CHARACTERISTICS OF IN VITRO SEED GERMINATION OF THREE BASIL GENOTYPES UNDER DIFFERENT NUTRITION

The present study was designed to investigate the effects of different basal medium composition on germination characteristics in three basil genotypes under in vitro conditions. Ocimum basilicum var. basilicum cv. Genovese, Ocimum basilicum var. purpurascens cv. Dark Opal and Ocimum x citriodorum seeds were grown in media with a quarter, half, and full-strength of macroand micronutrients and under different levels of nitrate alone or in combination with different levels of ammonium. Evaluated parameters included germination percentage, mean time to germination, rate of germination, and germination uniformity. Seeds of the studied basil genotypes demonstrated significant differences in germination characteristics depending on the availability of nutrients, but also the nitrogen form used. It was concluded that in order to obtain a high level of germinated seeds with significant speed and uniformity of germination under in vitro conditions culture media composition must be adjusted according to the investigated genotype. The results of this study could improve overall tissue culture conditions for a particular basil genotype.


INTRODUCTION
Plant tissue culture systems supply all necessary nutrients, water, and energy for plant growth through the basal medium and allow maintenance of whole plants, as well as organs, tissues, or cells under aseptic controlled laboratory conditions. Numerous investigations in this field formed two main distinct trendsefforts to optimize the basal medium for specific species or genotypes and screening large numbers of in vitro germinated seedlings for a particular genotype (PHILLIPS and GARDA, 2019).
Seed germination and consequent elongation of radicle and hypocotyl are among the most significant stages of the life cycle of the plant. The time frame for the duration of the germination process (from the moment of passive water uptake to seed coat decomposition and the appearance of radicle) is genetically conditioned, but the differentiation of embryos and zygotes in this phase is very sensitive to environmental conditions (CHACÓN et al., 2013). Although desiccated seeds are generally resistant to the stressful conditions, germinating 136 seeds are not, and consequently requiring defensive strategies to stressful conditions in order to ensure survival. The answer to these conditions depends on the type and intensity of the stress, as well as the plant species and genotype (MACOVEI et al., 2017). Prevention of stressful conditions during the germination period and development of young seedlings is a very important aspect for the successful production of economically significant plant species (BALABUSTA et al., 2016). However, one of the basic problems encountered during the cultivation process is control of mineral nutrition conditions in basal medium, which further may result in an unsuccessful plant tissue system, so unless mineral nutrition conditions are clearly defined, nutritional stress can lead to significant losses (DA SILVA et al., 2017).
One of the most important elements of mineral nutrition in plants is nitrogen. Nitrogen is a constituent of amino acids and proteins and it regulates the enzyme activities required for energy metabolism (HUSSAIN et al., 2016). Plant growth depends on an appropriate supply of nitrogen in order to synthesize the amino acids, nucleic acids, and other cellular constituents which are further necessary for plant development (SÁNCHEZ et al., 2004). Nitrate (NO3 -) and ammonium (NH4 + ) are primary nitrogen sources (SUNG et al., 2015) and plants produce visible changes in their growth and development if growing on the medium containing nitrate or ammonium under deficit or excess (POLESSKAYA et al., 2006). In recent years there is an increased interest in ammonium-based nutrition due to the high energy cost of nitrate absorption and assimilation, however, this form of nutrition is been reported as stressful to many important crops and may trigger oxidative imbalance in plants (JAKOVLJEVIĆ et al., 2017). Additionally, it is very difficult to differentially quantify NO3 − /NH4 + utilization in tissue culture media when both nitrate and ammonium are simultaneously available to plants (ZHANG et al., 2019).
The cultivation of the species from the genus Ocimum L. (basil) has been intensified globally, because of their pharmaceutical and nutritional importance as well as their long-term traditional use and wide range of adaptability at different soil types and climatic conditions. Among applications in the food industry, basil plants are valuable sources of essential oils and phenolic compounds with significant antioxidant, antimicrobial, cytotoxic and other biological activities (JAKOVLJEVIĆ et al., 2019). The alterations of characteristics in basil plants due to cultivation conditions raising the need to develop a system that is true to type, effective, and enable reproducible plant screening (SRIVASTAVA et al., 2014).
In the present paper, three different basil genotypes Ocimum basilicum var. basilicum cv. Genovese, Ocimum basilicum var. purpurascens cv. Dark Opal and hybrid Ocimum x citriodorum have been tested in order to study the effects of different basal medium composition and nitrogen source on in vitro germination characteristics. The results of this study may provide valuable information about the nutrient management regime for basil tissue culture and can contribute to a better yield of this important crop plant.

Plant material and experimental design
The seeds of three basil cultivars were used in this study: green-leaved Genovese basil (Ocimum basilicum var. basilicum cv. Genovese), purple-leaved Dark Opal basil (Ocimum basilicum var. purpurascens cv. Dark Opal), and lemon basil (Ocimum x citriodorum). Seeds were obtained from commercial sources ("Semesemena", Belgrade). Since it was demonstrated that basil physiological responses under tissue culture conditions depend on the type and straight of the media, as well as cultivar used (JAKOVLJEVIĆ et al., 2017; 2019) this experiment was designed to investigate genotype-related differences in the first week of in vitro growth of basil cultivars under different mineral nutrition. The surface sterilization of seeds 137 was done with a method that does not affect seed germination (JAKOVLJEVIĆ et al., 2017). After sterilization single seed was inoculated in a 25 ml volumetric glass tube containning 10 ml of appropriate media.
All media were supplemented with 7 g/l agar. The pH of all media was adjusted to 5.7-5.8 before autoclaving (121 °C, 15 min). After seed inoculation, the cultures were incubated in a plant growth chamber at 25 ± 2 °C at 60% relative humidity and 16/8 h photoperiod (cool-white fluorescent lamps with the irradiance of 70 µm m -2 s -1 ).

Germination characteristics
For every media used at least 200 seeds were inoculated and the results were obtained after three repetitions. Seeds were considered germinated after radicle appearance (at least 2 mm). The recording of sprouted seeds was done daily until the number of germinated seeds was constant. Germination percentage (GP), the mean time for germination (MTG), rate of germination (RG) and germination uniformity (U) were calculated according to ESPANANY et al.

RG = (3)
G= the percentage of seed germination at one day intervals and t is the total germination period.

Statistical analysis
Data are presented as mean ± S.E. (standard error) of three measurements. The program SPSS v.20 for Windows was used for the statistical evaluation of the results with an analysis of variance (ANOVA) and Tukey's multiple range tests (p ≤ 0.05).

RESULTS AND DISCUSSION
Nitrate and ammonium are the main sources of nitrogen for crop plants, and when it comes to the tissue cultures, nitrogen is generally supplied as NO3at concentrations close to 10 mM (KIFERLE et al., 2014; ZERVOUDAKIS et al., 2015). However, the extreme levels of nitrogen and other main nutrients can lead to toxicity symptoms and consequently to the death of plants. When it comes to basil germination studies, the in vitro culture technique can be useful since it does not compromise future growth and seedling development, especially in prolonged studies. The seeds from basil genotypes investigated in this study were capable to germinate and to develop seedling in different nutrient conditions, still, these processes demonstrated significant differences.

Ocimum basilicum var. basilicum cv. Genovese
The results obtained by examining the germination characteristics of "Genovese" basil indicate a significant effect of mineral nutrition on the development of Ocimum basilicum var. basilicum cv. Genovese during the first week (Tab. 1). The highest percentage of germination (GP = 89.36%) was observed for full strength media with nitrate alone (1a). However, a statistically significant difference was found between this media and full-strength media with nitrate and ammonium, since the presence of ammonium in the media significantly reduced the germination percentage (GP = 78.82%). The 1a media is also a treatment in which the seeds of Ocimum basilicum var. basilicum cv. Genovese showed the highest germination rate (RG = 73.06). However, the highest degree of germination uniformity (U = 35.70), as well as the fastest germination (MTG = 2.49) was achieved in half-strength media with nitrate (1/2a), while significantly slower germination was observed in the presence of ammonium.

Ocimum basilicum var. purpurascens cv. Dark Opal
The first week of in vitro germination of the genotype Ocimum basilicum var. purpurascens cv. Dark Opal is accompanied by significant differences in germination characteristics regarding the concentration of mineral elements and the form of available nitrogen in basal medium. The results are presented in Tab. 2. The highest percentage of germinated seeds (GP = 93.75%) was observed in the treatment with the minimum concentration of mineral elements and the nitrate form of nitrogen (1/4a media), and the results indicate that the increase in the concentration of mineral elements decreases the percentage of total germinated seeds. Opposite, ammonium presence and high nutrient content significantly reduced the germination percentage with the lowest values under full strength media with ammonium (GP = 64.91%). Although there are no statistically significant differences in the rate of germination between the media used, the lowest RG values were obtained at the highest nutrient concentrations. The highest and the lowest percentages of germinated seeds were followed by the highest and the lowest uniformity of germination (U = 31.99 for 1/4a treatment; U = 22.46 for 1b treatment). When it comes to the rate of germination, there are no significant differences between the media used, whereas the seeds germinated faster under half strength media with nitrogen (MTG = 2.73). The results are presented as the mean of three independent measurements ± standard error. Different letters indicate significant differences (p < 0.05) between treatments based on Tukey-test. The results are presented as the mean of three independent measurements ± standard error. Different letters indicate significant differences (p < 0.05) between treatments based on Tukey-test.

Ocimum x citriodorum
"Lemon" basil genotype is characterized by the highest percentage of total germinated seeds, since in each basal medium used the percentage of germinated seeds was greater than 90% (Tab. 3). The highest percentage of germinated seeds was observed in media 1/4b (GP = 97.78%) and was statistically significant from all other basal media used. The medium with the highest germination percentage is also the medium with the most uniformed germination (U = 42.70). In addition, significant uniformity in germination is achieved by applying 1/2a media (U = 40.92), while in other media used significantly lower values of uniformed germination were observed. The most suitable treatment for achieving rapid and uniformed germination was half strength media with nitrate (in media 1/2a MTG = 2.24). The highest rate of germination is also measured with the application of this medium (RG = 74.31), significantly different from other media used. Similarly, rapid (MTG = 2.29) and uniformed germination (U = 42.70) was noted for 1/4b treatment. The results are presented as the mean of three independent measurements ± standard error. Different letters indicate significant differences (p <0.05) between treatments based on Tukey-test.
Seeds of the studied basil genotypes show significant differences in germination characteristics depending on the availability of nutrients, but also the nitrogen form used. Among tested genotypes, seeds of the hybrid genotype Ocimum x citriodorum showed the highest germination percentage (almost 98%) at a minimal nutrient concentration and under ammonium presence. The seeds of Ocimum basilicum var. purpurascens cv. Dark Opal germinated in the highest percentage at a minimum concentration of nutrients, but without ammonium and under nitrate as the only nitrogen source. Opposite, Ocimum basilicum var. basilicum cv. Genovese seeds germinated in the highest percentage in full strength basal medium with nitrate alone. However, the total germination of seeds only reflects the ability of seeds to germinate, without the indications of speed, synchronization, and rate of germination, i.e. to the vital factors that must be involved in the characterization of the seed, not just with horticultural and agronomic points, but in order to define physiological, Mean germination time, a significant indicator of emergence, can be defined as the length of lag germination phase from the beginning imbibition to the penetration of the radicle (MAVI et al., 2010). Since MTG represents the mean time from germination initiation to its completion, a lower MTG value represents faster germination (FERNANDEZ et al., 2015). According to MTG, tested genotypes demonstrated the following order: Ocimum basilicum var. purpurascens cv. Dark Opal < Ocimum basilicum var. basilicum cv. Genovese < Ocimum x citriodorum, whereby MTG of Ocimum basilicum var. purpurascens cv. Dark Opal and Ocimum basilicum var. basilicum cv. Genovese was significantly longer in the presence of NH4NO3. Opposite to the MTG, higher values of rate of germination indicate faster germination (FERNANDEZ et al., 2015). According to this parameter, the same order is observed: Ocimum basilicum var. purpurascens cv. Dark Opal < Ocimum basilicum var. basilicum cv. Genovese < Ocimum x citriodorum, and, in general, the germination rate is lower when the seeds germinate in the presence of NH4NO3. Uniformity germination indicates variability among seeds over the mean time germination of samples (RANAL and SANTANA, 2006). The same trend in germination uniformity for tested genotypes was observed as in the case of MTG and RG. 141 Regardless of the requirements that individual genotypes have in terms of nutrient concentration, and in relation to achieving the best germination characteristics, it has been shown that when the basil seeds are in contact with different concentrations of NH4NO3 the germination process is significantly affected. The effects of the presence of ammonium ions have been demonstrated by PÈREZ-FERNÀNDEZ et al. (2006), and the authors suggested a significant delay of the germination process with the treatment including NH4NO3. Positive effects of KNO3 on germination processes are shown in the case of in vitro culture of smallleaved basil genotype O. basilicum var. minimum (JAKOVLJEVIĆ et al., 2017).
Based on the results obtained in this investigation, it can be concluded that seeds of the studied basil genotypes show significant differences in germination characteristics depending on the availability of nutrients, but also the nitrogen form used. Although different modifications of basal media are available from commercial sources, for many plant species or different types of tissue culture, it is necessary to prepare media using individual chemicals and chemical components as stock solutions, and it is very important to create a reproducible media for diverse genotypes. The results of this study provide information's regarding the best basal medium composition and concentration for three basil genotypes for rapid and uniform germination and could improve overall tissue culture conditions for this valuable plant.