RESEARCH ARTICLE: AGRICULTURAL BIOTECHNOLOGY AND FOOD REVISTA DE CIENCIAS AGRÍCOLAS

Volumen 35(2):5-13 ISSN Impreso 0120-0135 e-ISSN 2256-2273

doi: http://dx.doi.org/10.22267/rcia.183502.86

In vitro arrow cane (Gynerium sagitatum Aubl.) multiplication in double phase medium

Multiplicación in vitro Gynerium sagitatum Aubl.) en medio doble fase de caña flecha (

Claudia López D.1; Isidro E. Suárez2

1 , [email protected]. 2 Investigadora, Instituto de Biotecnología Aplicada para el Caribe (IBAC), Universidad de Córdoba, Montería, Colombia, [email protected]. Docente, Ph.D. Instituto de Biotecnología Aplicada para el Caribe (IBAC), Universidad de Córdoba, Montería,

In vitro Gynerium sagitatum Aubl.) multiplication Revista de Ciencias Agrícolas Citar: Lopez, C. & Suarez, I. (2018). arrow cane ( in double phase médium. . 35(2): 5-13 doi:http://dx.doi.org/10.22267/ rcia.183502.86

Received: august 25 2017. Accepted: september 24 2018. ABSTRACT

Gynerium sagitatum

Arrow cane ( Aubl.) is a species used as fiber source to make traditional and valuable handmade craftsmanship by indigenous communities in Northern Colombia. Since no commercial crops are established fiber needs are taken from natural populations affecting ecosystem. A micropropagation protocol to clonally multiply large quantities of arrow cane plant material for planting commercial crops has been developed; however, micropropagated are costly compared to naturally extracted plant material. To reduce micropropagated plants costs, in the present research a double phase medium formulation along with continuous shoot culture with no periodic transfers to fresh medium was compared to semisolid medium system with subculture every four weeks with respect to multiplication rate and costs of micropropagated plants. The results showed that continuous culture of explants with double phase medium and no periodic transfers resulted in higher multiplication rates and larger shoots compared to shoots cultured using the conventional semisolid medium system and transfer to fresh medium every four weeks. Plants from both, semisolid and double phase culture system, fully adapted and recovered when transferred to ex vitro conditions. Keywords:The cost analysis In vitro showed that double phase cultured shoots are ≥20% lessex vitro expensive..

propagation, double phase, axillary meristems, BAP, UNIVERSIDAD DE NARIÑO Rev. Cienc. Agr. Julio - Diciembre 2018, 35(2): 5-13 6 López y Suárez - In vitro multiplication of cane arrow

RESUMEN

Gynerium sagitatum Aubl.) es una especie de la familia Poaceae utilizada como

Caña flecha ( necesidadesfuente de fibra de parala industria elaborar son tradicionales tomadas de y plantacionesvaliosas artesanías naturales por afectando comunidades el ecosistema. indígenas Parade la proveerCosta Norte material Colombiana. vegetal paraDebido la siembraa que no de existen cultivos cultivos comerciales, comerciales, un protocolo la fibra para para las la

multiplicación clonal masiva de plantas de caña flecha ha sido desarrollado; sin embargo, las plantas micropropagadas resultan costosas comparadas con los propágulos extraídos periódicasde las poblaciones a medio naturales.fresco comparado Con el fin con de el reducir sistema los convencional costos de las en plantas, medio semisólidoen el presente con transferenciasestudio se evaluó cada el cuatrouso de semanasmedio doble a medio fase fresco,y cultivo con continuo respecto de a brotesla tasa sinde multiplicacióntransferencias

doble fase sin transferencias periódicas a medio fresco resultó en tasas de multiplicación más y costos de las plantas micropropagadas. Los resultados mostraron que el cultivo en medio

altas y brotes de mayor longitud al compararlos con los brotes obtenidos en medio semisólido trasplantadosy transferencias a condiciones cada cuatro ex semanas. vitro Tanto los brotes cultivados en medio doble fase como los cultivados en medio semisólido se adaptaron y establecieron normalmente cuando fueron mediante transferencias mensuales .a El medio análisis fresco. de costos mostró que los brotes multiplicados en medio doble fase son ≥20% menos costosos y se recuperan ex vitro similar a los obtenidos Palabras clave: Propagación in vitro ex vitro.

, doble fase, meristemos axilares, BAP,

INTRODUCTION

A Gynerium sagitatum Aubl.) is a Poaceae Arrow cane propagates by sexual and clonal meth and,- species native to West India and distributed ods; plants are dioceaus and flowers can be -1 wind rrow cane ( pollinated, seeds index is around 1.7°C. million Flowering Kg usu when viable, seeds can germinate from three to sev- from México through in the American en days after imbibition at 20-30 - continent. The plant is well adapted to inter tropical ally occurs at the stem terminal in 18-20 months old zone conditions with a better growth rate in wet low stem; however, under Humid Caribbean conditions sexual propagation does not occur because seeds lands, organic soils and altitude up to 1600m above are unviable. Clonal propagation is the current way sea level (GRIN, 2013). Cultivation and processing Zenú Indian group, most of for plant multiplication and dissemination; the new of arrow cane have been the main income source shoots emerge from underground rhizomes that for communities of the expand radially up to 20m from the main stem. The them dedicated to make craftsmanship products newly grown stems eventually mature and flower from the plant´s central nerve becoming the most becoming a new source for basal growing shoots. famous handmade Colombian products (DANE, This growth and propagation habit is not only very 2005). Recent studies evidenced the potential for efficient for colonization new territorieset al but also landfill phytoremediation using arrow cane alone an effectiveet way al to preserve wetlands and shores or in association with other plant species (Madera- from erosion and degradation (Kalliola ., 1992; Parra, 2015a, 2015b). Araméndiz ., 2005). UNIVERSIDAD DE NARIÑO Rev. Cienc. Agr. Julio - Diciembre 2018, 35(2): 5-13 López y Suárez - In vitro multiplication of cane arrow 7

A rate and costs variables. rrow cane plant extraction from wild populations micropropagation with respect to multiplication to make craftsmanship (hats, rings, shoes, etc.), MATERIALS AND METHODS homes, musical instruments and ornamental products happens at a very high rate since Plant material. Plant material consisted of in vitro commercially crops are not available. It is estimated that more than 50% of arrow cane becomingnatural populations a cultural, economic in the Colombian and environmental Northern established arrow cane plants cultivar “Criolla” Coast have been eliminated in the past 10 years cultivated for 12 months with monthly subcultures. The culture medium was MS (Murashige-1 and issue. Several studies have been conducted aimed Skoog, 1962)® supplied with (in mg® L ) myo inositol (100), sucrose (30.000), thiamine HCl (0.4) and at developing an efficientet propagation al methodet foral., natural restoration and commercial crops for fiber Phytagel (3.000) (Sigma Co .). The explants production (Araméndiz ., 2005, Suarez consisted of clusters with three 3-4 cm long shoots obtained from established plants after four weeks 2013). of subculture. The cultures were s stored). at 25°C with 12 h photoperiod provided-2 -2 by cool white Micropropagation is a clonal propagation technique periods of time, reduced space and under aseptic fluorescent tubes (40-50 µmol m that allows massive plant multiplication in short Shoot multiplication. et al. 3 Clusters with three stems conditions (Sinhg , 2013; Waikhom and were established3 into 750cm polycarbonate flasks Louis, 2014). Micropropagated plants are cultured containing3 double phase medium consisting of from explants, established in closed containers with nutrient media and hormone supply that 100cm MS semisolid bottom medium added with result in high levels of genetic and phenotypic 30cm liquid MS medium on top of the semisolid uniformity. Usually, micropropagation requires phase; four explants were established in each container and the liquid phase was re-plenished fully equipped labs, relatively high amount ofet to avoid contamination. For conventional every two weeks inside of a laminar flow hood al.,reagents, well trained people and many labor micropropagationhours which results strategiesin high plant for costs arrow (Shinde cane 3 MS semisolid medium in order 2016). to Several produce studies massive related plant to material developing for containedsemisolid in system, 125cm 3 similarly processed explants were cultivated in 30cm et al. polycarbonate flasks; a single Riveraplanting et commercialal cropset haveal. been reported explant was established in each container and total (Pastrana and Suarez, 2009, Suarez , 2009; explants were transferred to fresh medium of the planting material., 2009; from Suarez natural populations., 2017); however, Double same formulation every four weeks. micropropagated plants results in higher costs than to reduce costs of Vitis vinifera et al., The medium formulation for both treatments was phase Ananas medium comosus system have been implementedet al., MS (Murashige-1 and Skoog, 1962) supplied with

Whitania somnifera (Couseloet al (in mg L ) mio inositol (100), sucrose® (30.000), 2006), (Scherwinski-Pereira thiamine HCl (0.4) and BAP (0.5); the semisolid 2012) and (Singh ., 2016) phase was added with Phytagel (3.000) (Sigma® micropropagated plants by means of reduced Co.). All flasks were covered with heavy duty gelling agents, subculture, reagents and labor hours cultures were stored at 25 aluminum foil and sealed with Nescofilm . The in the process. In the present research, double °C with 12 h photoperiod phase medium with continuousGynerium shoot growthsagitatum in s the same recipient was compared to conventional provided-2 by-2 cool white fluorescent tubes (40-50 µmol m ) during 120 days. UNIVERSIDADsemisolid DEmedium NARIÑO system for Rev. Cienc. Agr. Julio - Diciembre 2018, 35(2): 5-13 8 López y Suárez - In vitro multiplication of cane arrow

3 containers and semisolid Treatments, consisting of double3 phase medium phenol emission or contamination symptoms; no dispensed in 750 cm incallus vitro tissue or adventitious shoot growth was medium dispensed in 250 cm , each treatment was toobserved. BAP supplied This growth medium. performance Cultures isadapted typical to of replicated 10 times for a total of 20 experimental established cultures when transferred units distributed with a complete randomized in vitro adapted cultures design. After 120 days in culture, the total number in vitro conditions respond accordingly to PGR of shoots, shoots per flask, newlyT formed shoots supply in contrast non- per explant and shoot length µ were registered. usually show a non-synchronicet al., growth with mean,Data wereβ analyzed using test based on the large asymmetric organs, phenol emission and 𝑢𝑢 𝑗𝑗 𝑗𝑗 𝑗𝑗𝑗𝑗𝑗𝑗 𝑌𝑌container = µ and+ 𝛽𝛽 Ɛ + Ɛ here was the overall necrotic tissues (Suárez 2009; Pastrana was the effect of medium consistency and and Suárez, 2009). T multiplication ratewas and the costexperimental of elements error. used Costs for data allowed to detect statistical differences for of shoot production were quantified based on the The results of the studentPr test applied to the Pr total number of shoots ( = 0.0001), number of Mediumeach system. sterilization. Pr shoots per flask ( Pr= 0.0011), mean number of The pH of all media was newly formed shoots per explant ( = 0.0148) adjusted to 5.7-5.8 prior to addition for of15 gellingmin. and shoot length ( = 0.0001) as a result of agent when indicated. Media were-2 sterilized by the applied treatments (Table 1). The analysis showed that continuous culture of arrow cane Transferautoclaving to atex 120 vitro °C conditions. and 1.2 kg cm Micropropagated explants in double phase medium significantly increases the total number of shoots, the number shoots were washed with sterile distilled water to of new shoots per explant, the number of shoots remove medium residues and transplanted into 72- in each individual flask and the length of shoots compared to the conventional semisolid medium plug containers filled with® andpeat irrigation as substrate. provided The Figure 1). containers were placed in a shade house with 20% system in the same time frame (90 days) (Table 1, light coverage with Saram by a fog system every 20 min for 30 seconds during® and the first two weeks. After two weeks, plants were The cost analysis of micropropagated shoots transferred to 50% light coverage with Saram at a rate of 6.8 new shoots per explant using a irrigated three times a day two minutes each. Seven conventional semisolid medium system with four week transfer to fresh medium estimates a cost of normalweeks after conditions transferring was recorded to ex vitro and conditions, survival rate the US$0.18 per shoot (López, 2016). The results of calculated.number of plants that survived and adapted to the present research showed that multiplication rate in double phase medium can be about two RESULTS AND DISCUSSION folds (13) compared to semisolid medium system. Considering the medium quantity needed for each system, gelling agent amount, labor related to New shoots formed from established explants subculture and washing glassware; it is estimated were visible after two weeks in culture from both that double phase medium system can lower the double phase and semisolid medium cultures. New total cost of plants by 20% compared to semisolid medium cultures system (Table 1). UNIVERSIDADshoots showed DE NARIÑO a healthy growth pattern with no Rev. Cienc. Agr. Julio - Diciembre 2018, 35(2): 5-13 López y Suárez - In vitro multiplication of cane arrow 9

Table 1. In vitro multiplication of Gynerium sagitatum

Aubl. Shoots in double phase and Total shoots Shoots per Shoots per Shoot Shoot cost Medium producedsemisolid mediumflask after 120 daysexplant in culture.length (cm) (US$) Double phase 855 A 47.50 A 13.00 A 7.6 A 0.15

Semisolid 516 B 6.88 B 6.88 B 5.2 B 0.18 Values with the same letter are not different according to T test (α = 0.05)

Figure 1. cultured in semisolid medium Upper row: Explants cultured in double phase medium, Lower row: Explants

UNIVERSIDAD DE NARIÑO Rev. Cienc. Agr. Julio - Diciembre 2018, 35(2): 5-13 10 López y Suárez - In vitro multiplication of cane arrow

Culture media for in vitro tissue culture can be in vitro propagated plants grow important for plant survival during transfer to ex etformulated al. as semisolid, liquid or double phase svitro conditions; -2 (liquid on top of semisolid) (Akhtar, 2013; Mehta under-1 very low light intensities (<60 µmol m time , for 2014; subculture Litwińczuk, because 2013). slow Semisolid nutrient media and ) that prevents photosynthesis activation and favor explant stability and allow to expand the therefore, energetic compoundin vitro supply, usually sucrose, in needed, creating a heterotrophicet al. water release to the tissues; in contrast, they are (photomixotrophic)et al. model etfor al., plant growth usually more expensive because of gelling agents and development (Kapchina-Toteva , 2014; absencecosts and ofnutrient gelling absorption agents, nutrientby tissues and is slower; plant toNunes ex vitro , 2016; Chavan 2014). Because of in contrast, liquid media are less expensive for this external energetic support, when transferred et conditions, plants must have plenty of al.regulators absorptionet al., by explants is faster, carbohydrate reserves in organs, such as leaves and especially in cell suspension systems; (El-Hawaz stems, that serve as “life boat” effect before newly , 2015; Perera 2015). For liquid cultured photosynthetic active leaves are formed. Therefore, tissues subcultures are more frequent because of micropropagated plants with larger organs will nutrient depletion, tissue vitrificationet al. and phenol store more nutritional reserves and will have more leakage in the medium that negatively affects opportunities toet successfully al., withstandet al. adaptation growth and survival (Dong , 2016; Jones and when transferring to natural environment (Kane, Saxena, 2013). Double phase medium formulation 1996; Resende 2016; Zakavi , 2016). combines the benefits of semisolid and liquid media: explant stability is provided by semisolid transferPlants cultured to ex vitro in both systems had a complete phase; rapid nutrient uptake is favored by liquid survival percentage (100%) seven weeks after phase, the subcultures are eliminated by periodical conditions; however, plants addition of liquid medium which reduces cost from cultured in larger containers/double phase/ gelling agents, labor and support elements such as no transfer were in average larger (13cm) than sealing material (Litwińczuk, 2013; López, 2013). those cultured in semisolid media with monthly reservetransfers accumulation to fresh medium and rapid (5cm) allocation (Figure for new 2); In semisolid medium system, subculture of plant difference that may be the result of the higher tissue to fresh medium is necessary in order to rates.provide During constant subculture, adequate nutrientstems and levels, leaves to avoid are organ formation in the first; however, more studies tissue decay and to maintain high multiplication are recommended to deep into this topic. separatedcut back tot maintain the shoot/container size ratio accordingly; additionally, shoot clusters are o scale up propagule number to keep high multiplication rates. The data collected in this research showed that cultures grown continuously with no transfers to fresh medium and absence of shoot cutting resulted in shoots >40% larger than those sequentially severed, separated and transferred to fresh medium periodically during the 120 days of the research; this may indicate Figure 2. Ex vitro acclimatized plants from that subculture induces some stress levels by structure severinget al. and high nutrient saturation on fresh medium that negatively affect shoot semisolid (left) and double phase (right) growth (Shin , 2013). Plant size is especially UNIVERSIDAD DE NARIÑO multiplication Rev. Cienc. media, Agr. Juliorespectively. - Diciembre 2018, 35(2): 5-13 López y Suárez - In vitro multiplication of cane arrow 11

CONCLUSSION Dong, Ys., Fu, Ch., Su, P., Xu, Xp., Yuan, J., Wang, S., Zhang, M., Zhao, Cf. & Yu, LJ. (2016). MechanismsPhysiologia and The present study has shown that micropropagation Plantarumeffective control of physiological browning phenomena in plant cell cultures. of arrow cane shoots using double phase medium decrease micropropagated plant costs at least . 156(1):13-28. doi: 10.1111/ resulted in higher multiplication rates that may ppl.12382. In Vitro Curcuma longa L. in Response 20% compared to those micropropagated using El-Hawaz, R., Bridges, W. & Adelberg, J. (2015). semisolid medium system. Likewise, doubleex phase vitro Growth of Plos One. cultured shoots were 40% larger which may have to Five Mineral Elements and Plant Density a beneficial impact on plant adaptation to in Fed-Batch Culture Systems. Conflictand growth of when interest transferred: to field conditions. 10(4). doi: https://doi.org/10.1371/journal. pone.0118912. The authors declare that there is noBIBLIOGRAPHIC conflict of interest. REFERENCES GRIN - Germplasm Resources InformationRecuperada Network. (2013). National Germplasm Resources Laboratory, Beltsville, Maryland. de http://www.arsgrin.gov/cgibin/npgs/

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