartha et al. (1982) for cryopreservation of shoot tips of cassava (Manihot esculenta). Basically, in this procedure, shoot tips are precultured with an osmotic agent, loaded with loading solution, dehydrated with plant vitrification solution 2 (PVS2, Sakai et al., 1990, see the composition of PVS2 in the following section) and placed individually into 4–15 μl droplets of PVS2 made on a piece of aluminum foil, which is then directly immersed in LN. Aluminum foils with cryopreserved shoot tips are thawed directly and unloaded in liquid medium enriched with sucrose. Following that, shoot tips are cultured on a post-culture medium for survival and regeneration (Panis and Lambardi, 2006; Sakai and Engelmann, 2007; Benson, 2008; Sakai et al., 2008). Successful cryopreservation of sweetpotato shoot tips by dropletvitrification was first reported by Towill and Jarret (1992). Shoot tips (0.5–0.7 mm long) with 3 to 4 LP were excised from axillary buds of in vitro 8- to 12-week old stock plants and subjected to cryopreservation. Ninety-one percent and 26% of survivals of cryopreserved shoot tips were obtained for genotype PI 508515 and PI 290657, respectively. However, results were poorly reproducible, with shoot regrowth varying widely among replicates (0–38%) and experiments (38–64%). In addition, survived shoot tips formed callus first and then started to develop shoots from the central portion of the callus and not all survived shoot tips were able to develop shoots from the callus, resulting in significantly lower shoot regeneration. Based on the previously discussed protocol proposed by Towill and Jarret (1992), Pennycooke and Towill (2000) developed a modified droplet-vitrification procedure. Apical shoot tips (0.5–1.0 mm in length) with 2–3 LP were excised from 4- to 8-week old in vitro stock plants (genotype PI 290657) immediately after the 8-h dark period. Shoot tips precultured with 0.3 M sucrose were loaded with a loading solution consisting of 2 M glycerol and 0.4 M sucrose in MS medium, and then dehydrated by exposure to PVS2 at 22 °C. PVS2 contains 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulfoxide (DMSO) and 0.4 M sucrose in MS medium (Sakai et al., 1990). Following dehydration, droplet was made by placing

dehydrated single shoot tips in 10 μl of PVS2 on thin strips (40 × 2 mm) of sterile aluminum foil, prior to a direct immersion into nitrogen slush (–208 °C) for 15–30 min and then in LN. Thawing was conducted in liquid MS medium containing 1.2 M sucrose for 20 min at 22 °C, and then the shoot tips were post-cultured on a recovery MS medium containing 0.2 mg/l NAA, 1.1 mg/l BA and 0.2 mg/l mg kinetin for shoot regeneration. With optimized parameters, about 66% of shoot tips survived after cryopreservation, all surviving shoot tips developed shoots with only minimum callus formation, and the results were reproducible. Pennycooke and Towill (2001) further found that shoot regrowth following droplet-vitrification (Pennycooke and Towill, 2000) could be largely improved by placing the cryopreserved, thawed shoot tips on NH+ 4 -free medium for the initial 5 days of post-culture and then transferred to NH+ 4 containing medium for regrowth. Using this post-culture medium, shoot regrowth of cryopreserved shoot tips was 3 times of those postcultured directly on NH+ 4 -containing MS medium. Furthermore, shoots developed directly from cryopreserved shoot tips without callus formation. Shoot regrowth rates of all genotypes tested were high: 93% for PI 290657, 83% for PI 573324, 63% for W-235 and 62% for PI 508515. Post-culture of cryopreserved sweetpotato shoot tips on NH+ 4 -free medium for the initial days to increase recovery has been repeatedly reported with other cryogenic procedures for sweetpotato such as encapsulation–dehydration (Pennycooke and Towill, 2001) and encapsulation–vitrification (Wang and Valkonen, 2008b), as will be addressed in the following sections. Improved survival of cryopreserved shoot tips or cells post-cultured on NH+ 4 -free medium has also been observed in many other plant species such as Oryza sativa (Kuriyama et al., 1989), Lavandula vera (Kuriyama et al., 1996), Betula pendula (Ryynänen and Häggman, 1999, 2001) and Holostemma annulare (Decruse and Seeni, 2002; Decruse et al., 2004). These data indicated that NH+ 4 is toxic to frozen cells and survival of cryopreserved shoot tips or cells can be largely improved by exclusion of NH+ 4 from the post-culture medium in the first few days of recovery. The major advantage of droplet-vitrification lies in that it can establish very high cooling-warming rates by usage of minimum volume

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of droplets made on aluminum foils, a high conducting material (Sakai and Engelmann, 2007; Benson, 2008). The droplet-vitrification technique is characteristic of high reproducibility and wide applications to different tissues and plant species, thus being at the present time one of the most widely applied cryopreservation methods (Sakai and Engelmann, 2007; Benson, 2008). Up to date, the vitrification-droplet technique has been routinely employed for long-term preservation of potato genetic resources (446 accessions) at the International Potato Center (CIP) in Peru (Panta et al., 2006) and banana germplasm (306 accessions) at Katholieke University, Leuven, Belgium (Panis et al., 2005). For sweetpotato, this technique has been applied to cryopreservation of at least 10 genotypes (Table 1), and therefore, can be considered a most promising method for long-term storage of sweetpotato germplasm. Yet, extra studies are still needed to test its applications to a wide range of sweetpotato genotypes. 2.2.3. Encapsulation–dehydration This procedure is also a vitrification-based method, using the technology developed for production of synthetic seeds. With the high desiccation of explants, most or all freezable water is removed from cells, and vitrification of internal solutes occurs upon rapid freezing in LN, thus avoiding lethal intracellular ice crystallization (Engelmann, 1997; Engelmann et al., 2008). The procedure requires encapsulation of shoot tips in 2–3% sodium alginate solution, preculture of encapsulated shoot tips in sugar-rich medium to increase tolerance of the shoot tips to dehydration and subsequent freezing, followed by evaporative desiccation by either air-drying or with silica gel. After then, dehydrated shoot tips are directly plunged into LN. Cryopreserved shoot tips are thawed by either at ambient temperatures (slow thawing) or at 35–38 °C (fast thawing), followed by placing on post-culture medium for survival and shoot regeneration (Wang and Perl, 2006; Panis and Lambardi, 2006; Benson, 2008; Engelmann et al., 2008). Pennycooke and Towill (2001) reported successful cryopreservation of sweetpotato shoot tips by encapsulation–dehydration, according to Dereuddre et al. (1990). Shoot tips (0.5–1.0 mm) with 2–3 LP excised from apical buds of 4–8 weeks old in vitro stock cultures (genotype PI 290657) were encapsulated into beads (4–5 mm in diameter), as described by Dereuddre et al. (1990). The beads were stepwise precultured in liquid MS medium containing increasing sucrose concentrations of 0.25 M, 0.5 M and 0.75 M, with one day for each concentration. Following dehydration by air drying in a laminar flow to reduce water content of the beads to about 18.1%, beads were plunged directly into nitrogen slush (–208 °C) for 15–30 min, and then transferred to LN. Beads containing cryopreserved shoot tips were rapidly thawed in liquid MS containing 0.06 M sucrose at 30 °C for about 5 s, and then, were post-cultured on NH+ 4 -free solid MS medium containing 0.2 mg/l NAA, 0.1 mg/l BA, 0.2 mg/l kinetin and 0.09 M sucrose. Following 2 days of incubation in the dark, the shoot tips were extracted from the beads and cultured on the same fresh medium in the light for 3 days. Finally, the shoot tips were transferred on normal MS medium in light for recovery. With this protocol, 67% of cryopreserved shoot tips regenerated shoots. This technique is easy to handle, can be used for simultaneous treatment of a large number of samples and usually results in high survival, rapid and direct regrowth of cryopreserved shoot tips (Wang and Perl, 2006; Benson, 2008; Engelmann et al., 2008). More importantly, it utilizes sucrose as the only cryoprotectant, thus avoiding the toxic effect of other cryoprotectants such as DMSO. The only disadvantage is time extensive involved in desiccation step. Up to date, the technique has been successfully applied to shoot tips of more than 70 different plant species ranging from temperate and tropical regions (Benson, 2008; Engelmann et al., 2008). However, there existed only one report on cryopreservation of sweetpotato shoot tips by encapsulation–dehydration (Table 1). Therefore, this technique should be tested for other sweetpotato genotypes.

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2.2.4. Encapsulation–vitrification The encapsulation–vitrification procedure, originally developed by Tannoury et al. (1991) for cryopreservation of shoot tips of carnation (Dianthus caryophyllus), resembles a combination of alginate encapsulation and PVS2 vitrification. In this technique (Wang and Perl, 2006; Sakai and Engelmann, 2007; Benson, 2008; Sakai et al., 2008), alginate-encapsulated shoot tips are precultured with sucrose-rich medium, loaded with a loading solution, followed by dehydration with vitrification solution, and then directly immerged in LN. After thawing, cryopreserved shoot tips are treated with unloading solution to remove vitrification solution, and then cultured on post-culture medium for survival and regeneration (Wang and Perl, 2006; Sakai and Engelmann, 2007; Sakai et al., 2008). Hirai and Sakai (2003) were the first to describe an encapsulation– vitrification protocol for successful cryopreservation of 3 Japanese sweetpotato cultivars: Beniazuma, Chikou-1gou and Kogane-sengan. Nodal segments (about 8 mm long) were removed from in vitro stock cultures that were maintained on MS medium supplemented with 0.09 M sucrose, 1 g/l casamino acids, 0.5 mg/l BA at 25 °C under a 16 h photoperiod condition. Following 10- to 14-day incubation, shoot tips (1 mm in length) with 3–4 LP were excised from apical buds and encapsulated into beads (4 mm in diameter), according to Dereuddre et al. (1990). Beads were pre-incubated in liquid MS medium supplemented with 0.09 M sucrose and 1 g/l casamino acid for 24 h at 25 °C on a rotary shaker (90 rpm), then transferred into liquid MS medium containing 0.3 M sucrose and precultured for 16 h. Precultured shoots tips were loaded with liquid MS medium supplemented with 2 M glycerol and 1.6 M sucrose for 3 h on a rotary shaker (60 rpm) at 25 °C, dehydrated by exposure to PVS2 for 60 min on a rotary shaker (60 rpm) at 25 °C, followed by a direct immersion into LN. Cryopreserved shoot tips were rapidly warmed at 38 °C for 2 min, unloaded with 1.2 M sucrose solution for 20 min at 25 °C, and placed on a post-culture MS medium containing 0.5 mg/l BA and 1 mg/l GA3 for 7 days and then transferred to fresh MS medium containing 0.5 mg/l GA3 without BA. Morphologically normal shoots with roots regenerated in 21 days of post-culture, without intermediary callus formation. Average rate of shoot regrowth of cryopreserved shoot tips of three cultivars tested exceeded 80%. This procedure has been successfully applied to 13 sweetpotato cultivars, with some modifications including an 8-h dark incubation period of nodal sections prior to excision of shoot tips, adjustments in thawing duration and minor changes in post-culture medium (Jenderek et al., 2008). At present, 20 sweetpotato genotypes are maintained in cryostorage at the Fort Collins which is the collection of sweetpotato germplasm in USA (Gaba and Singer, 2009). More recently, the encapsulation–vitrification procedure described by Hirai and Sakai (2003) was also tested for Chinese sweetpotato cultivars (Wang et al., unpublished), with some modifications specific to postculture medium as described by Wang and Valkonen (2008b, see the following sections). Briefly, 1.0–1.5 mm shoot tips with 3–4 LP excised from apical buds were used. After thawing, shoot tips were postcultured on NH+ 4 -free MS medium supplemented with 0.5 mg/l BA. After 5–7 days of post-culture, surviving shoot tips were transferred onto shoot regrowth medium for shoot regeneration. Shoot regrowth medium was composed of MS medium supplemented with 5–10 mg/ l GA3. With this modified protocol, shoot tips of all 5 cultivars tested (Lizixiang, Lushu 8, Xushu 18, Jishu 15 and Beijing 553) survived and regenerated into shoots without callus formation following cryostorage, with the highest survival (85%) and lowest (43%) obtained for cv. Lushu 8 and cv. Beijing 553, respectively. Morphologies of plants regenerated from cryopreserved shoot tips were identical to those of the control. This protocol is currently being tested for a wide range of Chinese sweetpotato genotypes. As with encapsulation–dehydration, encapsulation–vitrification is easy to handle and can be used for simultaneous treatment of a large number of samples. In addition, this technique avoids of the lengthy desiccation involved in the encapsulation–dehydration method. Until

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now, the encapsulation–vitrification technique has been successfully applied to at least 9 sweetpotato genotypes (Table 1) and, therefore, can be considered as another promising method for sweetpotato. 3. Cryopreservation of sweetpotato embryogenic tissues 3.1. Role of somatic embryogenesis in sweetpotato biotechnology Sweetpotato somatic embryogenesis has great potential applications to micropropagation (Bieniek et al., 1995; Gaba and Singer, 2009), synthetic seed production (Chée et al., 1992), virus elimination (Wang et al., 2003a), germplasm conservation (Gaba and Singer, 2009) and breeding of induced mutants (Luan et al., 2007; He et al., 2009), and transformation studies (Kreuze et al., 2009; Zhang et al., 2009). Since the first reports on plant regeneration via somatic embryogenesis (Tsay and Tseng, 1979; Liu and Cantliffe, 1984; Jarret et al., 1984), great efforts have been made in studies on somatic embryogenesis (Kreuze et al., 2009; Zhang et al., 2009). To date, somatic embryogenesis has been established using various explants including anthers (Tsay and Tseng, 1979), leaves (Liu and Cantliffe, 1984; Zheng et al., 1996; Luan et al., 2007), petioles (Zheng et al., 1996), shoots (Liu and Cantliffe, 1984), buds (Cavalcante Alves et al., 1994; Al-Mazrooei et al., 1997; Sihachakr et al., 1997) and meristems (Liu and Cantliffe, 1984; Chée and Cantliffe, 1988a; Desamero et al., 1994; Liu et al., 1992, 1993, 1997, 2001; Torres et al., 2001; Chen et al., 2006). Somatic embryogenesis has been widely used for genetic transformation (Gama et al., 1996; Choi et al., 2007; Min et al., 2006; Yi et al., 2007; Yu et al., 2007; Kim et al., 2009; Kreuze et al., 2008, 2009; Zhang et al., 2009). 3.2. Problems involved in establishment and maintenance of somatic embryogenic cultures For somatic embryogenesis, explants were generally cultured on solid medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (Chée and Cantliffe, 1988a,b; Zheng et al., 1996; Torres et al., 2001; Liu et al., 1992, 1993, 1997, 2001) or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (Al-Mazrooei et al., 1997), to induce embryogenic callus formation. Embryogenic calli induced can be transferred into liquid medium supplemented with 2,4-D, to develop embryogenic cell suspensions (Chée and Cantliffe, 1988b, 1989; Torres et al., 2001; Liu et al., 1997, 2001). Formation of somatic embryos occurred when embryogenic calli were transferred onto solid medium containing 2,4-D (Chée and Cantliffe, 1988a,b; Torres et al., 2001; Liu et al., 1997, 2001). Maturation and germination of somatic embryos were achieved by culture of somatic embryos on solid medium without plant growth regulators (Al-Mazrooei et al., 1997; Sihachakr et al., 1997; Chen et al., 2006) or containing ABA (Liu et al., 1992, 1993, 1997, 2001; Torres et al., 2001). The whole procedure of somatic embryogenesis from initiation of culturing explants to plant regeneration required about 20 weeks (Liu et al., 1992, 1993, 2001; Chen et al., 2006). There have been, however, a number of difficulties associated with the development of suitable embryogenic tissues. Firstly, cultivar-specific response is very common. Production of high frequencies of somatic embryogenic tissues was restricted to one or a few cultivars, and some were found to be still recalcitrant (Liu et al., 1992, 2001; Cavalcante Alves et al., 1994; Al-Mazrooei et al., 1997; Kreuze et al., 2009), although promising results have been obtained that embryogenesis could be induced in 15 Chinese sweetpotato genotypes (Liu et al., 2001) and 14 out of the 16 sweetpotato genotypes tested (Al-Mazrooei et al., 1997), respectively. Nevertheless, production of high-quality embryogenic tissues for a wide range of genotypes is still a skilled task (Kreuze et al., 2009). Secondly, considerable time is required to produce high-quality embryogenic tissues, and once produced, these tissues must be proliferated and maintained by frequent subculture (Chée and Cantliffe, 1988a,b; Liu et al., 2001). As with many plant species like Vitis (Martinelli and Gribaudo, 2001), Musa (Panis et al., 2000) and Citrus (Olivares-Fuster

et al., 2000), retention of the morphogenetic potential of sweetpotato embryogenic tissues during their long-term maintenance is difficult (Cavalcante Alves et al., 1994; Al-Mazrooei et al., 1997; Sihachakr et al., 1997; Liu et al., 2001). With increasing times of subculture, embryogenic cultures reverted irreversibly into friable fast-growing non-embryogenic callus and lost their morphogenetic potential (Cavalcante Alves et al., 1994; Sihachakr et al., 1997; Liu et al., 2001). Thirdly, in vitro maintenance of embryogenic tissues by frequent subcultures increases risks of contamination and genetic variation, and is laborious and costly (Engelmann, 1997; Wang and Perl, 2006).

3.3. Cryogenic techniques 3.3.1. Encapsulation–dehydration Detailed information on this technique, including mechanism, procedure, advantage and disadvantage, has been addressed in the previous sections. Blakesley et al. (1995) were the first to successfully cryopreserve sweetpotato embryogenic tissues by encapsulation–dehydration. Embryogenic tissues of two sweetpotato genotypes, TIB 10 and Nemanete, were used in their study. Embryogenic tissues (1.5–2.0 mm in diameter) were first-step precultured with 0.1 M sucrose for 3 days at room temperature, and then encapsulated into beads (4.5–5.5 mm in diameter), according to Fabre and Dereuddre (1990). The beads were second-step stepwise precultured with 0.4 M sucrose for 3 days and 0.7 M sucrose for 2 days at room temperature. And then, the beads were dehydrated by air drying, followed by rapid freezing or two-step freezing, which was achieved by slow freezing from the ambient to 0 °C at −10 °C min− 1, and then to −40 °C at −0.5 °C min− 1, prior to immersion into LN. Following thawing in water bath at 35 °C, the beads were post-cultured on 0.1 M sucrose at 25 °C for survival and plant regeneration. With rapid freezing, maximal survival was obtained for TIB 10 (92.8%) without dehydration, while no survival was found for Nemanete without dehydration. Best survival (59.3%) was achieved for Nemanete when the precultured tissues were dehydrated for 4 to 5 h at 18–19% of water content of the beads. For both genotypes, proliferation of cryopreserved callus started within 4 days of post-culture. However, high percentage (64.3%) for TIB 10 and all for Nemanete of recovered calli lost their embryogenic competence and became non-embryogenic tissues. In contrast with rapid freezing, slow freezing not only resulted in high survivals (100% for TIB 10 and 77.8% for Nemanete), but also significantly improved percentages (74.1% for TIB 10 and 25.9% for Nemanete) of embryogenic tissues recovered from surviving tissues. All embryogenic tissues recovered retained their embryogenic competence and produced new compact globular embryogenic structures. Similar results were also obtained by Bhatti et al. (1997) for other 9 sweetpotato genotypes from different geographic regions including Asia, Africa and the Americas. The reason for this has not yet been understood. Bhatti et al. (1997) also found that different genotypes required different sucrose preculture and time of dehydration. With two-step freezing, a preculture treatment of 0.1 M sucrose for 3 days, 0.4 M sucrose for 3 days and 0.7M sucrose for 2 days was found suitable for genotypes 865M, 1023M, 209M, TIB10 and Papota, while genotypes 30MT, 207M and 132M required preculture of 0.1 M sucrose for 3 days, 0.4 M sucrose for 3 days, 0.7 M sucrose for 2 days, followed by 1.0 M sucrose for 2 days. Optimal dehydration time was 3 h for genotypes 207M, 209M and 132M; 4 h for 30TM, 1023M, TIB10 and Papota; and 5 h for 865M. Working on the encapsulation–dehydration protocol described by Blakesley et al. (1995), however, Golmirzaie et al. (2000) at CIP did not obtain any survival of cryopreserved embryos in all five cultivars tested, regardless of using either two-step freezing or fast freezing. These results confirmed again that cryopreservation of sweetpotato embryogenic tissues was strongly genotype-dependent (Blakesley et al., 1995, 1996, 1997; Bhatti et al., 1997).

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As previously discussed, encapsulation–dehydration is easy to handle, and can be used for simultaneous treatment of a large number of samples. Lengthy desiccation is a major disadvantage of the technique. Until now, this technique has been applied to successful cryopreservation of 9 sweetpotato genotypes (Table 2), indicating that it can be considered a promising method applicable to a wide range of sweetpotato genotypes.

3.3.2. Pregrowth–desiccation Pregrowth–desiccation, also belonging to one of vitrification-based techniques, combines pregrowth and desiccation procedures and is very similar to encapsulation–dehydration, with the only difference lying in that it eliminates encapsulation step. Samples are first cultured on pregrowth medium containing cryoprotectants and then desiccated, Table 2 A list of studies on cryopreservation of sweetpotato embryogenic tissues. Cryogenic procedure

Cultivar or genotype

Frozen method

Survival (%) Total

ET

Non-ET

Encapsulation– dehydration

TIB 10

TIB10

FFc SFd FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF SF FF

92.9 100 59.3 77.8 NDe ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 0 0 100

28.6 74.1 0 25.9 19 ND 38 ND 28 ND 29 ND 5 ND 14 ND 29 ND 0 ND 4 ND – – – – – – – – – – 88.9

64.3 25.9 59.3 51.9 0 76 0 66 20 66 0 71 0 33 0 43 64 71 0 14 53 ND – – – – – – – – – – 11.1

María Angola Jonathan Chugoku Morada Inta Tanzania TIB 10 Papota 30MT 865M 209M 1023M Kokei-14

FF FF FF FF FF FF FF FF FF FF FF FF

0 0 0 0 0 ND ND ND ND ND ND 92f

– – – – – 57 23 37 83 10 37

– – – – – 43 77 63 17 90 60

Nemanete 30MT 209M 1023M 865M 132M 207M TIB10 Papota Nemanete María Angola Jonathan Chugoku Morada Inta Tanzania Pregrowth– desiccation

Pregrowth

a

a

Ref. b

Blakesley et al. (1995)

Bhatti et al. (1997)

Golmirzaie et al. (2000)

Blakesley et al. (1995) Golmirzaie et al. (2000)

Blakesley et al. (1997)

Shimonishi et al. (2000)

ET = embryogenic tissues. Non-ET = embryogenic tissues. FF = fast freezing. d SF = slow freezing. e ND = not determined. f In this study, somatic embryos were used for cryopreservation, and 92% of embryos survived following freezing cryopreservation. b c

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followed by direct immersion in LN (Engelmann, 1997; Wang and Perl, 2006). This method was first introduced for cryopreservation of sweetpotato embryogenic tissues by Blakesley et al. (1996). Embryogenic tissues (9–12 mg fresh weight) were stepwise precultured on increasing sucrose concentrations of 0.1 M for 3 day, 0.4 M for 3 day and 0.7 M for 2 day at room temperature. Precultured embryogenic tissues were dehydrated using silica gel, followed by a direct immersion into LN. Thawing was conducted at 35 °C for 2 min. After that, cryopreserved embryogenic tissues were post-cultured at 25 °C in the dark for 48 h and then transferred to the light for recovery. With this method, maximum survivals (83.3–88.9%) of embryogenic tissues were obtained following dehydration for 2–2.5 h with the water content at 27–37% and subsequent fast-freezing in LN. All survived embryogenic tissues retained their embryogenic competence and produced new globular embryogenic structures from the surface of the surviving tissues. Whole plants regenerated when somatic embryos were transferred to the regeneration medium. However, when this protocol was tested for cryopreservation of embryos of 5 sweetpotato cultivars at CIP, none of them survived (Golmirzaie et al., 2000), thus confirming again that cryopreservation of sweetpotato embryogenic tissues was strongly genotype-dependent (Blakesley et al., 1995, 1996, 1997; Bhatti et al., 1997). The pregrowth–desiccation technique is one of the simplest and most cost-effective cryopreservation methods. However, manipulation of non-encapsulated embryogenic tissues and precise determination of water content in samples are difficult (Blakesley et al., 1996; Wang and Perl, 2006). In addition, it can be applied only to the plant species or tissues with high tolerance to desiccation (Engelmann, 1997; Benson, 2008). To date, this technique has been successfully applied to only one sweetpotato genotype, while failed in the cryopreservation of some other genotypes tested (Table 2).

3.3.3. Pregrowth In this method, samples are usually pregrown on cryoprotectantcontaining medium for various periods of time, depending on the plant species, followed by direct freezing in LN (Engelmann, 1997; Wang and Perl, 2006). Pregrowth using sucrose preculture alone without desiccation was introduced by Blakesley et al. (1997) for cryopreservation of sweetpotato embryogenic tissues. In this study, 6 genotypes originating from Nigeria, Peru and China were subjected to cryopreservation. Embryogenic tissues (9–12 mg fresh weight) were precultured on MS medium supplemented with 0.1 M sucrose for 3 days, 0.4 M for 6 days and subsequently 0.7 M for 3 days or 6 days at 25 °C, depending on genotypes. Following fast freezing in LN, cryopreserved tissues were thawed at 35 °C for 2 min and then post-cultured directly on 0.1 M sucrose medium, or first on 0.4 M sucrose medium for 3 days and then on 0.1 M sucrose medium for recovery, depending on genotypes. With this protocol, embryogenic tissues of all 6 genotypes tested survived following cryopreservation. Survival of cryopreserved embryogenic tissues ranged from 83% for genotype 865M to 10% for 209M with an average of 41.2% obtained for the 6 genotypes. Surviving embryogenic tissues retained their embryogenic competence and produced mature embryos and whole plantlets upon transfer to a hormone-free medium. Pregrowth was also tested for cryopreservation of somatic embryos of Japanese sweetpotato cv. Kokei-14 (Shimonishi et al., 2000). Somatic embryos at early stages were precultured with 5–10 mg/l ABA. Precultured somatic embryos were cryoprotected by either a mixture of 10% DMSO and 10% sucrose, or a mixture of 10% DMSO, 10% sucrose and 5% glycerol. The cryoprotected samples were then prefrozen to − 30 °C at 0.3 °C min− 1, followed by direct immersion into LN. Cryopreserved somatic embryos were rapidly thawed at 40 °C, washed with 10% sucrose and then post-cultured for recovery. With this protocol, about 92% of somatic embryos survived after cryopreservation.

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In sweetpotato, ABA has frequently been added to the medium to improve development and maturation of somatic embryos (Liu et al., 1992, 1993, 1997, 2001; Torres et al., 2001). Therefore, somatic embryos that had been treated with ABA-containing medium were thought to have already obtained freezing tolerance strong enough to survive in LN, without additional treatments (Yoshinaga and Yamakawa, 1994; Shimonishi et al., 2000). ABA-induced tolerance to freezing has also been observed in many plant species such as Begonia x erythrophylla (Burritt, 2008), Eucalyptus (Padayachee et al., 2008), Manihot esculenta (Danso and Ford-Llyod, 2008) and Ginkgo biloba (Popova et al., 2009). The pregrowth technique is the simplest and most cost-effective method among cryopreservation methods. However, manipulation of non-encapsulated embryogenic tissues is difficult. So far, this technique has been applied to successful cryopreservation of 7 sweetpotato genotypes (Table 2), and therefore, may be considered an alternative promising method for long-term storage of sweetpotato embryogenic tissues. 4. Cryotherapy of shoot tips for pathogen eradication 4.1. Yield loss caused by pathogens to sweetpotato and needs for production of pathogen-free plants Plant pathogens including viruses and phytoplasma severely limit yield and quality of sweetpotato production worldwide (Loebenstein et al., 2003, 2009; Zhang et al., 2009; Wang et al., 2010). Studies conducted in the main sweetpotato-growing areas in China showed that sweetpotato viruses caused an average yield loss of about 20–30% (Gao et al., 2000), with the most severe loss up to 78% (Shang et al., 1996). Although more than 20 viruses have been reported to infect sweetpotato worldwide, Sweetpotato feathery mottle virus (SPFMV) and Sweetpotato chlorotic stunt virus (SPCSV) are among the most common sweetpotato viruses (Loebenstein et al., 2003, 2009; Njeru et al., 2004; Tairo et al., 2005). Infection with SPFMV or SPCSV alone did not significantly reduce tuber yield (Milgram et al., 1996; Gutiérrez et al., 2003). However, co-infection of SPFMV and SPCSV, and the consequent synergistic interactions of these two viruses, which resulted in the development of the severe sweetpotato virus disease (SPVD) (Schaefers and Terry, 1976), caused reduction of the tuber yield down to 90% (Ngeve and Bouwkamp, 1991; Milgram et al., 1996; Gibson et al., 1998; Karyeija et al., 1998; Gutiérrez et al., 2003; Mukasa et al., 2006). The Sweetpotato little leaf phytoplasma (SPLL) (Candidatus Phytoplasma aurantifolia), a highly damaging disease of sweetpotato, occurred in most of sweetpotato-growing areas (Loebenstein et al., 2003, 2009). SPLL phytoplasma disease reduced size and number of root tubers (Pearson et al., 1984; Wang and Valkonen, 2008a) and may cause significant yield loss up to 50% (Pearson et al., 1984). In practice, usage of pathogen-free propagation materials is an efficient means for control of virus and phytoplasma diseases in sweetpotato (Loebenstein et al., 2003, 2009). Meristem culture has been widely used for production of sweetpotato virus-free stock plants (Gao et al., 2000; Gaba and Singer, 2009; Zhang et al., 2009; Wang et al., 2010). In meristem culture, the frequency of success in virus eradication is inversely proportional to the size of excised meristems, while the regeneration ability is positively proportional to the size of the meristems (Faccioli and Marani, 1998). Therefore, the size of excised meristem is a key factor limiting success of virus elimination. Recently, cryotherapy of shoot tips, i.e. a brief treatment of shoot tips in LN (Wang et al., 2009; Wang and Valkonen, 2009; Yin et al., in press), was found to efficiently eradicate sweetpotato viruses (Wang and Valkonen, 2008b) and phytoplasma (Wang and Valkonen, 2008a). To date, cryotherapy of shoot tips has been successfully applied to production of plants free of pathogens including viruses (Brison et al., 1997; Helliot et al., 2002; Wang et al., 2003b, 2006, 2008; Wang and

Valkonen, 2008b), phytoplasma (Wang and Valkonen, 2008b) and bacterium (Ding et al., 2008). Cryotherapy of shoot tips is becoming a novel biotechnology for plant pathogen eradication (Wang et al., 2009; Wang and Valkonen, 2007, 2009; Yin et al., in press).

4.2. Cryotherapy techniques for sweetpotato pathogen eradication 4.2.1. Virus eradication The sweetpotato genotype 199004.2 infected with sweetpotato feathery mottle virus (SPFMV), infected with sweetpotato chlorotic stunt virus (SPCSV) or co-infected with both viruses were subjected to cryotherapy for virus elimination (Wang and Valkonen, 2008b). Single-node segments (1 cm in length) were taken from young shoots of the stock plants grown in a greenhouse. Following surface sterilization, the explants were cultured on survival medium consisting of a basic medium containing 0.5 mg/l BA. The basic medium was composed of MS medium supplemented with 2 mg/l calcium panthotenate, 200 mg/l ascorbic acid, 20 mg/l putrescine, 100 mg/l arginine, 10 mg/l calcium nitrate and 0.09M sucrose. After 3 weeks of culture, shoot tips (1.0–1.5 mm in size) with 3–4 LP were excised from apical buds of the in vitro cultures and subjected to cryotherapy by an encapsulation–vitrification protocol as described in detail by Hirai and Sakai (2003) (see previous section), with some modifications only in post-culture step for survival and shoot regrowth. Following thawing, the beads were post-cultured for survival on NH+ 4 -free MS medium containing 0.5 mg/l BP for 5–7 days. Surviving shoot tips were then transferred onto NH+ 4 -containing MS supplemented with 5–10 mg/ l GA3 for shoot regrowth. With this protocol, about 83–87% of shoot tips (1.5–2.0 mm) survived following cryotherapy and 87% of the surviving shoot tips regenerated to normal shoots with welldeveloped roots 2 months after post-culture. Virus status of plants regenerated from cryo-treated shoot tips was detected by grafting on indicator plant I. setosa, TAS-ELISA and RT-PCR. Results showed that SPFMV and SPCSV can be eradicated at 100% efficiency from sweetpotato plants using cryotherapy, regardless of the size of shoot tips (0.5–1.5 mm) and infection status (single or co-infection) of the plants. However, with shoot tip culture without cryotherapy, although all shoot tips of different sizes survived and regenerated shoots, virusfree frequency decreased with increasing size of shoot tips: culturing of 1.5 mm shoot tips with 4 LP resulted only in 10% of SPFMV-free shoots from SPFMV-infected plants and 7% of SPFMV-free shoots from plants co-infected with SPFMV and SPCSV.

4.2.2. Phytoplasma eradication Stock plants of the sweetpotato genotype 199004.2 infected with SPLL phytoplasma (Candidatus Phytoplasma aurantifolia) were maintained in greenhouse conditions (Wang and Valkonen, 2008a). In vitro diseased stock cultures were established, using the same protocol as described previously by Wang and Valkonen (2008b). Shoot tips (1.0–1.5 mm long) containing 3–4 LP were excised from apical buds of 3-weeks-old in vitro stock cultures and used for cryotherapy based on the procedure described in detail by Hirai and Sakai (2003) (see previous section), with some modifications special to post-culture step as previously described in detail (Wang and Valkonen, 2008b). High percentages (85–90%) of the shoot tips (1.0–1.5 mm) survived following cryotherapy and 80–86% of surviving shoot tips regenerated into plantlets. All of the regenerated plants were found to be free of phytoplasma. Although survival (100%) and regrowth (94–100%) of shoot tip culture were higher than those of cryotherapy, only a few regenerated plants (7–10%) were phytoplasma-free. The phytoplasmafree plants obtained by cryotherapy and grown in the greenhouse were morphologically identical to the original pathogen-free plants. The main steps involved in pathogen eradication by cryotherapy of sweetpotato shoot tips are illustrated in Fig. 1.

C. Feng et al. / Biotechnology Advances 29 (2011) 84–93

Establishment of in vitro diseased stock cultures

Establishment of pathogen-free nuclear stock plants

Excision of shoot tips

Indexing of pathogen in plants regenerated from cryotherapy

Cryotherapy

Post-culture for plant regeneration

Fig. 1. Main steps involved in production of pathogen-free sweetpotato plants by cryotherapy of shoot tips.

4.3. Mechanism for pathogen eradication by cryotherapy of shoot tips Sweetpotato viruses and phytoplasma in infected shoot tips before cryotherapy and surviving cells in shoot tips after cryotherapy were localized, in order to understand the mechanism as to why cryotherapy can efficiently eradicate these pathogens (Wang and Valkonen, 2008a,b). Histochemical immunolocalization as described by Karyeija et al. (2000) was used to localize SPFMV and SPCSV. In plants infected with SPFMV alone and co-infected with SPFMV and SPCSV, SPFMV was detected in the LP5 and 4, but not in the LP3, 2, 1 and the AD (Wang and Valkonen, 2008b). However, SPCSV was observed only in the LP5, not in the less developed LP (1, 2, 3 and 4) and the AD in plants infected with SPCSV alone and co-infected with SPFMV and SPCSV. Transmission electron microscopy (TEM) was used to observe distribution of SPLL phytoplasma in infected plant. Results showed that phytoplasma was present in sieve elements of vascular tissues in the LP3, 4 and 5, but not in the AD and the LP1 and 2 (Wang and Valkonen, 2008a). Cells in the AD and the youngest LP (1 and 2) were small, contained small vacuoles, and had a large nucleo-cytoplasmic volume ratio (Wang and Valkonen, 2008a,b). In contrast, cells in the basal part of the meristem and LP3 and 4 were bigger, contained larger vacuoles, and had a smaller nucleo-cytoplasmic volume ratio. These results were consistent with those obtained in Musa shoot tips (Helliot et al., 2003). Following cryotherapy, surviving cells were observed only in the AD and in the youngest LP (1 and 2), while cells in the basal part of AD, in LP3 and other older tissues were killed. This surviving pattern has been observed in various plant species like Musa (Helliot et al., 2002, 2003), Cosmos atrosanguineus (Wilkinson et al., 2003), Rubus idaeus (Wang et al., 2005, 2008) and Solanum tuberosum (Kaczmarczyk et al., 2008). The previously discussed data provided sound explanations as to why cryotherapy of shoot tips could eliminate the pathogens: when infected shoot tips are subjected to cryotherapy, cells in the basal part of AD and more developed LP are killed by freezing in LN and these cells are generally infected by plant pathogens especially like viruses, according to a concept of uneven distribution of virus inside the plant (Holmes, 1948). In contrast, cells in the upper part of AD and the youngest LP1 and 2 survived following cryotherapy, and these cells are free of the pathogens or contain very low concentration of them (Holmes, 1948). Subsequently, plants regenerated from cryotherapy can be free of pathogens (Brison et al., 1997; Helliot et al., 2002; Wang et al., 2003a,b, 2006; Ding et al., 2008; Wang and Valkonen, 2008a,b; Wang et al., 2008). 5. Conclusion and prospects for further studies Various protocols have been successfully established for cryopreservation of sweetpotato shoot tips and embryogenic tissues. The former explants are preferred for long-term conservation of sweetpotato genetic resources, while the latter are valuable for sweetpotato genetic improvement. Cryopreserved embryogenic tissues were able to resume growth, develop normal somatic embryos and finally regenerate into

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whole plants. Therefore, the competence of sweetpotato embryogenesis can be retained by cryopreservation. Cryopreservation procedures are easy to implement, do not require special equipment in addition to those typically available in a plant tissue culture laboratory, and facilitate treatment of a large number of samples. However, the main limitation lies in genotype-specific response to cryopreservation protocols. Therefore, further studies are still needed to develop widespectrum protocol applicable to cryopreservation of a wide range of sweetpotato genotypes. In addition, special attentions should be paid to determination of genetic stability of plants regenerated from cryotreated explants including shoot tips and embryogenic tissues, to ensure that these plants are true-to-type, because cryopreservation involves not only freezing in LN, but also tissue culture procedures. Compared with conventional meristem culture, cryotherapy of shoot tips yields much high frequency of pathogen-free plants and avoids the difficulties associated with excision of very small meristems and plant regeneration. Thus, cryotherapy of shoot tips provides an alternative, efficient means for production of pathogenfree plants. By confirmation of phytosanitary status and genetic stability of plants regenerated from cryotherapy, cryo-treated shoot tips can be considered to be safer for exchange of germplasm between countries and regions. Acknowledgements The authors would like to acknowledge the financial supports from Department of Science & Technology of Shaanxi Province through a key project “13115” (2009ZDKG-10) and from the President Foundation of Northwest A & F University. References Al-Mazrooei S, Bhatti MH, Henshaw GG, Taylor NJ, Blakesley D. Optimization of somatic embryogenesis in fourteen cultivars of sweet potato ([Ipomoea batatas (L.) Lam.]). Plant Cell Rep 1997;16:710–4. Bajaj YPS. Storage and cryopreservation of in vitro cultures. In: Bajaj YPS, editor. High-tech and micropropagation. Biotechnology in agriculture and forestryBerlin, Heidelberg, New York: Springer; 1991. p. 361–81. Benson EE. Cryopreservation of phytodiversity: a critical appraisal of theory & practice. Curr Rev Plant Sci 2008;27:141–219. Bhatti MH, Percival T, Davey CDM, Henshaw GG, Blakesley D. Cryopreservation of embryogenic tissue of a range of genotypes of sweet potato (Ipomoea batatas [L] Lam.) using an encapsulation protocol. Plant Cell Rep 1997;16:802–6. Bieniek ME, Harrell RC, Cantliffe DJ. Enhancement of somatic embryogenesis of Ipomoea batatas in solid cultures and production of mature somatic embryos in liquid cultures for application to a bioreactor production system. Plant Cell Tissue Organ Cult 1995;41:1–8. Blakesley D, AI-Mazrooei S, Henshaw GG. Cryopreservation of embryogenic tissue of sweet potato (Ipomoea batatas): use of sucrose and dehydration for cryoprotection. Plant Cell Rep 1995;5:259–63. Blakesley D, Al Mazrooei S, Bhatti MH, Henshaw GG. Cryopreservation of nonencapsulated embryogenic tissue of sweet potato (Ipomoea batatas). Plant Cell Rep 1996;15:873–6. Blakesley D, Percival T, Bhatti MH, Henshaw GG. A simplified protocol for cryopreservation of embryogenic tissues of sweet potato (Ipomoea batatas (L.) Lam.) utilizing sucrose preculture only. Cryo Lett 1997;8:77–80. Brison M, de Boucaud MT, Pierronnet A, Dosba F. Effect of cryopreservation on the sanitary state of a cv. Prunus rootstock experimentally contaminated with plum pox potyvirus. Plant Sci 1997;123:189–96. Burritt DJ. Efficient cryopreservation of adventitious shoots of Begonia x erythrophylla using encapsulation–dehydration requires pretreatment with both ABA and proline. Plant Cell Tissue Organ Cult 2008;95:209–15. Cavalcante Alves JM, Sihachakr D, Allot M, Tizroutine S, Mussio I, Servaes A, et al. Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.). Plant Cell Rep 1994;13:437–41. Chée RP, Cantliffe DJ. Somatic embryony patterns and plant regeneration in Ipomoea batatas Poir. In Vitro Cell Dev Biol Plant 1988a;24:955–8. Chée RP, Cantliffe DJ. Selective enhancement of Ipomoea batatas Poir. embryogenic and non-embryogenic callus growth and production of embryos in liquid culture. Plant Cell Tissue Organ Cult 1988b;15:149–59. Chée RP, Cantliffe DJ. Composition of embryogenic suspension cultures of Ipomoea batatas Poir. and production of individualized embryos. Plant Cell Tissue Organ Cult 1989;17:39–52. Chée RP, Leskovar DI, Cantliffe DJ. Optimizing embryogenic callus and embryo growth of a synthetic seed system for sweetpotato by varying media nutrient concentrations. J Am Soc Hortic Sci 1992;117:663–7.

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