In vitro direct organogenesis in response to floral reversion in lily

Our previous study indicated that the tiger lily (Lilium lancifolium var. Flore Pleno) has a great ability to produce inflorescence bulbils in nature as a form of natural phenomenon of floral reversion in plants. This present research was carried out to investigate the artificial floral reversion in in vitro culture of two lilies (Asiatic hybrid cv.”Black out"), and (Lilium longiflorum cv "White heaven") based on the type and developmental stage of explants plus the different concentrations of naphthalene acetic acid (NAA) and benzyl aminopurine (BA). Developmental changes were observed in both lilies in response to floral reversion which was enhanced by growth regulators under in vitro condition. The regeneration of vegetative organs was associated with certain degeneration of floral organs. Large bulblets and multiple shoots were formed only in specific regions in floral organs, precisely in two attached points: the boundary region between the receptacle with other floral organs and the branching point where the peduncle joins the pedicel. This direct organogenesis was highly dependent on type of lily, type and developmental stage of explants in addition to the concentration of BA and NAA in in vitro culture. However, 1 mg/L BA combined with 0.1 mg/L NAA was the optimum for regenerating shoots and bulblets in in vitro culture of both lilies after six weeks.


INTRODUCTION
In nature, there are more than 250,000 species of flowering plants, and they represent the most wide spread groups of plants. Flowers are important sexual reproductive organs of flowering plants and source of fruit and seed for completing plants' life cycle.
However, in some species, the phenomenon of floral reversion occurs rarely in nature in response to adverse environment and it is affected by photoperiod and hormones. Furthermore, this phenomenon can be efficiently induced in some species in vitro under optimal concentrations of auxins and cytokinins (Tooke et al., 2005;lashman and Kamenetsky, 2006;Supriyo et al., 2013). It can also be an excellent method for propagating some ornamental geophytes. It has been shown that it is a great alternative to explants of underground storage organ for overcoming the problem of heavy contamination which usually occurs in these organs (Ziv and Lilien-Kipnis, 2000;Poluboyarova et al., 2011) E-mail: Hassan.asker2@gmail.com.
Creative Commons Attribution Author(s) agree that this article remains permanently open access under the terms of the License 4.0 International License Various floral organs such as pedicel and filament were taken from some geophytes as explants to regenerate shoots and bulblets in in vitro culture (Kumar et al., 2006;Nhut et al., 2001). The study of Liu and Burger (1986) observed that the explants which were taken very close from the receptacle and the most distilled section of the pedicel produced the greatest number of buds in in vitro culture (Lilium longiflorum).
In recent years, the histological study of Allium altissimum (Poluboyarova et al., 2014) showed that the morphogenic tissue in the fused area of stamens and sepals had the potency to regenerate shoot in in vitro culture. Moreover, several molecular genetic studies confirmed that the pedicel parts are different in their developmental processes in the main model plant (Arabidopsis thaliana) (Douglas and Riggs, 2005) and are genetically regulated by several genes (Cho and Cosgrove, 2000;Kirik et al., 1998;Song and Clark, 2005;Ragni et al., 2008).
The objective of the current study was to investigate the artificial floral reversion of two lilies based on the type and developmental stage of explants in addition to the concentrations of naphthalene acetic acid (NAA) and benzyl aminopurine (BA) in in vitro culture.

MATERIALS AND METHODS
This study was designed to investigate the organogenetic response of two lilies, Asiatic hybrid cv."Black out" and L. longiflorum cv "White heaven" to floral reversion process in in vitro culture. Three cultural experiments were done in vitro. The inflorescence segments were collected from plants grown in computerized greenhouses at the school of biological sciences, University of Plymouth during the year 2014. The explants were carefully washed and sterilized with 10% v:v sodium hypochlorite for 15 min. They were washed three to four times with sterilized distilled water before culturing. The explants were then cultured on Murashige and Skoog (MS) basal medium containing (30 g L -1 ) sucrose (8 g L -1 ) agar, pH 5.7, supplemented with different concentrations of NAA and BA. All cultures were incubated in a Gallenkamp growth cabinet under 16 h photoperiod, provided by cool-white fluorescent lamps with an irradiance of 100 μmol m -2 s -1 at a constant temperature of (25°C).
Three types of explants were taken for this study: explants of receptacle, explants of the branching point where the peduncle joins the pedicel and explants of whole flower bud. Four concentrations of BA and NAA: (1 mg/L BA+1 mg NAA), (1 mg/L BA+0.5 mg NAA), (1 mg/L BA+0.1 mg NAA) and (0 mg/L BA+0 mg NAA) as control were used. Two different developmental stages of explants were chosen: young explants were taken when the size of floral bud was 4 to 6 cm and mature explants were taken at fully mature stage. The receptacle explants were prepared by cutting the portion of receptacle into two identical pieces and the half piece was cultured horizontally on agar ; otherwise, all other explants were placed vertically on the agar.
Each experimental treatment was carried out with at least 15 explants per treatment. The experiment was arranged in a completely randomized block design. The number and weight (g) of bulblets and roots per explant, the number, weight (g) and length (cm) of shoots and the weight of ovary per explant were recorded after six weeks of culture in vitro.
The statistical analysis SAS system (SAS, 2012) was used to show the effect of different factors on the study parameters. Significant difference (LSD) test was used in this study to compare between means at the 0.05 level of significance.

RESULTS
Plate 1 (for Asiatic hybrid lily cv. Black out) and Plate 2 (for L. longiflorum cv. White heaven) shows that the processes of floral reversion after six weeks in in vitro culture, converting floral to vegetative organogenesis resulted in the formation of a wide range of vegetative organs using young and mature receptacle explants with different concentrations of naphthalene acetic acid (NAA) and benzyl aminopurine (BA) (Plates 1 and 2).
Plate 3 records the in vitro culture of Asiatic hybrid lily after six weeks using whole floral buds explants with different concentrations of (NAA) and (BA). The bulblets and shoots appeared on specific points in floral organs: receptacle boundary and in branching points of pedicelpeduncle of explants. Plate 4 shows the in vitro culture of Asiatic hybrid lily using young and mature branch explants of pedicel-peduncle with constant concentration of 1 mg/L BA combined with 0.1 mg/l NAA. Plate 5 shows the in vitro culture of longiflorum lily after six weeks using mature whole floral bud explants and mature receptacle explants with constant concentration of 1 mg/L BA combined with 0.1 mg/L NAA. Figure 1 records the results of the in vitro cultures of Asiatic hybrid lily for six weeks using different type and developmental stage of explants plus different concentrations of NAA and BA.
The explants of inflorescence stalk branch have higher ability to regenerate bulblets and shoots than receptacle ones ( Figure 1A). The young explants produced more shoots but less bulblets compared to the mature ones ( Figure 1B). 1 mg/L BA combined with 0.1 mg/L NAA was the optimum for regeneration of shoots and bulblets compared to others ( Figure 1C). The interaction effect of all these experimental factors is indicated in Figure 1D and the results confirm that the regeneration ability of explants is dependent on all these factors. Figure 2 shows the results of in vitro cultures of both lilies for six weeks using different concentrations of NAA and BA with young receptacle explants. Longiflorum lily has higher ability to regenerate bulblets and shoots than Asiatic hybrid lily (Figure 2A). The interaction effect between the type of lily and concentrations of NAA and BA is recorded in Figure 2B, and the results confirm that the regeneration ability of explants is clearly dependent on these experimental factors.      The results of Figure 3A confirm that the whole floral bud explants had higher ability to regenerate bulblets, shoots and roots compared to receptacle explants. Figure 3B indicates that the regeneration ability of explants is highly dependent on all these experimental factors. However, the ovary enlargement was clearly observed in treatment of the mature receptacle of longiflorum lily.

DISCUSSION
Our previous study was concerned with the natural phenomenon of floral reversion in tiger lily L. lancifolium var. Flore Pleno, with high ability to produce inflorescence bulbils in nature (Asker, 2105). This present study investigates the artificial floral reversion processes in both lilies: Asiatic hybrid cv."Black out" and L. longiflorum cv "White heaven". They were enhanced by growth regulators under in vitro condition. Thus, many developmental changes were observed in response to this reversion when the regeneration of vegetative organs was associated with the degeneration of floral organs.
Large bulblets and multiple shoots appeared in specific regions in floral organs, precisely in two attached points: the boundary region between the receptacle with other floral organs and the branching point where the peduncle joins the pedicel. This is in line with an histological study on A. altissimum (Poluboyarova et al., 2014) which reported that the morphogenic tissue in the area of fusion between stamens and sepals had the highest potency for direct shoot regeneration. This also agreed with the study of Ziv and Lilien-Kipnis (1997), which indicated that the pedicel -peduncle junction had high shoot regeneration in some geophytes. That generated vegetative organs which appear only in specific points may be because the pedicel parts are different in their development process. Douglas and Riggs (2005) reported that the proximal portion and bulged distal region are different in their development in the model plant (Arabidopsis thaliana). Moreover, it was found that pedicle development process is under genetically control by several genes (Cho and Cosgrove, 2000;Kirik et al., 1998;Song and Clark, 2005;Ragni et al., 2008). The current results also showed that the direct Figure 3A. Effect type of explants on the regeneration ability of explants after 6 weeks of of culture in vitro of both lilies using the concentration of 1mg/ 1BA combined with 0.1mg/1 NAA. Using LDS test (p≤0.05). Figure 3B. Interaction effect of type of lily and the type and developmental stage of explants on the bulblets, shoot and roots regeneration after 6 weeks of culture in vitro using the concentration of 1mg/ 1BA combined with 0.1mg/1 NAA. Using LDS test (p≤0.05).