Vanoverberghia sepulchrei Merr. (Zingiberaceae) by rhizome ...

* Corresponding author: [email protected]

วารสารพฤกษศาสตรไทย 8 (1): 111–126. 2559.

THAI JOURNAL OF BOTANY 8 (1): 111–126. 2016.

ABSTRACT. The pantropical Zingiberaceae are considered among the most threatened of the monocotyledonous fl ora in the Philippines as many of its endemic species are now on the brink of extinction. The fl ora includes the monotypic Leptosolena haenkei C. Presl listed under the vulnerable (VU) category of the Threatened Plants of the Philippines and Vanoverberghia sepulchrei Merr., which is under the other threatened species (OTS) category. This study examined the morphoanatomical features of these two species and produced a vegetative propagation scheme by determining the viability of the rhizome explants using 1-naphthalene-acetic acid (NAA) and n-2-furanylmethyl-1H-1-purine-6-amine (Kinetin) as plant growth hormones. They were treated with different concentrations and combinations of NAA and Kinetin. All treatments induced the viability of the rhizome explants. The results revealed that low concentration produced more viable and fairly viable rhizome explants than high concentration of NAA or Kinetin. Also, L. haenkei is said to be more viable to V. sepulchrei in all of the eight treatments, X2 (2, N = 160) = 22.71, p < 0.05, and this is because of many factors such as phenotypic variations among the two gingers and microclimatic factors.

KEYWORDS: endemic, kinetin, Leptosolena, NAA, Philippines, Vanoverberghia, viability

Morphoanatomy and vegetative propagation of the Philippines endemic Leptosolena haenkei C. Presl and Vanoverberghia sepulchrei Merr. (Zingiberaceae) by rhizome cutting using NAA and Kinetin

RUDOLPH VALENTINO ABRENICA DOCOT1, 2, *, MARIANE GLADYS MATIAS GOMEZ1, ROVEN BRYAN COMPERADA AQUINO1, ANTONIO BARONGAN BARRETTO III1, SHINRI MATEL ORIHARA1 & CYNTHIA BOBADILLA MINTU1

1 Department of Biological Sciences, Institute of Arts and Sciences, Far Eastern University, Nicanor Reyes Sr. Street, Sampaloc, 1008 Manila, Philippines2 The Graduate School, University of Santo Tomas, España Boulevard, Sampaloc, 1015 Manila, Philippines

INTRODUCTION

The monotypic and endemic to the Philippines, Leptosolena haenkei C. Presl (Figs. 1A–D), was presumed extinct in the wild for the past century (Kress et al., 2002). The systematics of this ginger was fi nally resolved when Hidenobu Funakoshi and Leonardo Co rediscovered it on Mountain Province, Northern Luzon (Funakoshi et al., 2005). Because of their long narrow corolla tube which is exerted from the calyx for more than half its length and by the absence of fl oral bracts and bracteoles, this ginger is considered exceptional in the tribe Alpinieae, making them a distinct genus in the family (Smith, 1990; Funakoshi et al., 2005). The former monotypic Vanoverberghia sepulchrei Merr. (Figs. 1E–H), also an endemic species, loses its monotypic status when a new species from Lanyu Island, Taiwan, V. sasakiana H. Funak. & H. Ohashi was discovered (Funakoshi & Ohashi, 2000). Moreover, the third species of Vanoverberghia has been already noticed also in Northern Luzon (personal communication with Hidenobu Funakoshi). This genus is also exceptional inside the tribe because of their fi liform lateral staminodes, lateral corolla lobes that are basally connate to each other and to the middle of the labellum, and by the absence of bracteoles (Larsen et al., 1998). Recent phylogenetic analyses revealed that these two genera were nested within the paraphyletic and polymorphic Alpinia eubractea (Kolowratia) clade. It’s composed of species from Pacifi c Islands including the Philippines, Australia and Oceania, and it was proposed by Kress et al. (2007) to have new generic name when apomorphic

characters are circumscribe and complete taxa sampling are done.

Because of the non-stop conversion of their natural habitats into alienable and disposable land, and occurrence of natural catastrophes in the country, it’s not surprising that these gingers are listed in the Threatened plants of the Philippines (Madulid, 1991 & 1996). Leptosolena haenkei is under vulnerable (VU) category, while Vanoverberghia sepulchrei is under other threatened species (OTS) category (Fernando et al., 2008). In elucidation for this alarming depletion of these endemic gingers, this study produced a vegetative propagation scheme using “Macropropagation”, a method of multiplying plants using specialized or non-specialized vegetative parts as alternative to the much expensive “Micropropagation” or tissue culture. The viability of the rhizome explants were interpreted by observing its change in color, appearance, turgidity, and possible bud formation on the nodes after 30 consecutive days using 1-naphthalene-acetic acid (NAA) and n-2-furanylmethyl-1H-1-purine-6-amine (Kinetin) as plant growth hormones. This study also made a morphoanatomical study for additional taxonomic characters and to help understand the result of the propagation.

MATERIALS AND METHODS

Plant collection

Field collections of the specimens were made in Mt. Data, Barangay Alab Oriente, Bontoc, Mountain Province. At least 5 individuals each species were obtained and used for herbarium preparation and spirit

112 Rudolph Valentino Abrenica Docot et al.

specimens. Each plant samples were folded in newspapers and was placed in a polyethylene bag. Denatured alcohol was poured into the bag to prevent abscissic acid activity (Victor et al., 2004), therefore preserving the plants for processing. Ephemeral characteristics such as color, odors etc. were also observed. Since the fl owers of gingers are ephemeral (Kress, 1990), the infl orescence were fi rst fi xed in Formalin Acetic Acid (FAA) for more than 48 hours and were transferred in 70% alcohol for a well preserved spirit specimens.

Morphoanatomical analyses

For morphological analyses, digital image of voucher specimens from different foreign herbaria and the vouchers from Philippine National Herbarium (PNH), University of the Philippines Herbarium (PUH), and our collection deposited in University of Santo Tomas Herbarium (USTH), were used for morphological analyses. For anatomical analyses, transverse section of different parts such as leaves, pseudostem, roots and rhizomes were fi rst fi xed in FAA for more than 48 hours. After fi xing, the matured parts were passed through a graded solutions of ethanol (50%, 70%, 80%, 90%; absolute grade) and xylene (50%; absolute grade). The preserved specimens were then embedded in paraffi n and cut using a rotary microtome. Sections mounted onto slides, stained with Safranin O and counterstained with Fast Green. The sections were sealed with Canada balsam and viewed under light microscopy. A survey interview was also conducted about facts on the economic uses of the two gingers.

Vegetative propagation

Since the rhizome contains the majority of the meristematic cells (Remashree et al., 1997) and are capable of initiating shoots (Leong-Škorničková & Newman, 2015), it was used as the explant. Macropropagation by rhizome cuttings is the most convenient and easiest method and usually preferred (Schmidt, 1993). The gingers were acclimatized for one month to adapt to the new environment. After acclimatization, the rhizomes were removed from the plant and cut into segments which have at least 1–3 nodes. The scales were removed to prevent growth of microorganisms that were lodged into the scales. The rhizome explants were treated with Benlate (fungicide) for a minute to further prevent microorganism accumulation and were washed using distilled water. Finally, the explants were soaked for 3 minutes in different concentrations and combinations of plant growth hormones, namely NAA and Kinetin. Ten replicates of rhizome explants were used in each treatment. Sterilized saw dusts were used as substrate, and it was sprayed with SNAP (Simple Nutrient Addition Program) solution to provide the nutrient sustenance of the explants (Santos & Ocampo, 2005). All explants were sprayed with distilled water once a week for 30 consecutive days. The whole propagation set-up was placed in an improvised small-scale greenhouse located in Sampaloc, Manila, Philippines.

Characterization of rhizome explants

Commercial fertilizer (14-14-14) was used as positive control (C1) because this fertilizer is commonly used in propagating

Morphoanatomy and vegetative propagation oݦ the Philippine endemic 113Leptosolena haenkei and Vanoverberghia sepulchrei by rhizome cutting using NAA and Kinetin

plants (Hoang Hoah, 1992). For the negative control (C2), SNAP solution was used. The results from C1 and C2 were used in creating the criteria for evaluating the rhizome explants: [1] Not viable if the explant has presence of contaminants, color became black or dark brown, and appearance became dry and flaccid; [2] Fairly viable if the explant retained its color, turgidity, and there is absence of contaminants, and; [3] Viable if the explant retained its color, turgidity, there is absence of contaminants, and presence of bud(s) on the nodes. Chi-square test of independence was used in interpreting the results by PASW Statistics v.18 (Allen & Bennett, 2010).

RESULTS AND DISCUSSION

Morphoanatomical Features

Leptosolena haenkei C. Presl in Reliq. Haenk. 1: 11, t.18. 1827. Type: Philippines, Luzon, Haenke s.n. (lectotype PR [digital image!], isotypes PR, PRC).– Alpinia leptosolenia (C. Presl) K. Schum. in Pfl anzenr. IV 46: 312. 1904.– Leptosolena insignis Ridl. in Publ. Bur. Sci. Govt. Lab. 35: 84. 1906.– Leptosolena auriculata Elmer nom. nud. in Leafl . Phil. Bot. 10: 3808. 1939.

Terrestrial herb, 3–4 m, erect. Rhizome fl eshy-brown, aromatic, covered with thin brown scales, 1.5–2 cm wide. Pseudostem green, terete, 1.5–3 cm wide, bulbose at the base. Plane of distichy of leaves perpendicular to rhizome. Ligule whitish, oval, apex rounded, 5–8 × 6–7 mm. Leaves sessile, glabrous on both side, subcoriaceous above, linear, 39–43 × 2.5–5 cm, base cuneate, apex attenuate, margin entire. Inflorescence

terminal panicle, 14–20 cm long, peduncle with 2–4 brown sterile bracts; rachis green, 7–10 cm long; pedicels 3–4 mm long; fl owers snow-white; floral bracts and bracteoles absent; calyx tube green, apex 2–3 dentate, ciliated at the apex, 6.8–7.5 cm long; corolla tube white, glabrous, extremely long and narrow, 11–13.2 cm long; dorsal corolla lobes white, oblong-ovate, apex rounded to obtuse , 1.7–2.6 × 1–1.3 cm; lateral corolla lobes white, oblong–ovate, apex obtuse, 1.4–2 × 1–1.3 cm; labellum white, oblong-ovate, crisped, glabrous, recurved at anthesis, 5.4–6 × 3.3–4 cm; lateral staminodes white, petaloid, ovate, bilobed, apex of lobes acute, 6–7 mm long; fi lament not observed; anther creamy-white, oblong, slightly pubescent, crest small, 8–13 × 5–7 mm; stigma creamy-yellow, cupular, pubescent along the edge of hollow, about 1 mm wide; style white, 14–15 cm long; nectaries 2, yellow, located at the base of style, about 2 mm long; ovary green, glabrous, 3-locular, axile placentation, 6–8 × 4–5 mm. Fruits green, oblong, glabrous and subcoriaceous, calyx remained at apex, 4.5–7 × 3–6 cm. Seeds white, globose or subglobose, about 2 mm wide.

Transverse Section. Leaves. The shape of adaxial and abaxial epidermis is rectangular. Beneath the adaxial epidermis is a layer of hypodermis. The palisade layer is composed of elongated parenchymatous cells, while the spongy layer is composed of irregularly arranged cells where the vascular bundles are found. The vascular bundle is composed of prominent metaxylems and phloem which are composed of sieve tube elements and companion cells. Adaxial and abaxial sclerenchyma girders or pillars


are present. Stomata openings are also observed in the abaxial epidermis (Fig 2B). The midrib is composed of 5 prominent central vascular bundles that are located more abaxially (Fig. 2A). Pseudostem. The vascular bundles are scattered throughout the ground tissue and with greatest abundance on the periphery. Each vascular bundle is composed of large air spaces and metaxylems that are associated with phloem and enclosed by sclerenchyma cells (Fig 2C & 2D). Rhizome. The internal structure of the rhizome is separated by the endodermoidal (intermediate) layer into two distinct regions; the cortex (outer zone) and the stele or central cylinder (inner zone) (Fig. 2E). Numerous vascular bundles are scattering and embedded within the two zones, becoming more abundant in the stele. Vascular bundles located at the cortex are termed as cortical vascular bundles, while in the stele are stellar vascular bundles. They are greatly concentrated near the endodermoidal layer and are composed of thick layers of sclerenchyma cells, phloem and fairly angular metaxylems (Fig. 2G). Root. A multiple epidermis or velamen is observed, and it’s composed of 5–6 layers of rectangular shaped cells. The cortex is composed of isodiametric cells and beneath it, is the endodermis which is composed of cells that has thick cell walls. A multiple layer of thin-walled cells called pericycle is located beneath the endodermis. The vascular bundles are immediately located after the pericycle which has prominent metaxylems and sclerenchymatous cells. The pith is also composed of isodiametric cells (Fig 2F & 2H).

Habitat.― Along open rocky slopes at low altitudes of 300–1,300 masl.

Distribution.― Aurora, Benguet, Cagayan, Ilocos Norte, La Union, Mountain Province, Nueva Vizcaya, Quezon, and Zambales.

Etymology.― The genus is from the Greek word “Lepto-solen” meaning “slender pipe” (Smith, 1990; Funakoshi et al., 2005) and the specifi c epithet honors Thaddäus Haenke (1761–1816), a Czech geographer and explorer, who is part of the 1792 Malaspina expedition in the Philippines, where the ginger was fi rst collected.

Common Names.― Banai (Ilk.), Panauil (Bon., Knk.), Kalawin (Bon.), Alpugan, Dapuyan and Lapugan (SblPn.) (Madulid, 2001).

Phenology.― The fl ower blooms from April–July and emits sweet fragrance during its nocturnal anthesis (Funakoshi et al., 2005). Fruiting is from July–November.

Economic Uses.― The fl owers (together with the corolla tube) are collected as vegetable ingredients for Filipino cuisines such as “Sinigang” and “Adobo”. The fruits are eaten by the villagers which has sweet-sour fl avour (Fig. 1C).

Specimens examined.―PHILIPPINES. Luzon. s. loc., Haenke s.n. (PR [digital image!]). Benguet: Twin Peaks, May 1904, Elmer 6428 (K [digital image!], NY [digital image!], P [digital image!], US [digital image!]). Cagayan: Mount Tabuan, May 1929, Ramos 77052 (NY [digital image!]). Ilocos Norte: Burgos, July 1918, Ramos 32863 (P [digital image!]). Laguna: Cultivated in Mount Makiling, Los Baños, June–July 1917, Elmer 17977 (NY [digital


image!], P [digital image!]). La Union: National Forage Park, Damortis, 24 May 1972, D.A Madulid & E. Hamoy 849 (PNH!). Mountain Province: Bontoc, November 1912, Vanoverbergh 1898 (P [digital image!]); Mount Data, 5 May 1953, Steiner 2198 (L [digital image!]); Sitio Dandanak, Barrio Ban–aw, Besao, Bontoc, 5 May 1953, J. V. Santos 5674a (PUH!); Barangay Alab, Bontoc, 3 May 2002, H. Funakoshi & L. Co 2006 (PUH!); Barangay Balili, Bontoc, 2 September 2002, Funakoshi 2025 (PUH!); Mount Data, Brgy. Alab Oriente, Bontoc, 17˚04’01.4”N, 120˚56’57.0”E, 380 m, 1 November 2013, Docot 0002 (USTH!); 3 July 2015, Docot 0003 (USTH! incl. spirit - 2 sheets). Zambales: Mount Pinatubo, Villar, May 1929, R.B. Fox 230 (PNH!).

Vanoverberghia sepulchrei Merr. in Philipp. J. Sci. 7: 76. 1912.

Terrestrial herb, 4–8 m, drooping. Rhizome red, robust, strongly aromatic when cut and exposed, covered with thick brown scales, 3–4 cm wide Pseudostem green, terete, glabrous, bulbose at the base, 2.4–3.7 cm wide. Plane of distichy of leaves perpendicular to rhizome. Ligule reddish up to the petiole and beginning of midrib, oval, apex rounded, glabrous and coriaceous, 7–13 × 6–10 mm. Petiole short, about 1 cm long. Leaves dark green above, paler beneath, glabrous and subcoriaceous, oblong, immense in size, 30–45 × 12–17 cm, biggest are located in the superior portion, base rounded, apex attenuate to caudate with fl agellate tip that are twisted, 3–5 cm long, margin entire and undulate. Infl orescence

terminal raceme, hanging, varying from colors of red, pink and white, 18–26 cm long; peduncle deep red, terete, 8–13 cm long, with 2–3 brown persistent sterile bracts; rachis 6–9 cm long; pedicels pink, 2–5 mm long; fl owers numerous, white to pink, 6.7–8 cm long; floral bracts pinkish-white at the base and brown at the apex, spathaceous or tubular at the base, oblong when dissected, apex acuminate, pubescent at the apex, 2.5–3 × 1.5–2 cm; bracteoles absent; calyx tube white, funnel-shaped, 2–3 dentate, pubescent at the apex, 1.5–2 × 1–1.3 cm; corolla buds white, the apex yellow; corolla tube white, glabrous, 1.2–1.6 cm long; dorsal corolla lobe linear-oblong, apex rounded and slightly bilobed, 22–27 × 5–6 mm; lateral corolla lobes white, linear–oblong, apex rounded, 20–32 × 4–5 mm; labellum white, connate to the base of lateral corolla lobes, free part bifi d or split into two subulate lobes, 17–23 × 2–3 mm, apex of lobes entire; filament white, channeled on the inside, enclosing the style half its length, 30–35 × 3–6 mm; anther creamy–white, oblong, pubescent, crestless, 15–20 × 4–5 mm; style white, slender, 4–5 cm long; stigma white, cupular, pubescent along the edge of the hollow, about 1 mm wide; nectaries 2, yellow, located at the base of the style; ovary deep-red, subglobose, glabrous and coriaceous, 3-locular, axile placentation, 4–5 × 2–3 mm. Fruits ellipsoid to subglobose, deep-red, 2–2.5 × 1.5–1.9 cm. Seeds white to brown, globose to subglobose, with aril, about 2 mm long and wide.


Transverse Section. Leaves. The shape of both adaxial and abaxial epidermis is rectangular. A three layered hypodermis is present in the entire lamina, which is composed of large isodiametric cells that serve as protection against the cold temperature. The palisade mesophyll is composed of a layer of small elongated cells, while the spongy mesophyll is consisted of fairly rectangular cells where numerous vascular bundles are embedded. Adaxial and abaxial sclerenchymatous girders are also prominent. Each vascular bundle has a distinct metaxylem, phloem cells and sclerechymatous cells. Beneath the spongy mesophyll is a three layered hypodermis that is composed of rectangular cells (Fig. 3A). The midrib is composed of prominent central vascular bundles and small bundles (Fig. 3B). Pseudostem. The vascular bundles are scattered throughout the ground tissue and with abundance on the periphery (Fig. 3C). The vascular bundles located at the periphery have prominent metaxylems, while vascular bundles at the center have 2–3 prominent metaxylems (Fig. 3E). Rhizome. The internal anatomy of the rhizome is also divided into two zones, the cortex and the stele by the endodermoidal layer (Fig. 3F). Numerous starch grains, varying in size, number and shape are distributed in both zones of the rhizome. Oil cells are very persistent throughout the rhizome (Fig. 3G). Stellar vascular bundles are numerous than cortical vascular bundles, and they are more situated near the endodermoidal layer. Vessel elements that are in scalariform were also observed (Fig. 3H). Root. A multiple

layered epidermis or velamen composed of 5–6 layers of cells is observed. Beneath is the thick cortex that is composed of parenchymatous cells and an endodermis which is composed of thick walled cells. A multiple layer of thin-walled cells called pericycle is located beneath the endodermis. The vascular bundles are immediately located after the pericycle which has prominent metaxylems and sclerenchymatous cells. The pith is composed of isodiametric cells (Fig 3D).

Habitat.― Found in clumps on thick dense forest and steep slopes along streams at medium altitudes of 1,000–1,600 masl.

Distribution.― Antique, Aurora, Benguet, Camarines Sur, Ifugao, Mountain Province, Quezon, and Sorsogon.

Etymology.― Merrill (1912) established the genus to honor Father Morice Vanoverbergh for the discovery of the ginger and to his intensive and enthusiastic botanical exploration at Bauko, Mountain Province. The specific epithet honors Father Jules Sepulchre, who established the Bauko mission, and rendered assistance to Father Vanoverbergh and in their missionary work, and in his botanical explorations.

Common Names.― Agbab (Bon.), Akbab (Bon., Knk.), Barapat, Paddapad and Chakchakil (Igt.) (Madulid, 2001).

Phenology.― The fl ower blooms from September–January. Fruiting is from February–July.

Economic Uses.― The fruits are edible. It has a sweet–sour taste where seeds are eaten and the pericarp is disposed (Fig 1G).


Specimens examined.―PHILIPPINES. Luzon. Ifugao: Bayninan, 6 March 1963, H.C. Conklin & Buwaya I-984 (L [digital image!], PNH!). Mountain Province: Bauko, Bontoc, 17 August 1911, Vanoverbergh 956 (US [digital image!], P [digital image!]); Mount Data, Barangay Alab Oriente, Bontoc, 17˚03’54.5”N, 120˚57’08.4”E, 1 November 2013, Docot 0001 (USTH! - 2 sheets); 14˚01.828”N, 121˚01.802”E, 1456 m, 9 January 2016, Docot 0027 (USTH! incl. spirit - 3 sheets). Sorsogon: Mount Bulusan, Irosin, August 1916, Elmer 19095 (P [digital image!]); September 1916, Elmer 17383 (U [digital image!]).

Vegetative propagation

The viability of rhizome explants treated with different concentrations and combinations of NAA and Kinetin were observed after 30 consecutive days (Table 1). The result of the Chi-square test of independence revealed that Leptosolena haenkei and Vanoverberghia sepulchrei had a signifi cant difference at X2 (2, N = 160) = 22.71, p < 0.05. This means that L. haenkei is more viable, where 100% of viable and 57.4% of fairly viable explants of the experiment are found, compared to V. sepulchrei, where 71.4% of not-viable explants are found (Table 2).

TABLE 1. Total number of viable, fairly viable, and not viable rhizome explants in each treatments of the respective plants.

Treatments

Leptosolena haenkei Vanoverberghia sepulchrei

ViableFairly Viable

Not Viable

ViableFairly Viable

NotViable

T1 3 7 0 0 6 4

T2 2 7 1 0 4 6

T3 1 7 2 0 5 5

T4 0 8 2 0 3 7

T5 3 5 2 0 5 5

T6 1 7 2 0 6 4

T7 2 8 2 0 6 4

T8 0 5 5 0 5 5

Legend: T1: 8 mg/L NAA; T2: 12 mg/L NAA; T3: 8 mg/L Kinetin; T4: 12 mg/L Kinetin; T5: 8mg/L Kinetin + 12 mg/L NAA; T6: 8 mg/L NAA + 12 mg/L Kinetin; T7: 8 mg/L Kinetin + 8 mg/L NAA; T8: 12 mg/L Kinetin + 12 mg/L NAA.


TABLE 2. The result of the Chi-Square independence test between all the rhizome explants of Leptosolena haenkei and Vanoverberghia sepulchrei.

PlantRatings

TotalViable Fairly Viable Not Viable

Leptosolena haenkeiCount 10 54 16 80

% Viability 100% 57.4% 28.6% 50%

Vanoverberghia sepulchreiCount 0 40 40 80

% Viability 0% 42.6% 71.4% 50%

Total10 94 56 160

100% 100% 100% 100%

Treatments comparison

Treatment 1 and 2. T1 (8 mg/L NAA) and T2 (12 mg/L NAA) of Leptosolena haenkei showed no signifi cant difference at X2 (2, N = 20) = 1.33, p > 0.05, same as with Vanoverberghia sepulchrei at X2 (1, N = 20) = 0.18, p > 0.05. Islam et al. (2004) and Cencki et al. (2009) in their study of Curcuma longa L. and Thermopsis turcica Kit Tan, Vural & Küçük., respectively, stated that low concentrations of NAA can produced best results of survival rate. This supports the result of T1 on both gingers which produced higher number of viable and fairly viable explants than their T2. Appearance of bud was observed in one explant of L. haenkei in T1 (Fig. 4A).

Treatment 3 and 4. T3 (8 mg/L Kinetin) and T4 (12 mg/L Kinetin) of Leptosolena haenkei showed no signifi cant difference at X2 (2, N = 20) = 1.13, p > 0.05, same as with Vanoverberghia sepulchrei at X2 (1, N = 20) = 0.43, p > 0.05. Most of the T3 explants of both gingers gained more number of viable

and fairly viable explants than their T4. These results were in accordance with the fi ndings of Sharma & Singh (1997) in their study of in vitro propagation of Zingiber offi cinale Rosc. were they concluded that low concentration induces more growth than high concentration of kinetin.

Treatment 5 and 6. T5 (8 mg/L Kinetin + 12 mg/L NAA) and T6 (8 mg/L NAA + 12 mg/L Kinetin) of Leptosolena haenkei showed no significant difference at X2

(2, N = 20) = 1.33, p > 0.05, same as with Vanoverberghia sepulchrei X2 (1, N = 20) = 0.20, p > 0.05. The number of viable and fairly viable explants in T5 and T6 of both gingers are higher than their non-viable explants. The combination of different concentrations of NAA and Kinetin favored the growth of the explants. Appearance of buds was observed in explant of L. haenkei in T5 and T6.

Treatment 7 and 8. T7 (8 mg/L Kinetin + 8 mg/L NAA) and T8 (12 mg/L Kinetin + 12 mg/L NAA) of Leptosolena haenkei


showed no significant difference at X2

(2, N = 20) = 3.38, p > 0.05, same as with Vanoverberghia sepulchrei X2 (1, N = 20) = 0.20, p > 0.05. The results were in favored with Sharma & Singh (1997), Islam et al. (2004) and Cencki et al. (2009) conclusion that low concentration of NAA or Kinetin can induce growth and development. The results were also in covenant with Inden et al. (1988) who concluded that high concentrations of growth hormones inhibit growth and development, and this is also the reason why few of the rhizome explants of both gingers on T8 were decayed and considered not viable (Fig. 4D).

Factors that affects the viability of the rhizome explants

One factor that greatly affects the viability of the rhizome is the phenotypic variations among the two gingers. The anatomy of the rhizome greatly affects the translocation of hormones to the vascular tissues (Bell, 1980). The rhizome of Vanoverbegrhia sepulchrei is more robust (Fig. 1H) and the cortex is thicker (Fig. 3F) compare to Leptosolena haenkei (Figs. 1D & 2E). Meristematic tissues of rhizomes are generally located at the endodermoidal or intermediate layer that is beneath the cortex (Remashree et al., 1997) (Figs. 2E & 3F), therefore translocation of hormones in V. sepulchrei is slower, which made them gained 71.4% of not viable explants of the experiment.

Vegetative propagation material is in general much more sensitive to desiccation and other damages than seeds (Schmidt, 1993). Microclimatic factors such as relative humidity, light, and temperature may have affected also the development of the explants. Cuttings should be rooted under shade, and the requirements and tolerances in terms of light and temperature varies according the genotypic variations of species (Schmidt, 1993). Leptosolena haenkei are found mostly on open slopes at low altitudes, while Vanoverberghia sepulchrei are common on shaded forest at medium to high altitudes. This will explain why most explants from Vanoverberghia sepulchrei died and most explants from Leptosolena haenkei survived, because the set-up is located on urban place where the temperature is relatively high.

Contamination is also a major factor that affects the mortality rate of the explants. This is may be due to molds, fungi, bacteria etc. present on the substrate or lodged in the remained scales and nodes. Several authors agreed that contamination of underground rhizomes is very high and is difficult to avoid (Islam et al., 2004). Though all explants were treated with fungicide (Benlate), contamination was observed. This is because of some unwanted mechanical disturbances on the set-up such as when watering the gingers that made the accumulation of contaminants. Molds are observed in explant of L. haenkei on T5 (Fig. 4B).


FIGURE 1. A.–D. Leptosolena haenkei C. Presl: A. Inflorescence (Funakoshi © 2006); B. Flower (Tandang © 2013); C. Fruit; D. Rhizome. E.–H. Vanoverberghia sepulchrei Merr.: E. Infl orescence; F. Flower (Docot © 2016); G. Fruits; H. Rhizome (Docot © 2015).


A

B C

E

F

HD

G

FIGURE 2. A.–H. Transverse sections of Leptosolena haenkei C. Presl: A. Midrib; B. Lamina; C & D. Pseudostem; E & G. Rhizome; G. Cortical vascular bundle; F. & H. Roots. Abbreviations: ab: abaxial epidermis, abg: abaxial sclerenchyma girder, ad: adaxial epidermis, adg: adaxial sclerenchyma girder, as: air space, c: companion cell, co: cortex, cvb: central vascular bundles, ed: endodermoidal layer, end: endodermis, ep: epidermis, gt: ground tissue, hyp: hypodermis, mx: metaxylem, per: pericycle, ph: phloem, pi: pith, sc: sclerenchyma cells, st: sieve tube elements, ste: stele, sto: stomata, svb: stellar vascular bundles, vb: vascular bundle, ve: velamen.

AB

C D

G

H

FE


FIGURE 3. A.–H. Transverse sections of Vanoverberghia sepulchrei Merr.: A. Lamina; B. Midrib; C. & E. Pseudostem; D. Roots; F, G & H: Rhizome. Abbreviations: ab: abaxial epidermis, abg: abaxial sclerenchyma girder, ad: adaxial epidermis, adg: adaxial sclerenchyma girder, c: companion cells, co: cortex, cvb: central vascular bundles, ed: endodermoidal layer, end: endodermis, gt: ground tissue, mx: metaxylem, oc: oil cells. per: pericycle, ph: phloem, pi: pith, px: protoxylems, sbv: stellar vascular bundle, sc: sclerenchyma cells, sg: starch granules, stv: sieve tube elements, vb: vascular bundle, ve: velamen, ves: vessel elements.

A

D E

G

HF

B C


CONCLUSION

The study successfully updated information and pioneered a transverse sectional anatomical study on the gingers that can be used for future taxonomic studies. The result of the propagation revealed that low concentration produce higher number of viable and fairly viable rhizome explants than high concentration of NAA and Kinetin. Combination of NAA and Kinetin such as in T5, T6 and T7 also favored the growth of rhizomes explants, except for T8 because of the high concentration of hormones. Different factors may also affect the viability of the explants such as phenotypic variations, microclimatic factors, and contaminations. Based on the morphoanatomy of the two gingers, the rhizome of Vanoverbegrhia sepulchrei is more robust and the cortex is thicker compared to Leptosolena haenkei, therefore, L. haenkei is easily propagated than V. sepulchrei. For future studies, we recommend to use different concentrations and combinations of plant growth hormones.

A B C D

FIGURE 4. A.–B. Leptosolena haenkei: A. Viable rhizome explant from T1 (8 mg/L NAA) that has appearance of bud on node and retained its turgidity and color; B. Rhizome explant from T5 (8 mg/L Kinetin + 12 mg/L NAA) that were decayed due to accumulation of molds; C.–D. Vanoverberghia sepulchrei: C. Fairly viable rhizome explant from T3 (8 mg/L Kinetin) that retained its turgidity and color; D. Decayed rhizome explant from T8 (12 mg/L Kinetin + 12 mg/L NAA) due to high concentration of hormones.

ACKNOWLEDGEMENTS

The authors are thankful to our parents for the support and to Dr. Hidenobu Funakoshi for his educative advices. We wish to express our gratitude to Ms. Ofelia B. Lopez, our offi cial guide in Mount Data during the fi eld collection, and to all the kind people of Bontoc, Mountain Province, especially “Barangay Alab Oriente”.

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