Bulb Induction on Madonna Lily Stems and Three Years Growing Performance of the Bulblets

Currenly, Madonna lily (Lilium candidum L.) is considered as one of the most important ornamental and perfumery plants. Madonna lily is currently propagated via seed or bulb scales. However, seed derived plants reaches sexual maturity at the end of five years and flower production is interrupted in vegetative propagation via bulb scales because fertile bulbs are needed as source of scale. To overcome the two obstacles, stems of mature plants were used as material for bulb production in the current study. For this purpose, the stems, obtained from plants at the end of flowering were stored in cardboard boxes under the conditions of darkness, normal temperature and pressure (20°C and 1 atm) during four months. At the end of this period, it was observed that 14 bulbs per stem formed on the stored stems. The stem derived bulbs were cultivated in pots and were observed to determine their development performances and some morphological characters until reaching flowering during three years. An increase of 226% in bulb weight, 51.2% in bulb diameter and 58.9% in bulb circumference were observed at the end of the three years. In this study, a new and effective method for Madonna lily propagation was described for the first time. Performance of the


INTRODUCTION
The plants of Lilium (Liliaceae) are one of the oldest plants humans have used for centuries with different purposes. Historical records have indicated that these plants were cultivated thousands of years ago in Mediterranean basin (Fornaris et al., 2011). Ancient Egyptians used lilies as wreath in funeral ceremonies and Romans used them as ornament to decorate their palace gardens (Mammadov et al., 2017). Furthermore, lily gardens were also mentioned in Sumerian tablets, prepared about 5000 years ago (Ozen et al., 2012;Dhiman et al., 2018). Today, lilies are cultivated as ornamental plants throughout the world, as well as used as important perfumery plants.
Lilium genus consists of 120 species growing mainly wild in temperate zone of Northern Hemisphere and is represented in Turkey by the presence of seven species, one of which is Lilium candidum L. (Demir and Celikel, 2017). This perennial bulbous plant grows in sparse populations on rocky slopes and maquis shrubland of Mediterranean basin. Its distribution areas include Balkan countries, Greek islands, Lebanon, Palestine, Israel, Syria and the coastal regions of southwestern and western parts of Anatolia. In Turkey, L. candidum spreads naturally in territory of coastal cities of Mediterranean and Aegean geographic regions, namely Aydın, İzmir, Mersin, Muğla, Antalya, Balıkesir, Çanakkale, İstanbul. It prefers especially maquis shrublands and rocky shades which receive sufficient sunlight but are not exposed to sun directly as habitat (Kahraman, 2014(Kahraman, , 2015Ocak et al., 2014).
Thanks to its large and shining white flowers, L. candidum has been accepted as the symbol for innocence in worldwide and is named as Madonna lily (Fornaris et al., 2011). In Turkey, this plant is known under the local names "akzambak, beyaz zambak, mis zambak, desti zambağı ve bey zambak" (Ocak et al., 2014). Madonna lily has been used traditionally in folk medicine for the treatment of inflamed and purulent wounds, ulcers, skin inflammations, burns and various injuries since ancient times (Pieroni, 2000;Zaccai et al., 2020). Like the other lilies, it includes many of chemical compounds with a wide range of bioactivities. However, it has been recognized in worldwide as an ornamental and cut flower in recent years. Today, the most popular using way of Madonna lily is production of ornamental plants. It is also an economically important perfumery plant with its pleasant odor (Royandazagh and Pehlivan, 2016;Patocka and Navratilova, 2019).
Today, L. candidum is one of the species that are in danger of extinction. Harvesting its bulbs is forbidden and heavy fines are levied from ones collecting the wild bulbs. In addition, this prominent flower is listed in the species under protection according to CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora). Export for L. candidum bulbs is only permitted in cultivation areas.
For that reason, it is necessary to describe cultivation practices for this species with the aim of protecting the flora and increasing its trade value.
Madonna lily is currently propagated via seed and bulb scales generatively or vegetatively (Alam et al., 2013;Kahraman et al., 2017). Seeds are dormant in the winter season and produce one small bulblet by germinating in spring. The number of bulb scale and bulb diameter increase significantly in consecutive years and the bulb has reached the capacity of flowering approximately at the end of five years (Akcal and Kahraman, 2016). Thus, propagation via seeds takes a longer time than propagation via bulb scales (Balge et al., 2020). In vegetative production, the bulb scales that are obtained only from large bulbs are used as material and flower production is interrupted as a result. The large bulbs, consisting of many scales with various dimensions are divided into their scales and each scale produces a new plant. Bulblet of the scale-derived plantlet develops enough to blossom after three years (Altan and Burun, 2017;Royandazagh et al., 2015). Several efforts have also been undertaken for in vitro production of L. candidum (Khawar et al., 2005;Bakhshaie, 2016;Mazor et al., 2021).
Alternatively, stems were used as propagation material instead of seeds and bulb scales in the present study. To the author"s knowledge, there is no report on bulb formation on stems and development performance of the stem derived bulbs in the current literature. Thus, we stored the stems, obtained from plants at the end of flowering in cardboard boxes and observed bulb formation on these stems during storage period. Then, we cultivated the stem-derived bulbs in pots during three consecutive years and determined their performance as propagation material. In this way, it is the first time we have described here a new and effective method for Madonna lily propagation.

MATERIALS and METHODS
In the present study, it was aimed to induce bulbs on stems and to describe development performance of the stem-derived bulbs until they reached flowering.
The study was conducted out in two phases; firstly, bulb formation on stems and secondly determination of growth and development performances of the stem derived bulbs during cultivating in pots. The stems that were used as material were kindly provided by lily producers from the Bafra district of Samsun province, Turkey. and dry summers, cool and rainy winters, reflected typically general climate characters of Black sea region and is semi-humid. Most precipitation falls in the autumn and winter months. The spring and summer months are relatively less rainy. The snow cover stays in the region for a short time or for some years it does not snow at all. The first frost occurs in November and the last frost in April. The average temperature is 12.5 °C as mean of long years, the highest average temperature was recorded in July and August (22.1-22.5 °C) and the lowest average temperature was recorded in January and February (2.1-3.4 °C). On average, relative humidity rate is 65.4% and total amount of annual precipitation is 520.2 mm (Yildirim, 2020).
In the first stage, stems of plants at the end of flowering were harvested on June 06, 2017. Total of 100 stems were stored in cardboard boxes under the conditions of darkness, normal temperature and pressure (20 °C and 1 atm) for four months. Then, the bulbs formed on stems at the end of storage were counted and separated.
In the second stage, the stem derived bulbs were transferred into the pots, 15 cm in diameter and 3 liters in volume on October 02, 2017 to observe the growth and development performances of individuals until reaching flowering during three years. The pots were filled with a mixture of 80% soil + 15% commercial peat + 5% burnt barn manure. The study was conducted out in the open under semi-shaded conditions during three consecutive years. As experimental data, bulblet number per stem, root length (cm), root weight (g), bulb weight (g), bulb diameter (cm), bulb circumference (cm), scale number per bulb, and total fresh weight were recorded at the end of each vegetation period. Fresh weight of basic leaf (g) and basic leaf number per plant in the first two years and plant height (cm), leaf number per plant, stem weight (g) and stem diameter (cm) in the last year were also recorded.

RESULTS and DISCUSSION
Bulblet production phase (June 01, 2017 -October 01, 2017) The stems, picked from mature plants at the end flowering were stored as mentioned previously and bulblet formation was observed on these stems after four months of storage (October 01). The stems in cardboard boxes before storage are shown in Figure  1a and the bulblets formed on these stems are shown in Table 1b. The stem-derived bulblets were separated from stem tissue by bistoury and photographed after sorted from big to small for exhibition (Figure 1c-d).
According to the results, the tested stems produced a number of large and small bulblets. As shown in Figure 1, bulblet formation occurred on stems from bottom to top and lower bulblets were larger, but upper ones were smaller. It is also noteworthy to note  that the bulblets started to root before separated the stems. This is especially true for the robust bulblets from bottom part of stems ( Figure 1). Bulblet number per stem varied with 7-12 and the tested stems produced 14 bulbs on average. Eken and Sirin (2018) searched the most suitable growing media for different Lilium spp. and reported 8.3-17.5 bulblets per stem depending on species. Main bulb bigness, stem thickness and suitability of growing medium are thought to affect bulblet formation. It is obvious that large main bulbs have thick stems and, as shown in In the second phase of the study, the stem derived bulbs were cultivated in pots and observed until flowering during three years. As shown in Figure 1, these bulbs varied with their largeness. Accordingly, the plants they produced also varied with their dimensions. It was observed that 33, 27, 18 and 7% of the tested bulbs produced 1, 2, 3 and 4 basal leaf, respectively at the end of first year of cultivation in pots. Some pictures of the first-year plants and data for the morphologic observation and measurements tested are shown in Table 1 and 2. First year planted bulbs thrived in accordance with their largeness and larger bulbs produced higher number of scales. The stem derived bulbs with single scale produced bulbs with 3-8 scales in first year. These bulbs varied in terms of diameter (1.40-3.87 cm), weight (1.01-9.15 g) and circumference (2.73-8.89 cm). Besides, these bulbs produced the plants, 1.41-12.69 g in fresh biomass and these plants provided the leaves, the smallest and biggest of which were 2.06 x 5.79 cm and 2.78 x 10.02 cm, respectively in dimensions (Tables 1-2). The first vegetation period was ended with leaf drying in June 10 and the harvested bulbs were stored for the second-year cultivation.
Growth performance of the bulbs in the second year (September 05, 2018 -May 30, 2019) The bulbs, stored during the summer season, were retransferred into pots on September 05 and cultivated until the end of vegetation period. As shown in Table  3, some morphologic measurements, each one represented 10 individuals, were taken at the end of the second cultivation year. The size of the pot-grown plants varied with bulb largeness and the photograph of mean-size individual representing plant growth and development in the second year is shown in Table  3. It was observed that basal leaf number per plant was 6.52, bulb diameter was 3.77 cm, scale number per bulb was 11.97, and total fresh biomass 53.80 g as mean values at the end of the second cultivation season. Total fresh biomass and scale number per bulb doubled when compared to the first-year data. But no individual blossomed at the end of the second year and for that reason, plant height could not be measured. In the experimental region, pollination and insemination are insufficient due to the misty and humid climates. Thus, seed formation is weak and wild lily populations are also under pressure because of bulb harvesting for commercial purposes (Arslan et al., 2019). This phenomenon has forced to development of different economic and ecological production techniques. Generally, previous studies in this context provided similar results. Ozen et al. (2012) reported that wild L. candidum plants produced 2-12 basal leaves per plant dimensions of which varied with 1.5-5 x 14-35 cm but these leaves became lost completely at flowering. As mean values, the width and height of L. candidum basal leaf were 3.4 and 19.7 cm, respectively. Our data in the present study did not confirm those of the former authors as we observed 1-4 basal leaves per plant, dimensions of which varied with 2.58 x 7.10-  The leaves that dried completely at flowering were smaller than basal leaves and have become smaller from bottom to top on stems (Ozen et al., 2012). In the current study, leaf number per plant could be measured in the third year and was found to be 47.4-80.3. Besides, plant height and stem width could also be measured in third year. Mean values were found to be 67.0-102.2 cm for plant height and 0.8-1.5 cm for KSU J. Agric Nat 25 (2): [258][259][260][261][262][263][264][265][266]2022 Araştırma Makalesi Research Article stem width. In concomitant with our results, plant height was reported as 43-150 cm (Ozen et al., 2012) and 105.3-120.4 cm (Ozel and Erden, 2010). Ucar and Kazaz (2015) detected plant height as 26.4-74.7 cm and stem width as 5.0-9.5 mm. Kahraman (2015) used L. candidum bulbs, 4 cm in diameter as propagation material and reported plant height and stem width as 38.07-39.50 cm and 7.03-7.33 cm, respectively. The same author also searched the effects of different solid culturing media on some plant characters of greenhouse-grown L. candidum and reported plant height as 47.63-64.64 cm and stem width as 0.64-0.75 cm (Kahraman, 2014 L. candidum has yellowish-white bulbs, 1.2-7.6 x 0.8-2.0 cm in dimensions and the bulbs bear approximately 50 scales (Ozen et al., 2012). In the present study, scale number per bulb was 3.00-8.84 depending on basal leaf number per plant in the first year and this value was recorded as 10.18-13.57 and 23.3-34.2 in second and third years, respectively, as expected ( Figure 2). As mentioned in previous reports, main bulb size has affected significantly plant growth and development. We observed that our experimental plants could blossom hardly in the third year and bulb weight exhibited a great variation between 1.01 and 57.5 g during experiment ( Figure 2 Figure 2) and this parameter was reported to be 0.8-2.0 cm (Ozen et al., 2012), 4.7-7.2 cm (Kahraman, 2014) and 5.92 cm (Kahraman, 2015). Similar to Kahraman (2015) who observed an increase of 48% in bulb diameter between main bulb and the harvested bulb, we also detected an increase of 51.2% in the same parameter ( Figure 2). As for bulb circumference, this parameter exhibited the same change trend as bulb diameter and weight. Bulb circumference increased significantly at the rate of 58.9% when mean values of second and third years were compared and varied with 3.1 and 20.9 cm (Figure 2). Uysal and Kaya (2013)  In the previous efforts, generative or vegetative propagation methods via seeds and bulb scales and several in vitro techniques were tested. But two main obstacles were indicated in these previous reports. First, flower production is interrupted because fertile bulbs are needed as source of scale in the vegetative propagation of lilies via bulb scales. Second, seedderived plants can reach sexual maturity hardly at the end of five years in generative propagation via seeds. When sufficient bulblets induction on stems were provided as described in the present study, a sustainable flower production can be continued without needing bulbs by re-using the stems of harvested plants as propagation material. In this study, we report for the first time that lily stems could produce 14 bulbs per stem on average and can be used effectively as source of bulb to use as propagation material. Considering these findings as well as the stem-derived bulbs that could transform into reproductive plants within three years, it is obvious that our results have made a significant contribution to the current knowledge on the propagation of Madonna lily that is under protection in accordance with CITES.

Researchers Contribution Rate Declaration Summary
Authors declares the contribution of the authors is equal.

Conflicts of Interest Statement
Authors have declared no conflict of interest.