Propagation of Ornamental Plants Vol. 5, № 3, 2005: 156-161 A WOUNDING METHOD AND LIQUID CULTURE IN PAPHIOPEDILUM DELENATII PROPAGATION Duong Tan Nhut1*, Phan Thi Thuy Trang1, Nguyen Hong Vu1, Dang Thi Thu Thuy1, Dinh Van Khiem1, Nguyen Van Binh1 and K. Tran Thanh Van2 1 Dalat Institute of Biology, 116 Xo Viet Nghe Tinh, Dalat, Lam Dong, Vietnam, *Tel. +84-63-831056, *Fax. +84-63-831028, *E-mail: 2 Institut de Biotechnologie des Plantes, Université de Paris-Sud, UMR 0569,
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  156 Propagation of Ornamental PlantsVol. 5,   № 3, 2005: 156-161  Received: June 16, 2005 Accepted: August 15, 2005 INTRODUCTION  Paphiopedilum is a terrestrial orchid genus, whichgrows from the Himalayas in Southeast Asia to Papua New Guinea (Teoh 2005). The name of this genus is de-rived from the Greek word “pedilon”, meaning “sandal”or “slipper”, giving rise to the common name “slipper orchid” (Sheehan 2001).  Paphiopedilum delenatii ,an endangered species recognized and protected bythe Convention on International Trade of EndangeredSpecies of Wild Flora and Fauna (CITES), has been afavorite potted plant for centuries due to its attractivecolor and distinctive shapes. However, the increasingmarket demand, together with the low multiplication rateof conventional propagation methods has endangered thesurvival of this unique orchid. Consequently, plant celltissue culture through which a large numbers of plantletscan be obtained within a short period has become an idealsolution for preserving this genus from extinction.Though  Paphiopedilum is a high-value potted plant,the success of   Paphiopedilum micropropagation is rela-tively limited due to the difficult bacterial and fungaldecontamination of  ex vitro -derived explants and the poor development of explants that survive under  in vitro conditions (Stewart and Button 1975, Huang 1988).Different methods for   Paphiopedilum micropropagionhave been reported, including axillary shoot inductionand plantlet regeneration from shoot tips (Huang 1988)and shoot multiplication from seedlings (Huang et al.2001). Recently, plantlets were successfully regeneratedthrough protocorm-like bodies (PLBs) from totipotentcalli (Lin et al. 2000), and direct shoot bud formation wasobtained from in vitro leaf explants (Chen et al. 2004).In this paper, the appropriate medium for  in vitro germination of   P. delenatii seeds was determined.Moreover, a practical method for   P. delenatii propagation by wounding the seedlings bases in combination withliquid culture was established. MATERIALS AND METHODS  Plant materials Pollen grains were manually dropped onto the stig-mata of   Paphiopedilum delenatii flowers in the green-house of the Dalat Institute of Biology. Three-, six-, or nine-month-old fruits were collected for the germinationexperiment.  In vitro six-month-old seedlings, 2.5 – 3.0cm in height, were used as explants for shoot induction by a wounding method.  Nutrient media Knudson C (Knudson 1946), basal MS (Murashigeand Skoog 1962), and 1/2 MS (half-strength MS A WOUNDING METHOD AND LIQUID CULTURE IN  PAPHIOPEDILUM DELENATII  PROPAGATIONDuong Tan Nhut 1 *, Phan Thi Thuy Trang 1 , Nguyen Hong Vu 1 , Dang Thi Thu Thuy 1 , Dinh Van Khiem 1 ,Nguyen Van Binh 1 and K. Tran Thanh Van 2 1 Dalat Institute of Biology, 116 Xo Viet Nghe Tinh, Dalat, Lam Dong, Vietnam,*Tel. +84-63-831056, *Fax. +84-63-831028, *E-mail: 2 Institut de Biotechnologie des Plantes, Université de Paris-Sud,UMR 0569, F-91405 Orsay Cedex, France. Abstract An innovative method for mass shoot propagation of   Paphiopedilum delenatii (‘Pink’ slipper orchid), anendemic and endangered species of Vietnam, was established in this study. Nine-month-old seeds culturedon Knudson C medium was the optimal condition for  in vitro germination. Seeds began swelling and becamegreenish in color after 30 to 40 days of culture. Then, rounded bodies, known as protocorm, were observed.Leaves and roots developed after 90 – 100 days of subculturing on the same medium. The combination of wounding and liquid culture was shown to be appropriate for highly frequent adventitious shoot formation.Shoot regeneration rate of 5.2 per wounded seedling could be obtained in MS liquid media supplemented with1.0 mg l -1 TDZ via this method. Key words: germination, liquid culture,  Paphiopedilum delenatii , TDZ, wounding  157medium) media were used for the experiment on thegermination of   P. delenatii seeds.Basal MS medium supplemented with differentconcentrations of Thidiazuron (TDZ 0.5, 1.0, or 3.0 mgl -1 ) or TDZ (0.25, 0.5, 1.0, or 2.5 mg l -1 ) in combinationwith 0.5 mg l -1 α-naphthaleneacetic acid (NAA), 20 gl -1 sucrose, and 8 g l -1 agar (for solid media only) wereused for investigating the shoot regeneration on woundedseedlings.Basal MS medium supplemented with 0.5 mg l -1   NAA, 2.0 mg l -1 6-benzyladenine (BA), 1 g l -1 activatedcharcoal (AC), 20% (v/v) coconut water, 30 g l -1 sucrose,and 8 g l -1 agar was used as a fresh medium for subcultur-ing shoots from wounded seedlings.The rooting medium was a basal MS medium sup- plemented with 0.5 mg l -1 NAA, 1 g l -1 AC, 10% (v/v)coconut water, and 25 g l -1 sucrose.The pH of all media was adjusted to 5.8, prior to theaddition of AC and agar. Seventy ml of solid mediumor 30 ml of liquid medium were added in each 250-mlErlenmeyer flask covered by a two-layer polyethylenecap before autoclaving at 121ºC, 1 atm for 20 minutes.A 2 x 2 cm piece of filter paper (OSI, France), whichacted as a support keeping the explants from completelysubmerging in the medium, was added in the liquid-containing flask.  Effect of medium and seed age on germination rate  P. delenatii fruit at different ages (Fig. 1a, 2a) weresterilized by rubbing the entire capsule surfaces in 70%(v/v) ethanol and dipping in 0.1% (w/v) HgCl 2 for 10min, before rinsing thoroughly with sterile distilled wa-ter. Seeds were then released by longitudinally splittingcapsules in sterile conditions using a sterilized scalpel,and evenly spread on nutrient media (Fig. 1b).  Effect of wounding on shoot regeneration in solid media Roots of six-month-old in vitro seedlings (Fig. 2b)were carefully removed and the seedling bases were pierced three or four times using a sharp needle, whosediameter was 0.3 mm (Fig. 1m, m 1 ). Three woundedseedlings were then placed onto one media-containingflask (Fig. 1m 2 ’, m 2 ’’). Three non-wounded seedlings (per flask) were placed onto the same media as a control.  Effect of wounding method on shoot regeneration inliquid media The experiment was conducted in the same way asdescribed above, but solid media was replaced by liquidmedia (Fig. 1m 1 ’, m 1 ’’). Each explant was placed in thecentre of the piece of filter paper.  Acclimatization Individual shoots formed at the wounded region of seedlings were separated before subculturing to freshmedia. Vigorous shoots were then transferred to rootingmedium (Fig. 1e). Rooted plantlets were obtained after 3 months of culture (Fig. 1f), and then acclimatized inthe greenhouse. Fig. 1. Diagram of the wounding and liquid culture used for shoot regeneration of   Paphiopedlium delenatii cultured in vitro a) Nine-month-old fruit of   P. delenatii , b) Seeds spread in vitro , c) Three-month-old seedlings, d) Six-month-old seedlings, e, f) Nine-month-old seedlings, g) Two-year-old plantlet flowering in greenhouse , m, m1) Wounded plantlets, m1’, m1’’)  Wounded plantlets cultured in liquid medium, m2’, m2’’) Wounded plantlets cultured in solid medium.  Duong Tan Nhut et al.  A wounding method and liquid culture in Paphiopedilum delenatii propagation  158 Culture conditions All in vitro cultures were placed at 25 ± 2 o C, 70 – 80% relative humidity and a 12-hour photoperiodat 45 μmol m -2 s -1  photosynthetic photon density flux,which was provided by fluorescent tubes (Rang Dong,Vietnam).Rooted plantlets were planted on styrofoam trayswith a fern substrate and placed under natural light/dark circle of sunlight at 27 ± 3 o C and 75 – 85% relative hu-midity. Plantlets were placed on shade in the greenhouseand were softly watered every 2 days with tap water.  Statistical analysis Each treatment was conducted with 12 flasks. Seedgermination rate, shoot survival rate and the number of shoots per explant were recorded after 3 months of  Fig. 2. Wounding method and liquid culture effects on  Paphiopedilum delenatii propagation. A) Flowers and fruits of   P.delenatii cultured in the greenhouse, B) Six-month-old in vitro seed-derived plantlets, C) Shoots regenerated from woundedseedlings in liquid medium, D) Shoot cluster regenerated from wounded base of  in vitro P. delenatii after 3 months of culture,E) Shoot cluster developed from wounded base of  in vitro P. delenaii on fresh medium after 3 months, F)  In vitro rooted plantlets after 3 months.Propagation of Ornamental PlantsVol. 5,   № 3, 2005: 156-161 ACEFDB  159culture. Data were analyzed for significance by Duncan’smultiple range test (Duncan 1995). RESULTS AND DISCUSSION  Effect of medium and seed age on germination rate In this experiment, the highest germination rate(90.1%) and the most vigorous seedlings were obtainedon nine-month-old seeds cultured on Knudson C medium(Table 1). After 30 to 40 days of culture, seeds beganswelling and became greenish in color. Then, rounded bodies, known as protocorm, were observed. Leaves androots developed after 90 – 100 days of subculturing onthe same medium. It has been reported that immatureseeds germinate better than mature ones, due to their distended testa cell and metabolically awakened embryos(Linden 1980). Mature seeds, in contrast, are recalcitrantto germination primarily due to the change in the qualityof their food reserves and the dormancy or inhibitoryfactors that are present inside their cells. However, veryyoung orchid ovules are not appropriate for germina-tion because they may need time for organogenesis or synthesis of nutrients that help embryos recognize thestimulating agents, which are present in the mediumand germinate. Young seeds from 6-month-old fruitsdeveloped rather slowly on Knudson C. Seed-derived protocorms were hard to recognize and no seedlings wereobtained from 3-month-old seeds in this treatment. Theseresults suggest that the appropriate age for collecting  P.delenatii fruits is 9 months.Orchid seeds in natural environments have a poor nutrient supply even at maturity. Hence, they requirea suitable stimulus by a fungus which is believed toaugment the carbohydrate, auxin, and vitamin transportfor their germination in nature (Arditii et al. 1982).Knudson (1922) demonstrated that the fungal require-ment of orchid seeds can be bypassed during in vitro germination by adding an appropriate carbohydrate inthe culture medium. Since then, many nutrient mediafor orchid seed culture have been proposed, includ-ing Knudson C, MS and Morel (Morel 1964). In thisstudy, MS medium seemed inappropriate for germina-tion of   P. delenatii seeds. MS medium may inhibit  P.delenatii germination due to its high nutrition content.Decreasing the micro- and macro-organic, as well asthe inorganic components in the MS medium may havea positive effect on seed germination (Table 1). Thenutrition-poor Knudson C medium was determined to be appropriate for germination of   P. delenatii seeds inthis study.  Effects of wounding method on shoot regenerationin solid medium As shown in Table 2, no shoot formation was ob-tained in control treatments with non-wounded shootsin all kinds of media. The highest survival rate was MediaSeed age (months)Germination rate (%)Knudson C3068.8990.1MS99.01/2 MS73.0 Table 1. Effect of medium and seed age on germinationrate. recorded from wounded seedlings cultured on MS solidmedium supplemented with 0.5 mg l -1 TDZ, withoutexternal auxin. An average of 2.3 green and vigorousshoots were obtained on medium containing 0.25 mgl -1 TDZ and 0.5 mg l -1 NAA. There is no doubt that thewounding step is a prerequisite for shoot formationin seedlings. Wounded cells at the damaged site may be responding to stimulating agents (e.g. certain plantgrowth regulators (PGRs)) that are present in nutrientmedia by differentiating into novel organs, such asadventitious shoots. No shoot formation was recordedon non-wounded seedlings in control treatments, sug-gesting that intact cells of these seedlings were notaffected by the stimulators.Explant differentiation depends significantly on me-dium components including minerals, nutrients, sugar,vitamins, and PGRs. TDZ, a non-purine, cytokinin-likecompound has been shown to exhibit stronger effectsthan any other conventional cytokinin over a wide rangeof species and to be effective for shoot proliferationand adventitious shoot organogenesis (Huetteman andPreece 1993). Тхе mode of action of TDZ may be at-tributed to its ability to induce cytokinin accumulation(Victor et al. 1999) and enhance the accumulation andtranslocation of auxin within TDZ-exposed tissues(Murch and Saxena 2001). Therefore, TDZ promotesthe induction of shoot formation of wounded seedlingsdue to its optimal characteristics. TDZ, when supple-mented to the medium at an appropriate concentration,enhanced shoot formation. TDZ was applied success-fully for   Paphiopedilum micropropagation in several Table 2. Effect of the wounding method on shoot regenera-tion in semi-solid medium after three months of culture. PGRs (mg l -1 )Survival rate (%)Shoots per explantTDZ NAA0.250.5752.3 a y 0.50.5751.2 c1.00.57502.50.55000.5-852.1 a1.0-801.1 c3.0-801.6 b y Different letters within a column indicate significant differencesat α = 0.05 by Duncan’s multiple range test.  Duong Tan Nhut et al.  A wounding method and liquid culture in Paphiopedilum delenatii propagation
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