In vitro Propagation of Cut Flower Variety Muscari armeniacum Leichtl. ex Bak. Through Direct Bulblet Proliferation Pathways

Muscari armeniacum is one of the important ornamental cut flower in floriculture industry which native to Southern Europe, North Africa, Western Asia and Asia Minor. In this study, bulb explants (basal plate of bulb having meristem), bulb scales and leaf segments from in vitro derived bulblets were culture in Murashige and Skoog (MS) medium with different plant growth regulator concentrations and combinations to assess growth regulators effect on different bulblet organogenesis pathways in vitro. The results demonstrated that cytokinin in combination with auxin is required for both axillary and adventitious bulblet regeneration.  Benzyladenine (BA) - α-naphthalene acetic acid (NAA) combination, showed significant effects compared to other growth regulator combinations tested. 4.0 µM BA + 2.0 µM NAA was the found to the best for axillary bulblet formation from bulb explants. Likewise, bulb and leaf segments showed the best response in adventitious bulblet organogenesis when they were cultured in BA-NAA combinations. Out of several concentrations of BA with NAA, 4.0 µM BA + 1.0 µM NAA was optimum for adventitious bulblet regeneration. Bulblets, properly isolated from both axillary and adventitious proliferation systems, showed maximum percentage of rooting on half strength MS medium containing 2.0 µM Indole-3-butyric acid (IBA). However, the higher concentration of all the auxins showed either callus formation at the base of shoots or malformation of roots. All the in vitro regenerated plantlets were successfully acclimatized under ex vitro environment in the garden soil with 60% survival rate.


INTRODUCTION
systems) was cut into small pieces and subsequently sub-cultured on the MS medium supplemented with 2.0, 4.0 and 6.0 μM BAP or Kn in combination with 1.0, 2.0 μM NAA or IBA for the proliferation of adventitious bulblets.

In vitro rooting
Regenerated bulblets (3.0 -4.5 cm) were excised from the in vitro grown culture and transferred to half strength MS medium supplemented with either IBA or NAA in concentration of 0.5, 1.0, 2.0, 3.0 and 4.0 μM. The cultures were maintained under a 16h photoperiod at 25 ºC (±2 ºC) and observed after six weeks of incubation. Observation was recorded after six weeks of incubation.

Hardening and acclimatization
In vitro regenerated plantlets were taken out from the culture tubes and washed thoroughly under running tap water. Small plastic pots were kept ready filled with garden soil and compost (1:1). Taking special care not to damage the roots, plantlets were then planted one in each pot and the potted plantlets were transferred to culture room condition under artificial light and temperature (25 ± 2 º C) and 70 -80% relative humidity. The polythene cover was removed periodically in order to gradual acclimatization of plantlets. After three weeks of indoor acclimatization potted plantlets were transferred to outdoor laboratory conditions.

Culture observation, data collection, and data analysis
Five cultures were used per treatment and each experiment was repeated thrice times. The data on axillary/adventitious bulblets proliferation were recorded after 8 weeks of culture whereas the data on adventitious rooting were recorded after 6 weeks of culture. The effect of different treatments was determined with respect to number and length of bulblets and roots. Data were statistically analyzed by the Duncan's multiple Range Test at the P < 0.05 level of significance using a statistical software-SPSS.

Axillary bulblet proliferation
In the present investigation, attempts were taken to produce large number of bulblets through axillary proliferation from the bulb explants containing pre-formed meristem. Explants were cultured on MS medium supplemented with different concentrations and combinations of cytokinin (BA and Kn) and auxin (NAA and IBA). Concentrations of cytokinins were usually 2.0, 4.0, 6.0 µM, whereas auxins were used in 1.0, 2.0 µM. Cultured explants were started to form bulblets at axil of the bulbs after 1 weeks of incubation ( Fig. 1 A), and the proliferated bulblets attained maturity within 8 weeks (Fig 1B.). Callus was seen on the basal portion of the explants when the cytokinin and auxin were used in approximately equal concentration, and hence the numbers of bulblets were found to be reduced.
When the explants were cultured on 4.0 µM BA + 2.0 µM NAA containing medium, 5.6 ± 0.40 bulblets with the maximum height (5.2 ± 0.13 cm) were observed per culture (Table 1). No callus was formed in this growth regulator combination. During this experiment, bulb explants were primarily cultured on only BA or Kn containing medium, but no significant shoot proliferation was observed. In addition, NAA was found to be the best compared to IBA for axillary proliferation in combination with cytokinin. On the other hand, BA exhibited better proliferation than Kn. These findings clearly indicated the synergistic effect of cytokinin-auxin growth regulators on bulblet proliferation in M. armeniacum.

Direct adventitious bulblet induction and proliferation
Two different explants-bulb scale and in vitro derived leaf sheath segments, which don't contain any pre-formed meristems, were used in this experiment with the same cytokinin-auxin combinations used in previous experiment to explore their potentiality for direct adventitious bulblet organogenesis without intermediate callus formation.

Effect of auxins and cytokinins on direct adventitious bulblet proliferation from the bulbscale explants
In this experiment bulb-scale explants were cultured on MS medium supplemented with cytokinin and auxin with different concentrations. Adventitious bulblets were formed in most of the treatment within 2 weeks of culture ( Fig. 1 C), however, significant difference was observed in the number and length of bulblets. In MS medium containing 4.0 µM BA + 1.0 µM NAA the cultured explants showed best results for all the parameters evaluated. In this medium formulation, bulb-scale explants produced the highest number of bulblets (8.4 ± 0.24) as well as the longest bulblets (5.2 ± 0.12 cm) after 8 weeks of culture (Fig. 1D). The media containing BA always showed better performance enhancing the proliferation than that of containing Kn, whereas NAA was observed as a potent auxin compared to IBA (Table 2). During this study, 4.0 µM Kn + 1.0 µM NAA exhibited the second highest performance regarding direct bulblets induction in bulbscale explants when it produces 6.8 ± 0.37 bulblets with 4.5 ± 0.14 cm length. A higher concentration of BA or Kn with NAA or IBA did not result in any improved effect on adventitious bulblets induction. It was remarkable to note that there was no response on the adventitious bulblets initiation on the medium supplemented with BA or Kn, even at significantly higher concentrations of both.

Effect of auxins and cytokinins on direct adventitious bulblet proliferation from the leaf explants
Leaf explants were taken from the in vitro proliferated shoots and they were cultured on MS medium supplemented with different cytokinin-auxin combinations for direct adventitious shoot organogenesis. Direct adventitious shoot induction was occurred in different extent in all the treatments ( Table 2). The number of adventitious bulblets per explant was recorded after 8 weeks. During this experiment, no calluses were formed in MS medium augmented with 4 μM BA and 1μM NAA. The highest number of bulblets/explant (10.8 ± 0.58) and the highest mean length (4.2 ± 0.14) of bulblets/explant were observed in the medium containing 4 μM BA and 1μM NAA ( Fig. 1 E & F) followed by 4 μM BA and 2μM NAA containing medium. When BA was increased gradually, explants produced fewer bulblets with fewer lengths. Like axillary proliferation, BA was also found to be a potent cytokinin for direct bulblets induction, however BA showed the best performance when it was combined with NAA.

Rooting of in vitro proliferated bullblets
Bulblets regenerated from bulb-scale and leaf explants need roots to acclimatize and establish them in the ex vitro condition. Investigations were also carried out on adventitious rooting, and transplantation of rooted bulblets on to the soil and acclimatize them successfully under the field condition. In this experiment, no rooting was observed from the base of the bulblets before 7 days of culture. Rooting frequency increased gradually with the incubation period, and it was reached to 100 % after 15 days of culture on rooting medium. Percentages of root induction, number of root per shoots were greatly controlled by the concentrations and type of the auxin. IBA was found to be more effective in root induction of M. armeniacum bulblets. The maximum percentage of culture that regenerated roots was 100 % when the bulblets were cultured on ½ MS media having 2.0 µM IBA (Fig. 1 G). Media containing NAA was not good as that contained IBA. Only 40-80 % of rooting was observed on the media supplemented with 0.5-4.0 µM NAA. The highest numbers and maximum length of the longest roots per bulblets 4.6 ± 0.68 and 4.2 ± 0.38 cm, respectively on the ½ MS medium containing 2.0 µM IBA (Fig. 2).

Hardening and acclimatization
Plantlets obtained from different rooting media, which had well developed root system i.e. no basal callusing and no malformation of roots (Fig. 1 G) were used for this experiment. The plantlets washed with tap water were transferred on to the small plastic pots containing sterilized soil mix (garden soil and compost in 1:1 ratio). Transferred plantlets were maintained 25-30 days  in culture room conditions and then transferred to the outdoor condition. After transferring them to outdoor condition approximately 60 % plantlets were survived well ( Fig. 1 H).

DISCUSSION
The results presented in the foregoing section reveal that bulb and leaves segments of M. armeniacum are successfully established for producing axillary and adventitious bulblets through in vitro technique. However, in vitro responses were found to be different, and the possible causes of these differences have been discussed in below.
In this study, contamination-free cultures with expected survivability of the explants were primarily achieved by treating the explants with 0.1% HgCl 2 solution for 15 minutes where 80% bulb segments produced contamination free explants. There are also many other reports using HgCl 2 for surface sterilization of explants from field grown plants (Apurva and Thakur, 2009;Biswas et al., 2009;Robinson et al., 2009;Sudersan and Aboel-Nil, 2002).
Axillary bulblets were produced directly from bulb-scale explants after 7-8 weeks of culture. BA-NAA combination was found to be better growth regulator combination than BA-IBA, Kn-NAA and Kn-IBA combinations, and 4.0 µM BA with 2.0 µM NAA enhanced maximum axillary bulblets formation from the bulb explants. The variable increase in the bulb size could be attributed to the variable effects of plant growth regulators in the culture medium. Ozel et al. (2007) reported that the BAP-NAA combination had significant effects on axillary shoot multiplication of M. macrocarpum. They obtained highest number (12.64) of shoots per explants from bulb-scale explants on medium containing 2.0 mg/l BA in combination with 2.0 mg/l NAA. In Muscari mirum, Nasircilar et al. (2011) reported MS medium supplemented with 4.0 mg/l BA and 0.25 mg/l NAA as the best growth regulator combination for the highest percentage of bulb formation (23.5 per explant) from bulb-scale explants. Recently, Uzun et al. (2014) reported that MS medium with 4.0 mg/L BA and 0.50 mg/L NAA showed the best bulblets regeneration after 1 year of culture condition. Ozel et al. (2015) also observed, the best results of in vitro bulblet regeneration of M. muscaimi using twin scale explant on MS medium containing 17.76 µM BAP with 10.74 µM NAA.
The type of explants is extremely important in the establishing of as efficient micropropagation and regeneration system (Koroch et al., 2002;Uranbey et al., 2003;Basalma et al., 2008;Uranbey, 2011). Two types of explants viz. bulb-scale and leaf were used during this investigation to test the effect of explant type on direct adventitious bulblets proliferation. The results indicated  that an auxin-cytokinin ratio 1:4 is the best combination for producing maximum number of adventitious bulblets per culture. For bulb explant, among the various concentration of BA with NAA, the maximum number (8.4 ± 0.58) of adventitious bulblets per explants were found on MS medium containing of 4.0 µM BA + 1.0 µM NAA. Whereas, leaf explants produced the maximum number (10.8 ± 0.24) of adventitious bulblets per explants containing MS medium with 4.0 µM BA + 1.0 µM NAA. Our study indicates that leaf explants of M. armeniacum have a high regenerative capacity than bulb explants for producing adventitious bulblets directly. This finding is in agreement with several previous reports (Suzuki and Nakano, 2001;Wang et al., 2013). On the basis of the present results between the two cytokinins BA in combination with NAA was found to be better than Kn with NAA for proliferating bulblets from the leaf and bulb-scale explants. Uranbey (2010) also found similar observation of Muscari azurium. In conclusion, this technique could be utilized for preparing largescale plant materials with a view to field trial and to establish this plant as a new cut flower variety. It also could be an optimum method for developing clonal variation in laboratory conditions.

CONCLUSION
In conclusion, the present study described a successful regeneration and acclimatization protocol of M. armeniacum using bulb scale and leaf explants through direct organogenesis pathways. Among different plant growth regulators, BA-NAA combination was found to the best for both axillary and adventitious bulblet proliferation. Results also demonstrated the leaf-derived explants as the best explant for adventitious bulblet proliferation than that of bulb-derived explant. However, for in vitro rooting, IBA in half strength MS medium was recorded to be the best. Taken together, the developed in vitro protocol could be helpful for commercial production of M. armeniacum and subsequently for its acclimatization in the new environment.