Recovery of Cryopreserved Buds of Chinese Yam (Dioscorea Polystachya Turcz.) Observed under Scanning Electron Microscope (Sem)

Cryopreservation Procedure

A combination of vitrivication and droplet cryopreservation protocol was imple- mented in this study. The detail protocol was described in Leunufna S, et al [8]. In vitro plantlets of Yam 21 (Dioscorea polystachya) cultivated for 2 months in Murashige and Skoog (MS) [9] medium supplemented with 2.0 µM Benzylaminopurine (BAP), 0.1 µM Naphtaleneacetic acid (NAA) (M2 medium) with 3% sucrose, 0.2 mg/l activated charcoal (AC) and 1% agar, in a culture room of 25/27°C, 60-80 µmol cm-2s-1 light intensity and 16h/8h light/dark periods using fluorescent lamps. Selected explants were further cold acclimated in a growth chamber at an alternating 28°C/5°C and 12h thermo-photo-periods with light intensity of 60-80 µmol cm-2s-1 provided by fluorescent lamps. Following cold acclimation for 3 weeks period, the plantlets’ apical buds were dissected under a light microscope (binocular) to 2-4 mm in length and further preculture in M2 medium with 15% sucrose.

The explants were further loaded for 20 min in loading solution followed by dehydration in plant vitrification solution (PVS) 2 for 20 min and immersion into liquid nitrogen (LN) in droplets of 7.5 μl PVS2. After rewarming for 3 min in MS, 3% sucrose solution and unloaded in M2, 40% sucrose for 5 min, they were cultivated in M2 medium followed by cultivation in MS medium without hormone one week after.

Sample Preparation for SEM Observation

Total samples used for the main experiment were 50 explants including 20 cryo- preserved (+LN), 10 PVS2 treated (-LN), and 10 normal in vitro (control) explants.

Survival rates obtained were 60% for cryopreserved, 90% for PVS2 treated and 100% for control explants. In addition to the main experiment, other small experiments were conducted to obtained additional samples. Overall, 3 to 6 samples were observed for each treatment at each stage of development. Observation was conducted at 3 days, 7 days and 16 days after re-culture. Before preparation for the SEM, general conditions of the explants were recorded under the light microscope (binocular).

Samples for SEM were trimmed to about 2 mm and fixed overnight in a fridge (4°C) in 0.05 M cacodylate buffer containing 2% glutaraldehyde and 2% formal- dehyde at pH 7.2. Fixed samples were than stored in a fridge in 0.05 M cacodylate buffer until further use. Samples were than degassed before washed twice, each for 15 min with fresh 0.05 M cacodylate buffer followed by dehydration in a series of solution comprising 30%, 50%, 70%, 90% and 100% ethanol. Dehydration process was conducted 2 times and each treatment lasted 15 min. Samples were then transferred into a Bal-Tec CPD030 critical point dryer (Bat-Tec Ag, Liech- tenstein) and critical point dried. Dried samples were gold coated in an Edwards S150B sputter coater (Edwards high vacuum inc., Crawley, West Sussex, UK) and examined in a Hitachi S4100 SEM (Hisco Europe, Ratingen, Germany).

Natural Shoots and Buds Morphology, and Developmental Pattern

Visual observation of morphological features of yams natural shoot development revealed that beside the terminal shoot, a number of buds can arise from the axillary node (Figure 3).

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Figure 1: In vivo (from glass house grown plants) shoot developmental pattern of the four yams species and in vitro explants used for cryopreservation. Terminal shoot (a-e), main and serial buds (f, g, i, j), aerial tuber’s primordium (arrow head - h) and in vitro buds to be cryopreserved (k, l). D. bulbifera (a, f, h, k), D. polystachya (b, g, l), D. alata (c, i) and D. cayenensis (d, e, j). Bars indicate 1 mm.

These buds have been designated as main bud (one) and more than one serial buds [10] (Figure 2), which include the buds developed to form an aerial tuber and at the aerial tuber. The development of new shoots after cryopreservation resembles this system since the explants used were buds at the upper most node (Figure 3 k,l).

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Morphological Features at Three Days After Re-culture

The new emergence of the cryopreserved explants appeared at two sites: at the axil between the young leaf (leaf primordium) and the petiole (inner core of the bud), and at the center or near apex of the leaf primordium (Figure 3).

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Figure 3: Morphological features of D. polystachya explants three days after re-culture. Surviving cryopreserved explants showing structures developed at the axil (main bud – a, b), swelling of the leaf primordium (c), and green (under light microscopic observation) original leaf primordium with a small swelling at the side, which eventually turn brown in colour and die (d). Surviving PVS2 treated explant (green in colour under light microscope - e), and dead explant (f - brown in colour under light microscope). sp shoot pri- mordium, bp bump (green structure under light microscope), s swelling. Bars indicate 300 µm (a), 400 µm (b-e), 100 µm (f).

These new growths took the form of shoot at the axil (Figure 3a), bump at the axil (Figure 3b) or swelling of the original leaf primordium (Figure 3c) and could already be observed at three days after re-culture. Most of the original young leaf did not survive, show- ing brown in colour. In some cases, the original leaf promordium remained green with or without a small swelling (Figure 3d) but eventually turned brown. PVS2 treated explants mostly remained green at 3 days after re-culture and exhibited similar features as that before treatment (Figure 3e), however, some explants showed suppressive growth resulting in the appearance of a swelling near the apex of leaf bud similar to that shown by survival cryopreserved explant (picture not shown).

Although relatively very low in number in comparison to those of cryopreserved ones, dead explants were also observed after PVS2 treatment (Figure 3f). There were no noticeable changes on the in vitro (control) explants at three days after culture (they remained green in colour).

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Figure 4: Morphological features of surviving and dead explants at three days after recul- ture. Dead cryopreserved (a) and PVS2 treated (b) leaf primordium (explants) show small wrinkles at various sites at the tissue’s surface and at trichomes, whilst surviving PVS2 treated (c) and in vitro culture (d) explants exhibit turgened (normal) cells at surface (epidermis) of the tissues. Newly developed axillary structure of cryopreserved explants exhibit highly wrinkled surface (d, f); tw, wrinkle. Bars indicate 100µm (e), 40µm (the rest).

Remarkable morphological differences were not observed on the surface tissue of original leaf buds (explants) between the survived and the dead explants. However, closer look indicated small wrinkles appeared at various sites of the tissue surface and at trichomes of the death cryopreserved (Figure 4a) and PVS2 treated (Figure 4b) leaf buds. These features did not or very little appeared on the surviving leaf buds of PVS2 treated (Figure 4c) and on in vitro cultured leaf buds (Figure 4d). On the tissues’ surface of the new axillary developed structures of cryopreserved explant, however, highly wrinkled tissues’ surface were present (Figure 4e, f).

Development at 7 Days after Re-culture

The emergence of new axillary buds was one of the features observed at seven days after re-culture on cryopreserved explants (Figure 5a). The previously developed axillary structure showed further elongation and enlargement (increase in size – Figure 5b). Newly developed fresh and smooth tissues structures were added to the previously developed callus from near apex of the original leaf bud (Figure 5c). Surviving old tissues of cryopreserved leaf buds seven days after rewarming also showed a disappearance of wrinkles in comparison to that of three days after re- warming (Figure 5d, e). Although the leaf buds of PVS2 treated (-LN) explants were still alive seven days after re-culture, their development was slow (Figure 5f). Rapid growth was shown at the base part and later took over (replaced) the slowly developed old leaf buds (Figure 5g). Other explants showed normal growth and development. In vitro cultured explants showed normal enlargement of the original leaf buds followed by initiation of new leaf buds primordium from the axil after seven days of culture (Figure 5g,h).

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Figure 5: Morphological features one week after re-culture (a-i). An axillary leaf bud developed from the dead original leaf primordium (a), enlargement of the axillary developed structure (b) and development of new tissues on the previously developed tissue structures from the near apex of the original leaf primordium (c) of the surviving cryopreserved explants. Fresh and smooth epidermal tissues observed on the seven days re-cultured surviving cryopreserved explants (e) in comparison to several wrinkled and broken trichomes on the surface of three days old surviving explants (d). Slowly developed surviving original leaf primordium (f) and rapidly elongated tissue from the base part of the leaf primordium (g) after PVS2 treatment. Elongation and enlargement of the original leaf primordium (h) and newly developed axillary bud primordium (i) exhibit by in vitro cultured explants; xb, axillary bud, s, newly developed tissue structures, tw, wrinkled trichomes, sw, slightly rough (wrinkled) tissue surface, t, trichome, x, axilary bud pri- mordium. Bars indicate 20 μm (d), 40 μm (e) and 200 μm (the rest).

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Figure 6: Explants development 16 days after re-culture (a-f). Advanced lateral swelling of leaf primordia surrounding the apical dome (a) and development of a smooth green structure (shoot) following the advanced growth of the leaf primordium (b) mark the initial stages of shoot formation and eventually plantlet development from the previously developed green structures of cryopreserved explants. Further development of the PVS2 treated explants indicating initial shoot growth at the axil (c) and bud with two leaf pri- mordia at the near apex (terminal) of the original leaf (c and d). An axillary bud with two leaf primordia (e) and a terminal bud with four leaf primordia (e and f) are shown by the in vitro cultured explants. p, leaf primordium, m, apical dome, s, shoot, x, axillary bud, y, terminal bud. Bars indicate 100µm (f), 200µm (a), 400 µm (b, d), 500 µm.

Development at 16 Days after Re-culture

Various stages of growth and development were exhibited by the explants of the three different treatments. The latest development of cryopreserved explants showed differentiation into the formation of new shoot at the previously developed green structure, initiated with the advanced lateral swelling on the sides of the structure indicated the development of leaf primordia surrounding the meristematic dome (Figure 6a). A more advanced development was indicated by the formation of small, fresh and smooth green structure on the previously developed tissue structure (leaf primordium–Figure 6b). These types of structures later developed into shoots and eventually into plantlets. The most advanced development of PVS2 treated explants was shown by the presence of new shoot with two leaves developed at the terminal leaf (near apex of the original leaf – Figure 6c,d). New shoot was also observed at the axil of the original leaf but with lesser advanced in development. The difference in development between axillary and terminal (developed from the original leaf) buds was also seen on the in vitro culture explants (Figure 6e,f). The terminal bud at 16 days after culture produced shoot with four leaves whilst axillary bud was on the two leaves stage. The terminal bud of the in vitro culture explant marked the most advanced developmental stage of all explants at 16 days after treatment.