Nutritional compositions of the media used for micropropagation of Hasanbey and Cinikiz melons (value per 100 g of edible portion) *From USDA Nutrient Database, July 2012 
Cucurbits are an economically important family of plants. The majority of the vegetable production in Turkey, for example, derives from the species beloning to the family
1.1. The family cucurbitaceae
1.2. The importance of melon production in the world
Melons are grown in both temperate and tropical regions. Due to various morphological variations in its fruit characteristics such as size, colour, shape, taste and texture, melons are hence described an extensive diverse group. In addition,
Although Africa is the origin of the melon, the diversification center for this fruit encompasses all Asian countries from Turkey to Japan. China, Turkey, Iran, and USA produce 57% of the melon annual production in the world [3-6].
|Total Lipid (Fat) (g)||0.10|
|Fibre, total dietary (g)||0.9|
|Sugars, total (g)||5.69|
|Vitamin K (µg)||2.5|
|Vitamin C (mg)||21.8|
|Vitamn B6 (mg)||0.163|
|Vitamin E (mg)||0.05|
Melons provide several nutrients involving protein (0,6-1,2%/100 g) vitamin E,, vitamin C, and Vitamin K for human metabolic reactions in daily dietary [9, 10]. Melon fruits are used in production of deserts, such as jam, ice cream, yogurt as well as soup (from the juice), pickling and cosmetics .
A characteristic skin color and aroma for melons are the primary traits sought by melon breeders. In addition, the development and ripening of melon fruits are very complicated due to the many biochemical and physiological changes comprising cell wall degradation, alteration in pigment biosynthesis, aromatic compounds, and increasing of sugar content. Therefore, performing the ex-situ regeneration of melon is very important for the research focused on improving the agronomic traits of melon
1.3. The importance of melon production in turkey
Turkey is an important country for cultivation of the economically important plant family
In Turkey, the most popular cultivars are Yuva, Kirkagac, Kislik Sari Kuscular, Hidir, Cumra, Cinikiz and Hasanbey and melons are cultivated on landraces of less than 5 ha in size. Due to climatic conditions, melon harvesting in Turkey can change based on cultivation regions, but generally it is done from June to September .
1.4. The importance of in vitro propagation of melon varieties
1.5. Two important turkish melon cultivars: Hasanbey and Cinikiz
The local melon genotypes are the primary production resources of melon production in Turkey . Hasanbey (Figure 3) and Cinikiz melon (Figure 4) varieties are the domestic farmgate
Hasanbey melon is commonly grown in Western Anatolia in Turkey. This variety is round and dark green with a long shelf life. The Hasanbey melon cultivar is harvested from August to September due to its late ripening period . Cinikiz melons are grown in the Central Anatolia in Turkey. This melon group has the highest ascorbic acid, sweetness and sugar content. The immature fruits of the Cinikiz melon have a light green skin color with dark green spots; mature fruits, a yellow colored skin [16, 18].
Individual plants of the Hasanbey or Cinikiz melon genotypes under
In the light of these facts, we hypothesize that an efficient regeneration method of economically important Turkish Cultivars, Cinikiz and Hasanbey, allows comparison of two different melon cultivars in regard to regeneration ability under an identical artificial medium. In the present study, two Turkish melon varieties were tested for
2. Meterials and methods
2.1.1. Seed sources
Mature seeds of
2.1.2. Media and culture conditions
The achievement of
After surface sterilization, for the initiation of seed cultures, 10 seeds were placed into 100x15mm petri dishes containing basal MS basal medium with MS vitamins, 3% (w/v) sucrose and 0.75% (w/v) agar for three days. The pH level of the medium was adjusted to 5.7 prior to adding gelling agents. The media were sterilized by autoclaving at 121°C for 20 minutes.
Cultures were incubated in a growth room at 25±2°C at dark for three days. In vitro grown cotyledon pieces of mature seeds were transferred into regeneration medium containing different concentrations of IAA (0.0, 2.5, 5.0 mg L-1), Kin (0.0, 2.5, 5.0 mg L-1) and NAA (0.0, 0.5 mg L-1) for organogenesis. Cotyledon explants were incubated at 25±2°C under 16 h photoperiod provided by cool white fluorescent lamps.
2.2.1. Surface sterilization of seeds
Melon seed coats were removed and the seeds were dipped into 70% ethanol for ten minutes and kept in 20% sodium hypochlorite with 2 drops of Tween-20 per 100 ml solution with occasional shaking for 10 minutes. The seeds were then rinsed three consecutive times with sterile distilled water and blotted dry in a laminar flow cabinet. After straining the water, the seeds were placed directly on the culture medium under sterile conditions.
2.2.2. Seed germination
For seed germination, 10 seeds in each of the 100x15mm petri dishes containing the culture medium containing MS salts , MS vitamins and 3% (w/v) sucrose, gelling agent were placed.
Cultures were maintained on a hormone free MS medium for five days in a growth chamber at 25±1°C in darkness.
2.2.3. Plant regeneration treatments
The explants obtained from the part proximal to the apex of the seedling were taken to induction medium. All media were sealed with parafilm and maintained in a growth room at 25±2°C under dark conditions. Every combination of growth regulators was used in the medium for each melon genotype. The experiment was set as a total of 22 treatments for both of the melon genotypes; each treatment was carried out in triplicates containing ten explants in each culture medium.
The regeneration ability of each genotype was then scored weekly for a period of 6 weeks. The data on seed germination and plant regeneration were collected and regenerated plants less than 1 mm in length were not taken into consideration.
3. Results and discussion
3.1. Seed germination
The germination ability of the melon seeds can be affected by both internal and external factors. Since seed size is considered important for better germination [45-47], seeds in similar size were selected for both genotypes. Germination ratio of the Cinikiz melon seeds was found higher (85%) than that of the Hasanbey seeds (78%) under the same culture conditions.
In vitroplant regeneration
In the study presented here, cotyledons from
Different concentrations and combinations of IAA (0,0, 2,5, 5,0 mg L-1), Kin (0,0, 2,5, 5,0 mg L-1) and NAA (0.0, 0.5 mg L-1) were investigated to optimize regeneration of two comercially important Turkish melon varieties: Hasanbey and Cinikiz.
There were significant differences between two melon varieties based on the growth regulator concentrations. According to our findings, comparison of the genotypes showed that the Cinikiz melon cultivar has better regeneration abilitythan did the Hasanbey melon. In addition, the maximum shoot regeneration was achieved on MS medium supplemented with 2,5 mg L-1 IAA and 2,5 mg L-1 Kin was determined the best regeneration medium for both cultivars. Moreover, NAA was foun to be the best growth regulator in the induction of callus of both melon variaties. NAA alone induced direct callus formation, while Kin exhibited synergism with IAA for induction bud formation for both two varieties.
To date propagation of
In our study, the media containing 0,5 mg L-1 NAA showed that supplying NAA in the medium increased the callus formation, although the IAA and Kin concentration (5 mg L-1) was the identical to that of previous study. The addition of NAA alone in our experiment stimulated formation of the callus. The best callus formation ratios were observed from the media containing 0,5 mg L-1 NAA, 5 mg L-1 IAA and 5 mg L-1 Kin for Cinikiz and Hasanbey melon cultivars 100% and 87%, respectively. The medium containing 0,5 mg L-1 NAA, 5 mg L-1 IAA and 5 mg L-1 Kin stimulated callus growth for Hasanbey melon cultivar. On the contrary, the frequency of callus formation for Cinikiz melon genotype due to NAA concentration was significant. In all medium containing 0,5 mg L-1 NAA, high callus formation (100%) from Cinikiz melons’ cotyledons was obtained (Table 3).
The calli were white to yellowish; their surface showed structures such as shoot formation and the calli were rarely regenerative. Our result agrees with the reported callus formation from the plants regenerated
In an earlier study on regeneration of melon, shoot buds were obtained at high rates in cotyledon explants  and well-developed shoots were observed from calli growing MS media containing 1,5 mg L-1 IAA and 6,0 mg L-1 Kin.
According to previous study,  Kin was essential for shoot formation.
Of the growth regulators tested, NAA was the most effective at inducing callus creation from the cotyledons of both two melon genotypes presented here (Table 3).
Development in regeneration from the melon Hasanbey cotyledon explants on the medium added 2,5 mg L-1 IAA and 2,5 mg L-1 Kin was found to be slower than with melon Cinikiz.
After 4 weeks in the culture, there was little further development on the explants of Hasanbey melon cultivar compared to that of Cinikiz.
The best results for shoot regeneration were obtained from MS medium supplemented with 2,5 mg L-1 IAA and 2,5 mg L-1 Kin for Cinikiz and Hasanbey melon genotypes 75% and 50%, respectively. The results of the regeneration tests are summarized in Table 3.
The regeneration efficiency of Hasanbey and Cinikiz melon using cotyledon explants was evaluated in terms of regenerated plants. Cotyledon organogenesis was induced during incubation and bud formations located along the cut basal edge of the explant were visible on 8 day-old cultures of both of the two melon varieties. On the other hand, the first shoots formed 12 day-old explants of Cinikiz melon. According our results, the Cinikiz melon cultivar gave better results than did the Hasanbey cultivar (Figure 6).
Furthermore, the most important difference was observed in the number of bud induced between the two melons. In the Cinikiz melon young tuberances often clustered in the point of regeneration and shoot meristems developed into leaves(Figure 6-A). In the Hasanbey melon, the first regenerated shoots were observed in 15 day-old cotyledon explants and regenerated shoots were originated from the epidermal layer of the explants (Figure 6-B). These results as those of the previous studies show the clear cut effect of the genotype on regeneration.
For the Hasanbey genotype, explants which were cultured on the MS medium free from plant growth regulators, showed callus formation with different coloration and appearance after 2 weeks. Most of them were white to yellowish and friable. A mass of small cells initiated the regeneration of the shoot meristems form directly on explants
After 3 weeks on the medium, explants developed into buds and about a month on culturing, root formation was observed. As they continued to grow, they became yellow and did not develop into shoots. (Figure 7-A). In addition, some demonstrated necrosis. Shoot formation (50%) for Hasanbey melon genotype was obtained on a MS medium consisting of 2,5 mg L-1 IAA and 2,5 mg L-1 Kin after 2 weeks. Explants after 3 weeks of culture formed shoot structures, continued to grow, bud formation was not observed at this stage (Figure 7-B).
As a result, it was observed that the melon possessed the ability to regenerate by means of direct organogenesis from cotyledon explants. After 4 weeks in culture the explants enlarged.
Control groups of Cinikiz melon cultivar developed on the MS medium free from plant growth regulators showed developing callus and roots (Figure 8-A). On the media containing 2,5 mg L-1 IAA and 2,5 mg L-1 Kin, the highest percentage of shoot regenerants from Cinikiz melon cultivar after 2 weeks was obtained (Figure 8-B).
In contrast by involving the control groups of Cinikiz melon cultivar on the MS medium free from plant growth regulators, shoot formation was observed. After 2 weeks, some young protuberances clustered and developed; after 4 weeks, finger-like structures were observed (Figure 8-B). After 4 weeks, no difference could be determined between protuberances that became leaves from Cinikiz samples.
Our in vitro propagation results from these two melon varieties, Hasanbey and Cinikiz, were selected as research material for the study presented here due to their importance for the Turkish agricultural production. Due to open pollination, melon varieties can be more or less stable against environmental factors from one generation to the next. In addition, as can be seen from the results presented here, Hasanbey and Cinikiz melon genotypes can be
On the other hand, further studies are needed to analyze other Turkish melon varieties and identify the optimum regeneration medium for each genotype. In addition, if the response of the two melon cultivars selected in this study is observed from the point of view of breeder significance, experimental fields should be conducted in future studies. Understanding the role of growth regulators in the development of selected melon cultivars has highly facilitated melon production under controlled environments. Moreover, the resulting information can be also used for research on melon developmental physiology, an intensive continuation of
The authors are grateful to Laboratory of Plant Biotechnology in Horticulture Department, Agriculture Faculty, Cukurova University, Adana, Turkey for kindly provided the germplasms used in the study. We also thank Nancy Karabeyoglu for critically reading this manuscript and providing valuable comments for its improvement.