Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

Cryopreservation of carnation (Dianthus caryophyllus L.) shoot tips by encapsulation-vitrification

Shoot tips excised from in vitro cultured plants of Dianthus caryophyllus L. (cv. Pallas, cv. Pink Candy and cv. Wanessa) were successfully cryopreserved using an encapsulation-vitrification method. Shoot tips (2–3 mm in length) were encapsulated in sodium alginate, precultured on liquid Murashige and Skoog (1962) medium supplemented with various sucrose concentrations (0.25, 0.5, 0.75, 1.0 M) for 24 h or 48 h and dehydrated with the vitrification solution PVS2 (up to 4 h) at 24 °C or 0 °C prior to direct immersion in liquid nitrogen (−196 °C). A maximum of shoot regeneration from cryopreserved shoot tips was obtained with the following combinations: preculture in 0.5 M sucrose and 180 min dehydration treatment at 0 °C for cv. Pallas (60% shoot formation), or preculture in 0.75 M and 200 min dehydration at the same temperature for cv. Pink Candy (66.6% shoot formation) and cv. Wanessa (73% shoot formation).     

Comparing encapsulation-dehydration and droplet-vitrification for cryopreservation of sugarcane (Saccharum spp.) shoot tips

In this study, in vitro shoot tips of two sugarcane clones were successfully cryopreserved using encapsulation-dehydration and droplet-vitrification with two vitrification solutions, PVS2 and PVS3. For both clones, encapsulation-dehydration induced significantly higher recovery, reaching 60% for clone H70-144 and 53% for clone CP68-1026, compared with droplet-vitrification in which recovery was 33–37% for clone H70-144 and 20–27% for clone CP68-1026. Optimal conditions included preculture of encapsulated shoot apices for 24 h in liquid medium with 0.75 M sucrose and dehydration with silica gel to 20% moisture content (fresh weight basis) before direct immersion in liquid nitrogen. With both protocols employed, regrowth of cryopreserved samples, as followed by visual observation, was always rapid and direct.     

Cryopreservation of Malus cultivars: Comparison of two droplet protocols

Two droplet procedures, droplet-vitrification (PVS2) and droplet (DMSO) were applied for cryopreservation of in vitro cultured apple (Malus domestica Borkh., cvs. Florina, Idared, Colmar and Rebra) plants. The highest post-thaw regrowth rates (70% for cv. Florina, 66% for cv. Idared, 63% for cv. Colmar and 60% for cv. Rebra) were achieved after using the droplet-vitrification (PVS2) protocol. The excised shoot tips (2–3 mm in length) were precultured in 0.5 M sucrose enriched media for 24 h. Subsequently they were transferred in PVS2 vitrification solution for 30 or 40 min (depending on cultivar) at 24 °C and then immersed in liquid nitrogen. Rewarming was performed in liquid MS medium at 24 °C. Plants regenerated from cryopreserved shoot tips did not display any sign of morphological alteration or abnormalities in growth in comparison with control plants.     

Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices (Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.     

香蕉茎尖的玻璃化法超低温保存及其植株再生

 以香蕉(Musa spp. ) 为试材, 对其离体培养茎尖玻璃化法超低温保存影响因素进行研究。结果表明, 不定芽在MS + 3.0～5.0 mg/L 6-BA + 0.1 mg/L NAA的培养基上分化较好。香蕉茎尖超低温保存较佳体系是: 2.0～3.0 cm的茎尖在含0.4 mol/L蔗糖培养基上预培养2 d, 剥取带1～2个叶原基的茎尖(长1.0～1.5 mm) , 室温(25℃) 下装载液(MS + 2 mol/L甘油+ 0.4 mol/L蔗糖) 装载20～30 min, 然后用玻璃化溶液( PVS2 ) 于0℃下处理40 min, 换1次PVS2后迅速投入液氮。保存至少1 h后, 在40℃水浴中化冻90 s, 用1.2 mol/L蔗糖培养液洗涤2次, 每次10 min, 然后转入含0.3 mol /L蔗糖的MS培养基上,暗培养10～15 h后转移到含0.5 mg/L 62BA的MS培养基中, 暗培养1周后转移到正常光下, 3个香蕉品种(巴西蕉、广东香蕉2 号、广东粉蕉1 号) 的成活率分别为75.9%、40.0%和69.6% , 再生率分别为63.4%、35.0%和63.4%。再生植株生长和分化正常, 生根后可移栽成活。     

Phloroglucinol enhances recovery and survival of cryopreserved Dendrobium nobile protocorms

The aim of the present study was to evaluate the effect of phloroglucinol in the recovery and survival of cryopreserved Dendrobium nobile protocorms. The exposure of protocorms to 2 M glycerol osmoprotective solution for 20 min followed by 10 min in PVS2 vitrification solution with 1% phloroglucinol resulted in the highest protocorm recovery and survival (68%). A positive effect of phloroglucinol was observed when combined with glycerol and PVS2. Phloroglucinol added at 1% provided an increase of over 100% in protocorm recovery and survival as compared to the same treatment without phloroglucinol. However, when sucrose was added to treatments, a negative effect was observed with a reduction in survival by 90%. Protocorms that survived cryopreservation were successfully regenerated into plants and acclimatized with 100% survival in greenhouse. Survival of cryopreserved D. nobile protocorms was a determining factor for seedling survival and growth into normal and fully functional plants. This study demonstrated an efficient procedure for cryopreservation and subsequent recovery and survival of cryopreserved D. nobile protocorms using phloroglucinol as a cryoprotectant additive.     

Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange

Shoot tips obtained from in vitro grown plantlets of guava (Psidium guajava L.) were encapsulated in calcium alginate beads for short-term storage and germplasm exchange. A gelling matrix of 3% sodium alginate and 100 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Maximum percent response for conversion of encapsulated shoot tips into plantlets was obtained on growth regulator free full strength liquid MS medium. The regrowth ability of encapsulated shoot tips was affected by medium strength and sucrose concentrations in the medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 30 days with a survival frequency of 25%. After 60 days of storage under minimal growth conditions (sucrose lacking medium), about 75% encapsulated shoot tips were converted into plantlets when subcultured on 3% sucrose containing medium. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

马铃薯茎尖小滴玻璃化法超低温保存及其再生植株的遗传稳定性

以马铃薯试管苗为试材,对其茎尖小滴玻璃化法超低温保存的影响因素进行了研究,并对再生植株进行了遗传稳定性检测。结果表明,马铃薯茎尖依次在含有0.3 mol · L-1和0.5 mol · L-1蔗糖的液体MS培养基中预培养各1 d后,在0 ℃下PVS2处理30 min,转到铝箔条上PVS2小滴上（约15 μL）,将粘有茎尖的铝箔条在液氮里蘸一下,然后直接装入盛满液氮的冷冻管中,投入液氮至少保持1 h。室温下用含有1.2 mol · L-1蔗糖的MS液体培养基解冻并洗涤30 min后,接种到MS + 0.5 mg · L-1 Zeatin + 0.1 mg · L-1 NAA＋1.0 mg · L-1 GA3恢复培养基上,存活率和再生率最高达79.91%和62.52%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。     

Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L.

Shoot tip explants obtained from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100 mM calcium chloride for the production of synthetic seed in Solanum nigrum L., a medicinally important plant. Morphogenic responses of encapsulated shoot tips to various sowing media (full or half-strength 0.8% agar-solidified or liquid MS medium or full-strength MS medium containing BAP) were evaluated in vitro. Of the six media evaluated, maximum conversion was obtained on 0.8% agar-solidified growth regulator free full-strength MS medium. The addition of MS nutrients in alginate matrix had a pronounced effect on the length of shoots that emerged from alginate beads. Encapsulated shoot tips also converted when directly sown in sterile soil moistened with liquid MS medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

银条茎尖玻璃化法超低温保存及其植株再生

 为长期稳定保存银条种质, 建立了玻璃化法超低温保存其茎尖的技术体系。选择继代4次的 银条无菌壮苗, 5 ℃低温驯化14 d; 剥取5 mm的茎尖, 在含有0.5 mol·L ^{- 1}蔗糖的MS (无Ca^{2 +} ) 液体培养基预培养1 d; 25 ℃条件下经改良MS + 2PVS₂装载20 min; 在- 20 ℃, 95%乙醇浴中以PVS₂脱水处理4h; 更换新鲜的PVS₃后投入液氮, 保存24 h后取出冷冻管在40 ℃水浴中化冻1 min; 以含有1.2 mol·L ^{- 1}蔗糖的改良MS (无Ca^{2 +} ) 溶液洗涤2 次, 每次10 min; 冻存后的茎尖相对存活率超过70%。将冻存后的茎尖转接到再生培养基上, 暗培养20 d后转入正常光照条件下培养, 存活率达63.7%; 继续培养得到正常分化和生长的再生植株, 生根后可移栽成活。     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

A micropropagation system for cloning of Bambusa tulda Roxb.

The communication describes standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from field grown culms of Bambusa tulda. Administration for 10 min of 0.05 and 0.1% mercuric chloride to explants collected in winter and summer seasons, respectively facilitated optimum culture establishment and bud break. 0.1–0.2% mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud break due to toxicity to explants. MS liquid medium enriched with 100 μM glutamine, 0.1 μM indole-3-acetic acid and 12 μM 6-benzylaminopurine supported maximum in vitro shoot multiplication rate of two-fold. The proliferated shoots were successfully rooted on MS liquid medium supplemented with 40 μM coumarin resulting in a maximum of 98% rooting. The procedure requires 45 days cycle for the in vitro clonal propagation (15 days for shoot multiplication and 30 days for root induction) and 80 days for acclimatized plantlet production.     

Encapsulation for in vitro short-term storage and exchange of ginger (Zingiber officinale Rosc.) germplasm

Synseeds of ginger (Zingiber officinale) were produced using aseptically proliferated 2-week old encapsulating explants (microshoots) upon complexation of 4% sodium alginate prepared in Murashige and Skoog (1962) medium (MS) and 100 mM calcium chloride. Conversion of synseeds into plantlets (conversion) was recorded as 66% and 53% on MS (3% sucrose) and on MS (3% sucrose) + 2.5 mg/l BA media, respectively. However, shoots/synseed were significantly higher on MS (3% sucrose) + 2.5 mg/l BA medium. For short-term storage of germplasm, sucrose-dehydrated synseeds were found better than air-dehydrated or fresh synseeds. Synseeds dehydrated in 0.25 M sucrose liquid medium for 16 h and stored in cryovials (with out medium) at 25 °C for 8 weeks and 12 weeks exhibited 53% and 13% conversion, respectively, on MS (3% sucrose) + 2.5 mg/l BA medium. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plants. This synseed protocol could be useful for short-term storage and exchange of germplasm of ginger between national as well as international laboratories.     

Effect of carbohydrates on in vitro low-temperature storage of shoot cultures of apricot

Procedures for cold storage of in vitro cultures can delay subculturing, reducing production costs and risks of contamination and somaclonal variation. The present work investigates the effects of media with sorbitol (116.8 mM, medium SO) or sucrose (58.4 mM) alone (medium SU), or the latter in combination with mannitol (58.4 mM, medium M) on 7-month storage at 5 °C of apricot shoots, cv San Castrese and Boreale. Shoots in SO survived in lower percentages and grew less than in the other treatments during storage, and died in large numbers after transfer to standard culture conditions. In comparison to other treatments, survival was 100% in the presence of M and both shoot weight and number of surviving proliferated axillary shoots was increased. Moreover, M improved regrowth compared to SU under standard culture conditions. The SOD and CAT activity confirmed the higher stress of shoots stored in SO than controls, and in contrast, the low stress of shoots in M.     

通过玻璃化超低温处理脱除草莓轻型黄边病毒(SMYEV)研究

以草莓品种明宝为材料,取茎尖做初代培养,继代扩繁5次后,取2mm左右的茎尖,利用玻璃化超低温技术对SMYEV进行脱除,并利用结合内标的多重RT-PCR技术对再生植株进行病毒检测。结果表明,在病毒脱除过程中,预培养蔗糖浓度为0.5mol/L,处理3d;装载处理为25℃,60min;玻璃化处理为0℃,120min;液氮处理60min后,进行40℃水浴2min,草莓茎尖的存活率为76%,而草莓轻型黄边病毒的脱除率为95%。只有通过液氮处理才可以脱除该病毒,液氮处理前的玻璃化处理脱毒率为0。利用超低温脱毒法可以简便有效地脱除SMYEV。     

食用百合种质的玻璃化法超低温保存技术初探

以离体茎尖为试材 ,采用玻璃化法 ,对食用百合离体超低温保存技术进行了初步研究。结果表明 ,用2～ 3mm茎尖 ,在MS +0 .5mol·L-1蔗糖浓度的培养基上预培养 1～ 2d ,室温下植物玻璃化溶液 (PVS2 )处理 2 0min ,换入新鲜的PVS2 ,迅速投入液氮中 ,2d后取出 ,在 4 0℃水浴中解冻 2min ,再在 2 5℃水浴中解冻 10min ,用1.2mol·L-1蔗糖液体培养基洗涤 2 0min ,接种在 6 -BA 0 .5mg·L-1+NAA 0 .1mg·L-1+GA3 0 .3mg·L-1+蔗糖 30 g·L-1+琼脂 7g·L-1的MS培养基上 ,2 5℃弱光培养 1周后转为正常光下培养 ,2周后再生率达到 5 2 .6 %。     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.