Indian Journal of Biotechnology Vol 3, October 2004, pp 584-588 Rapid in vitro regeneration of Gerbera jamesonii (H. Bolus ex Hook. f.) from different explants Purnima Tyagi and S L Kothari* Botany Department, University of Rajasthan, Jaipur 302 004, India Received 12 May 2003; accepted 22 September 2003 Rapid in vitro multiplication of shoots has been described using capitular sections and leaf explants in Gerbera jamesonii. Three types of basal media regimes were used including MS and two of its modified forms MSI and MSA. Shoot induction was achieved on all three regimes but MSI medium supplemented with Kn (4 mg/l) and IAA (0.5 mg/l) was ideal for shoot bud initiation (8-11) from capitular sections and Kn (4 mg/l) and IAA (0.1 mg/l) from leaves (5-8 shoot buds). MSI medium with 2 mg/l Kn+0.5 mg/l PAA was used for rapid multiplication of organogenic callus; 20-25 shoot buds developed from this callus. Rooting of in vitro shoots was achieved on MS medium with 0.5 mg/l IAA. Plantlets were transferred to pots where they matured and flowered. Keywords: Gerbera jamesonii, capitulum, leaf, in vitro regeneration IPC Code: Int. Cl. 7 A 01 H 4/00, 5/00 Introduction Gerbera jamesonii is an important commercial cut flower crop. It is one of the top ten traded cut flowers in the world. Its vegetative propagation is slow and other conventional methods of propagation are inadequate for the production of large number of uniform propagules for commercial cultivation 1. Over the years many workers have attempted tissue culture propagation of Gerbera from different explants such as shoot tips 2, inflorescence 3-7 and ovule 8,9. The demand for tissue cultured plants is increasing more as compared to conventional production of plants. In this paper, the authors have successfully attempted regeneration from capitulum, leaf and in vitro shoot explants. From one capitulum explant, 2200 shoots were obtained in 13 weeks, which is the highest rate of multiplication achieved so far. Materials and Methods Mature explants were taken from commercial single yellow coloured variety obtained from Pushpawali nursery, Jaipur. Leaves (1.5-2.8 cm) with petiole and capitulum (0.3-0.7 cm diam) at unopened stage were taken from pot grown plants. In vitro shoots were obtained from actively growing cultures. *Author for correspondence: Tel: 91-141-2703439; Fax: 91-141-2703439 E-mail: slkothari@lycos.com Both young leaves and unopened capitula were collected from potted plants. They were rinsed in running tap water and then washed with detergent Extran (20% v/v). The explants were surface sterilized with 0.1% (w/v) aqueous mercuric chloride solution for three min and were rinsed with sterile distilled water for at least 5 times. After sterilization, involucral bracts were removed and capitula sectored into 20 pieces while leaves were inoculated intact. In vitro shoots taken from shoot multiplying cultures required no sterilization. In Gerbera, the stem is compressed and compact and leaves arise from it on long petioles, this stem is termed heart. From growing cultures the hearts with leaves were removed. Leaves were excised and hearts used for culturing. 12 replicates per explant were taken for each treatment. Cultures formed from these explants at the time of subculturing were divided into pieces of approximately 500 mg. The nutrient media chosen for these studies were: I, Murashige and Skoog's (1962) medium 10 with 30 gm/l sucrose-(ms); II, 1/2 MS macro salts+heller s micro salts+80 mg/l adenine sulphate and sucrose (10-40 gm/l)-(msa); and III, MS salts with altered concentrations of MnSO 4, MgSO 4, ZnSO 4 and Thiamin HCl, Pyridoxine HCl, Nicotinic acid with addition of Tyrosine, Adenine sulphate, NaH 2 PO 4 and 30 gm/l sucrose-(msi).
TYAGI & KOTHARI: RAPID IN VITRO REGENERATION OF G. JAMESONII 585 Different auxins (IAA, IBA, NAA, PAA, 2,4-D) and cytokinins (Kn and BAP) were incorporated in the above mentioned media singly or in combinations. The ph of medium was adjusted to 5.8 and for gelling 0.8% agar was added. MS, MSA and MSI media incorporated with BAP or Kn were used for callus induction and multiplication of shoots. Callus produced on different combinations was transferred on MS and MSA media with BAP (2,4 mg/l)+iaa (0.1 mg/l) and adenine sulphate or MSI medium with 4 mg/l Kn and 0.5 mg/l IAA for shoot initiation. Cultures obtained from the explants were regularly subcultured for maintenance and multiplication of shoots. During subculturing care was taken to remove pale and necrotic tissues, as their presence was inhibitory for cultures. MSI with Kn 4 mg/l and IAA 0.5 mg/l was used for multiplication of shoots. Later on multiplication was done on MSI medium with 2 mg/l Kn + 0.5 mg/l PAA. Regenerated shoots (>2.5 cm) were separated individually and transferred to rooting medium. MS medium supplemented with IBA, IAA (0.2-0.5 mg/l) was used for rooting. Plantlets with fully developed root system were transferred to pots containing peat moss and garden soil (1:1). Data were analyzed using a one-way analysis of variance (ANOVA) and comparisons between the mean values of treatments were made by the least significant difference test. Results and Discussion Effect of Basal Salt Medium The basal salt medium used for regeneration studies of G. jamesonii has been varied many times in different studies. Murashige et al 2 used MS medium with modifications while Huang and Chu 3 used only MS medium. Laliberte et al 11 used Heller's mineral salts along with MS mineral salts. Mandal and Datta 4 also used MS and Heller's salts to induce shoot buds from capitular sections. The authors have also used combination of MS macro and Heller's micro salts with some more additional changes (MSA) but this was not as effective as the modification of MS along with additives (MSI). In the present study, MS, MSA and MSI were used and best results in all the three explants were observed on MSI medium (Table 1). MSA medium promoted formation of callus from the receptacles of capitular sections and swelling of flowers was initiated which also callused after 2 weeks. MSI medium supported best callus formation and multiplication. MS gave intermediary results. Effect of Growth Hormones Capitulum The sectored capitula when cultured on the three basal salt regimes fortified with different auxins (IAA, IBA, PAA, NAA, 2,4-D) in various concentrations (0.1-5 mg/l) induced callus. The callus induction started from the cut ends of capitular sections. Although on all the three media callus induction occurred but maximum callus formation (approximately 800 mg) occurred on MSI medium (Table 1). The soft pale green callus turned brown after four weeks. Rhizogenesis was observed on IAA (0.1-2 mg/l) and IBA (2-3 mg/l) supplemented media. The root formation occurred on the cut ends of capitular sections. On IAA the root length reached upto 7 cm while on ABA the roots were 2 to 3 cm and covered with root hairs. Organogenic callus (Fig. 1A) was formed in response to BAP or Kn (2-5 mg/l) + IAA (0.1-0.5 mg/l) or PAA (0.1 mg/l) and induction of shoot bud was observed within 6 to 8 weeks of inoculation. The MSI medium supplemented with Kn (4 mg/l) and IAA (0.1, 0.5 mg/l) was ideal for shoot bud formation (Fig. 1B) (Table 1). MSA and MS media produced upto 4 shoot buds on Kn (4 mg/l) and IAA (0.1,0.5 mg/l) after 8 weeks of inoculation. Combination of IAA (0.1, 0.5 mg/l) with BAP (5 mg/l) and 80 mg/l adenine sulphate in MSA medium induced upto 5 shoot buds from the capitular sections. Same combination of BAP and IAA in MSI medium was less effective and induced only 2 to 3 shoot buds. In this study, effect of Kn was found to be superior to BAP in combination with IAA. However, Mandal and Datta 4 reported regeneration of flower heads on BAP and IAA combination. In literature the regeneration was generally reported using BAP or Kn along with IAA. In this report we studied the effect of PAA with BAP and Kn and observed some remarkable improvements. Shoot buds formed on PAA and BAP or Kn supplemented media were thin and vitrified in morphology initially (Fig. 1C) but after subculturing on the Kn (4 mg/l) and PAA (0.1 mg/l) supplemented MSI media these buds developed into normal shoots approximately 10 in number. Callus obtained from different combinations with initiating shoot buds or abnormal shoot buds were transferred on MSI medium with 4 mg/l Kn and 0.5 mg/l IAA for recovery of shoots and further multiplication. Several workers have used capitulum explants as these have remarkable advantage over shoot tip explant, which costs the life of plant used in
586 INDIAN J BIOTECHNOL, OCTOBER 2004 Table 1 Effect of different media regimes on shoot initiation from leaves, capitulum sections and in vitro shoots of G. jamesonii (Culture period 6-8 weeks) Basal media Kn IAA Leaves* Capitulam In vitro shoots MS 2 0.1 2.5± 0.3 b 0 a 8.3±1.1 a 0.5 2± 0 c 2.17±0.2 b 11±2.4 b 1.0 0 a 0 a 6.3±4.2 c 4 0.1 5.5±0.3 d 2.5±0.3 c 10±4.4 d 0.5 2.5±0.3 b 4±0.4 d 8±16 a 1.0 0 a 1.8±0.2 e 6±0.8 c MSA 2 0.1 0 a 0 a 5±0.8 e 0.5 0 a 0 a 5.5±0.3 e f 1.0 0 a 1±0.4 f 3.7±1.8 g 4 0.1 3±1.6 e 2.5±0.3 c 9.2±7.7 h 0.5 1.8±.9 f 3.5±1.9 g 7.5±3.9 i 1.0 0 a 2.3±0.2 h 6±5.6 c f MSI 2 0.1 0.7±0.6 g 0 a 11.2±6.9 b 0.5 0±0 a 3±0.4 i 10.8±9.7 b 1.0 0±0.4 a 2.2±0.2 b 6±2 c f 4 0.1 6.8±0.4 h 7±1.2 j 11±12 b 0.5 3.6±0.2 i 10±1.2 k 10.8±8.1 b 1.0 0 a 5±1.2 l 6.5±3.1 c f Means followed by the same letter do not differ significantly at the 0.05 level of significance *The response was observed after 12 weeks experiment. Pierik et al 5 quartered the mature capitulum and from one capitulum 8 to 12 shoots were procured in 8 weeks. Laliberte et al 11 also utilized capitulum but at immature stage and divided it into 20 pieces and each piece produced 12 shoots after 12 weeks. Mandal and Datta 4 using 12 sections of immature capitulum obtained 4-5 shoots per section and on subculturing obtained 8-10 shoots from these sections. The authors also used immature capitulum and found that on MSI medium with 4 mg/l Kn and IAA (0.1, 0.5 mg/l) 200 shoots were produced in 8 weeks and 2200 shoots could be obtained on MSI and Kn (2 mg/l) + PAA (0.1, 0.5 mg/l) in 13 weeks. Leaves 2,4-D (1-2 mg/l) incorporated media initiated formation of semicompact callus from leaves. BAP or Kn (0.5-5 mg/l) initiated swelling of leaves and increase in thickness, by the end of fourth week they became cup shaped but callusing was not observed. Callus formation was observed on BAP (2-5 mg/l) or Kn (2-5 mg/l) in combination with IAA (0.1-1.0 mg/l) or PAA (0.1-0.5 mg/l) incorporated media. At the time of initiation callus was brown green and compact. MSA medium did not support good growth of leaves in comparison to MS and MSI media. MSI Fig. 1 In vitro regeneration of G. jamesonii: (A) Callus formation from capitular section on MSI medium supplemented with Kn (4 mg/l) and IAA (0.1 mg/l); (B) Shoot bud development on MSI medium with Kn (4 mg/l) and IAA (0.1 mg/l); (C) Callus formation and abnormal shoot buds on MSI medium with Kn (4 mg/l) and PAA (0.1 mg/l); (D) Shoot multiplication on MSI medium with Kn (2 mg/l) and PAA (0.1 mg/l) & (E) Rooting of shoot on MSI medium with 0.5 mg/l IAA.
TYAGI & KOTHARI: RAPID IN VITRO REGENERATION OF G. JAMESONII 587 medium promoted proliferation of yellowish green soft and watery callus that turned black after three weeks and, therefore, it was subcultured regularly at 2-3 weeks interval. MS medium supported growth of green to dark green callus, which was harder than the callus formed on MSI. Shoot bud organogenesis was observed on MS medium with 4 mg/l BAP and 0.1 mg/l IAA added with 80 mg/l adenine sulphate and on MSI with 4 mg/l Kn and 0.1 mg/l IAA. On MS medium fortified with 4 mg/l BAP, 0.1 mg/l IAA with 80 mg/l adenine sulphate, 2-3 shoot buds were observed after 12 weeks of maintenance in 20% cultures. These shoot buds did not elongate or increase in number on this combination thus the cultures were transferred onto other combinations such as BAP (2-5 mg/l) with IAA (0.1-0.5 mg/l) and Kn (2-5 mg/l) with IAA (0.1-0.5 mg/l). Kn (4 mg/l) and 0.1 mg/l IAA promoted multiplication (8-10) and elongation (1-3 cm long) of shoot buds. On MSI medium with 4 mg/l Kn and 0.1 mg/l IAA shoot bud formation (5-8) was observed after 12 weeks and on regular subculturing the number of shoots increased (8-10). Leaf explants were slow to be influenced for shoot multiplication in comparison to capitulum and in contrast to the report by Jerzy and Lubomski 12 they required both cytokinin and auxins for shoot organogenesis. In vitro Shoots From growing cultures the hearts with leaves were removed. Leaves were excised and hearts were used for culturing on MS and MSI media with different combinations of BAP or Kn (5 mg/l) and IAA, IBA, NAA, PAA (0.1, 0.5 mg/l) with or without adenine sulphate. A multiplication rate of upto 8-15 shoots from one in vitro shoot every 4 weeks was obtained on MSI medium with 4 mg/l Kn and 0.1, 0.5 mg/l IAA. Kn was better for shoot multiplication than BAP. Callus obtained on MS medium was dark green nodular while callus on MSI medium was light green and more in amount than on MS medium 12. Maintenance of Cultures Cultures obtained from the explants were regularly subcultured for maintenance and multiplication of shoots. During subculturing care was taken to remove pale and necrotic tissues, as their presence was inhibitory for cultures. For multiplication of shoots MSI medium with Kn 4 mg/l and IAA 0.5 mg/l was used. Callus obtained from different combinations with initiating shoot buds or abnormal shoot buds was transferred on MS or MSI medium with 4 mg/l Kn and 0.5 mg/l IAA for recovery of shoots and further multiplication. Initially this combination was used for multiplication. IAA has been the main auxin, which is reported to be supporting shoot regeneration in G. jamesonii tissue culture studies 2,4,11. The authors observed that apart from IAA, PAA was also supporting shoot initiation. Thus, its effect on maintenance of cultures was checked and BAP or Kn (2,4 mg/l) were combined with PAA (0.05-2.5 mg/l) where Kn was more efficient with PAA (Table 2) than BAP. On 2 mg/l Kn+0.5 mg/l PAA the cultures multiplied rapidly and 20-25 shoot buds developed (Fig 1D); 12-25 shoot buds were observed in organogenic cultures with 2 mg/l Kn and 0.1 mg/l PAA. Effect of PAA in improvement of elongation and normal shoot recovery has been well established in other plant systems such as Phaseolus vulgaris 13 Capsicum annuum 14 and Helianthus annuus 15. In the present study, PAA showed improvement in shoot multiplication and facilitated healthy shoot recovery. These cultures were regularly subcultured for two years. In this study one capitulum was sectored in 20 pieces, each of which produced average 10 shoots after 6 weeks. Thus from one capitulum 200 shoots were produced in 8 weeks. Each of these 20 cultures were sectored into 4 cultures after 8 weeks and each culture produced average 13.8 shoots on MSI medium with Kn and IAA and 22.5 shoots on Kn and PAA supplemented medium within 5 weeks. As a result one capitulum would form 200 shoots in 8 weeks and later 2000 shoots after 5 weeks. Rooting and Plantlet Formation Rooting was observed on different levels of IAA supplemented in MSI medium. On 0.5 mg/l IAA Kn Table 2 Maintenance of shoot organogenic cultures of G. jamesoni on different basal media and PGR regimes PGRs PAA IAA Basal media MS MSA MSI 1 0.1-9.2±0.2 a 6.1±0.5 a 10.3±2.2 b 0.5-10±0.4 b 6±0.4 a 13.8±4.1 a - 0.01 7.8±0.9 c 4.3±0.2 b 6±0.4 c - 0.5 9.3±0.2 a 5.1±0.5 c 8±4 d 2 0.1-9.2±0.9 a 6±0.4 a 19.3±3 e 0.5-11.3±0.7 d 7.6±2.2 d 22.5±1.5 f - 0.1 10.6±1.8 e 6±1.6 a 12±1.6 g - 0.5 13.5±4.3 f 5.8±1.7 a 14.16±0.5 a Means followed by the same letter do not differ significantly at the 0.05 level of significance
588 INDIAN J BIOTECHNOL, OCTOBER 2004 containing medium best rooting was observed within 4 weeks (Fig. 1E). However, rooting on hormone free basal medium has been obseved 4. The plantlets were transferred to pots containing peat moss and garden soil where they survived and grew to maturity. This regeneration protocol could be used to obtain large number of plantlets from this value flower plant. Acknowledgement This work was supported by the CSIR project No- 38(900)/95/EMR II. References 1 Das P & Singh S P K, Gerbera in Commercial flowers, edited by T K Bose and L P Yadav (Naya Prokashan, Kolkata) 1989, 601-622. 2 Murashige T, Serpe M & Jones J B, Clonal multiplication of Gerbera through tissue culture, Hortsci, 9 (1974) 175-180. 3 Huang M C & Chu C Y, A scheme for commercial multiplication of gerbera (Gerbera hybrida Hort) through shoot tip culture, J JPN Soc Hortic Sci, 54 (1985) 94-100. 4 Mandal A K A & Datta S K, Introduction of Gerbera cultivation in Lucknow Agro-climate through tissue culture of young flower heads, Indian J Biotechnol, 1 (2002) 212-214. 5 Pierik R L M, Steegmans H H M, Verhaegh J A M & Wouters, Effect of cytokinin and cultivar on shoot formation of Gerbera jamesonii in vitro, Neth J Agric Sci, 30 (1982) 341-346. 6 Pierik R L M, Steegmans H H M & Maredis J J, Gerbera plantlets from in vitro cultivated capitulum explants, Sci Hort, 1 (1973) 117-119. 7 Pierik R L M, James J L M, Maasdam A & Binnendij K, Optimization of Gerbera plantlet production from excised capitulum explants, Sci Hort, 3 (1975) 351-357. 8 Cagnet-Sitbon M, Recherches peliminaires sur la production d'haploides de Gerbera jamesonii par culture d'anthers et d'ovules non fecondes in vitro,theses de 3e cycle. Plantes Universite Paris-Sud. Centre d Orsay (1980). 9 Meynet J & Sibi M, Haploid plants from in vitro cultures of unfertilized ovules of Gerbera jamesonii, Z Pflanzenzuchtg, 93 (1984) 78-85. 10 Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962) 473-497. 11 Laliberte S, Chretien L & Vieth J, In vitro plantlet production from young capitulum explants of Gerbera jamesonii, Hortsci, 20 (1985) 137-139. 12 Jerzy M & Lubomski M, Adventitious shoot formation from ex vitro derived leaf explants of Gerbera jamesonii, Scientia Hortic, 47 (1991) 136-143. 13 Small D K & Morris D A, Promotion of elongation and acid invertase activity in Phaseolus vulgaris L. internode segments by phenyl acetic acid, Plant Growth Regulat, 9 (1990) 329-340. 14 Husain S, Jain A & Kothari S L, Phenyl acetic acid improves bud elongation and in vitro plant regeneration efficiency in Capsicum annum L., Plant Cell Rep, 19 (1999) 64-68. 15 Dhaka N & Kothari S L, Phenyl acetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L., Plant Cell Rep, 21 (2002) 29-34.