Axillary Shoots Derived from Thin Cell Layer and Adenine Sulphate Application in in vitro Mass Propagation of Gerbera [Gerbera jamesonii (H. Bolus ex Bolus f.)]



A new route of in vitro mass propagation protocol of Gerbera jamesonii (H. Bolus ex Bolus f.) derived from application of thin cell layers (TCL) and adenine sulphate (AS) was successfully developed and established. Shoot tip explants and half-strength MS medium containing 0.25 mg/l N6-benzylaminopurine (BAP), 20 g/l sucrose and 7 g/l Swallow agar were used as explant source and basic medium. Different TCL of transversal TCL (tTCL) and longitudinal TCL (lTCL) in four slicing positions of 1, 2, 3 and 4; varieties and clones i.e. G. jamesonii ‘Black Jack’, ‘Carambole’, ‘Nuance’, ‘Violente’, 01.098 and 11.46 clone; AS concentrations viz. 0, 20, 40, 60, 80 and 100 mg/l were tested in the study. Each step of in vitro culture established had unique and specific results. In the initiation stage, first slicing position of ‘Black Jack’ shoot tip tTCL was the most optimal combination treatment to produce 7.0 shoots per explant with 13.5 leaves. The first slicing position on shoot tip explants of 01.098 clone tTCL and 20 mg/l AS in half-strength MS medium containing 0.25 mg/l BAP were the most optimal combination treatment in obtaining the highest number of shoots produced per shoot up to 9.4 shoots per shoot with 34.1 leaves and 2.37 cm length of leaves in the proliferation stage, however the treatment did not give significant effect compared to control. Under periodical subcultures on the basic medium, number of shoots and leaves increased gradually from the initial culture with 3-6 shoots per shoot and 9.4-11.6 leaves till the fourth subculture with 6-11 shoots per shoot and 16.7-28.8 leaves and declined thereafter. Subculturing of shoots in accordance to produce qualified shoots for planting materials could be carried out till sixth to seventh subculture. The highest shoot multiplication rate (SMR) was established on 01.098 clone with as high as 7.3. The well shoots were easily rooted on half-strength MS medium supplemented with 0.1 mg/l BAP, 0.05 mg/l NAA and 1.5 g/l AC. Plantlets were then transferred to ex vitro condition for acclimatization on a mixture of burned-rice husk and organic manure (1:1, v/v) with 85-100% survivability. The ‘Black Jack’ and 11.46 clone were the best genotypes on the acclimatization stage with 100% survivability of plantlets. Results of the study have implication that first slicing position of shoot tip tTCL can be applied in establishing of in vitro propagation protocol for other gerberas.


adenine sulfate; Gerbera jamesonii; in vitro culture stages; thin cell layer (TCL)

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Akter N, Hoque MI, Sarker RH (2012). In vitro propagation in three varieties of gerbera (Gerbera jamesonii Bolus) from flower bud and flower stalk explants. Plant Tissue Culture and Biotechnology 22(2):143-152.

Arias AMG, Valverde JM, Fonseca PR, Melara MV (2010). In vitro plant regeneration system for common bean (Phaseolus vulgaris): effect of N6-benzylaminopurine and adenine sulphate. Electronic Journal of Biotechnology 13(1):1-8.

Aswath CR, Choudhary ML (2002). Rapid plant regeneration from Gerbera jamesonii Bolus callus cultures. Acta Botanica Croatica 61(2):125-134.

Awal A, Ahmed ABA, Taha RM, Yaacob JS, Mohajer S (2013). Effect of adenine, sucrose and plant growth regulators on the indirect organogenesis and on in vitro flowering in Begonia x hiemalis Fotsch. Australian Journal of Crop Science 7(5):691-698.

Bantawa P, Roy OS, Ghosh P, Mondal TK (2009). Effect of bavistin and adenine sulphate on In vitro shoot multiplication of Picrorhiza scrophulariiflora Pennell.: an endangered medicinal plant of Indo-China Himalayan Regions’. Plant Tissue Culture and Biotechnology 19(2):237-245.

Bhargava B, Dilta BS, Gupta YC, Dhiman SR, Modgil M (2013). Studies on micropropagation of gerbera (Gerbera jamesonii Bolus). Indian Journal of Applied Research 3(11):8-11.

Bui VL, Hang Phuong NT, Anh Hong LT, Tran Thanh Van K (1999). High frequency shoots regeneration from Rhynchostylis gigantea (Orchidaceae) using thin cell layer. Plant Growth Regulation 28:179-185.

Cardoso JC, Teixeira da Silva JA (2013). Gerbera micropropagation. Biotechnology Advances 31:1344-1357.

CBI (2016). CBI Trade Statistics: Cut Flower and Foliage. pp: 1-9.

Dahab, TAMA (2006). In vitro propagation of Hydrangea macrophylla Thunb’, Arabian Journal of Biotechnology 10(1):161-178.

Deepika R, Kanwar K (2010). In vitro regeneration of Punica granatum L. plants from different juvenile explants. Journal of Fruit and Ornamental Plant Research 18(1):5-22.

Gabriela V (2011). Effect of adenine sulfate (AdSO4) on the in vitro evolution of white clover variety (Trifolium repens L.). Analele Universitatii din Oradea, Fascicula Protectia Mediului 17:203-210.

Gantait S, Mandall N, Das PK (2009). Impact of auxins and activated charcoal on in vitro rooting of Dendrobium chrysotoxum Lindl. cv. Golden Boy. Journal of Tropical Agriculture 47(1-2):84-86.

Gök KM, Şan B, Bayhan AK (2016). Micropropagation of Gerbera (Gerbera jamesonii Bolus) under different color of light‐Emitting diodes. Journal of Natural and Applied Sciences 20(3):468‐474.

Khan MK, Misra P, Sharma T, Shukla PK, Ramteke PW (2014). Effect of adenine sulfate on in vitro mass propagation of Stevia rebudiana Bertoni. Journal of Medicinal and Plant Research 8(13):543-549.

Khatun MM, Khatun H, Khanam D, Al-Amin MD (2010). In vitro root formation and plantlet development in Dendrobium orchid’. Bangladesh Journal of Agriculture Research 35(2):257-265.

Kanwar JK, Kumar S (2008). In vitro propagation of Gerbera - A review’. Horticulture Science (Prague) 35(1):35-44.

Kshirsagar K, Braganz, VJ (2012). Micropropagation of Rosa damascena Mill. through transverse thin cell layer culture. South Asian Journal of Experimental Botany 2(4):184-189.

Kumar S, Kunwar JK (2006). Regeneration ability of petiole, leaf and petal explants in gerbera cut flower cultures in vitro. Folia Horticulturae Annual 18(2):57-64.

Kumar S, Kunwar JK (2007). Plant regeneration from cell suspensions in Gerbera jamesonii Bolus. Journal of Fruit and Ornamental Plant Research 15:157-166.

Le BV, Nhut DT, Tran Thanh Van K (1999). Plant production via shoot regeneration from thin cell layer pseudo-bulblet explants of Lilium longiflorum in vitro. Comptes Rendus de l’Academie des Sciences-Series III-Sciences de la Vie 322(4):303-310.

Mathur A, Mathur AK, Verma P, Yadav S, Gupta ML, Darokar MP (2008). Biological hardening and genetic fidelity testing of micro-cloned progeny of Chlorophytum borivilianum Sant. et Fernand. African Journal of Biotechnology 7(8):1046-1053.

Murthy KSR, Kondamudi R (2011). Rapid shoot regeneration from thin cell layer explants of an endangered medicinal asclepiad Ceropegia spiralis L.’. Plant Tissue Culture and Biotechnology 21(1):63-73.

Mohamed SA, Özzambak ME (2014). Shoot regeneration capacity of in vitro cultures of some Gerbera (Gerbera jamesonii Bolus) explants. Sudanese Journal of Agriculture Sciences 1: 24-29.

Nandagopal S, Kumari BDR (2006). Adenine sulfate induced high frequency shoot organogenesis in callus and in vitro flowering of Cichorium intybus L. cv. Focus- apotent medicinal plant. Acta Agriculture Slovakia 87(2):415-425.

Naz S, Naz F, Tariq A, Aslam F, Ali A, Athar M (2012). Effect of different explants on in vitro propagation of gerbera (Gerbera jamesonii). African Journal Biotechnology 11(37):9048-9053.

Nhut DT, Hai NT, Huyen PX, Huong DTQ, Hang NTT (2005). Thidiazuron induces high frequency shoot bud formation from Begonia transverse thin cell layer culture. Propagagation of Ornamental Plants 5:151-157.

Nhut DT, Ana TTT, Huonga NTD, Dona NT, Haia NT, … Vua NH (2007). Effect of genotype, explant size, position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture. Scientia Horticulturae 111(2):146-151.

Nhut DT, Hai NT, Phan MX (2010). A highly efficient protocol for micropropagation of Begonia tuberous. In: Jain SM, Ochatt SJ (Eds.). Protocols for In vitro Propagation of Ornamental Plants, Springer protocols, Humana Press, pp 15-20.

Noiton D, Vine JH, Mullins MG (1992). Effects of serial subculture in vitro on the endogenous levels of indole-3-acetic acid and abscisic acid and rootability in microcuttings of ‘Jonathan’ apple. Plant Growth Regulation 11:377-383.

Saha M, Phatak A, Chandra N (2004). In vitro culture studies in four dioecious varieties of Carica papaya L. using axillary buds from field-grown plants. Journal of Cell and Tissue Research 4(2):211-214.

Schmildt O, Schmildt ER, Amaral JAT (2007). Sulfato de adenina na multiplicação in vitro de mamoeiro “Tainung 01”. Scientia Agraria 8(2):141-147.

Shabanpour K, Sharifi A, Bagheri A, Moshtaghi N (2011). Effect of genotypes and culture medium on shoot regeneration and proliferation of Gerbera jamesonii. African Journal of Biotechnology 10(57):12211-12217

Shabbir K, Ahmad T, Hafiz IA, Hussain A, Abbasi NA, Ahmad J (2012). In vitro regeneration of Gerbera jamesonii cv. Sunglow. African Journal of Biotechnology 11(42):9975-9984.

Sharma T, Modgil M, Thakur M (2007). Factors affecting induction and development of in vitro rooting in apple rootstocks. Indian Journal of Experiment Biology 45:824-829.

Shylaja MR, Sashna P, Chinjusha V, Nazeem PA (2014). An efficient microprogation protocol for Gerbera jamesonii Bolus from flower buds. International Journal of Plant, Animal and Environmental Sciences 4(3):641-643.

Son NV, Mokashi AN, Hegde RV, Patil VS, Lingaraju S (2011). Response of gerbera (Gerbera jamesonii Bolus) varieties to micropropagation. Karnataka Journal of Agriculture Science 24(3):354-357.

Stefaniak B (1994). Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort). Plant Cell Reports 13:386-389.

Teixeira da Silva JA (2003a). Thin cell layer technology in ornamental plant micropropagation and biotechnology. African Journal of Biotechnology 2(12):683-691.

Teixeira da Silva JA (2003b). Thin cell layer technology for induced response and control of rhizogenesis in chrysanthemum. Plant Growth Regulation 39:67-76.

Teixeira da Silva JA (2004). The effect of carbon source on in vitro organogenesis of chrysanthemum thin cell layers. Bragantia, Campinas 63(2):165-177.

Teixeira da Silva JA, Van KTT, Biondi S, Nhut DT, Altamura MM (2006). Thin cell layers: developmental building blocks in ornamental biotechnology. FOB 1(1):1-13.

Teixeira da Silva JA, Fukai S (2003). Chrysanthemum organogenesis through thin cell layer technology and plant growth regulator control. Asian Journal of Plant Science 2:505-514.

Teixeira da Silva JA (2008). Plant thin cell layers: challenging the concept. International Journal of Plant Development Biology 2(1):79-81.

Tran Thanh Van K (1980). Control of morphogenesis by inherent and exogenously applied factors in thin cell layers. International Review of Cytology 32:291-311.

Tyagi P, Kothari SL (2004). Rapid in vitro regeneration of Gerbera jamesonii (H. Bolus ex Hook. f.) from different explants. Indian Journal of Biotechnology 3:584-588.

Winarto B (2012). In vitro proliferation study of three Indonesian Dendrobiums protocorm-like bodies (plbs) on different fertilizer media. Prosiding Seminar Nasional Anggrek 2012, Jln. H. Adam Malik, No. 5, Medan, Sumatra Utara, 21 Juni 2012, pp 154-169.

Winarto B, Setyawati AS (2014). Young shoot nodes derived organogenesis in vitro in mass propagation of Ruscus hypophyllum L. South Western Journal of Horticulture, Biology and Environment 5(2):63-82.

Wróblewska K (2012). The influence of adenine and benzyladenine on rooting and development of Fuchsia hybrida cuttings. Acta Agrobotanica 65(4):101-108.

Van Staden J, Zazimalova E, George EF (2008). Plant growth regulators II: Cytokinins, their analogues and antagonist. In: George EF, Hall M, De Kleck G.J. (Eds.) Plant Propagation by Tissue Culture. Vol. 1, The Background, Springer, The Netherlands, pp 205-226.

Vardja R, Vardja T (2001). The effect of cytokinin type and concentration and the number of subcultures on the multiplication rate of some decorative plants. Proceeding of Estonian Academy of Science Biology and Ecology 50(1):22-32.

Vujovic T, Ruzic Dj, Cerovic R (2012). In vitro shoot multiplication as influenced by repeated subculturing of shoots of contemporary fruit rootstocks. Horticulture Science (Prague) 39(3):101-107.

Vyas S, Guha S, Kapoor P, Rao IU (2010). Micropropagation of Cymbidium Sleeping Nymph through protocorm-like body production by thin cell layer culture. Scientia Horticultuare 123:551-557.

Zhao P, Wang W, Feng FS, Wu F, Yang QZ, Wang WJ (2007). High-frequency shoot regeneration through transverse thin cell layer culture in Dendrobium candidum Wall Ex Lindl. Plant Cell Tissue and Organ Culture 90:131-139.

Zibbu G, Batra A (2010). Effect of adenine sulfate on organogenesis via leaf culture in an ornamental plant: Thevetia peruviana (Pers,) Schum. International Journal of Pharmacy and Bioscience 1(2):1-9.



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