RAPID MULTIPLICATION of ORNAMENTAL BULBOUS PLANTS of Lilium Orientalis and Lilium Longiflorum

RAPID MULTIPLICATION OF ORNAMENTAL BULBOUS PLANTS OF Lilium orientalis AND Lilium longiflorum

Shumaila Khan1*, Muhammad Jafar Jaskani1, M. Zafar Iqbal2 and Ashifa Rafiq1

1Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan 2Department of Plant Pathology, Bahauddin Zakariya university, Multan, Pakistan *Corresponding author (e-mail: [email protected])

Abstract Lilium is one of the most important cut flowers all over the world. It ranks within the ten top most beautiful cut flowers because of its attractive and large showy flowers. Ornamental lily hybrids have been developed in very large amount. Effect of different growth hormones and different growth media was observed in this study. Bulb scales were used as an explant, cultured in vitro on Murashige and Skoog (MS) medium containing different concentrations of 6-benzyladenine (BA) and naphthalene acetic acid (NAA). Among different treatments used for plant culturing, the MS medium supplemented with 6-benzylaminopurine (BAP) 3.0 mg/L was found to be the best for shoot initiation from scales of bulbs. After that plants were transferred to different media for multiple shooting. Out of different concentrations used the medium with 1.0 mg/L BAP and 1.0 mg/L NAA increased frequency of shoot formation up to 90%. An average of about 10 ± 3.73 roots/explants with well-developed roots and bulblet formation were obtained in this medium.

Introduction of lilies consist of the insufficient accessibility of Today proliferation of numerous showy foliages via vigorous, disease-free foliage, and little tissue culture has come to be a known profitable exponentiation ratios (Van Aartrijk et al., 1990). exercise. Lilies first initiated in East Asia plus North Three to four bulbs can produce by scaling of lilies in America. Genus Lilium, have its place in family vitro according to size and variety. So through Liliaceae, exists in worldwide significant cut florets. scaling one can get 50 and 100 bulbs, to meet today Lilium used as showy foliage in place of centuries demand for lilies (Varshney, 2000). Lilies have principally for their enormous florets. In place of become economically significant, principally because profitable purposes, conversely, this is an of their enormous, attractive florets. Various varities unproductive proliferation system as the lilium has of hybrid lilies have superior potentialities in little exponentiation ratio over small time. In recent horticulture industry (Kapoor et al., 2008). Lilies are times, tissue culture approaches have been applied to widely used in floret trade and container plant as well produce lilies to promptly attain an enormous amount by using a method of scaling (Stimart 1981), leaves of selected foliage (Kanchanapoom et al., 2011). Lily (Stenberg 1977), stem sections (Ebrahim, 2004) and secures 7th position in cut flowers in demand and roots (Kumar et al., 2008). Scaling method is widely monitory returns, likewise as main container plant used in tissue culture (R. Kapoor et al., 2008). Bulbs additionally. Eighty out of 220 species are growing in can be produced directly from scales and from leaves northern hemisphere. Overview of lilium will after callus formation. (Nhut et al., 1998). In the definitely show a momentous role in contribution experiment different levels of NAA, 2,4-D and BA new proportions to the prevailing floriculture trade will use to examine their effect on bulblet which in consequence far functions on customary regeneration in different explants of two hybrid lily lines. The usage of tissue cultured foliage can cultivars. (Kapoor et al., 2008.) lily bulbs when take rationalize lilium cultures because direct planting into from greenhouse or from a field have very high pots without special manipulation in place of an contamination rate. Optimum application and enormous harvest of bulbs is possible. So treatment time of sterilizing agent is very important microproliferation via in-vitro approaches is of as it is different for different type of plants. Sucrose countless importance so that accelerate the is used to provide energy and penetration ability to proliferation ratio plus decrease the requirement of lily explant (Liu et al., 2005). Temperature is also bulblets (Ghaleb et al., 2010; Saifullah et al., 2010). play a significant role in propagating plants in vitro. The foremost limitations in conventional proliferation So keeping temperature optimum is necessary

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(Wang., 2006). Light is a very necessary physical 1.5 mg/L and 2 mg/L BA (6-Benzylaminopurine). factor for growth and development of plant in vitro. Each treatment was applied in three replications Certain amount of naphthalene acetic acid (NAA) having ten explants in each. The explants were will be added to check the root growth initiation and cultured in a growth room at 25°C and a 16 hour development (Liu et al., 2012). The main object of photoperiod under florescent light at the intensity of this experiment is to check the propagation of Lilium 100 μmol m-2 per second. The explants cultured under different culture conditions. without growth regulators served as control treatment. The Materials and Methods Cultures were transferred to fresh medium at 30 days Lilium bulbs were obtained from Institute of intervals. After ninety days, the following data were Horticultural Sciences, Universityof Agriculture recorded: percentage of explants producing bulblets, Faisalabad. Bulbs were pre-cooled for almost 6 average number of bulblets per an explant. All data weeks before culture. The optimum temperature for was analyzed using completely randomized design bulbs were kept at 2○C. bulbs were grown in (Gomez and Gomez, 1984). The statistical analysis glasshouse where temperature was maintained at based on mean values per an explant was made using 25+2°C for 30 days whereas humidity was 90 %. ANOVA (analysis of variance). The comparative After one month of culture some plants began LSD multiple range test (P = 0.05) was used to sprouting when leaves started unfolding .Different determine differ rences between treatments. types of explants e.g leaf portion, inner scales of a In the preliminary experiment different levels of bulb and lower scale portion of a bulb were separated NAA and BA alone or in all possible combinations in each cultivar, washed with tap water and then with were used to examine their effect on bulblet distilled water. After washing with tap waterfollowed regeneration, average number of bulblets in the by distilled water bulbs were surface sterilized by 5% explants of bulbscale, leaf,inner scales and lower NaOCl for 5 minutes and then washed 3 to 5 times portion of scales of Lilium. with double distilled water. Leaves were cultured on basal MS medium supplemented with 3% sucrose Results and Discussion and 8g/L agar for solidification. Medium pH was After inoculation of about one month, 70% to 80% maintained at 5.8. Scales were cultured after washing uncontaminated. After thirty days of inoculation, under tap water 6-7 times then sterilize with NaOCl about 70% to 80% uncontaminated cultures were followed by 2 drops of tween 20 cultured in basal MS obtained when the initial explants were surface medium supplemented with 30g/L and 8g/L agar. sterilized with 5% NaOCl. The analysis of the data The following growth hormones were added to revealed that the treatment with 2 mg/L NAA and medium separately or in combination with each 1.5 mg /L BA was the most effective in explant other. regeneration and stimulation of bulblet formation 1.0 mg/L and 2 mg/L NAA (naphthalene acetic acid) (Table. 1).

T a b l e 1 . Effect of NAA and BA on explant regeneration in different explants of two hybrid cultivars of Lilium Treatment (mg/L) Aslatic hybrid Oriental hybrid Explants Number of bulblets Explants producing Number of producing bulblets per explant bulblets % bulblets per % explant Control 75.3 1.3 0.00 0.00 NAA (1.0) 76.5 1.9 73.5 1.3 NAA (2.0) 77.1 2.1 74.9 1.9 BA (1.5) 76.8 1.7 0.00 0.0 BA (2.0) 78.7 2.2 68.2 1.2 NAA + BA (1.0 + 1.5) 76.7 2.7 74.8 2.0 NAA + BA (1.0 + 2.0) 77.5 3.3 57.0 1.9 NAA + BA (2.0 + 1.5) 80.5 3.9 76.1 2.6 NAA + BA (2.0 + 2.0) 78.7 2.6 75.5 1.8

LSD0.05** 2.8 0.3 0.8 0.4 Least significant difference at 5% level of significance

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Of the cultured explants, about 78% developed amethystina. The efficiency of root for multiplication bulblets in bulblscale explant, which differed of a large number of genetically identical plants significantly from other explants (Tab. 2). These makes it a potential source of explant for mass results are contrary to Niimi (1984) who reported that propagation (Ostazaki and Henson, 1965). The least the ability to regenerate bulblets was greatest in number of bulblets was recorded in the explants of explants of stem in Lilium rubellum but support leaves. Among the cultivars, ‘Oriental lily’ formed others, who reported that in vitro scale culture was greatest number of bulblets. The interaction between the best and most prolific method (Kumar et al., cultivars and explants revealed that the cultivar 2006). No significant differences were observed in ‘Oriental lily’ produced greater number of bulblets the explants producing bulblets among the cultivars. (4.0) in the explants of inner portion of bulbscales. The greatest average number of bulblets per an The explants of bulbscale produced the largest explant was observed in the explants of root (Tab. 3). bulblets, followed by leaf explants. Niimi (1984) Kumar et al. (2008) reported greatest number of reported heaviest bulblets in the explant of stem in L. bulblets with 2 mg/L NAA and 2 mg/L BA from in rubellum. The differences in the results may be due vitro explant of root in oriental hybrid lily. Cavallini to differences in cultural conditions and genotypes and Natali (1989) obtained somatic embryos and used. regenerated plants from root explant in Brimeura

Table 2. Effect of different concentrations of BAP + NAA on multiple shoot formation. Bulb regeneration rate (%) Explant Asiatic hybrids Oriental hybrids Explant mean Leaves 2.66+0.02 2.16+0.03 2.43 Inner bulbscales 4.00+0.06 2.75+0.14 2.78 Lower bulbscales 3.60+0.05 3.30+0.10 3.45 mean 3.02 2.73

Effect of BAP + NAA on shoot multiplication of supplemented with 0.1 mg/L NAA + 0.1 mg/L BAP regenerated plants: in all harvesting seasons proved to be superior to Previously regenerated plantlets were transferred to others. Naz et al., (2011) noticed that maximum MS medium supplemented with different shoot formation response i.e., 90% was shown by concentrations of BAP + NAA for multiplication of medium MS+NAA 18µmol and 80% in MS+ BAP 2 shoots. Best results were obtained in 0.1mg/L BAP + mg/l. MS+ BAP 2 mg/l appears to be the better 0.1mg/L NAA with 90% of frequency of shoot medium for the micropropagation of Riccinus regeneration (Table 3). Along with shoot communisas compared to other media combinations multiplication, this medium also induced highest applied. It is noticed that these concentrations of number of root formation. Length of roots formed bothauxins and cytokinins (BAP 1.0 + NAA 1.0) was also highest in the same medium. Similar mg/L enhanced multiplication rate in Lilium plants concentration of BAP + NAA was also reported by otherwise all other moderate and higher Azadi and Khi-Kosh (2007), they tested the various concentrations of these hormones would not show treatments to induce multiple shooting in Lilium In better results. vitro regenerated plants, the MS medium

Table 3 . Effect of explant, cultivar and their interaction on mean number of bulblets in hybrid lilies Plant hormones Shoot regeneration No. of shoots/ explant Number of roots/explant BAP (mg/L) NAA (mg/L) frequency % 1.0 1.0 90 8.0+2.51a 10.0+3.73a 2.0 2.0 50 1.0+0.59b 01.0+0.07d 3.0 3.0 40 1.0+0.73b 01.0+0.13d No. of test tubes cultured = 10, each value is mean of five replicate with standard error (mean ± S. E). Means within a column not sharing a common superscript differ significantly (p<0.05) according to LSD

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Effect of BAP and NAA on shoot regeneration Ghaleb, W.S., Sawwan, J.S., Akash, M.W., Al- from Lilium bulb scales: Abdallat, A.M., 2010: In vitro response of two As evident in the Table 3, cultures inoculated in BAP Citrus rootstocks to salt stress. International 1.0 mg/L showed highest rate of shoot regeneration Journal of Fruit Science 10, 40-53. of 90% along with roots with many root hairs. Gomez K.A., Gomez A.A. 1984. Statistical Concentrations of NAA did not response towards procedures for agricultural research. John Wiley shoot regeneration process. The promotive effect of & Sons, New York, 328-332. BAP was also noticed by Takayama & Misawa Han, B.H., J.Y. Hee, W.Y. Byeoun and Y.P. Kee. (1982) that tested the effect of NAA, BAP and 2004. In vitro propagation of Lilium longiflorum Kinetin on organ formation from bulb scales and ‘Georgia’ by shoot formation as influenced by observed that BAP has a stronger physiological effect addition of liquid medium. Scientia Hort., 103: on organ formation both for shoot formation and root 39-49. formation. Han et al., (2004) used bulb scales of L. Kanchanapoom, K., T. Ponpiboon., W. Wirakiat plus longiflorum cv. Georgia and cultured these on MS K. Kanchanapoom. 2011. Regeneration of lily medium with BA (benzyladenine) to induce shoots. (Lilium longiflorum ‘Easter lily’) via callus After 6 weeks, the frequency of shoot formation was derived from leaf exfoliage cultured in vitro. very high on the media with 2.2 µM BA was most Sci. Asia 37: 373–376. effective in inducing shoots from bulb scales. The Kapoor, R., S. Kumar., J. K. Kanwar plus P. K. highest results for shoot induction (98.33%) and Mahajan. 2008. In vitro bulblet productivity in number of shoots (22.07) were observed from same different exfoliage of hybrid lilies. J. of Fruit cormel sprout in Gladiolus on MS medium plus Showy Pl Research 16: 345-352. containing BAP 4 mg L-1 by Memon et al., (2013). Kumar S., Chaudhary V., Kanwar J.K. 2008. In vitro Sivanesan et al., (2012) observed that BA was found propagation of oriental lily from root explant. to be the most effective cytokinin for multiple shoot ADV.HORT. SCI. 22: 63-65. induction among the other cytokinins used in the Kumar S., Kanwar J.K., Sharma D.R. 2006. In vitro study. It is clearly evident from the work of other propagation of Lilium – review Paper. ADV. scientists that BAP has showing tremendous results HORT. SCI. 20: 181-188. among all other cytokinins. This is cleared from the Liu X., Liu Y. Q. and Chen Y. Q. (2005). In vitro done experiment and from results of the work carried plant regeneration from the immature seeds of out by other researchers. Cymbidium faberi. Plant Cell, Tissue and Organ Culture, 81: 247-251. Conclusion: Liu, X., Y. Diao., Y. Zhang plus Y. Lu 2012. In The present study demonstrates a simple and efficient vitro micro-proliferation of Longiflorum-Asiatic method for high frequency direct shoot regeneration (LA) hybrids lily (Lilium) cultivar ‘eyeliner’. from scales of 2 Lilium species. The system is rapid, Afr. J. Biotechnol. 11: 13506-13517. starting with the initiation of tissue culture. Such a Memon, N., M. Qasim, M.J. Jaskani, A.A. Khooharo, high regeneration frequency would be useful for mass Z. Hussain and I. Ahmed. 2013. Comparison of propagation and multiplication of this valuable plant various explants on the basis of efficient shoot in Pakistan. regeneration in Gladiolus. Pak. J. Bot., 45(3): 877-885. References Naz, S., F. Tabassum, S. Javad, S. Ilyas, F. Aslam, N. Azadi, P. and M. Khosh-Khui. 2007. Munir and A. Ali. 2011. Micropropagation and Micropropagation of Lilium ledebourii (Baker) callogenesis of a recalcitrant species Boiss as affected by plant growth regulator, Ricinuscommunis. Pak. J. Bot., 43(5): 2419- sucrose concentration, harvesting season and 2422. cold treatments. Elec. J. Biotechnol. 4: 0717- Nhut, D. T. 1998. Microproliferation of lily (Lilium 3458. longiflorum) via in vitro stem node and pseudo- Cavallini A., Natali L. 1989. Cytological analysis of bulblet culture. Foliage Cell Reports. 17: 913– in vitro somatic embryogenesis in Brimeura 916. amethystina Saliso (Liliaceae). PLANT SCI. 62: Niimi Y. 1984. Bulblet productivity of explants from 255-261. scale, leaves, stem and tepals of Lilium Ebrahim M.K.H. 2004. Comparison, determination rubellum Baker. Sci. Hort. 22: 391-394. and optimizing the conditions required for Ostazaki A., Henson P.R. 1965. Effect of rhizome and shoot formation and flowering of morphology of propagules on performance of in vitro cultured callus explants. Sci. Hort. 101: birdsfoot trefoil clones. CROP SCI. 5: 253-254. 305-313.

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