Celosia Argentea Var. Cristata) Kitti Bodhipadmaa, Sompoch Noichindaa, Intira Yadbuntunga, Waranya Buaeiama, David W.M

S HORT REPORT ScienceAsia 36 (2010): 68–71 doi: 10.2306/scienceasia1513-1874.2010.36.068 Comparison of in vitro and in vivo inflorescence of common cockscomb (Celosia argentea var. cristata) Kitti Bodhipadmaa, Sompoch Noichindaa, Intira Yadbuntunga, Waranya Buaeiama, David W.M. Leungb,∗ a Department of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangsue, Bangkok 10800 Thailand b School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand ∗Corresponding author, e-mail: [email protected] Received 7 Jul 2009 Accepted 17 Dec 2009 ABSTRACT: Inflorescence of common cockscomb (Celosia argentea var. cristata) was induced in vitro from plantlets regenerated from nodal explants cultured for 5 weeks on semi-solid basal MS medium supplemented with or without 0.5 mg/l benzyladenine under aseptic and light conditions. In vitro inflorescence of 12-week-old plantlets was compared with that formed under natural conditions. Florets from both sources had the same number of floral parts and exhibited similar characteristics (floret shape and percentage of pollen germination). However, the florets formed in vitro were obviously smaller than those formed under natural conditions. KEYWORDS: in vitro flowering, pollen germination, pollen morphology, sexual reproduction INTRODUCTION inflorescence obtained from both sources of plant in the family Amaranthaceae have never been evaluated. Common cockscomb (Celosia argentea var. cristata) Therefore, in this study, in vitro and in vivo inflores- is a herbaceous plant and has been classified as a cence of common cockscomb were investigated and member of the family Amaranthaceae. This annual compared for the first time to ascertain if the in vitro plant has a distinctive characteristic inflorescence inflorescence of common cockscomb would retain the which looks like the crest of a rooster or convoluted same dazzling features and potential for pollination as brain after it has developed fully 1. Owing to the the in vivo ones. dazzling and attractive inflorescence colour, it is a popular ornamental plant in all regions of Thailand. MATERIALS AND METHODS Additionally, this plant could produce some useful Seeds of common cockscomb, Celosia argentea var. chemicals such as antiviral proteins, betalains, and cristata, were purchased from TSA Ltd., Bangkok. anthocyanin 2–4 that can be applied in many beneficial Surface-sterilization of seeds began with immersing ways. the seeds in distilled water for 3 h and then soaking In vitro flowering has been reported for different for 10 min in 15% (v/v) Clorox (a commercial bleach kinds of flower morphology: solitary in Capsicum solution containing 5.25%, w/w, sodium hypochlorite annuum 5 and rose 6, or inflorescence in Amaranthus 7 as available chlorine) to which 2–3 drops of Tween-20 and bamboo 8, or even complex inflorescence as in were added. After this, they were rinsed 3 times with Chicory 9 and Pentanema indicum 10. To explore the sterile distilled water (5 min for each rinse) before potential practical applications of in vitro flowering, they were placed on basal MS medium 14 and kept in for example, for breeding valuable plants under con- a growth room for 3–4 weeks under 16 h illumination trolled, high-health conditions, an important prereq- from white fluorescent lamps (44.57 µmol m−2 s−1) uisite is to investigate if the flowers formed under in and 8 h darkness at 25 ± 2 °C. To increase the number vitro conditions are basically free of gross aberrations of nodes, shoot cuttings (1 cm long) were excised when compared to those formed on plants grown in from the axenically grown seedlings and placed on soil. Henceforth the former will be referred to as in plant growth regulator-free MS medium. After the vitro flowers and the latter as in vivo flowers. In vitro shoots had grown for 3–4 weeks, 3 to 4 nodal explants and in vivo flowers have previously been studied and (each 0.8 cm long) were excised and transferred to MS compared to only a limited extent 11–13. Furthermore, medium supplemented with 0 or 0.5 mg/l benzylade- www.scienceasia.org ScienceAsia 36 (2010) 69 nine (BA). Table 1 Comparison of size between in vivo and in vitro After inflorescence had developed on the 12- inflorescence of common cockscomb (Celosia argentea var. week-old in vitro plantlets, they were investigated and cristata). compared with those from pot plants obtained from Sources Inflorescence size (cm) a local market in Bangkok at the same age (lower part of ex vitro inflorescence had started producing width length seeds). Floret morphology was examined under both a In vivo 1.51 ± 0.14a 3.87 ± 0.34a stereomicroscope (EMZ-TR, Meiji Techno Co., Ltd.) In vitro MS 1.00 ± 0.11b 1.22 ± 0.25b and a compound microscope (ML2000, Meiji Techno In vitro MS + BA 0.96 ± 0.11b 1.19 ± 0.20b Co., Ltd.) and photographs were taken using a digital Values are means of 10 replications ± SD. Data marked camera (Olympus C-760). by same letter in a column are not significantly different 15 The modified medium of Mercado et al consist- (ANOVA, P < 0.05). ing of 0.1 mM boric acid, 1 mM calcium chloride,231 232 and 20% (w/v) sucrose was used for both in vitro and233 234 in vivo pollen germination tests. This medium was235 adjusted to pH 5.7, gelled with 0.8% (w/v) agar, and236 237 autoclaved at 121 °C and 15 psi for 20 min before238 it was poured into Petri dishes (80 mm diameter).239 240 Anthers in the in vitro and in vivo florets at anthesis241 242 from five different inflorescence were collected and243 placed on the pollen germination medium. Pollen244 245 grains from the dehiscent anthers were then left over246 247 Fig.Fig. 1 Appearance 1 Appearance of in vivo of (left), in vivoin vitro (left), MS (m iniddle) vitro and MSin vitro (middle) MS + BA and(right) the surface of the medium and incubated at 25 ± 2 °C248 inflorescence on plant and plantlets of common cockscomb. in a dark room. Germination of pollen from both249 in in vitro MS + BA (right) inflorescence on plant and plantlets 250 of common cockscomb. vitro and in vivo florets was examined under a light251 microscope (ML2000, Meiji Techno Co., Ltd.) over252 231253 a period of 24 h. At least 3 fields in each Petri dish232254 233255 inflorescence (Table 1 and Fig. 2). However, callus were examined to count pollen grains with or without234256 was formed on the cut end of the nodal explants pollen tubes and pictures were taken using a digital235257 236258 placed on MS medium supplemented with 0.5 mg/l camera (Olympus C-760). 237259 BA and the number of roots formed on this medium 238260 RESULTS AND DISCUSSION 239261 was also obviously less than that formed on plant 240262 growth regulator free-medium (Fig. 3). 241263 In vitro flowering in common cockscomb (Celosia242264 Both in vitro and in vivo florets have the same 265 argentea var. cristata) was first reported by Yamada243 number of bracts, tepals, stamens, ovaries, and styles 16 244266 et al . This was shown in various cultures including245267 (2, 5, 5, 1, and 1, respectively). Various parts of the seedlings (on MS basal medium), shoot apices (on246268 247269 Fig. in 1 vitroAppearance florets of in vivo were (left), generallyin vitro MS (middle) found and in to vitro be MS smaller + BA (right) 1/2 MS basal medium), nodal segments (on 1/2 MS248270 inflorescenceFig.than 2 Close-up those on viewplant of andof in in plan vivo vivotlets (left), florets, of common in vitro except cockscomMS (middle) theb. and ovary in vitro length MS + BA 249271 (right) inflorescence on plant and plantlets of common cockscomb. basal medium), and leaf segments (on MS medium250272 (Table 2). This suggests that development of in vitro supplemented with 3 mg/l kinetin and 0.3 mg/l IAA).251273 252274 florets proceeded in the same way as under natural In the present experiments, it was found that induction253275 of common cockscomb’s inflorescence in vitro from254276 255277 plantlets regenerating from nodal explants occurred on256278 257279 MS medium with or without 0.5 mg/l BA. This result 258280 confirmed that external cytokinin may not be neces-259 260 sary for in vitro inflorescence initiation in common261 cockscomb. The in vitro and in vivo inflorescence262 263 were of the same shape which was spicate with264 a crested terminal. Furthermore, the present study265 266 revealed that inflorescence of common cockscomb267 268 formed under in vitro and in vivo conditions were269 largely indistinguishable except for the floret size270 Fig.Fig. 2 Close-up 2 Close-up view ofview in vivo of (left), invivo in vitro (left), MS (m iniddle) vitro and MS in vitr (middle)o MS + BA 271 (right)and inflorescence in vitro MS on plant + BAand plantlets (right) of inflorescencecommon cockscomb. on plant and (Table 1 and Fig. 1). Adding BA to the medium had272 no effect on the formation and the characteristics273 of plantlets of common cockscomb. 274 275 276 277 www.scienceasia.org 278 279 280 281 282 283 284 285 286 287 288 289 281 70 ScienceAsia 36 (2010) 282 290 283 291 284 292 Table 3 Comparison of the pollen morphology and germi- 285 293 nation from in vivo and in vitro florets from 12-week-old 286 294 287 plants and plantlets of common cockscomb. PL = pollen 288 295 length in equatorial view; PG = pollen germination. 289 296 290 297 Sources PL (mm) PG (%) 291 298 a b 292 In vivo 0.0226 ± 0.0017 28.0 ± 7.3 299 a b 293 300 Fig.In vitro3 Number MS of root form 0.0219ation± on0.0015 in vitro MS26.9 plantle± 8.5t (left) was higher than in vitro 294 In vitro MS + BA 0.0217 ± 0.0019a 24.8 ± 6.2b 295 301 MS + BA plantlet (right) because of callus forming (arrow) on the cut end of nodal 296 302 segmValuesent.

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