International Journal of Current Biotechnology

Rajeshbabu P, M. Gopalakrishnan, B. Janarthanan and T. Sekar, An efficient and rapid regeneration protocol for micropropagation of Rosa bourboniana from nodal explants, Int.J.Curr.Biotechnol., 2014, 2(1):24-29. International Journal of Current Biotechnology ISSN: 2321-8371 Journal Homepage : http://ijcb.mainspringer.com An efficient and rapid regeneration protocol for micropropagation of Rosa bourboniana from nodal explants Rajeshbabu P, M. Gopalakrishnan, B. Janarthanan and T. Sekar* Post Graduate and Research Department of Botany, Pachaiyappa s College, Chennai 600 030, Tamil Nadu, India. A R T I C L E I N F O A B S T R A C T Article History: Received 12 January 2014 Received in revised form 18 January 2014 Accepted 20 January 2014 Available online 25 January 2014 Key words: Micropropagation, Plant Growth Regulators, Rosa bourboniana, Shoot multiplication, Nodal explants. Introduction Roses are best known as ornamental plants grown for their flowers in the garden and sometimes indoors. Rose is one of the most important commercial flower crop used in the floriculture and cut flower industry throughout the world including India. Among the 200 species of roses distributed throughout the temperate regions, only a few are scented such as, Rosa damascena, R. gallica, R. centifolia, R. bourboniana, R. chinensis, R. moschata and R. alba (Gudin, 2000). Commercial propagation of roses is usually done by cuttings, although they can also be propagated by budding and grafting (Horn, 1992), which is difficult un-desirable and tedious process. Similarly, these methods are not efficient to support the increasing demand for present day needs. The establishment of tissue culture system for various rose species has been described (Arnold et al., 1992; Telgan et al., 1992; Rout et al., 1999; Carelli and Echeverrigary, 2002). Micropropagation of plants through tissue culture has been considered as an important and very popular method to produce plants which are very difficult to propagate conventionally by seeds and other natural means. *Corresponding author. Email address: tsekar_bot@yahoo.com. The present study was aimed to establish an efficient and rapid protocol for in vitro propagation of Rosa bourboniana through nodal explants. In vitro shooting followed by multiplication response of nodal explants of Rosa bourboniana were observed on MS medium supplemented with individual and synergetic concentrations of plant growth regulators (BAP, NAA and IAA in a range of 0.25 to 2.0 mg/l). Among the concentrations tested, BAP at 1.00 mg/l showed a maximum of 71% of shoot proliferation with 17.33 ± 1.21 number of shoots per explant with 4.26 ± 0.18 cm mean height of individual shoot. The combination of BAP (1.00 mg/l) and NAA (0.10 mg/l) showed 70% of shooting response with 8.33±0.81 number of shoots per explant with 5.30 ± 0.80 cm mean height of individual shoot. The combination of BAP (1.00 mg/l) and IAA (0.15 mg/l) produced 56% of shooting response with 6±0.89 number of shoots per explants with a mean shoot height of 4.16±0.2 cm. After standardization of PGRs for shoot multiplication, the multiplied shoots were subjected for rhizogenesis using various concentrations of PGRs. IAA and IBA were tested in varying concentrations from 0.25-2.00 mg/l. IAA in a concentration of 1.00 mg/l recorded 92% of rooting response with a maximum number of 30.83 ± 1.16 root hairs in a mean root length of 2.38 ± 0.17 cm. IBA in a concentration of 0.25mg/l responded 86% of root formation with 12 ± 1.26 number of roots per explants with a mean root length of 1.73 ± 0.12 cm. The rooted plants were transferred to paper cups amended with red soil and vermiculite in the ratio of 1:1 and were kept in the humidity chamber for acclimatization. The established system is efficient enough to be used for mass production of healthy plants in a short period time. The study suggested that the protocol produced in the present study can be easily adopted for micropropagation of other rose varieties.. In the last few years, in vitro propagation technique has revolutionized commercial nursery business. The great benefit of in vitro propagation technique is the enormous multiplicative capacity to produce disease free plants in a relative short period of time with independent of seasonal factor in a cost effective manner. The plantlets developed through tissue culture will reduce input costs, increase effective management and enable market pricing because of contamination and disease free products. Although vegetative propagative method like cutting, layering, budding and grafting is a predominant technique in roses, yet it does not ensure healthy and disease free plants. Another main drawback in the conventional method is the seasonal dependence and slow multiplication rates of the plant. Significant features of in vitro propagation procedure are its enormous multiplicative capacity in a relatively short span of time, production of healthy and disease free plants, and its ability to generate propagules around the year (Dhawan and Bhojwani, 1986). There are several micropropagation protocols available for the mass production of Rosa bourboniana propagules through tissue culture (Kintzios et al., 1999; Ibrahim and Dobergh, 2001; Kim et al., 2003; Hameed et al., 2006; Drefahl et al., 2007; Previati et al., 2008) however only cost efficient protocol capable of producing a large number of healthy plantlets in several subcultures is successful. The ultimate aim of this study is to produce Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol. 24

Table 1. Individual and combined effect of BAP with NAA and IAA on in vitro direct regeneration from nodal explants of R. bourboniana Concentration of PGRs (mg/l) % of shoot proliferation No. of shoots per explant Mean shoot height (cm) 0 0 0 0 BAP 0.25 22 01.33±0.51 2.25±0.16 0.50 46 10.83±0.75 3.35±0.15 0.75 58 14.66±1.03 3.96±0.12 1.00 71 17.33±1.21 4.26±0.18 1.25 59 15.33±1.21 3.26±0.33 1.50 52 12.50±1.04 2.31±0.17 1.75 45 10.16±0.75 1.45±0.17 2.0 27 06.83±0.75 0.45±0.18 BAP NAA 0.25 0.0 22 1.33±0.51 2.25±0.16 0.50 0.025 53 5.83±1.16 3.65±0.18 0.75 0.05 67 6.83±0.75 4.08±0.07 1.00 0.10 70 8.33±0.81 5.30±0.80 1.25 0.15 63 6.33±1.03 4.08±0.19 1.50 0.20 60 4.33±0.81 3.4±0.34 1.75 0.25 49 3.16±0.75 3±0.28 2.0 0.30 36 1.33±0.51 1.66±0.30 BAP IAA 0.25 0.0 22 1.33±0.51 2.25±0.16 0.50 0.05 36 3.00±0.89 2.35±0.13 0.75 0.10 44 4.83±0.75 3.41±0.23 1.00 0.15 56 6.00±0.89 4.16±0.12 1.25 0.20 43 4.16±0.75 3.81±0.11 1.50 0.25 36 3.16±0.75 2.48±0.37 1.75 0.30 23 2.66±0.81 1.56±0.33 2.0 0.35 18 1.15±0.54 0.93±0.22 a suitable, reproducible and rapid protocol for micropropagation of R. bourboniana under in vitro conditions. Materials and Methods Plant material and sterilization The mother plants of Rosa bourboniana were collected from a progressive nursery farm located at suburban of Chennai during May, 2011. The collected plants were planted in mud pots amended with red soil and vermicompost and was established at the shade house of the Post graduate and Research Department of Botany, Pachaiyappa s college, Chennai, India. The plants were watered well and maintained for healthy growth. The explants for tissue culture were taken from these plants. Explants were collected from actively grown shoots of the mother plant without any disease symptoms using a sterile surgical blade. The explants were excised into 1 cm in length and were washed well in running tap water to remove the soil or sand particles adhering and also to reduce the microbial load in the surface of explants. After washing the plant materials, the explants were treated with tween 20 detergent solution for 5 minutes and rinsed in double distilled water for three times. Then the explants were treated with 70% ethanol for 30 seconds and washed with sterile distilled water for five times. Further the explants were immersed in 0.1% (w/v) mercuric chloride solution for 5 minutes. Finally the explants were repeatedly washed in distilled water for five times to remove the traces before it is inoculated on the culture medium. Culture medium and plant growth regulators Murashige & Skoog (1962) medium was tested and stock solutions of micro salts, vitamins (w/v) and plant growth regulators (PGRs) were prepared in sterile distilled water. All stock solutions were stored in a refrigerator at 4 C. During media preparation, macro salts were weighed separately and dissolved in distilled water one by one under continuous stirring. Stock solutions of micro salts, vitamins and PGRs were taken out from the refrigerator and allowed to attain room temperature. The required volumes of stock solutions of micro salts were taken using measuring cylinders. Vitamins and plant growth regulators like BAP, IAA, NAA and IBA were pipetted out; sucrose and other additives if any, were weighed, dissolved and the media is made up to the desired volume. The ph of the medium was adjusted to the suitable range (5.6±0.2) with 1N NaOH or 1N HCL before adding 0.8% agar and dissolving it by heating at 80 C in a water bath. Interaction between the in vitro raised plantlets with the gelling agent in culture medium is a dynamic process and the changes in gel consistency affect the regeneration of plants or tissues. Traditionally, 0.8% agar is added to the culture medium to increase its viscosity. The explants remain above the surface of the nutrient medium. Increasing the concentration of the agar beyond the critical limit inhibits organogenesis and shoots growth due to lack of water availability to cultures. Cultures supplemented with low input of agar concentration facilitates adequate contact between the plant tissue and the medium and better diffusion of medium constituents resulting in better growth and subsequent rooting. The 25 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014

Figure 1: Micropropagation of Rosa bourboniana Fig 1. A, B & C 7 days, three weeks & five weeks old plantlets in MS medium with 1.0 mg/l BAP; D - 6 weeks old plantlets ready for multiplication; E & F - well developed & 6 weeks old multiple shoots; G & H - root initiation and well developed roots in MS medium with 1.0 mg/l IAA; I in vitro grown plants in soilrite for hardening. Volume 2; Issue 1; Jan, 2014 Int.J.Curr.Biotechnol. 26

required media were dispensed in culture vessels (15 20 ml medium in 25 150 mm culture tubes and 50-60 ml medium in 250 ml culture flasks) and closed tightly with non-absorbent cotton plugs. The medium was sterilized by autoclaving at 1.06 kg/cm pressure at 121 C for 15 min. Cultures were incubated at 24±2 C under cool white florescent light (with quantum flux density of 40 µmol/m/ s) with 16-8h regime photoperiod. Results and Discussion Individual effect of BAP on in vitro direct regeneration from nodal explants The shooting response from nodal explants in MS medium supplemented with BAP is presented in table 1. BAP has been used for most experiments on in vitro flowering of a number of plants (Wang et al., 2002). BAP alone in the concentration of 1.0 mg/l promoted better shoot induction and multiple shoot development. Maximum number of 17.33±1.21 shoots emerged out from a single explant after six weeks on MS medium supplemented with 1.0 mg/l BAP with 71% of shooting response. Maximum shoot length was observed (4.26 cm) on MS medium supplemented with 1.0 mg/l BAP after 6 weeks (Fig. 1). Further shoot induction and multiple shoot development was observed when shoots were sub cultured in fresh medium once in two weeks on the same medium. The shoots were multiplied repeatedly by sub culturing the original explants on shoot multiplication medium (BAP 1.0 mg/l) after each harvest of new shoots (Fig. 1). Nodal explants on MS medium without PGRs did not showed any growth. The results obtained in the present study is comparable with earlier results (Hasegawa, 1980; Wulster and Sacalis, 1980) showing that inclusion of BAP at various concentrations in the agar solidified culture medium was essential for bud break and shoot multiplication of Rosa hybrida. Vijaya et al. (1991) also reported that BAP was the most effective growth regulator in stimulating shoot proliferation. Pati et al. (2001) successfully demonstrated the effect of BAP (2.0 mg/l) included in the static liquid MS medium for multiple shoot proliferation. Combined effect of BAP and NAA on in vitro direct regeneration from nodal explants The Synergistic effect of BAP (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00 mg/l) with NAA (0.025, 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30 mg/l) was studied for shoot initiation and multiplication in Rosa bourboniana (Table 1). BAP or NAA have been used for most experiments on shoot multiplication of a number of rose species (Wang et al., 2002; Vu et al., 2006; Drefahl et al., 2007). Vijaya et al. (1991) reported that the use of 3 auxins (NAA, IAA and IBA) in combination with BAP and found that NAA was more effective than IAA or IBA in the production of multiple shoots. Among the various concentrations and combinations of BAP and NAA tested, MS medium supplemented with the combinations of BAP (1.00mg/l) and NAA (0.10mg/l) resulted 70% shoot regeneration and producing highest number of shoots (8.33±0.81) with an average shoot length of 5.30±0.80 cm from nodal explants after six weeks of culture. The combination of BAP (0.75mg/l) with NAA (0.05mg/l) was found to show 67% response and producing 6.83±0.75 shoot lets per explant with an average shoot length of 4.08±0.07 cm. Only 36% of response was resulted at lower concentrations of both BAP and NAA. When the concentration of PGRs was increased, a gradual fall in the number of shoots per explants was recorded. Similarly on increasing the concentration of NAA, the response was low due to basal calli formation in the cut ends of the explants. The inclusion of auxins at low levels neither enhanced nor repressed shoot multiplication regardless of the BAP concentration. Bressan et al. (1982) reported maximum promotive effect with BAP as compared to 2-isopentyladenine (2-iP). Combined effect of BAP and IAA on in vitro direct regeneration from nodal explants The synergistic effect of BAP (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00 mg/l) with IAA (0.05, 0.10, 0.15, 0.20, 0.25, 0.30 and 0.35 mg/l) was studied for shoot initiation and multiplication in Rosa bourboniana. MS medium supplemented with BAP (1.0 mg/l) in combination with Table 2. Influence of IAA and IBA on rooting of in vitro formed shoots of R. bourboniana P la n t h o r m o n e ( m g /l) P e r c e n t a g e o f r o o t f o r m a ti o n N o. o f r o o t s p e r e x p la n t M e a n r o o t l e n g t h ( c m ) 0 0 0 0 I A A 0. 2 5 3 3 1 2. 8 3 ± 1. 1 6 0. 9 5 ± 0.1 8 0. 5 0 6 5 2 0. 0 0 ± 1. 1 4 1. 7 1 ± 0.1 3 0. 7 5 8 1 2 6. 5 0 ± 1. 0 4 2. 0 5 ± 0.1 0 1. 0 0 9 2 3 0. 8 3 ± 1. 1 6 2. 3 8 ± 0.1 7 1. 2 5 6 9 2 6. 5 0 ± 1. 8 7 1. 9 0 ± 0.1 4 1. 5 0 4 0 2 2. 8 3 ± 1. 6 0 1. 7 8 ± 0.1 1 1. 7 5 2 6 1 6. 6 6 ± 1. 3 6 2. 1 1 ± 0.1 4 2. 0 0 1 1 1 4. 1 6 ± 1. 7 2 1. 9 3 ± 0.0 8 I B A 0. 2 5 8 6 1 2. 0 0 ± 1. 2 6 1. 7 3 ± 0.1 2 0. 5 0 7 8 9. 1 6 ± 0. 7 5 1. 3 8 ± 0.0 7 0. 7 5 6 9 7. 3 3 ± 1. 2 1 1. 1 6 ± 0.0 8 1. 0 0 4 5 6. 1 6 ± 0. 7 5 0. 9 3 ± 0.1 0 1. 2 5 3 1 4. 3 3 ± 0. 8 1 0. 6 1 ± 0.0 7 1. 5 0 1 9 2. 8 3 ± 0. 7 5 0. 4 3 ± 0.0 8 1. 7 5 1 1 1. 6 6 ± 0. 5 1 0.2 ± 0. 0 6 2. 0 0 2 0. 5 0 ± 0. 5 4 0. 0 5 ± 0.0 5 27 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014

IAA (0.15 mg/l) resulted 56% of shooting response from the nodal explant (Table 1). The value of highest number of total shoots was 6 ±0.89 with an average shoot length of 4.16±0.12 cm after six weeks of inoculation. At lower concentrations of BAP and IAA, the percentage of shoot multiplication rate was very less (26% - 36%) with 1.33±0.51 mean average shoot with the size of the individual shoot ranging in 1.51±0.21 cm. Increasing the concentration of BAP and IAA resulted in poor response of explants in the culture and after two weeks, the growth was completely arrested. Bini et al. (1983) used BAP and Zeatin for multiplication of Rosa indica, whereas Singh and Syamal (1999) used BAP, NAA and GA3 to obtain more than 5 shoots per explant in rose explants. Influence of IAA and IBA on rooting of in vitro formed shoots In most of the earlier investigations, varying concentrations of different auxins were used for root induction. In the present study, the auxins like IAA and IBA were used for rooting from microshoots of R. bourboniana (Table 2). Individual micro shoots in a length of 5.30 cm were taken from in vitro multiplied shoots and inoculated on half strength MS medium supplemented with various concentrations of IAA and IBA individually for rhizogenesis. Among the various concentrations of IAA (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00mg/l) tested, maximum number of roots (30.83±1.16) with 92% response with an average root length of 2.38±0.17 cm was obtained in half strength MS medium supplemented with 1.00mg/l after six weeks of culture. Increasing the concentration of IAA decreased the number of root and its length (Fig. 1). The same effect prevailed as the concentration was decreased. Khosh- Khui and Sink (1982) reported that a combination of IAA and NAA was effective in induction of roots in R. hybrida cv. Bridal Pink. Rooting of in vitro grown shoots was also achieved by dipping the cut ends of shoots for a few hours in an aqueous solution of 1 mm IAA instead of being continuously cultured on auxin containing medium (Collet, 1985). Among the various concentrations of IBA (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00) tested, 0.25 mg/l concentration of IBA after six weeks of culture showed 86% of rooting response producing 12±1.26 roots per shoot with an average root length of 1.73±0.12 cm. Increasing the concentration of IBA resulted in decreasing the rate of root initiation (2%) with a least number of roots (0.5±0.54). No root was obtained in half strength MS Medium in the absence of PGRs. Sisko (2011) reported that, of the three genotypes of Rosa spp. studied, the second genotype developed 55.6% of rooting with full strength MS medium supplemented with 0.5 mg/l IBA. According to his results it showed that, the best rooting medium was very specific for each studied genotype. Horn et al. (1988) examined that the success of in vitro rooting of roses is strongly dependent on cultivar. Hardening and acclimatization The ultimate success of in vitro propagation lies on the successful establishment of plants in the soil. To acclimatize the micropropagated plants, different approaches were used so far to have a positive result in ex vivo condition. The rooted plants were removed carefully from culture vessels, washed under running tap water to remove the remains of agar and transferred to paper cups amended with red soil and vermiculite in the ratio of 1:1 and were kept in the humidity chamber under standard range of temperature, humidity, diffused light, carbon-dioxide concentration and controlled flow of air for acclimatization (Fig. 1). After acclimatization the plantlets were subjected to hardening. In the present study, the rooted plantlets were transferred to poly bags amended with red soil, charcoal chips, coconut coir and vermicompost in the ratio of 1:1:1:1. MS salt solution was used as nutrient source for one month thereafter MS salt solution was replaced by normal tap water. The plantlets were maintained in the poly house during the course of successful acclimatization. New shoots with tender leaves were found to be developed in the due course. After one month of time the poly bags were kept under direct sunlight and were watered regularly. The overall survival percentage of acclimatized plants was 86%. The Rose plantlets were watered near the root system avoiding flushing over the leaf with 1/ 10 th MS salt solution at one week interval, which proved to be beneficiary for better growth. The plantlets were further amended with ½ strength MS medium without vitamins and sucrose thrice a week. The plantlets were found healthy without any visible symptoms of wilting and necrosis. The successfully hardened plantlets were planted in mud pots for field transfer. Conclusion The results of the present investigation demonstrated that, a reproducible, rapid and efficient regeneration protocol for in vitro micropropgation of Rosa bourboniana has been established through nodal explants with BAP as a main PGR for shoot initiation and shoot multiplication. Disease-free plant propagation via tissue culture plays a vital role in commercial production. Therefore, further optimizations of the tissue culture protocols are crucial to integrate these technologies into commercial applications. It is, therefore, important to bring about further improvements in the existing tissue culture protocols. The study suggested that the protocol produced in the present study can be easily adopted for micropropagation of other rose varieties. Acknowledgements The authors are thankful to the Head of the Department of Botany, the Principal and the Management of Pachaiyappa s College, Chennai for providing laboratory facilities and encouragement. 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Gudin S, 2000. Rose: Genetics and breeding. In: Janick, J. (ed.), Plant Breeding Reviews, vol. 17, John Wiley and Sons, Inc. pp.159-189. Hameed N, Shabbir A, Ali A, Bajwa R, 2006. In vitro micropropagation of disease free rose (Rosa indica L.). Mycopath 4: 35-38. Hasegawa PM, 1980. Factors affecting shoot and root initiation from cultured rose shoot tips. J Amer Soc Hort Sci. 105: 216-20. Horn W, Schlegel G, John K, 1988. Micropropagation of roses (Rosa hybr.). Acta Hortic. 226:623-7. Ibrahim R, Debergh PC, 2001. Factors controlling high efficiency of adventitious bud formation and plant regeneration from in vitro leaf explants of roses (Rosa hybrida). Sci Hortic. 88: 41-57. Khosh-Khui M, Sink KC, 1982. Rooting enhancement of Rosa hybrida for tissue culture propagation. Sci Hortic. 17: 371 376. Kim SW, Oh SC, In DS, Liu JR, 2003. Plant regeneration of rose (Rosa hybrida) from embryogenic cell-derived protoplasts. Pl Cell Tiss Org Cult. 73: 15-19. Kumar A, Sood A, Plani UT, Gupta AK, Plani LMS, 2001. Micropropagation of Rosa damascenemill. from mature bushes using thidiazuron. J Hortic Sci Biotech. 76: 30-34. Pati PK, Sharma M, Ahuja PS, 2001. Micropropagation, Protoplast Culture and its implications in the improvement of scented roes. In: Proceedings of the Third International Symposium on Rose Research and Cultivation. Acta Hort. 547: 147-158. Previati A, Benelli C, Da Re F, Ozudogru A, Lambradi M, 2008. Micropropagation and in vitro conservation of virus-free rose germplasm. Prop Orna Plants 8: 93-98. Rout GR, Samoantaray S, Mottley J, Das P, 1999. Biotechnology of the rose: a review of recent progress. Sci Hortic. 81: 207 28. Singh SK, Syamal MM, 1999. Critical studies on the effect of growth regulators on in vivo shoot proliferation in Rosa hybrida L cv Sonia for micropropagation. J Appl Hort Lucknow 1: 91-93. Sisko M., 2011.Micropropagation of roses (Rosa spp.): The effects of different media on in vitro rooting. Agricultura 8: 19-22. Telgan H, Elagoz V, van Mill A, Paffen A, de Klerk G, 1992. Role of plant hormones in lateral bud growth of rose and apple in vitro. Acta Hort. 319: 137 42. Vijaya N, Satyanarayana G, Prakash J, Pierik RLM, 1991. Effect of culture media and growth regulators on in vitro propagation of rose.curr. Plant Sci Biotech Agric. 12: 209-214. Vu NH, Anh PH, Nhut DT, 2006. The role of sucrose and different cytokinins in the in vitro floral morphogenesis of rose (hybrid tea) cv. First Prize. Pl Cell Tiss Org Cul. 87: 315-320. Wang GY, Yuan MF, Hong Y, 2002. In vitro flower induction in roses. In Vitro Cell Dev Biol Plant 38: 513-518. Wulster G, Sacalis J, 1980. Effects of auxins and cytokinins on ethylene evolution and growth of rose callus tissue in sealed vessels. Hort Sci. 15: 736-737. 29 Int.J.Curr.Biotechnol. Volume 2; Issue 1; Jan, 2014