You are on page 1of 15

American Journal of Experimental Agriculture

5(2): 94-108, 2015, Article no.AJEA.2015.011
ISSN: 2231-0606

SCIENCEDOMAIN international

Comparative Growth Analysis and Acclimatization of
Tissue Culture Derived Cocoyam (Xanthosoma
sagittifolium L. Schott) Plantlets
Anne E. Sama1, Mohamed A. Shahba2*, Harrison G. Hughes1
and Mohamed S. Abbas2

Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins,
Colorado, 80523-1173, USA.
Department of Natural Resources, Institute of African Research and Studies, Cairo University, Giza,
12613, Egypt.
Authors’ contributions
This work was carried out in collaboration between all authors. Authors AES and HGH designed the
study and collected the data. Authors MAS and MSA managed the statistical analysis of the data and
wrote the first draft. Author MSA managed the literature searches. Author MAS managed the final
report writing while Author HGH managed the final editing. All authors read and approved the final
Article Information
DOI: 10.9734/AJEA/2015/10379
(1) Marco Aurelio Cristancho, National Center for Coffee Research, CENICAFÉ, Colombia.
(1) Anonymous, National Centre for Genetic Resources and Biotechnology, Nigeria.
(2) Anonymous, Jomo Kenyatta university, Kenya.
Peer review History:


Original Research Article

Received 26 March 2014
Accepted 24th April 2014
Published 12 September 2014

The current study was carried out to compare the external leaf structure of tissue culture-derived
and conventionally-propagated Cocoyam [Xanthosoma sagittifolium (L) Schott] plantlets and to
develop an efficient acclimatization protocol for these plantlets. Acclimatization studies were carried
out during winter and summer to ascertain seasonal influence relative to plant survival upon transfer
from in vitro to natural conditions. Results indicated that, cocoyam leaves have few stomates on
both abaxial and adaxial surfaces with fewer on the adaxial surface. High levels of epicuticular wax
(EW) found in vitro may have contributed to reduced transpiration rates. The reduced amounts of
EW on acclimatized plants could be attributed to the rapid cell enlargement in expanding leaves,
more rapid than the rate of wax formation. Acclimatization using humidity tent decreased leaf wilting
*Corresponding author: Email:;

Africa produces about 75% of the world production which is about 0. cocoyam has received very little research attention [34]. The objectives of this investigation were to determine an effective acclimatization protocol for micropropagated cocoyam plantlets through a comparison of the external leaf structure of tissue culture-derived plantlets and conventionally-propagated plants in terms of epidermal cells. sunflower [30]. SF: stomatal frequency. cormels. Flowering is rare. The stem has a starch rich underground structure. NAA: 1. 2. Previous studies have shown that shoot multiplication. and to determine an effective acclimatization protocol for cocoyam plantlets.4-D).canola[25]. It involves the use of defined growth media supplemented with appropriate growth regulators that enable morphogenesis to occur from naturally growing plant parts [11]. somatic embryogenesis and tuberization can be induced in shoot tips of cocoyam cultured in vitro on Murashige and Skoog medium [12] supplemented with various combinations of indol butyric acid (IBA). ABBREVIATIONS MS: Murashige and Skoog (1962) medium. MATERIALS AND METHODS 2. TDZ: Thidiazuron.. 1. AJEA. Mist-acclimatized plantlets produced about 50% fewer leaves than those acclimatized in a humidity tent. lentil [28]. AS: Adenine sulphate. rice [31] and banana [32]. 95 . winter squash [18]. date palm [27]. taro [20]. watermelon [21]. the inflorescence consists of a cylindrical spadix of flowers enclosed in a 12-15 cm spathe [1].011 and damage compared with the control treatment or with the mist treatment. animal feed [3-5] and of cash income for farmers [6]. but when it occurs. Article no. asparagus [23]. 2. cocoyam. stomatal frequency and stomatal index. the benefit of any micropropagation system can only be realized by the successful transfer of plantlets from tissueculture vessels to the field conditions [33]. Homestead. It is a staple food in the tropics and subtropics and one of the six most important root and tuber crops worldwide [2]. the corm. mainly because of a lack of knowledge concerning diseases. EC: Epidermal cells. groundnut [22]. A relatively high humidity (60-80%) for approximately two weeks reduced leaf injury from wilting and desiccation.17]. corm and cormels [10].Sama et al.4dichlorophenoxyacetic acid (2. Most species grown in vitro require an acclimatization process in order to ensure that a sufficient number of plants survive and grow vigorously when transferred to soil. 5(2): 94-108. stomatal frequency. Keywords: Tissue culture. The corm. BAP: Benzylaminopurine. and physiological determinants that may limit plant growth and development [35]. epidermal cells.naphthaleneacetic acid. cotton [26]. soybean[19]. Cocoyam breeding and production is labor intensive and requires large amounts of water [8]. proper management practices. common bean [29]. stomatal index.1 Source of Explants Cocoyam ‘South Dade’ white plants were obtained from the Tropical Fruit Company. The biochemical aspects of induction of in vitro organogenesis have been investigated in a number of plants including carrot [14]. black pepper [24]. In spite of its importance in many countries. BM: basal medium.45 million tons [7]. from which offshoots called cormels develop. Micropropagation is an efficient method to mass propagate good quality materials that may substantially improve production. EW: epicuticular wax. Similar results were obtained during winter acclimatization with a lower rate of leaf formation compared to summer acclimatization. acclimatization. The yield potential of cocoyam is seldom realized. 1naphthalene acetic acid (NAA). epicuticular wax. pea [15]. summer squash [16. Florida as sprouted corm sections. INTRODUCTION Cocoyam [Xanthosoma sagittifolium (L) Schott] is a monocotyledonous crop that belongs to the Araceae family. and leaves of cocoyam are an important source of carbohydrates for human nutrition.2015. It is highly susceptible to diseases such as cocoyam root rot disease caused by Pythium myriotylum [9] and Dasheen Mosaic virus found in the leaves. 2015. AJEA. However. SI: stomatal index. Benzylaminopurine (BAP) and kinetin [13].

the relative humidity was lowered to 96 and 94 and 92% respectively. Control plants were transferred directly to an open bench in the greenhouse. Whenever a semi-solid medium was desirable. High and low temperatures averaging 26 and 16oC respectively with corresponding relative humidities of 54% and 94%. A humidifier was used to provide an initial relative o humidity of 98% with a temperature of 25 C.4%. the average greenhouse relative humidity was 40 ± o 5%. thiamine HCl (10 mg L -1 1 ). perlite and vermiculite (1:1:0. washed and transplanted into the same soilless mix. the misting interval was increased to 16 minutes after the first two days for the remaining three days of acclimatization. On third. All plants were transferred to an open bench and grown under standard greenhouse conditions. perlite and vermiculite at a ratio of 1:1:0. Erlenmeyer flasks (125 ml) and test tubes (25 x 150 mm) were used for growing cultures.5 mm of corm tissue at the base were disinfected in a laminar flow hood using 1% (v/v) sodium hypochlorite solution for 10 minutes before transferred onto the culture medium. the epidermal cell layer was peeled with a Sections were potted in polyethylene pots (≅100 cm2) in a mix of peat. Temperature was maintained at 23± o 2 C. Kaput caps (Bellco Glass. Shoot-tips of 3-5 mm were excised and the apical meristem with 4-6 leaf primordia and approximately 0. Sucrose was provided at 30 g L . Flasks were stoppered with nonabsorbent cotton plugs. The modified component of B5 micro-salts was -1 MnSO4. leaf impressions were made using a thin film of transparent fingernail polish.01% dimethyl sulfoxide (DMSO) was used. To gradually reduce the humidity. Plants used for adaptation were previously proliferated in vitro in 2. Before transplantation. The media-containing vessels o were then autoclaved for 18 minutes at 121 C. the number of 96 . 3) humidity tent. After the dryness of the fingernail polish.0 µM TDZ multiplication medium.3 Acclimatizationof Cocoyam Plantlets Acclimatization studies were carried out during winter and summer. The transplants were then subjected to four different acclimatization treatments for five days as follows: 1) Control. 2) mist.011 leaves. These plants were maintained in a greenhouse under natural photoperiod. The number of stomates in tissue culture-derived and conventionally-propagated plants was examined to ascertain stomatal function and influence relative to plant survival upon transfer from in vitro to natural conditions.02. The plants subjected to the second treatment were placed under an automatic misting system set for six seconds at eight minutes intervals. AJEA. 2015.2015. fourth and fifth days. AJEA. the plants were taken out. During transplantation.Sama et al. After five days of acclimatization in the test tubes. Each treatment had at least 42 plants. 5(2): 94-108.5 by volume. Article no. and then covered with aluminium foil. Humidity was gradually reduced after the second day by partially opening the flaps of the chamber. and light intensities of approximately 400 µmol m-2 S-1. sprouts were collected. Eight weeks after planting. During winter.2 Basal Medium (BM) A modified Gamborg’s B5 mineral salts [36] supplemented with 0. Plants were watered as needed with tap water and fertilized with liquid fertilizer containing N:P:K at 20:10:20 by weight twice a week.4H2O at 10 mg L . trimmed to about 5 cm and washed under running tap water for 30-60 minutes. The initial temperature and relative o humidity were 25 C and 63% respectively. Inc.. N. type A) was added at a concentration of 0. 2.5 by volume). Plant height was measured from the basal plate to the lamina tip of the youngest fully expanded leaf. A thidiazuron (TDZ) solution containing 0. and then totally removed for the remaining three days of acclimatization to expose the plants to the natural environment while in the test tubes. The fourth treatment was conducted in culture vessels by partial uncapping. To count stomates. The third treatment placed plants in a locally constructed plastic humidity chamber with the dimension of 127 X 92 X 62 cm.7 ± 0. Plants were transplanted into 10 cm plastic pots containing pre-moistened non-sterile soilless substrate composed of peat.05 µM 1-naphthaleneacetic acid (NAA) was used throughout the study. and 4) test tube acclimatization by uncapping. 2. Organics consisted of -1 myo-inositol (100 mg L ). agar was gently washed off the roots with tap water. Aliquots of 25 ml and 15 ml were dispensed into the flasks and test tubes. The cultures were placed on a bench in the same environment as the control plants. respectively. Test tubes were covered with polypropylene closures. and the temperature was 22 ± 3 C. Caps were loosely opened for the first two days.. agar (Sigma agar. roots and plant height were recorded for each plant. nicotinic acid (1 mg L ) and pyridoxine HCl -1 -1 (10 mg L ). J). The pH of the medium was adjusted to 5. This was applied to peripheral sections on either side of the midrib and on both abaxial and adaxial surfaces of the lamina.

Sama et al.. 3). Epidermal cells (EC) of cocoyam leaves from the three sources urces were polygonal or irregular with undulate anticlinal walls (Fig. Article no.2015 transparent adhesive tape. 2A). Stomates from all leaf sources were scattered and at unequal distances from one another. with varying sizes on both sides. Also. 3. the epicuticular wax content on leaves from tissue culture--derived and conventionally propagated plants was compared. The cells were also varied in size and shape. All data were subjected to an analysis of variance using unequal replications due to contamination. and were morphologically similar to their conventionally propagated counterparts (Fig.5 respectively.1 Regenerated rated Plantlets Characteristics Tissue culture regenerated cocoyam plantlets did not show any obvious deviations. 2015.1 Stomatal frequency (SF) Cocoyam leaves from all sources were amphistomatic. Treatment ent means were separated by Tukey’s Multiple Range Test at a 5% level of significance [37]. 3. However. ECs and stomata along the veins were smaller and aligned in stream-like stream manner. 97 . Stomates of greenhouse plants were elliptical and sunken sunken. 3). 3). Almost twice as many stomates were found on the abaxialsurface (Fig. The average SF of adaxial and abaxial surfaces were 17. A morphological comparison between conventionally conventionally-propagated propagated and tissue cultured (3 months s in greenhouse) cocoyam plants growing in the greenhouse. AJEA. RESULTS 3. The imprints were then placed on microscope slides for observations.2.4 Data Analysis Experiments were laid out as a complete block design. However. 5(2): 94-108. The plantlets retained the characteristic sagitate leaves of conventionally-propagated ted plants without modifications of color or shape.2 Stomata in Tissue Culture Culture-Derived and Conventionally--Propagated Plants Fig.4 and 30. but below the level of epidermal cells (Fig. There was a significant significa difference in the average SF among control. acclimatized and conventionally conventionally-propagated plants (Table 1 and Fig.. 1). AJEA. 2. 1. 2A). Analysis of variance indicated that SF (the number of stomates per mm2) was significantly greater in the non-micropropagatedcontrol micropropagatedcontrol plants than in the acclimatized greenhouse plants (Table 1 and Fig. Abaxial and adaxial stomata were similar to one another.011 AJEA. the anticlinal walls were less distinct in ECs of cultured plantlets. 3. while those of in vitro plants were more spherical and raised.2015.

3 EpicuticularWax Content on Leaves of Culture-Derived and Conventionally-Propagated Plants 3. AJEA.0 < 0.001 < 0.2.0 195.0001 0.0001 < 0.0 2251. 4).2 Stomatal index (SI) Analysis of variance indicated no significant differences in stomatal index calculated as the number of stomata / number of epidermal cells 2 and stomata x 100 mm among various cocoyam plantlet sources (Table 1 and Fig. 3.6 for in vitro plants to 5.3 1335.1.0001 < 0.003 < 0.0 952.3 235.0 41 789.0001 < 0.0 952.0 3251.0001 < 0.0 2 95.05 0.0 0.001 < 0.35 2 1 2 41 152.14 3 2 6 41 252.015 < 0. Cocoyam plantlets showed no significant visual desiccation Analysis of variance indicated a significant difference among different plant sources in epicuticular wax (EW) formation on cocoyam 98 .0 10222.3 for control plants on the adaxial surface. Gravimetric determination showed that in vitro leaves had an average of 88.54 3 2 6 41 211.01 < 0. Cocoyam plantlets cultured in vitro were found to have greater deposits of EW.0 6 166.0001 0.1 µg/cm2 for plantlets transferred and grown in the greenhouse (Fig.0 466. 2015.0 1522.4.0001 0.Sama et al.011 Table 1. as compared to 50.4.4 Ttissue Culture-Derived Plantlets Behavior and Adaptation to Different Environmental Factors 3.3 935.0 789. Analysis of variance with mean squares and treatment significance of the effect of different cocoyam plant source and leaf surface on stomatal frequency and index and the effect of plant source on epicuticular wax content on cocoyam leaves and the effect of acclimatization procedures under different durations during summer on leaf damage.0001 < 0.0 15222. Values ranged from 8. leaf wilting.0001 0. 3. 2B).90 leaves (Table 1). Article no.0 351.1 Effects on leaves Analysis of variance indicated a significant difference among acclimatization procedures on leaf damage and leaf wilting (Table 1).0 241. leaf initiation and leaf shedding of tissue culture-derived cocoyam plants Source Stomatal frequency: Plant source (S) Leaf surface (F) SxF Rep Stomatal index: Plant source (S) Leaf surface (F) SxF Rep Cuticular wax: Plant source Rep Leaf damage: Acclimatization treatment (T) Duration (D) TxD Rep Leaf wilting: Acclimatization treatment (T) Duration (D) TxD Rep Leaf initiation: Acclimatization treatment (T) Duration (D) TxD Rep Leaf shedding: Acclimatization treatment (T) Duration (D) TxD Rep DF Mean squares P-value* 2 1 2 41 1052.44 2 41 4321. 5(2): 94-108.0 for in vitro propagated plants to 8.0 0.6 for control plants on the abaxial surface while it ranged from 4.0001 < 0.0 0.70 3 2 6 41 1252.0001 0.0 *Significant at P ≤ 0..005 < 0.24 3 122.0 0.0001 0. AJEA.1 Summer acclimatization 3.6 µg/cm2.0 0.2015.3 335.0001 0.

3. Two weeks after acclimatization. where SF was greater with in vitro plantlets than those that had been removed from culture. an average of 1. Donnelly and Vidaver. Plantlets acclimatized by uncapping the culture tube showed 14. plants continued to grow actively in the greenhouse with normal leaf and whole plant morphology. 5A and B). After two weeks of acclimatization.2 Effects on growth habit Analysis of variance indicated a significant difference among acclimatization procedures on leaf initiation and leaf shedding (Table 1).3 for the control plants (Fig. differences were observed in leaf wilting among different treatments.5 was associated with the previous leaf damage percentages respectively (Fig. After 24 hours. humidity tent plants shed only an average of 0. An average rate of one new leaf per plant was produced every two weeks. New leaves were produced by the second week after acclimatization and more grew after four weeks.6 leaves in mist-acclimatized and control plants. more wilting was observed as the percentage of damaged leaves increased significantly for open tube acclimatization (43. 2015. In similar findings. and apple [41]. AJEA. DISCUSSION The decrease in stomatal index and increase in epidermal cell size may affect plant growth physiology. In strawberry plants. 5C).2 Effects on growth habit The growth and development of plants was not affected by the method of acclimatization during shoot elongation. A significant difference among treatments was observed after two weeks and disappeared at four and six weeks. which were gradually reduced in subsequent weeks. [43] reported significantly reduced cell length in the upper epidermis of transferred ‘Pixy’ plum plants as compared to those aseptically and field grown. On the other hand. The rate of leaf formation was low as compared to summer acclimatized plants except for the mist treatment (Fig.2 leaves were produced from plants acclimatized in the humidity tent. 3. One week after acclimatization. Therefore. 5D). Article no. The reduced SF in leaves of acclimatized plants may have been due to their enlargement. 3. 5C). were observed in Linquidambar styraciflua [44.4. Plantlets acclimatized under mist and in the humidity tent had wilted leaves only after one week of acclimatization.5 Winter Acclimatization 3.45]. wilting assessment indicated that plantlets acclimated under mist suffered less wilting after 24 hours (Fig.7 %) as compared to that from the humidity tent which had a damage percentage of 15. Plantlets transferred directly fromculture to the open bench were more stressed after 24 hours compared when to other treatments. rather than the increase in cell number [42]. Mist-acclimatized plants produced fewer leaves than the other treatments after four and six weeks and was significantly different from those acclimatized in a humidity tent and uncapped tubes. the increase in size of persistent leaves was mainly the result of cell enlargement. but those acclimatized under 99 . 5B). AJEA.Sama et al.8 leaves as compared to 1. 5A).011 mist and humidity tent were able to regain turgidity. 5(2): 94-108. which was attributed to leaf expansion. [40] reported almost twice as many stomates on the abaxial surface in tissue cultured plantlets of Rubus idaeus. Blanke and Belcher [41] noticed a drastic decrease in SF of transferred apple plants.39]. On the other hand.5.5. one hour after transfer to the open bench. After acclimatization.. Brainerd et al. Comparable findings.6% in plantlets acclimatized under mist (Fig.9%.2015.9% leaf injury after 24 hours of acclimatization compared to only 0. Wilting assessment of 2. as compared to only 0.1 Effects on leaves Plantlet leaves wilted slightly during transplantation. altered leaf structures might be associated with poor field performance and increased disease susceptibility [38. 4.8 and 3. The number of leaves shed per plant per treatment was used as indication of the reverse of leaf production. The critical period for leaf injury was the first week after acclimatization. All plants survived in all treatments but less wilting and leave injury were associated with mist or humidity tent as compared with the control.1. Significant differences among the different acclimatization treatments were found in the number of new leaves formed in plants (Fig.

Vertical bars at the top represent standard errors 100 .011 50 Abaxial surface Adaxial surface a 40 Stomatal frequency A ab 30 a b ab 20 b 10 0 B 10 Stomatal index a a a 8 6 a a a 4 2 0 In Vitro Acclimatized Conventional Plant source Fig.Stomatal frequencies (A) and indices (B) of the leaves of in vitro.05 using Multiple Range Test for plant source comparison at abaxial and adaxial surfaces. 5(2): 94-108. 2.2015. AJEA.. acclimatized and conventionally propagated cocoyam. AJEA.Sama et al. Columns labeled with the same letter are not significantly different at P = 0. Article no. 2015.

Article no..2015 AJEA.2015.011 A B C 101 . 5(2): 94-108. AJEA. AJEA. 2015..Sama et al.

Quantitative variation has been described for many phenotypes including plant growth habit and agronomic performance [50-52]. Rosa multiflora [49]. The causes of somaclonal variations are believed to result from a range of genetic events during plant tissue culture. AJEA. Fig. 46]. suggested that the high SF of apple micropropagated plantlets was responsible for the higher water loss observed. stomates have a greater part in water loss of plantlets than epicuticular wax. when compared to 27. in vitro plants. in addition to their greater densities. 4. Quantitative variation is frequently found among regenerants derived from tissue culture and often indicates alteration of numerous loci [50]. Wetzstein and Sommer [45] found that stomata were also larger in vitro plantlets of sweet gum. which resulted in less wilting and high survival rates of cocoyam plantlets after transplantation. However.4 and 30. and Vitis sp. Article no. No significant differences in stomatal index among various cocoyam plantlets sources were found.011 Fig. Vertical bars at the top represent standard errors The average SF of 17.Sama et al. AJEA. acclimatized plants and C. but it is difficult to interpret somaclonal variation in a genetic mode [53-55]. ‘Valiant’ [46]. ‘Valiant’ [46] in comparisons made between in vitro and field grown plants. conventionally-propagated plants 100 g cm-2) 80 Epicuticular wax ( a 60 ab b 40 20 0 In Vitro Acclimatized Conventional Plant source Fig. B. Stomatal frequency on the abaxial surface of different sources of cocoyam plant as indicated by photomicrographs of the leave imprints (X 600). are relatively low.5 (adaxial only) for Vitis sp. Columns labeled with the same letter are not significantly different at P = 0. 5(2): 94-108.5 for adaxial and abaxial surfaces of cocoyam. Dami [46] found significantly greater stomatal densities in leaves of greenhouse-grown plants than in in vitro cultured leaves but found none when SI comparisons were made. As indicated by Brainerd and Fuchigami [47] and [48]. 2015. The rapid water loss could be due to stomatal malfunction [47] or the size of the stomates [45]. Zhao et al.05 using Multiple Range Test for plant source comparison. 13. [38] found that micropropagatedregenerants had produced a significantly lower stomatal index. Brainerd and Fuchigami [47].2015. but larger epidermal cell size than conventional plants when they investigated the alterations in leaf trichomes. These results agree with the idea that SI is a better estimate than SF in comparisons involving leaves with stomates of different sizes [48. respectively. A.. 3. 40.5 and 150 for Rubus idaeus [40]. These results corroborate previous findings that were reported for Solanum laciniatum [48]. This low SF may have contributed to low transpiration rates. 184. stomatal characteristics and epidermal cellular features in micropropagated rhubarb (Rheum rhaponticum L.). Effect of plant source on epicuticular wax formation on cocoyam leaves. In recent years the genetic analysis of plants regenerated from tissue culture has revealed that 102 . In contrast.

AJEA. 4 = intact) d Acclimatization treatment Fig. leaf wilting (B). AJEA. Vertical bars at the top represent standard errors 103 .2015. chrysanthemum [58]. strawberry [42].63] and 17/18 days in carnation [61]. there was lees wax deposits per unit area after transplantation. carnation [61]. Effect of acclimatization procedures during summer on leaf damage (A). Cocoyam plantlets cultured in vitro were found to have greater deposits of EW. Fabri et al. and grape [46]. Examples include cauliflower [60]. leaf initiation (C) and leaf shedding (D) of tissue culture-derived cocoyam plants after different durations.59]. The majority of morphological variants observed in tissue cultured plants were due to numerical and structural chromosome changes induced during culture [56. Wax formed after 10-14 days in Brassica oleracea [62.Sama et al.05 using Multiple Range Test for treatment effect comparison at different durations. while similar findings were observed in Solanum laciniatum acclimatized plants after a month [48]. 5.011 extensive genetic changes apparently occur during tissue culture. It was suggested that the decrease may be related to two possible causes: leaf enlargement that exceeded the synthesis of additional wax to cover the additional surface area. These results are in contrast to reports where more extensive wax deposits were observed in greenhouse and field plants than observed in vitro. The results obtained in this study showed a decrease in wax content per unit area in transferred plantlets at 9 and 12 weeks from transplantation. [42] observed an increase in EW deposits of transferred strawberry plantlets during the first 20 days. and wax metabolism during acclimatization. 2015. Columns labeled with the same letter are not significantly different at P = 0. The previous results indicate that wax deposition and breakdown are speciesdependent. Wax deposition after planlet transplantation occurs with time. Apparently. Sutter [58] found a similar phenomenon with apple plants.57].. with more EW in vitro and less after acclimatization. ab b C 4 2 weeks 4 weeks 6 weeks a ab b c 3 a a b b 2 a b 1 c c 0 4 ab a a a ab b b c ab b b c 3 2 Number of shed leaves 5 0 B 24 hours 24 hours 4 weeks D 2 weeks 4 weeks 6 weeks 3 a ab ab b 2 ab b 1 ab ab a a ab b 1 nt ro l be Co Tu i st Te M nt l ro nt Co Tu M Te be 0 i st 0 nt Leaves wilting (0 = dead. 5(2): 94-108. cabbage [61]. Article no. since previous studies have shown that wax biosynthesis and degradation is a continual and 40 a a 30 a b 20 a b c 10 c b 5 A 24 hours 2 weeks 4 weeks Number of initiated leaves Damaged leaves (%) 50 dynamic process [58.

Spence [69] observed that field grown cocoyam plants were wasteful in the manner in which they produced and maintained their leaves. The environmental conditions were not optimum [45. In this study. Cocoyam. AJEA. [76] obtained the highest tuberization rate (83%) of the white cocoyam cultivar with an inductive medium containing 6-benzylaminopurine (BAP) under Short day regime. [70] evaluated the success of a micropropagation system by the percentage of plants that are successfully transferred from culture to natural soil conditions. especially in areas such as the humid tropics with relatively high humidities. [71-73]. Tsafack et al [75] confirmed the findings of Gopal et al. 104 . The continuous turnover of large leaves reduced photosynthetic productivity of the plants. in areas with low relative humidities. unlike taro. 2015. The low wax content of acclimatized plants may have been caused principally by the rapid leaf expansion that supressed wax formation.68]. but did favor wax formation in vitro. and lower light intensities in winter conditions within the greenhouse. also obtained 100 % survival with different acclimatization studies.. Brainerd and Fuchigami [47] and Conner and Conner [48] showed that EW was less important than stomates in determining the amount of water loss in plants. The in vitro conditions in which cocoyam plantlets were grown seemed favorably for EW formation. Tsafack et al. Reduction in growth upon transplanting of tissue culture plantlets has been frequently reported in the literature [62. These losses are associated with rapid water loss and desiccation during the acclimatization phase. cannot withstand water-logging under natural conditions [8. This could be due to the use of growth regulators while in culture. The ability to successfully transfer cocoyam plantlets from culture at a relatively low cost with minimal loss is important to the micropropagation technique. Acclimatization procedures may be either unnecessary or just advantageous for a short period. may have been invaluable in conferring plantlet survival. It could also relate to the fact that cocoyam typically grows in high humidity.011 Sutter and Langhans [61] and Wezstein and Sommer [45] indicated that the environment in which a plant grows determines its morphology and chemical composition.67. [77] and Tsafack et al. Omokolo et al. The lag in growth in the case of winter acclimatization could be related to the low temperatures. The sunken and ellipsoidal stomata of cocoyam leaves in vitro. This high EW content may have contributed to plantlet survival upon transfer ex vitro. and that the wetness creates an environment favorable for microorganism growth. humidity. all cocoyam plants transferred from culture to in vivo conditions survived.66]. many tissue culture regenerated plants are lost during transfer to normal growth conditions. in addition to their high EW content. a humidity tent or cheaper method of maintaining a moderately high humidity is recommended. Another possible cause for the high amounts of EW observed in vitro may have been the dissolution of internal lipids from open stomata of in vitro plants to close upon removal from culture [45. Short et al. In general. On the other hand. Mist systems and humidity chambers are most commonly utilized in an attempt to mitigate plant loss [44]. Onokpise et al. [78] who reported that tubers could be induced in vitro without the use of plant growth regulators (PGRs). This could be true for cocoyam.47. However. may have been too wet to ensure normal growth. This probably slowed conversion from heterotrophic to autotrophic nutrition.2015. even without acclimatization.Sama et al. The relatively poor growth performance of plantlets acclimatized by mist system may be attributed to the wet conditions they were subjected to. [74] reported that rootless cocoyam shoots could be easily rooted and would rapidly develop into plantlets when transferred into soil. the number and weight of microtubers and the leaf weight were affected by day length and temperature. and thus may have wax production even under high humidities. Otherwise.65. [64] reported that nutrients are leached under a misting system. [75] mentioned that the tuberization rate. especially at the commercial scale. Article no. Continuous misting for a period of five days. Staritsky et al. The number of leaves shed was comparatively lower than that encountered from non-tissue culture derived plants under field conditions [69]. The use of media without PGRs was important to judge the innate capacity of genotypes to produce microtubers and to avoid the possibility of any undesirable carry-over effect of PGRs on morphogenesis and sprouting.61].48]. Griffis et al. the overall trend was that more leaves were produced than shed. rather than an expensive misting system. in addition to the high humidity. AJEA. and suggested the use of growth regulators to alleviate the shedding. 5(2): 94-108.

perspectives and future prospects. Marker proteins for embryogenic differentiation patterns in pea callus. 1994. 2004.85:479–487. 1998. 1995. Dadson R. Production.139161. Shoot production in squash (Cucurbita pepo) by in vitro organogenesis. Chen J. Cultivation of cocoyam. John Wileys and sons Ltd. Technology and Application. Paris HS. Purseglove JW. Phytopath. Sci. 1984. plant regeneration and tuberization in Xanthosoma sagittifolium cultured in vitro. Biol. 1978. Netherlands. 5(2): 94-108. Pacumbaba RP. Kanmegne G. C. Plant Cell Rep. no competing REFERENCES 1. U. 1992. R. J. In: Tropical root and tuber crops. 7. Acd. 2006. Nyochembeng LM. Adams MJ. Jacobsen HJ. 1995. Molecular characterization of an isolate of Dasheen mosaic virus from Zantedeschia aethiopica in China and comparisons in the genus Potyvirus. A wider-mouthed culture vessels should be used so that the mass of proliferated tissue can be removed easily. sweet potato. COMPETING INTERESTS Authors have interests exist. Food and agriculture organization statistical database: world production offruitsand vegetables. Tropical Crops: Monocotyledons. 5.2015. Nyochembeng L. Choi JH.2:19– 25. Ndzana X. declared that 2.234. Tsala GN. . Plant Cell. Two dimensional gel analysis of carrot somatic embryogenesis proteins. In vitro induction of multiple shoots.6:50–54. Article no. Tambong JT.1-13. Charles WB. Agyir-Sackey KE. Garton S. Sung ZR. FAO. Singer S. cocoyams. Chemical composition and effect of processing on oxalate content of cocoyam Xanthosoma sagittifolium and Colocasia esculenta cormels. Omokolo ND. Elman C. 8. Food Chem. (eds. Debergh PC. Zimmerman RH. Erlenmeyer flasks and test tubes did not prove to be the best culture vessels. Stirn S. Production of multiple shoots. Paris. 3. Murashige T. London. This study provides additional evidence of somaclonal variation in these regenerants. 10. C R Acad Sci. Plant Genetic Resources Newletters. CONCLUSION Evaluation of stomatal number showed that cocoyam leaves have few stomates on both abaxial and adaxial surfaces with fewer on the adaxial surface.112:49-54. Sciences de la vie/Life Sciences. 12. Onwueme IC. 2015. Micropropagation. High levels of epicuticular wax found in vitro may have contributed to reduced transpiration rates. 1992. Isolation and pathogenicity of rhizosphere fungi of cocoyam in relation to the cocoyam root rot disease. Read PE. ables/world. cassava. Available: http://www. Archives of Virology. Rep.Sama et al. A revised medium for rapid growth and bioassays with tobacco tissue culture. plant generation and tuberization from shoot tips of cocoyam. Xia X. 2003. Variability and germplasm loss in the Cameroon national collection of cocoyam (Xanthosoma sagittifolium Schott (L.21:739-46. more rapid than the rate of wax formation.318:773–778. 4. 1997.318:773-778.15:473–497.135:265–273. Plant Mol. Plant Physiol. Kluwer Academic Publishers. AJEA. The culture derived plants should be grown in the field under normal conditions to evaluate trueness-to-type.). FAO Plant Production and Protection Paper 126. 105 Sefa-Dedeh S. 6. 16. 1962. AJEA. Balange AP. 9. Evaluation of stomatal number showed that cocoyam leaves have few stomates on both abaxial and adaxial surfaces. Plant Cell Rep. The tropical tuber crops: Yams.146:1821-1829. 14. Further investigations on physiological parameters will be beneficial to understand the effect of altered leaf structure on plant growth and abnormal plants. 1987. Skoog F.pdf. Micropropagation. Wutoh JG.53:127134. 1991. The reduced amounts of EW on acclimatized plants could be attributed to the rapid cell enlargement in expanding leaves. 11.97-117. Onwueme IC. Longmans.ers. Tissue and Organ Culture. Gal-On A. In: Debergh PC. Gaba V.. Sama AE. Balange AP. Ndoumou DO. Tsala NG. 2001. K.)). Tambong JT. A relatively high humidity (60-80%) is required for approximately two weeks to prevent leaf injury resulting from wilting and desiccation. 13.usda.011 5. 15. Plant regeneration from cocoyam callus derived from shoot tips and petioles. Kanmegne G. Wutoh JG. Ananthakrishnan G. callus.

2015. 33. Anisuzzaman M. Miller RA. AJEA.50:151-158. Zorzoli R. Plantlet development through somatic embryogenesis and organogenesis in plant cell cultures of Colocasia esculenta (L. Watanabe KZ. Ezura H. .4:18-21.C. Chung W.25. 2002.). Exp. 22. Asian Journal of Agricultural Sciences. Khaleque MA. Zhao Y. 2006. Leaf anatomy of red raspberry transferred from culture to soil.) var. bras. Ojima K.18(5):1037-1048. Am. 2010. Valverde JM.164:413-418.2:47-50. Unexpected susceptibility of novel breeding lines of European rhubarb (Rheum rhaponticum L.) to leaf and petiole spot disease. Electronic Journal of Biotechnology 2010. 2008. 35. Bader SM. Nestares G. 2010. 39. 27. Plant Cell Rep. Grout BWW. Verma VM. Boykin LS. 2007. African Journal of Biotechnology. Vidaver WE.57:1123–1129. 18. agropec. 34. 37. Histology of organogenesis from callus cultures of black pepper (Piper nigrum L. Sujatha R. Acta Hort. 106 organogenesis. In vitro plant regeneration system for common bean (Phaseolus vulgaris): effect of N6benzylaminopurine and adenine sulphate. 2003. Cary. Plant Cell Rep. Alam I. Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch. Acta Hort.616:301–308. 28. Mol. The Open Plant Science Journal. Gamborg OL. Biol. Pyramid from mature cotyledons and embryos. SAS Institute. Int.6:861-867. 31. Krug MGZ.). Indirect organogenesis in summer squash (Cucurbita pepo L. 21.7:1145-1150. Sancak C.109:172176. Current Science. Journal of Plant Nutrition. Stipp LCL. 2003. Article no. Agric. Zhang XS. JIRCAS Working Reports. N. 2004.) Schott. Agric. Sharmin SA. 19.4:351–354. 1984. Uranbey S.18:167-170. Effect of thidiazuron on shoot regeneration from different explants of lentil (Lens culinaris Medik. J. Sarabi B. Crisp P. 2010. Challenges in biotechnology for abiotic stress tolerance on root and tubers. 2005.2015. 1985. Babu LC.40:861-865. In vitro response of different explants on callus development and plant regeneration in groundnut (Arachis hypogeae L. 1995. ActaHort. 5(2): 94-108. De Langhe E.) shoot tips. 36.. Plant Science. Soc. Brasília..31:63-70. Mendes BMJ.011 17.). For. Australian Journal of Agricultural Research. J.) via 29. Lee YK. Phenolic changes during in vitro organogenesis of cotton (Gossypium hirsutum L. 20. Mayor ML. Alam AKMM. 2010. 2006. AJEA. Micropropagation of date palm (Phoenix dactylifera L. Ozyigit II. Lodhi MA. 25. A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (Banana and plantain). explant type and nutrient medium components on canola (Brassica napus L. Pesq. AsPac J.. Inadvertent selection for unwanted morphological forms during micropropagation adversely affects field performance of European rhubarb (Rheum rhaponticum L.1:1-4. 2004. 2004.28:421426. yield and nutrient uptake of taro grown under upland conditions.736:213224.). Sci. Pal SP. Chardon U. Melara MV. Nutrient requirements of suspension cultures of soybean root cells. Banerjee N. Gatica Arias AMG. Crisp P. Analysis for combining ability in sunflower organogenesis-related traits. 38. Donnelly DJ. An YR. 2007. 26. Sarker KK. 32. Indirect organogenesis is useful for propagation of Iranian edible wild asparagus (Asparagus officinalisL. Hort. Nazeem PA.41:16-19. Li XG. 1968.75-83.) shoot in vitro organogenesis. Turk. In vitro organogenesis in watermelon cotyledons.] cv.13:1-8. Fonseca PR. Maktoom through direct organogenesis. Joyner EY. Goenaga R.). Grout BWW. 2007. Zhao Y.Sama et al. Growth. J. Cell Res. Almasi K.85:12. Khawar KM. Kamal GB. Hazarika BN. Su HY. SAS Institute. 2010. Expt. Andrés M. 24. Journal of Tropical Agriculture. Rodriguez APM. 2003. 30. Pistil induction by hormones from callus of Oryza sativa in vitro. Cho JJ. Khierallah HSM. African Journal of Biotechnology. Biotechnol.637:139–144. Acclimatization of tissuecultured plants. Effects of genotype. 23. Alam MF. SAS/STAT user’s guide. Asadollah A. Turk J Bot. Illich KG.23:448–452.). Callus induction and organogenesis in soybean [Glycine max (L.) Merr. Zcan S. 40. Picardi L.

Stomatal and cuticular water loss from apple. Plant Sci. 61. Hennen G. Article no. Sci. In: J. Lett. Cult. Caveness FE. Comparative water loss from leaves of Solanum laciniatum plants cultured in vitro and in vivo. Bot. Berttel RIS. 59. 52. Variation in morphology and disease susceptibility of micropropagated rhubarb (Rheum rhaponticum) PC49.43:179–188. Wardle K. Belcher AR. AJEA. 1986.33:618-622. Clark CS. Quinlan A. Biophys. PlantMol. J. Brainered KE. 65. Plant Cell Tiss.2015. Somaclonal variation: The myth of clonal uniformity. Fabbri A.82:357–361.3:73–112. 55. Colombia. Scanning electron microscopy of in vitro-cultured Liquidambar styraciflua plantlets during acclimatization. Amer. 44. Hort. Lett. Peter KV. Lessire R. Hahn SK. Plant Cell. 2004. Soc. Short KC. Sutter E. Tanner GJ.S. Dami I. Spindler LH. Am. Cassagne C. New York. Water loss and water transfer related to changes in leaf wax and to xylem regeneration. Duncan RR. Intl. Tissue culture-induced variation and crop improvement. Springer-Verlag.S. Variations among somaclones and its seedling progeny in Capsicum annum. Hort. Sci. I. J. Scientia Hort. Sommer HE.19:85–89. 1977. 1989.5:401-405. 62. CIAT. and cocoyam production. 2015. Acclimatization of aseptically cultured apple plants to low relative humidity. Studies on alkane biosynthesis in the epidermis of Allium porrum L. J. 1991. Banks PM. Cali. Fuchigami LH. Crisp P. 1979. Fuchigami LH. Ann.76:261–267. Tissue Organ Cult. 47. Kaeppler SM. Genome. 58. Sci. Acta Bot. In: Hohn B and E.215–245.) Plant Gene Research: genetic flux in plants.16:173-175. 1997. . Epigenetic aspects of somaclonal variation in plants. Soc. leaves. Environment influences anatomy of stomata and epidermal cells in tissue cultured Rosa multiflora. Am. Carulla C. 1990. Kaeppler HF. Leaf anatomy and water stress os aseptically cultured pixy plum grown under different environments. 63. Transplanting of cauliflower plants regenerated from meristem culture. CRC Crit. Res.115(1):141–145. Tissue Organ Cult.. J.). Oglesby RP. 1983. 53. Wetzstein HY. AJEA. 56. Conner LN. In vitro acclimatization of aseptically cultured plantlets to humidity. 46. Soc. Capellades M. An assay to measure the extent of variation in Begonia micropropagated plants of hiemalis. Grout BWW. Dobbs EB. Zhao Y. 1985. 1983. Sweet potato. 1989. Establishing tissue cultured plants in soil. var. 1975. 45. Hort. Langhans RW.108:475-480. Plant Cell. J. Kwiatkowski S. Ruan SA. Hort Sci. Soc. Hort. botrytis regenerated through apical meristem culture.17:1-7. Anu A. Plant Sci. Soc. 2005. yam. Brainered KE. Cock (ed. In vitro acclimatization of tissue cultured grape (Vitis sp. 57. 107 Larkin PJ.113:234-238. 49. Colorado State University. J. 1979. Thesis. Fontarnau R. Wardle K. Plant Prop. Sci. Wax development of leaf surfaces of Brassica oleracea var.108:386-389. 2000. 60. From somatic variation to variant plant: mechanisms and application. 64. 54. Plant Sci. Adv. 51. Global Workshop on Root and Tuber Crops Propagation. Stomata of apple leaves cultured in vitro. Biochem.106:515-518. 48.22-31. compared to conventional plants.36:241-246. Hort. Org. 1981. 1984. and sweet gum plants after removal from in vitro culture. Sci.Sama et al. 1981. M. Anatomical changes in persistent leaves of tissue-cultured strawberry plants after removal from culture. 43. 1990.165:274-280.31:705–711. Simpkins I. Aston MJ. Cytogenetics of plant cell and tissue cultures and their regenerates. ‘Valiant’) plantlets.39:145– 151. Davies PA. Proceedings of a regional workshop held in California. Amer. Agron. Bhati R. Grout BWW. 1988. Hort. Griffis Jr JL. Soc. Proc.104:493-496. Bouman H. Comb. currawong regenerated from meristem culture. cherry. 1983.43:745752.58:201–240. Scowcroft WR. Alvarez MN. D’Amato F. TerBrugge J. Dunston SJ. Hort. Soc. Grout BWW. 1986. 1985. Sci. Amer. Sutter E. Blanke MM. 13-16 September. Conner AJ. 5(2): 94-108. Debergh P. Babu KN. Scowcroft WR. Sutter E. Rhee Y.. 42. Dennis (eds. Epicuticular wax formation on carnation plantlets regenerated from shoot tip culture. 1983. Amer. Arch. 50.011 41. De Klerk GJ. Neerl. 1974. Abscisic acid and the regulation of water loss in plantlets of Brassica oleracea L. Biol. Rev.28:331-337.

Zandvoort EA. Boudjeko T. FAO plant macabo cocoyam germplasm in production and protection paper 87. Aston MJ. Dhaliwal HS. Millam S. 72. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. anatomy of cauliflower plantlets 74. Nzietchueng bananas in developing countries: S. Tropical Root and Tuber Wutoh JG. Hawaii. Microtuberization in potato (Solanum Agronomie. Root and tuber crops. In: L. In: Tropical root Omokolo ND. sucrose. Withers development of micropropagated and P.. 2015. Sci Horti. Alderson (eds. Gilbert PC.42:993-995. Omokolo ND.6:28–31. J Cam Tambong JT. sagittifolium L Schott (Cocoyam).98:337–345. Branchard M.). Mbouobda HD. Spence JA. tuberosum L.). Onokpise OU. Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain. Plant Cell Tiss Organ Cult. Schott). Butterworths.011 66.1016/s0304-4238(03)00066-9. Growth and development of 75. Effect of and tubers crops tomorrow 2. Agueguia A. Tsafack TJJ. Plant Cell Rep. Grout BWW. 70. Proceedings photoperiod and thermoperiod on nd of the 2 International Symposium on microtuberization and carbohydrate levels in Cocoyam (Xanthosoma sagittifolium L. 2009. in Cameroon.2015. provided the original work is properly cited. Article no. G.17:794–798.212:329-334. In vitro conservation of Bot. Staritsky G. 1985. Acta Hort. Tambong JT. AJEA. Tsafack TJJ. Onokpise OU. on news crops and news uses. Boudjeko T.394–396. Meboka MM. Mebeka MM. Grout BWW. Modified leaf Press. Effect of nitrogen nutrition on 1993. which permits unrestricted use. photoperiod. Ndzana X. Wilson JG.277-283. regenerated from meristem culture. 1987. Ashs 67. and reproduction in any Plantains and Nyochembeng L. Minocha JL. A. Acclimatization and flower induction of tissue culture derived cocoyam 77. 2004. 1992. Cameroon. Ann.0). Louwaars NP.55:129–131. Roberts AV. Wutoh JG. Bot. phytohormones. (Xanthosoma sagittifolium Schott) plants. 1970. In vitro tuberization of Xanthosoma chrysanthemum plantlets to humidity. In: Janick J (ed) Perspectives Italy. Evaluation of Challenges and opportunities. Tsafack TJJ. sagittifolium (L.) Schott: Effects of 71. Borns M. Short KC. Onokpise OU. 5(2): 94-108.4:337–344. in vitro tuberization of Xanthosoma 73. Eyango AS. tannia (Xanthosoma sp. Germplasm collection of macabococoyams 78.php?iid=665&id=2&aid=6075 108 . 1999. Honolulu. Omokolo ND. AJEA. 1998. 1986. Photosynthetic and under osmotic stress. Alexandra VA USA.12:193-199. Gopal J. culture and its agricultural applications. Ann. DOI:10. Dekkers AJ. Warburton J. London. distribution.47-52. 1978. Plant tissue strawberry plantlets following transplanting.. Rome. 2003. nitrogen and Nyochembeng L. FAO. Sama AE. aroid germplasm at reduced temperatures 68. _________________________________________________________________________________ © 2015 Sama et al. 69. Forum. In vitro hardening of cultured cauliflower and 76. African Tech.96:151-159. Hourmant A. Acad Sci.).Sama et al. 1988.