In-Vitro Mass Propagation of Limonium Sinuatum L. Mill. (Statice) (2004)

2019 Journal of Plant Resources Vol.17, No. 1 2019J. Pl. Res. Vol. 17, No. 1, pp 147-154, 2019Journal of Plant Resources Vol.17, No. 1

Srivastava, S. K., Rawat, A. K. S. & Mehrotra, S. Uprety, Y., Asselin, H., Boon, E. K., Yadav, S. & In-vitro Mass Propagation of Limonium sinuatum L. Mill. (Statice) (2004). Pharmacognostic evaluation of the root Shrestha, K. K. (2010). Indigenous use and bio- of Berberis asiatica. Pharmaceutical Biology, efficacy of medicinal plants in the Rasuwa Saraswoti Khadka*, Nabin Rana and Sabari Rajbahak 42(6), 467473. District, Central Nepal. Journal of Ethnobiology Department of Plant Resources, Thapathali, Kathmandu, Nepal *Email: [email protected] Thapa, L. B., Dhakal, T. M., & Chaudhary, R. (2014). and Ethnomedicine, 6(3). Wild edible plants used by endangered & WHO (1991) Basic Laboratory procedure in Abstract indigenous Raji tribe in western Nepal. Int J Appl clinical bacteriology. Geneva:World Health Limonium sinuatum L. Mill. (Statice), a rosulate plant with showy inflorescences is characterized Sci Biotechnol, 2(3), 243-252. organization. by its high ornamental value as a cut flower for both fresh and dry-flower arrangements with its increasing demand in the international markets. Nepalese farmers are dependent on Indian market Tiwari, P., Kumar, B., Kaur, M., Kaur, G. & Kaur, for ornamental plants with a huge investment but are not able to get quality plants. This research H. (2011). Phytochemical screening and work is focused to develop the protocol for in vitro mass propagation of Statice so as to provide extraction: A review. International the farmers with quality plants. Shoot tips were excised from the mother plant and were surface sterilized with freshly prepared 0.1% w/v aqueous solution of HgCl for 5 minutes. The explants pharmaceutical Science, 1(1). 2 were cultured in Murashige & Shook (1962) medium supplemented with different concentrations and combinations of BAP, KIN, NAA and IBA. During in vitro establishment of explants, concentration of BAP at 1.0 mg/L and NAA at 0.1 mg/L gave the best results of shoot induction and proliferation. About 94% of explants were established on MS medium enriched with BAP and NAA. Rooting was best induced on MS medium containing NAA 1mg/L. Thick and fibrous roots were developed within 30-35 days. Regenerated plantlets were acclimatized for 10 to 15 days in greenhouse at 20±5ºC. Plantlets were successfully established in sterile sand and were transferred into poly bag containing a mixture of garden soil, organic matter and sand in 1:1:1 ratio. Plantlets survival percentage was 100% in open field condition.

Keywords: Acclimatization, Explants, Micropropagation, Murashige & Skoog (MS), Tissue culture

Introduction stress-tolerant angiosperm (Aly et al., 2002). The excellent agronomic character such as flower color, Limonium sinuatum L. Mill. (Statice), a beautiful vigor and long-lasting quality makes it an ideal ornamental plant commonly known as sea flower in floriculture industry. Growers are involved lavender which belongs to Plumbaginaceae family in hybridization with breeding efforts, extending the (Kunitake et al., 1995), comprises 150 wild species variations in color and shape of flower (Henny & (Morgan et al., 1998; Aly et al., 2002; Lledo et al., Chen, 2004). Among the cut flower grown in the 2003). L. sinuatum, a perennial herb is native to the country, Statice is currently in high demand by new eastern Mediterranean are grown commercially investors for a large scale production owing to its around the world as a cut flower for both fresh and easy plant care requirement and good selling price dry-flower arrangements (Cohen et al., 1995). There in the auction market (Mellesse et al., 2013). are about 15 to 20 horticulturally cultivated species, Ethiopian Statice flower exported to the global involving L. sinuatum, L. bonduelli, L. dregeanum, market have increased five-fold between 2006 and L. sinense, L. latifolium, L. psylliostachys, L. 2008. In 2008 alone, Ethiopia earned 114 million bellidifolium, L. gmelinii and L. perezii (Jeong et dollars from the floriculture industry (Mellesse et al., 2001). These species were grown in borders and al., 2013). In context of Nepal, with the increasing rock gardens in European countries, a decade ago. number of nurseries, the number of floriculture Gradually, they have been produced as a cut flower shops/retailers are also increasing. The floriculture in Japan and the Netherlands with an advancement businesses are growing by 10-15% per year. The of mass-propagation techniques using plant tissue floriculture sector has already fetched investment culture (Kunitake, et al., 1995; Rout et al., 2006; over NRs. 375 million in infrastructure and planting Bose et al., 2017). Statice has become a popular materials. Nepal has already started exporting floral ornamental flower crop in recent years and are highly products just a few years back such as cut flowers

146 147 2019 Journal of Plant Resources Vol.17, No. 1 and flower buds suitable for bouquets or for Culture media and inoculation of explants ornamental purposes, dried, dyed, bleached, Shoots (clumps each having two to three leafy impregnated to India, USA, Japan, the Netherlands, microshoots) were inoculated in MS basal medium Norway, Australia, Taiwan, Italy, Germany and some supplemented with different concentration of plant of the Gulf countries (Gauchen et al., 2009). Nepal growth regulator (BAP+NAA, KIN+NAA, is giving hand on bulb, tubers, tuberous roots, corms, BAP+IBA and KIN+IBA) in various combination crowns and rhimzomes, in growth or in flower, and concentration. Sucrose 3% were used as a carbon chicory plants and roots, unrooted cuttings and slips sources and media was adjusted to pH 5.8 using (Thapa & Dhimal, 2017). Nepal is mainly dealing sodium hydroxide before autoclaving. Agar, plant with cut flower crops including Roses (Rosa tissue culture grade, Merck (0.8%) were used to hybrida), Gypsophila or Babys Breath (Gypsophila solidify the media and was autoclaved at 121°C for paniculata), Carnations (Dianthus caryophyllus) and 15 minutes. The cultures were incubated at 16 h Chrysanthemum (Chrysanthemum spp.) (Gauchen photoperiod with light intensity of 3000 lux using et al., 2009). Among them, Statice (Limonium spp.) fluorescent tube lights and temperature of 25± 2°C is widely commercially produced cut flower grown for 4 weeks (Rana et al., 2018). in high altitude (Getu, 2009). It is one of the most popular cut flowers used both as cut flower and In-vitro shoots proliferation potted plant. With the increasing number of After successful initiation of the shoot, newly formed horticulture business in Nepal, the demand for shoots were excised and sub-cultured on the MS Statice plants has also increased (Gauchan et al., medium supplemented with different hormonal 2009, Thapa & Dhimal, 2017). Farmers are concentration: BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, dependent on Indian market for plants with heavy 2.5mg/L, 3.0mg/L) and Kinetin (0.5mg/L, 1.0mg/ investment but are not able to get quality plants. This L, 2.0mg/L, 2.5mg/L, 3.0mg/L) with 0.1 mg/L NAA research is focused in developing the protocol for in as well as BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/ vitro mass propagation of Statice that could L, 3.0mg/L), Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, overcome the problem by producing quality plants. 2.5mg/L, 3.0mg/L) with 0.1 mg/L IBA. For each treatment, 15 explants were used and each Materials and Methods experiment were repeated three times.

Plant material collection In-vitro rooting of microshoots of Statice The plant materials were collected from Bhaisipati, MS medium with either NAA or IBA or IAA had Kathmandu on April, 2018, cultivated in green house profound effect on inducing early rooting. In-vitro of local farmer. rooting of micro shoots of Statice were observed on Surface Sterilization MS medium supplemented with different concentration of auxins: IBA (1.0 mg/L and 0.5 mg/ Shoot tips were excised from the mother plant and L), NAA (1.0 mg/L and 0.5mg/L), IAA (1.0mg/L were washed under running tap water for about 1 and 0.5mg/L), according to Echeverrigaray et al., hour with few drops of liquid detergent Tween 20. 2005. Among MS medium supplemented with The explants were thoroughly rinsed with distilled auxins IBA, IAA or NAA, the prominent root growth water for 4-5 times. The explants were surface was recorded on MS medium supplemented with 1 sterilized with freshly prepared 0.1% w/v aqueous mg/L NAA. solution of HgCl2 for 4, 4.5, 5, 5.5 and 6 minutes respectively to standardize the appropriate time and Sand rooting and Hardening were thoroughly rinsed for 4-5 times with sterilized After 4 to 5 successive in vitro shoot proliferation water. of explants, the cultured bottles were moved to green

148 2019 Journal of Plant Resources Vol.17, No. 1 2019 Journal of Plant Resources Vol.17, No. 1 and flower buds suitable for bouquets or for Culture media and inoculation of explants house for 10 to 15 days for acclimatization. The of shoots which are further subdivided into several ornamental purposes, dried, dyed, bleached, Shoots (clumps each having two to three leafy plantlets were removed from bottles with the help small clumps and is propagated for several impregnated to India, USA, Japan, the Netherlands, of sterile forceps and washed with distilled water to generations in a multiplying medium at the microshoots) were inoculated in MS basal medium for rooting at 3-4 weeks. After six weeks of transplantation, rooted plantlets were transferred to nursery Norway, Australia, Taiwan, Italy, Germany and some supplemented with different concentration of plant remove media from the plantlets. Then, the plantlets multiplication stage. Thirdly, an individual shoot of the Gulf countries (Gauchen et al., 2009). Nepal growth regulator (BAP+NAA, KIN+NAA, werepolybags inserted containing into sand garden trays soil, and organic covered matter with and sandfrom (1:1:1). the clump is placed in a rooting medium. The is giving hand on bulb, tubers, tuberous roots, corms, polythene hood to maintain moisture. The rooted plantlets are transferred to greenhouse for BAP+IBA and KIN+IBA) in various combination crowns and rhimzomes, in growth or in flower, and concentration. Sucrose 3% were used as a carbon temperature and humidity of the greenhouse was acclimatization and finally, were transplanted into Results and Discussion chicory plants and roots, unrooted cuttings and slips sources and media was adjusted to pH 5.8 using maintained at 20±5°C and 80% respectively. Plants the field. (Thapa & Dhimal, 2017). Nepal is mainly dealing were assessed for rooting at 3-4 weeks. After six sodium hydroxide before autoclaving. Agar, plant Surface sterilization Surface sterilization of explants were done using with cut flower crops including Roses (Rosa tissue culture grade, Merck (0.8%) were used to weeks of transplantation, rooted plantlets were As we know tissue culture techniques are often considered0.1% concentration to be free fromof HgCl microorganisms,2. Treatment time bacterial varied hybrida), Gypsophila or Babys Breath (Gypsophila solidify the media and was autoclaved at 121°C for transferred to nursery polybags containing garden paniculata), Carnations (Dianthus caryophyllus) and soil, organic matter and sand (1:1:1). from 4 to 6 minutes. Surface sterilization by 0.1% 15 minutes. The cultures were incubated at 16 h contamination has been a problem for both research andHgCl commercialat 5 minutes production was found of plant to be tissue suitable cultures for Chrysanthemum (Chrysanthemum spp.) (Gauchen 2 photoperiod with light intensity of 3000 lux using (Idowu et al., 2009). Endophytic bacteria are especiallyStatice. troublesome because these microorganisms et al., 2009). Among them, Statice (Limonium spp.) fluorescent tube lights and temperature of 25± 2°C Results and Discussion is widely commercially produced cut flower grown for 4 weeks (Rana et al., 2018). cannot be eliminated by external sterilization (Pierik,The 1988), effect andof growth their presence hormone in on cultured the shoot plantlets in high altitude (Getu, 2009). It is one of the most Surface sterilization frequently can be recognized only after prolonged subculturemultiplication (Leifert et al., 1991, Reed et al., 1995). popular cut flowers used both as cut flower and In-vitro shoots proliferation As we know tissue culture techniques are often potted plant. With the increasing number of Since internal infection by bacteria may cause poor Thegrowth addition and a ofdecrease sugar in in the the medium proliferation as a carbonrate of After successful initiation of the shoot, newly formed considered to be free from microorganisms, bacterial horticulture business in Nepal, the demand for source is a requisite for plant growth. Cytokinins shoots were excised and sub-cultured on the MS contaminationcultured plantlets has been (Pierik, a problem 1988), for it wasboth consideredresearch that leaf-tip necrosis of statice plantlets might be Statice plants has also increased (Gauchan et al., such as 6-benzylaminopurine (BA), kinetin (KN) are medium supplemented with different hormonal and commercial production of plant tissue cultures 2009, Thapa & Dhimal, 2017). Farmers are associated with endophytic bacteria (Liu et al., 2005).a classTherefore, of plant in hormonesorder to reduce which suchplays problems, an essential in concentration: BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, (Idowu et al., 2009). Endophytic bacteria are dependent on Indian market for plants with heavy role in plant morphogenesis and influences on the 2.5mg/L, 3.0mg/L) and Kinetin (0.5mg/L, 1.0mg/ especiallythis study, we troublesomehave described thebecause micropropagation these of the statice plant using tissue culture technique in investment but are not able to get quality plants. This formation of shoots and their relative growth rate L, 2.0mg/L, 2.5mg/L, 3.0mg/L) with 0.1 mg/L NAA microorganisms cannot be eliminated by external research is focused in developing the protocol for in four stages. Firstly, shoot tips explants are taken from(Debi a mother et al., plant, 2005). sterilized This study and asepticallyis also concern cultured to as well as BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/ sterilization (Pierik, 1988), and their presence in vitro mass propagation of Statice that could in vitro to establish culture. Secondly, the microshootsinvestigate are grown the to effects form ofa clump BA concentration, of shoots which the type are L, 3.0mg/L), Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, cultured plantlets frequently can be recognized only overcome the problem by producing quality plants. of supporting material as well as other hormones on 2.5mg/L, 3.0mg/L) with 0.1 mg/L IBA. For each afterfurther prolonged subdivided subculture into several (Leifert small et al., clumps 1991; Reedand is propagated for several generations in a multiplying growth and multiplication of Statice plantlets treatment, 15 explants were used and each et al., 1995). Since internal infection by bacteria may Materials and Methods medium at the ‘‘multiplication stage.’’ Thirdly, an individualcultured shoot and fromto assess the clump the possibilityis placed in ofa rooting shoot experiment were repeated three times. cause poor growth and a decrease in the proliferation ratemedium. of cultured The rooted plantlets plantlets (Pierik, are 1988), transferred it was to greenhousemultiplication for of acclimatization Statice plantlets and grown finally, on wereMS Plant material collection In-vitro rooting of microshoots of Statice considered that leaf-tip necrosis of statice plantlets medium. The effect of the different concentration transplanted into the field. The plant materials were collected from Bhaisipati, might be associated with endophytic bacteria (Liu of BAP and NAA is shown in table 2. The efficacy MS medium with either NAA or IBA or IAA had of combination of growth hormones was assessed Kathmandu on April, 2018, cultivated in green house profound effect on inducing early rooting. In-vitro etSurface al., 2005). sterilization Therefore, of explants in order were to reduce done using such 0.1% concentration of HgCl2. Treatment time varied of local farmer. problems, in this study, we have described the based on number of shoots and the height of the shoot rooting of micro shoots of Statice were observed on from 4 to 6 minutes. Surface sterilization by 0.1% HgCl2 at 5 minutes was found to be suitable for induced after inoculation. Shoot formation on MS medium supplemented with different micropropagation of the statice plant using tissue Surface Sterilization Statice. explants cultured on MS medium supplemented with concentration of auxins: IBA (1.0 mg/L and 0.5 mg/ culture technique in four stages. Firstly, shoot tips BAP 1.0 mg/mL + NAA 0.1 mg/L gave rise to Shoot tips were excised from the mother plant and L), NAA (1.0 mg/L and 0.5mg/L), IAA (1.0mg/L explants are taken from a mother plant, sterilized luxuriantly growing shoots within 2-3 weeks. were washed under running tap water for about 1 and 0.5mg/L), according to Echeverrigaray et al., and aseptically cultured in vitro to establish culture. Number of shoots/explant was 21.8±0.663 and hour with few drops of liquid detergent Tween 20. 2005. Among MS medium supplemented with Secondly, the microshoots are grown to form a clump The explants were thoroughly rinsed with distilled auxins IBA, IAA or NAA, the prominent root growth Table 1: Results of sterilization of Statice shoots using HgCl2 Table 1: Results of sterilization of Statice shoots using HgCl2 water for 4-5 times. The explants were surface was recorded on MS medium supplemented with 1 Treatment of Number of Number of Number of Number of sterilized with freshly prepared 0.1% w/v aqueous mg/L NAA. Concentration solution of HgCl for 4, 4.5, 5, 5.5 and 6 minutes time (minute) shoot treated browning shoot aseptic shoot contaminated shoot 2 47-- 7 respectively to standardize the appropriate time and Sand rooting and Hardening 4.5 7 - 1 6 were thoroughly rinsed for 4-5 times with sterilized After 4 to 5 successive in vitro shoot proliferation 0.1% 57-7 - water. 5.5 7 2 5 - of explants, the cultured bottles were moved to green 677- -

148 149 The effect of growth hormone on the shoot multiplication The addition of sugar in the medium as a carbon source is a requisite for plant growth. Cytokinins such as 6-benzylaminopurine (BA), kinetin (KN) are a class of plant hormones which plays an essential role in plant morphogenesis and influences on the formation of shoots and their relative growth rate (Debi et al., 2005). This study is also concern to investigate the effects of BA concentration, the type of supporting material as well as other hormones on growth and multiplication of Statice plantlets cultured and to assess the possibility of shoot multiplication of Statice plantlets grown on MS medium. The effect of the different concentration of BAP and NAA is shown in table 2. The efficacy of combination of growth hormones was assessed based on number of shoots and the height of the shoot induced after inoculation. Shoot formation on explants cultured on MS medium supplemented with BAP 1.0 mg/mL + NAA 0.1 mg/L gave rise to luxuriantly growing shoots within 2-3 weeks. Number of shoots/explant was 21.8±0.663 and maximum average height of shoots was 7.4±0.040 cm as shown in table 2. MS medium supplemented with BAP 2.5mg/L+0.1mg/L IBA gave rise to alternatively high number of shoots 21.2 ±0.374 but considerably shorter height of shoots 5.7±0.141 as shown in table 4. Data represented in table 2, 3, 4 and 5 showed that MS media with different concentration of BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L) and Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L) with 0.1 mg/L NAA as well as BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L), Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L) with 0.1 mg/L IBA. This variation may have resulted from the different requirements of shoot proliferation in lab such as light intensity and temperature given, pH measurement of media etc. Moreover, differences in stimulation of shoot proliferation may be related to differences in macronutrients (Matt & Jehle, 2005, Liu & Pijut, 2008, Ruzic &Vujovic, 2008). Increase and decrease in the concentration of BAP and Kinetin along with variation of IBA and NAA on MS medium is essence to observe. 2019 Journal of Plant Resources Vol.17, No. 1 Table 2: The effect of growth hormones (BAP+NAA) on the shoot multiplication * Average number of shoot and average height of shoots were given as mean±standard error Table 2: The effect of growth hormones (BAP+NAA) on the shoot multiplication

BAP+NAA No. of culture No. of weeks for shoot No. of shoot/ Average height of S.N. mg/L bottle proliferation explants* shoot (cm)* 1 0.5+0.1 5 6-8 17 ±1.224 6.74 ±0.132 2 1.0+0.1 5 6-8 21.8±0.663 7.4 ±0.040 3 1.5+0.1 5 6-8 15.8 ±1.157 6.36 ±0.128 4 2.0+0.1 5 6-8 13.4 ±1.886 7.04 ±0.050 5 2.5+0.1 5 6-8 15 ±1.581 6.7 ±0.126 * Average number of shoot and average height of shoots were given as mean±standard error

(A) (B) (C)

Figure 1: (A) Explant establishment on MS medium, (B) & (C) Induction of Statice shoots on MS medium supplemented with 1mg/L concentration of BAP and 0.1 mg/L NAA at 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 4 weeks (A) and 8 weeks (B) maximum average height of shoots was 7.4±0.040 Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, cm as shown in table 2. MS medium supplemented 3.0mg/L) with 0.1 mg/L IBA. This variation may with BAP 2.5mg/L+0.1mg/L IBA gave rise to have resulted from the different requirements of alternatively high number of shoots 21.2 ±0.374 but shoot proliferation in lab such as light intensity and considerably shorter height of shoots 5.7±0.141 as temperature given, pH measurement of media etc. shown in table 4. Moreover, differences in stimulation of shoot Data represented(A) in table 2, 3, 4 and 5 showed (B) that proliferation may be related (C) to differences in MS media with different concentration of BAP macronutrients (Matt & Jehle, 2005, Liu & Pijut, Figure(0.5mg/L, 1: (A) 1.0mg/L, Explant establishment 2.0mg/L, 2.5mg/L,on MS medium, 3.0mg/L) (B) & (C)2008, Induction Ruzic of &Vujovic,Statice shoots 2008). on MS Increase medium and supplemented decrease with 1mg/L concentration of BAP and 0.1 mg/L NAA at 16 hrs. photoperiodin the concentration with light intensity of BAP of and 3000 Kinetin lux and alongtemperature with ofand 25± Kinetin 2°C for (0.5mg/L, 4 weeks (A) 1.0mg/L, and 8 weeks 2.0mg/L, (B) 2.5mg/L, 3.0mg/L) with 0.1 mg/L NAA as well as BAP variation of IBA and NAA on MS medium is essence to observe. Table(0.5mg/L, 3: The 1.0mg/L, effect of growth 2.0mg/L, hormones 2.5mg/L, (KIN+NAA) 3.0mg/L), on the shoot multiplication * Average number of shoot and average height of shoots were given as mean±standard error Table 3: The effect of growth hormones (KIN+NAA) on the shoot multiplication

KIN+NAA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. mg/L bottle shoot proliferation explants* (cm)* 1 0.5+0.1 5 6-8 15.4 ±0.400 5.84 ±0.074 2 1.0+0.1 5 6-8 16.8 ±0.583 5.64 ±0.146 3 1.5+0.1 5 6-8 19.2±0.489 6.38 ±0.058 4 2.0+0.1 5 6-8 19 ± 0.547 6.28 ±0.058 5 2.5+0.1 5 6-8 20 ±0.547 6.36 ±0.092 * Average number of shoot and average height of shoots were given as mean±standard error *Culture* Culture condition: condition: 16 16 hrs. hrs. photoperiod photoperiod withwith light light intensityintensity ofof 30003000 luxlux andand temperaturetemperature ofof 25±25± 2°C2°C forfor 88 weeks.

Table150 4: The effect of growth hormones (BAP+IBA) on the shoot multiplication * Average number of shoot and average height of shoots were given as mean±standard error

Table 5: The effect of growth hormones (KIN+IBA) on the shoot multiplication * Average number of shoot and average height of shoots were given as mean±standard error

KIN+IBA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. mg/L bottle shoot proliferation explants* (cm)* 1 0.5+0.1 5 6-8 11.8±0.800 5.44±0.169 2 1.0+0.1 5 6-8 14.4±0.400 5.64±0.146 3 1.5+0.1 5 6-8 16.6±0.509 6.16±0.120 4 2.0+0.1 5 6-8 19.2±0.583 5.86±0.120 5 2.5+0.1 5 6-8 20±0.547 6.14±0.150 *Culture condition: 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8s weeks.

The addition of sugar in the medium as a carbon source is a requisite for plant growth. Cytokinins such as 6-benzylaminopurine (BA), kinetin (KN) are a class of plant hormones which plays an essential role in plant morphogenesis and influences on the formation of shoots and their relative growth rate (Debi et al., 2005). This study is also concern to investigate the effects of BA concentration, the type of supporting material as well as other hormones on growth and multiplication of Statice plantlets cultured and to assess the possibility of shoot multiplication of Statice plantlets grown on MS medium. The effect of the different concentration of BAP and NAA is shown in table 2. The efficacy of combination of growth hormones was assessed based on number of shoots and the height of the shoot induced after inoculation. Shoot formation on explants cultured on MS medium supplemented with BAP 1.0 mg/mL + NAA 0.1 mg/L gave rise to luxuriantly growing shoots within 2-3 weeks. Number of shoots/explant was 21.8±0.663 and maximum average height of shoots was 7.4±0.040 cm as shown in table 2. MS medium supplemented with BAP 2.5mg/L+0.1mg/L IBA gave rise to alternatively high number of shoots 21.2 (A) (B) (C) (A) (B) (C)

±0.374 but considerably shorter height of shoots 5.7±0.141 as shown in table 4. Figure 1: (A) Explant establishment on MS medium, (B) & (C) Induction of Statice shoots on MS medium supplemented Figure 1: (A) Explant establishment on MS medium, (B) & (C) Induction of Statice shoots on MS medium supplemented with 1mg/L concentration of BAP and 0.1 mg/L NAA at 16 hrs. photoperiod with light intensity of 3000 lux and temperature Data represented in table 2, 3, 4 and 5 showed that MS media with different concentration of BAP with 1mg/L concentration of BAP and 0.1 mg/L NAA at 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 4 weeks (A) and 8 weeks (B) of 25± 2°C for 4 weeks (A) and 8 weeks (B) (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L) and Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, Table 3: The effect of growth hormones (KIN+NAA) on the shoot multiplication Table 3: The effect of growth hormones (KIN+NAA) on the shoot multiplication 3.0mg/L) with 0.1 mg/L NAA as well as BAP (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L), Kinetin * Average number of shoot and average height of shoots were given as mean±standard error * Average number of shoot and average height of shoots were given as mean±standard error

(0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, 3.0mg/L) with 0.1 mg/L IBA. This variation may have resulted KIN+NAA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. KIN+NAA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. mg/L bottle shoot proliferation explants* (cm)* from the different requirements of shoot proliferation in lab such as light intensity and temperature mg/L bottle shoot proliferation explants* (cm)* 1 0.5+0.1 5 6-8 15.4 ±0.400 5.84 ±0.074 1 0.5+0.1 5 6-8 15.4 ±0.400 5.84 ±0.074 given, pH measurement of media etc. Moreover, differences in stimulation of shoot proliferation may be 2 1.0+0.1 5 6-8 16.8 ±0.583 5.64 ±0.146 2 1.0+0.1 5 6-8 16.8 ±0.583 5.64 ±0.146 3 1.5+0.1 5 6-8 19.2±0.489 6.38 ±0.058 3 1.5+0.1 5 6-8 19.2±0.489 6.38 ±0.058 related to differences in macronutrients (Matt & Jehle, 2005, Liu & Pijut, 2008, Ruzic &Vujovic, 2008). 4 2.0+0.1 5 6-8 19 ± 0.547 6.28 ±0.058 4 2.0+0.1 5 6-8 19 ± 0.547 6.28 ±0.058 5 2.5+0.1 5 6-8 20 ±0.547 6.36 ±0.092 Increase and decrease in the concentration of BAP and Kinetin along with variation of IBA and NAA on 5 2.5+0.1 5 6-8 20 ±0.547 6.36 ±0.092 *Culture condition: 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8 weeks. MS medium is essence to observe. *Culture condition: 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8 weeks.

2019 Journal of Plant Resources Vol.17, No. 1 2019 Table 4: The effect of growth hormones (BAP+IBA)Journal on ofthe Plant shoot Resourcesmultiplication Vol.17, No. 1 Table 4: The effect of growth hormones (BAP+IBA) on the shoot multiplication Table 2: The effect of growth hormones (BAP+NAA) on the shoot multiplication * Average number of shoot and average height of shoots were given as mean±standard error * Average number of shoot and average height of shoots were given as mean±standard error * Average number of shoot and average height of shoots were given as mean±standard error Table 2: The effect of growth hormones (BAP+NAA) on the shoot multiplication Table 4: The effect of growth hormones (BAP+IBA) on the shoot multiplication BAP+IBA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. BAP+IBA No. of culture No. of weeks for No. of shoot/ Average height of shoot BAP+NAA No. of culture No. of weeks for shoot No. of shoot/ Average height of S.N. mg/L bottle shoot proliferation explants* (cm)* S.N. mg/L bottle shoot proliferation explants* (cm)* mg/L bottle proliferation explants* shoot (cm)* 1 0.5+0.1 5 6-8 16.4±0.812 5.2±0.070 1 0.5+0.1 5 6-8 16.4±0.812 5.2±0.070 1 0.5+0.1 5 6-8 17 ±1.224 6.74 ±0.132 2 1.0+0.1 5 6-8 17.4±0.400 5.62±0.162 2 1.0+0.1 5 6-8 17.4±0.400 5.62±0.162 2 1.0+0.1 5 6-8 21.8±0.663 7.4 ±0.040 3 1.5+0.1 5 6-8 19.4±0.400 5.82±0.111 3 1.5+0.1 5 6-8 19.4±0.400 5.82±0.111 3 1.5+0.1 5 6-8 15.8 ±1.157 6.36 ±0.128 4 2.0+0.1 5 6-8 19.4±0.400 5.98±0.66 4 2.0+0.1 5 6-8 19.4±0.400 5.98±0.66 4 2.0+0.1 5 6-8 13.4 ±1.886 7.04 ±0.050 5 2.5+0.1 5 6-8 21.2 ±0.374 5.7±0.141 5 2.5+0.1 5 6-8 21.2 ±0.374 5.7±0.141 5 2.5+0.1 5 6-8 15 ±1.581 6.7 ±0.126 *Culture condition: 16 hrs photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8 weeks. *Culture condition: 16 hrs photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8 weeks. * Average number of shoot and average height of shoots were given as mean±standard error * Average number of shoot and average height of shoots were given as mean±standard error * Culture condition: 16 hrs photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8 weeks. Table 5: The effect of growth hormones (KIN+IBA) on the shoot multiplication Table* Average 5: The number effect of growthshoot and hormones average (KIN+IBA)height of shoots on the were shoot given multiplication as mean±standard error Table* Average 5: The number effect ofof shootgrowth and hormones average height(KIN+IBA) of shoots on the were shoot given multiplication as mean±standard error KIN+IBA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. KIN+IBA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. mg/L bottle shoot proliferation explants* (cm)* 1mg/L 0.5+0.1 bottle5 shoot proliferation6-8 11.8±0.800explants* 5.44±0.169(cm)* 12 0.5+0.1 1.0+0.1 55 6-86-8 11.8±0.80014.4±0.400 5.44±0.1695.64±0.146 23 1.0+0.1 1.5+0.1 55 6-86-8 14.4±0.40016.6±0.509 5.64±0.1466.16±0.120 34 1.5+0.1 2.0+0.1 55 6-86-8 16.6±0.50919.2±0.583 6.16±0.1205.86±0.120 Induction45 2.0+0.1 2.5+0.1 of healthy root 55 system from the6-86-8 regenerated shoots19.2±0.58320±0.547 is an essential part5.86±0.1206.14±0.150 of successful 5 2.5+0.1 5 6-8 20±0.547 6.14±0.150 * *CultureAverage numbercondition: of 16shoot hrs. and photoperiod average height with light of shoots intensity were of given 3000 as lux mean±standard and temperature error of 25± 2°C for 8s weeks. *Culture condition: 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8s weeks. * developmentCulture condition: of 16plantlets. hrs. photoperiod The concentration with light intensity and ofsource 3000 lux of andauxin temperature (IBA/IAA/NAA) of 25± 2°C for also 8s weeks.has a significant

influence on root initiation and development. Here, for root induction, regenerated shoots were cultured Rooting of microshoots of Statice and survival of MS medium supplemented with auxins IBA, IAA (A) (B) (C) plantletson MS in medium polybag supplemented with different concentrationor NAA, the of good IBA, root IAA growth and was NAA recorded according on MS to Figure 1: (A) Explant establishment on MS medium, (B) & (C) Induction of Statice shoots on MS medium supplemented with InductionEcheverrigaray of healthy et al., 2005.root MSsystem medium from enriched the medium with NAA supplemented 1mg/L shows with 100%1 mg/L of NAA shoot in table forming 6. 1mg/L concentration of BAP and 0.1 mg/L NAA at 16 hrs. photoperiod with light intensity of 3000 lux and temperature of 25± regeneratedroots. Roots shoots were isinitiated an essential at 15-20 part days of successful and were developedPlantlets intowere thick acclimatized and fibrous for within 10 to 30-35 15 days days in as 2°C for 4 weeks (A) and 8 weeks (B) development of plantlets. The concentration and shown in figure 2 (A). Among MS medium supplementedgreenhouse with auxins at 20±5ºC. IBA, IAAPlantlets or NAA, with the established good root maximum average height of shoots was 7.4±0.040 Kinetin (0.5mg/L, 1.0mg/L, 2.0mg/L, 2.5mg/L, source of auxin (IBA/IAA/NAA) also has a roots were successfully established in sterile sand cm as shown in table 2. MS medium supplemented 3.0mg/L) with 0.1 mg/L IBA. This variation may significantgrowth was influencerecorded on onMS root medium initiation supplemented and with(98%) 1 mg/L in figure NAA 2 in(B table & C) 6. and were transferred in development. Here, for root induction, regenerated with BAP 2.5mg/L+0.1mg/L IBA gave rise to have resulted from the different requirements of Plantlets were acclimatized for 10 to 15 days in greenhousepoly bag atcontaining 20±5ºC. aPlantlets mixture ofwith garden established soil, organic roots alternatively high number of shoots 21.2 ±0.374 but shoot proliferation in lab such as light intensity and shoots were cultured on MS medium supplemented matter and sand in 1:1:1 ratio in figure 3 (A & B). considerably shorter height of shoots 5.7±0.141 as temperature given, pH measurement of media etc. withwere different successfully concentration established of IBA,in sterile IAA sandand NAA (98%) inAfter figure 2 months,2 (B & C) field and trial were of transferredplantlets was in polydone bag in shown in table 4. Moreover, differences in stimulation of shoot accordingcontaining to a mixtureEcheverrigaray of garden et soil, al., organic 2005. matterMS andthe sandgarden in 1:1:1of Department ratio in figure of Plant 3 (A Resources& B). After in 2 proliferation may be related to differences in medium enriched with NAA 1mg/L shows 100% of figure 3 (C). Plantlets survival percentage was 100% Data represented(A) in table 2, 3, 4 and 5 showed (B) that (C) months, field trial of plantlets was done in the garden of Department of Plant Resources in figure 3 (C). shoot forming roots. Roots were initiated at 15-20 in open field condition. For recommendation, routine MS media with different concentration of BAP macronutrients (Matt & Jehle, 2005, Liu & Pijut, daysPlantlets and were survival developed percentage into thick was and 100% fibrous in openobservation field condition. of the environmental For recommendation, condition should routine Figure(0.5mg/L, 1: (A) 1.0mg/L, Explant establishment 2.0mg/L, 2.5mg/L,on MS medium, 3.0mg/L) (B) & (C)2008, Induction Ruzic of &Vujovic,Statice shoots 2008). on MS Increase medium and supplemented decrease with 1mg/L concentration of BAP and 0.1 mg/L NAA at 16 hrs. photoperiodin the concentration with light intensity of BAP of and 3000 Kinetin lux and alongtemperature with within 30-35 days as shown in figure 2 (A). Among be noted until inflorescence. ofand 25± Kinetin 2°C for (0.5mg/L, 4 weeks (A) 1.0mg/L, and 8 weeks 2.0mg/L, (B) 2.5mg/L, observation of the environmental condition should be noted until inflorescence. 3.0mg/L) with 0.1 mg/L NAA as well as BAP variation of IBA and NAA on MS medium is essence to observe. TableTable 6: 6: Effect Effect of of different different auxins auxins on on root root formation formation in inStatice Statice Table(0.5mg/L, 3: The 1.0mg/L, effect of growth 2.0mg/L, hormones 2.5mg/L, (KIN+NAA) 3.0mg/L), on the shoot multiplication Growth regulators % of shoots forming Days to root Days to root * Average number of shoot and average height of shoots were given as mean±standard error Nature of the roots Table 3: The effect of growth hormones (KIN+NAA) on the shoot multiplication (mg/L) roots initiation development

IBA(0.5) 55 20-25 40-45 Thick &fibrous KIN+NAA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. IBA0(1) 65 20-25 40-45 Thick &fibrous mg/L bottle shoot proliferation explants* (cm)* IAA(0.5) 35 20-25 40-45 Thick &fibrous 1 0.5+0.1 5 6-8 15.4 ±0.400 5.84 ±0.074 IAA(1) 45 20-25 40-45 Thick &fibrous 2 1.0+0.1 5 6-8 16.8 ±0.583 5.64 ±0.146 NAA(0.5) 65 15-20 30-35 Thick &fibrous 3 1.5+0.1 5 6-8 19.2±0.489 6.38 ±0.058 NAA(1) 100 15-20 30-35 Thick &fibrous 4 2.0+0.1 5 6-8 19 ± 0.547 6.28 ±0.058 5 2.5+0.1 5 6-8 20 ±0.547 6.36 ±0.092 * Average number of shoot and average height of shoots were given as mean±standard error *Culture* Culture condition: condition: 16 16 hrs. hrs. photoperiod photoperiod withwith light light intensityintensity ofof 30003000 luxlux andand temperaturetemperature ofof 25±25± 2°C2°C forfor 88 weeks.

Table150 4: The effect of growth hormones (BAP+IBA) on the shoot multiplication 151 * Average number of shoot and average height of shoots were given as mean±standard error

BAP+IBA No. of culture No. of weeks for No. of shoot/ Average height of shoot S.N. mg/L bottle shoot proliferation explants* (cm)* 1 0.5+0.1 5 6-8 16.4±0.812 5.2±0.070 2 1.0+0.1 5 6-8 17.4±0.400 5.62±0.162 3 1.5+0.1 5 6-8 19.4±0.400 5.82±0.111 4 2.0+0.1 5 6-8 19.4±0.400 5.98±0.66 5 2.5+0.1 5 6-8 21.2 ±0.374 5.7±0.141 (A) (B) (C) Figure 2: Hardening, acclimatization and sand rooting of plantlets (A) The roots of statice shoots cultured on MS medium *Culture condition: 16 hrs photoperiod with light intensity of 3000 lux and temperature of 25± 2°C for 8 weeks. supplemented with NAA (1mg/L), (B) Statice plantlets transferred to the sand, (C) Statice plantlets with developed roots.

Table 5: The effect of growth hormones (KIN+IBA) on the shoot multiplication * Average number of shoot and average height of shoots were given as mean±standard error

2019 Journal of Plant Resources Vol.17, No. 1

(A) (B) (C) Figure 2: Hardening, acclimatization and sand rooting of plantlets (A) The roots of statice shoots cultured on MS medium supplemented with NAA (1mg/L), (B) Statice plantlets transferred to the sand, (C) Statice plantlets with developed roots.

(A) (B) (C)

Figure 3: Statice plantlets transferred on polybags after 2 weeks (A) Plantlets of Statice after 4 weeks at 20±5°C temp and 80% humidity (B) Plantlets of Statice after 2 months at 20±5°C temp and 80% humidity, (C) Open field trial on the garden of department of plant resources.

Conclusion greenhouse at 20±5ºC. Plantlets were successfully grown in the garden of Department of Plant The sterilization of shoot tips with 0.1% Resources and the survival percentage was 100%. concentration of HgCl2 for 5 minutes was the ideal condition for the surface sterilization of the explant. Acknowledgements MS medium supplemented with 1.0 mg/L BAP plus 0.1 mg/L NAA, was suitable condition for maximum It is our privilege to express our sincere thanks deep number of shoot proliferation. Tissue culture technique sense of gratitude to Director General Mr. Sanjeev of the Statice was established (94%) on MS medium Kumar Rai, Deputy Director General Ms. Jyoti Joshi enriched with BAP and NAA. Thick and fibrous Bhatt, Deputy Director General Mohon Dev Joshi roots were observed on MS medium enriched with for facilitating lab, making it suitable to conduct this NAA 1mg/L within 30-35 days. Plantlets were research successfully. We sincerely thank to Suman successfully established in sterile sand and were Shrestha for providing us with sampling materials adapted ex vitro with surviving rate up to 98% in for in-vitro propagation of statice. We acknowledge

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Scientific Officer Mrs. Januka Pathak, Assistant Idowu, P. E., Ibitoye, D. O., & Ademoyegun, O. T. Botanist Mrs. Ganga Rijal and Mr. Aashis Shrestha (2009). Tissue culture as a plant production for their kind support in lab work as well as we thank technique for horticultural crops. African Journal to Lila Rai for her kind support for the of Biotechnology, 8(16). acclimatization of plantlets in greenhouse, Last but not the least we are thankful to Mrs. Kamala Idowu, P. E., Ibitoye, D. O., & Ademoyegun, O. T. Bhandari and Nirmala Bhandari for sterilization (2009). Tissue culture as a plant production process. technique for horticultural crops. African Journal of Biotechnology, 8(16). References Jeong, J. H., Murthy, H. N., & Paek, K. Y. (2001). High frequency adventitious shoot induction and Aly, M. A., Rathinasabapathi, B., & Kelley, K. (A) (B) (C) plant regeneration from leaves of statice. Plant (2002). Somatic embryogenesis in perennial cell, tissue and organ culture, 65(2), 123-128. Figure 2: Hardening, acclimatization and sand rooting of plantlets (A) The roots of statice shoots cultured on MS medium statice Limonium bellidifolium, Plumbaginaceae. supplemented with NAA (1mg/L), (B) Statice plantlets transferred to the sand, (C) Statice plantlets with developed roots. Plant cell, tissue and organ culture, 68(2), 127- Kunitake, H., Koreeda, K., & Mii, M. (1995). 135. Morphological and cytological characteristics of protoplast-derived plants of statice (Limonium Bose, S., Karmakar, J., Fulzele, D. P., Basu, U., & perezii Hubbard). Scientia horticulturae, 60(3-4), Bandyopadhyay, T. K. (2017). In vitro shoots from 305-312. root explant, their encapsulation, storage, plant recovery and genetic fidelity assessment of Leifert, C., Ritchie, J. Y., & Waites, W. M. (1991). Limonium hybrid Misty Blue: a florist Contaminants of plant-tissue and cell plant. Plant Cell, Tissue and Organ Culture cultures. World Journal of Microbiology and (PCTOC), 129(2), 313-324. Biotechnology, 7(4), 452-469. Cohen, A., Harazy, A., Rabinowitch, H. D., & Stav, Liu, T. H. A., Hsu, N. W., & Wu, R. Y. (2005). R. (1995). Selection for early flowering in blue Control of leaf-tip necrosis of micropropagated statice (Limonium sinuatum Mill.). Ornamental ornamental statice by elimination of endophytic (A) (B) (C) Plant Improvement: Classical and Molecular bacteria. In Vitro Cellular & Developmental 420, 118-124. Biology-Plant, 41(4), 546-549. Figure 3: Statice plantlets transferred on polybags after 2 weeks (A) Plantlets of Statice after 4 weeks at 20±5°C temp and 80% humidity (B) Plantlets of Statice after 2 months at 20±5°C temp and 80% humidity, (C) Open field trial on the garden of department Debi, B. R., Taketa, S., & Ichii, M. (2005). Cytokinin Liu, X., & Pijut, P. M. (2008). Plant regeneration of plant resources. inhibits lateral root initiation but stimulates lateral from in vitro leaves of mature black cherry root elongation in rice (Oryza sativa). Journal of (Prunusserotina). Plant Cell, Tissue and Organ Conclusion greenhouse at 20±5ºC. Plantlets were successfully plant physiology, 162(5), 507-515. Culture, 94(2), 113-123. grown in the garden of Department of Plant The sterilization of shoot tips with 0.1% Resources and the survival percentage was 100%. Gauchan, D. P., Pokhrel, A. R., Pratap, M., & Lama, Lledó, M. D., Erben, M., & Crespo, M. B. (2003). concentration of HgCl2 for 5 minutes was the ideal P. (2009). Current status of cut flower business Myriolepis, a new genus segregated from condition for the surface sterilization of the explant. Acknowledgements in Nepal. Kathmandu University Journal of Limonium (Plumbaginaceae). Taxon, 52(1), 67- MS medium supplemented with 1.0 mg/L BAP plus Science, Engineering and Technology, 5(1), 87- 73. 0.1 mg/L NAA, was suitable condition for maximum It is our privilege to express our sincere thanks deep 98. number of shoot proliferation. Tissue culture technique sense of gratitude to Director General Mr. Sanjeev Matt, A., & Jehle, J. A. (2005). In vitro plant of the Statice was established (94%) on MS medium Kumar Rai, Deputy Director General Ms. Jyoti Joshi Getu, M. (2009). Ethiopian floriculture and its regeneration from leaves and internode sections enriched with BAP and NAA. Thick and fibrous Bhatt, Deputy Director General Mohon Dev Joshi impact on the environment. Mizan Law Review, of sweet cherry cultivars (Prunusavium L.). Plant roots were observed on MS medium enriched with for facilitating lab, making it suitable to conduct this 3, 240-270. cell reports, 24(8), 468-476. NAA 1mg/L within 30-35 days. Plantlets were research successfully. We sincerely thank to Suman Henny, R. J., & Chen, J. (2004). Cultivar Mellesse, B., Kassa, N., & Mohammed, A. (2013). successfully established in sterile sand and were Shrestha for providing us with sampling materials development of ornamental foliage plants. Plant Yield and quality of statice [Limonium sinuatum adapted ex vitro with surviving rate up to 98% in for in-vitro propagation of statice. We acknowledge breeding reviews, 23, 245-290. (L.) Mill.] as affected by cultivars and planting

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densities. African Journal of Plant Science, 7(11), Reed, B. M., & Tanprasert, P. (1995). Detection and 528-537. control of bacterial contaminants of plant tissue cultures. A review of recent literature. Plant tissue Morgan, E. R., Burge, G. K., Seelye, J. F., Hopping, culture and Biotechnology, 1(3), 137-142. M. E., & Grant, J. E. (1998). Production of inter- specific hybrids between Limonium perezii Rout, G. R., Mohapatra, A., & Jain, S. M. (2006). (Stapf) Hubb. and Limonium sinuatum (L.) Tissue culture of ornamental pot plant: A critical Mill. Euphytica, 102(1), 109-115. review on present scenario and future prospects. Biotechnology advances, 24(6), 531-560. Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with Ruiæ, D. V., & Vujoviæ, T. I. (2008). The effects tobacco tissue culture. Physiol. Plant, 15,473- of cytokinin types and their concentration on in 497. vitro multiplication of sweet cherry cv. Lapins (Prunusavium L.). Horticultural Science, 35(1), Pierik, R. L. M., Sprenkels, P. A., Van Der Harst, B., 12-21. & Van Der Meys, Q. G. (1988). Seed germination and further development of plantlets of Thapa, M. B., & Dhimal, S. (2017). Horticulture PaphiopedilumciliolarePfitz. in vitro. Scientia development in Nepal: Prospects, challenges and Horticulturae, 34(1-2), 139-153. strategies. Universal Journal of Agricultural Research, 5(3), 177-189. Rana, N., Khadka, S., & Rajbahak, S. (2018). In- vitro Propagation of Lavender (Lavandula Xiao, Y., & Kozai, T. (2006). In vitro multiplication angustifolia Mill.). Journal of plant resources, of statice plantlets using sugar-free media. 16(1), 112. Scientia horticulturae, 109(1), 71-77.

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