In vitro laser radiation induces mutation and growth in Eustoma grandiflorum plant

Eustoma grandiflorum (family, Gentianaceae) is considered cut flowers in the international flowers market. The plant is a moderately cold-tolerant annual or biennial plant native to the southern part of the USA and Mexico (Roh and Lawson 1988). This plant attains to 50–75 cm in height with 20–40 flowers. By nature, Eustoma grandiflorum initially forms a rosette and grows very slowly during the winter, stems elongate in the spring, and it flowers in summer (Roh et al. 1989). Eustoma grandiflorum is commonly propagated by seed or cutting. A large number of seedlings can be produced by seed propagation but the quality is not uniform due to variations in flowering time, plant height, and the number of flowers. Propagation of Eustoma grandiflorum by tissue culture technique is relatively low. Several factors like genotype, media, plant growth regulators, and type of explants should effect the success of the micropropagation method, and most of plant growth regulators that have been used were 6-benzyle amino purine (BA), n-6-foural adenine (KIN), naphthalene acetic acid (NAA), and indole butyric acid (IBA) (Pati et al. 2005; Nhut et al. 2010).

Laser has been discovered in the past century and has been applied in the society from its conception until today. Among its application is its use in agriculture as a biostimulator device. The laser light at low intensity produces biostimulation when used on seeds and seedling plants (Chen et al. 2005). The basis of laser stimulation mechanism in any plant physiological stage is the synergism between the polarized monochromatic laser beams and the photoreceptors (Bielozierskich and Zolotariewa 1981; Koper et al. 1996). There are many facts that indicate the biostimulating action of laser radiation on various organs and tissues in plants (Anisimov et al. 1997). Plants absorb light via their photoreceptors and control all stages of plants development (Spalding and Folta 2005).

Protein molecular weight determination via SDS-PAGE is universally used method and it can be economically used for assessing genetic variation (Ranjan et al. 2013; Awatef 2017).

Mutagenesis experiment permits to increase possibilities of variability creation with high ornamentation (Cantor et al. 2002). Mutation is a natural process which creates changes in DNA sequences. The genetic variation created is useful because it helps population to survival and change over time. Mutagenesis is the process where changes occur in the genetic information of an organism not caused by genetic segregation but induced by chemical and physical agents (Roychowdhury and Tah 2011).

The experiment was conducted from 2014 to 2016 at the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza and Tissue Culture Laboratories of Ornamental Plants and Woody Trees and Biotechnology Departments, National Research Centre (NRC), Egypt to investigate the effect of laser as physical mutagens on in vitro and in vivo propagation behaviors as well as flowering of acclimatized plants, biochemical and cytological behaviors of Eustoma grandiflorum plant.

The tallest branches (11, 11.16, and 12.16 cm) resulted from different types of laser radiation such as green, blue, and red for various times (10, 5, and 20 min., respectively) as compared to control (1.86 cm).

The highest number of leaves per branch (39.67 leaf/branch) was resulted with green laser for 25 min as compared to control (17.67 leaf/branch).

Data presented in Table 3 pointed out that the highest leaf area (3.70 cm²) was resulted from using green light of laser for 20 min compared to control (0.962 cm²) and other treatments.

Shootlets with various types and different laser radiation led to delay bloom bud formation. The longest duration (192.33, 192, 203.67, and 192 day) was observed with 25 min blue light, 5 min green light, 5 min red light, and 10 min red light laser irradiation, respectively with no significant difference between them and control (183.33 day).

Considering the flowering percentage of Eustoma grandiflorum plant, Table 4 showed that among different treatments, shootlets exposure to blue and red light laser radiation for 20 min gave the best results (59.25%, 4.83 cm and 72.07%, 4.5 cm) of flowering percentage and flower diameter compared to control (18.51% and 2.33 cm) and other treatments respectively.

The highest number of flower buds per plant and the highest number of flowers per plant (12.33 buds/plant and 8.33 flower/plant) were resulted with blue laser radiation for 20 min, while control treatment formed (2.33 bud/plant and 1.67 flowers/plant) respectively.

The longest bloom stem length (29 cm) and fresh weight of flower (1.42 g) were obtained with 20 min of blue light of laser radiation followed by 25 min of red light of laser radiation which reached to 14 cm in height and 1.33 g, while the shortest bloom stem length (2.3 cm) and fresh weight of flower (0.03 g) was obtained with 5 min of red light of laser radiation as compared to control (6.5 cm and 0.41 g) respectively.

The longest peduncle length (9.7 cm) was obtained with 25 min red light of laser radiation as compared to control (3.67 cm) while the shortest peduncle (2.9 cm) was observed with 5 min and 10 min of red light of laser radiation. The peduncle length was increased gradually with increasing time exposure times.

The treated shootlets with 20 min of blue light laser radiation delayed the flower senescence to 12.67 day and recorded the highest petal area (7.58 cm²) as compared to control (3 days and 3.76 cm²) respectively. While the maximum number of petals (24 and 21.67 petal/flower) were observed with 20 min of green light and 25 min of red light laser radiation as compared to control (11 petal/flower).

The maximum number of stamens (seven stamens/flower) was recorded with 10 min of green laser. While the best results of dry weight also was observed with 25 min of each of blue and red light of laser rays comparing with control and other treatments.

The changes range of flower color and form were varied as shown in (Fig. 1) as follows.

The flowers of unirradiated plants were characterized by violet (light purple) color in the middle of the petals, dark purple in the flower center, and white or brown color on the edges of the petals; the ovary is closed with green color and the stamens is yellow (Fig. 1a). The irradiation with green laser for 10 min resulted in very light purple flowers, wide and big stamens, and small light yellow ovary (Fig. 1b). While exposing plants to the same type of laser (green laser) for 20 min (Fig. 1c) caused variation in colors in the flowers such as the first flower was very light purple, the second one was very dark purple, but increasing this time exposure to 25 min (Fig. 1d) changed the color to light purple flowers with arranged and compact petals. Blue irradiation laser treatment for 5 min (Fig. 1e) resulted in light purple flowers planned with light white color; the petals were varied in the shape from triangle to round and sometimes serrate. The ovary was small and greenish yellow. Further, 10-min (Fig. 1f) exposer of blue laser led to very light purple flowers brushed with white color in the edges of external petals, while increasing this time exposure to 20 min (Fig. 1g) was a very important factor to produce two different colors which were creamy white and dark purple with big petals. The third type of laser (red) resulted in dark purple flowers for the exposure time of 20 min (Fig. 1h), but increasing this time to 25 min (Fig. 1i) led to flowers varied in the darkness of purple color from light to dark purple reddish and planed in white color.

The goal of the present work was to investigate the influence of laser irradiation Invitor on growth, anatomy, flowering, chemicals composition, and gene mutagenesis.

According to data presented in vitro growth ability, we can notice that most of laser types (green, blue, and red laser radiation) for any exposure times (5, 10, and 25 min) led to increase in shoot multiplication of Eustoma grandiflorum except for blue laser radiation for 5 min when compared with control (1.22). The data go in line with those obtained by Danaila et al. (2011) on Petunia hybrid and Dianthus caryophyllus plants and Hwida et al. (2012) on Balanites aegyptiaca and Cotoneaster horizontalis. Lobna et al. (2014), Rania et al. (2015), and Hwida et al. (2012) mentioned that the maximum shootlets number per explant of Balanites aegyptiaca were observed with red laser treatments. Therefore, the mechanism influence of laser irradiation is most likely attributed to light and electromagnetism effects. Longest shootlets were produced from exposing Eustoma grandiflorum shootlets to green and blue laser for short time exposure 5 min produced as well as long time exposure of red laser (20 min) as compared to control. These results were in agreement with Lobna et al. (2014), Sahar et al. (2014), and Ali et al. (2014). The cell elongation resulted by laser treatments increased gibberellic acid which increased the cell vacuoles (Mahmoud and brahem 2000).

The highest number of leaves per shootlet appeared with red laser for 5 min as compared to control. These results were confirmed by researchers such as Danaila et al. (2011), Hwida et al. (2012) Lobna et al. (2014), and Rania et al. (2015). Using He-Ne laser beam stimulation related to higher activity of some enzymes in treated biological material Dobrowolski et al. (1987).

All laser radiation treatments had not significant effect on rooting percentage of Eustoma grandiflorum plant as compared to control. Some studies similar with our study like Hanna and Babelewski (2014)mentioned that the laser radiation did not affect percentage of rooted cutting. This was due to the first reason type and concentration of auxin.

The highest number of roots was obtained with blue laser for 5 min. However, the minimum of root/shootlet was observed with red laser for 25 min as compared to control. These results were confirmed by Hwida et al. (2012), Metwally et al. (2013) and Rimal et al. (2014).

Regarding the effect of laser radiation on length of roots as affected by different types of laser radiation and various times exposure, data showed that the longest roots were resulted from irradiation of shoots with green laser for 10 min as compared to control. Our study was confirmed by Lobna et al. (2014), Hwida et al. (2012), and Metwally et al. (2013).

In this investigation, results in acclimatization stage also showed that the best results for the number of branches per plant were obtained from the green laser for the long time exposure (25 min) and red laser for short time exposure (5 min). These results similar to Osman et al. (2009) and Aguilar et al. (2015) reported that laser radiation could cause enhancement of enzyme activity. Also may be the endogenous content of GA and role in cell elongation, where GA may cause cell elongation by induction of enzymes that weaken the cell wall (Macleod and Millar 1962).

The tallest branches were resulted from different types of laser radiation such as green, blue, and red for various times (10, 5, and 20 min, respectively) as compared to control. Our study agree with (Aladjadjiyan 2002) who mentioned that the He-Ne laser irradiation could raise the activities of superoxide dismutase (SOD) and Ascorbate peroxidase (Apx) enzymes in plants which resulted in accelerating the plant physiological metabolism and increased plant growth.

The highest number of leaves per branch was resulted with green laser for 25 min as compared to control. Osman et al. (2009) found that the best number of leaves and number of branches produced from application of laser treatments for 20 min for both seasons. Noha and El Ghandoor (2011) expressed the stimulated seedling length, average number of leaves for longer treatment time with laser application for 30–120 min. The highest leaf area was resulted from using green light of laser for 20 min compared to control and other treatments. These results were confirmed by some researchers such as Rybiñski and Garczyñski (2004), Al-sherbini et al. (2015), and El-Kereti et al. (2013) who revealed that the increase in leaf area may be reflected by the effect of rays on cell division which continues to all parts of plant at vegetative stage or may be the main biological active gibberellic acid formation is promoted by laser radiation.

It is evident from our result that red laser radiation for 5 min delayed bud flower initiation. The longest period was resulted from red light laser treatments for 5 min. Contrary, the shortest was obtained with blue light of laser for 20 min. These results were agreed with Podleoeny (2002) and Metwally et al. (2013).

Considering the flowering percentage of Eustoma grandiflorum plant, results showed that among different treatments, shootlets exposure to blue and red light laser radiation for 20 min gave the best results of flowering percentage and flower diameter compared to control and other treatments.

The highest number of flower buds per plant and the highest number of flowers per plant were resulted with blue laser radiation for 20 min. These finding were in agreement with El-Tobgy et al. (2009) and Osman et al. (2009).

The longest bloom stem length and fresh weight of flower were obtained with 20 min of blue light of laser radiation as while the shortest bloom stem length and fresh weight of flower was obtained with 5 min of red light of laser radiation as compared to control.

The longest peduncle length was obtained with 25 min red light laser radiation as compared to control while the shortest peduncle was observed with 5 min and 10 min of red light laser radiation. The peduncle length was increased gradually with increasing time exposure times.

The treated shootlets with 20 min of blue light laser radiation delayed the flower senescence to 12.67 day and recorded the highest petal area as compared to control these results online with Metwally et al. (2013) and Ritambhara and Girjesh (2013). While the maximum number of petals were observed with 20 min of green light and 25 min of red light laser radiation as compared to control.

The maximum number of stamens was recorded with 10 min of green laser. Mohammed (2005) found that irradiated Salvia officinalis plant with different doses and time exposure of He-Ne laser produced higher yield of herb compared to other types of laser. While the best results of dry weight also was observed with 25 min of each of blue and red light of laser rays comparing with control and other treatments. These results were agreed with Sahar et al. (2014).

Data presented indicated that shootlets were irradiated with short exposure time of green laser radiation resulted the best results for pigments content as compared to control and other treatments. The maximum values of chlorophyll (a, b), carotenoids, and total chlorophyll (a + b) were resulted from green light of laser for 5 min as compared with control. Our results were agreed with some researchers such as Lobna et al. (2014) Al-sherbini et al. (2015), Rania et al. (2015), and Dziwulska et al. (2016).

Our data indicated that anthocyanin content of Eustoma grandiflorum petals were significantly increased by using dose of laser radiation and reached up to maximum values with both doses (green for 20 min and red light laser for 25 min) as compared to control. Kurata et al. (2000) found that blue and red laser radiation was able to stimulate anthocyanin production.

The results presented indicated that the highest thickness of midvein, thickness of lamina, number of xylem rows, number of vessels, and diminution of vascular bundle (length − wide) were obtained with 20 min cadmium and 25 min helium neon laser as compared with control and other treatments. The results were mentioned by Hwida et al. (2012) and Bedour et al. (2012).

In general, the plant growth is associated with some factors such as enzymes and hormones like cytokinin and gibberellic acid (GA₃). The primary study observed that the red light of laser radiation have important role on GA₃ formation and the endogenous content of GA₁ according to Kamiya and Martinez (1999). The GA₃ response for cell elongation and other effects such as weaken the cell wall, production of proteolytic enzymes, increase of auxin content, increase of concentration of sugar, raising the osmotic pressure in cell soap. This elongation of cell which is treated with laser radiation led to increase of plant height, number of branches, and number of flower according to Lobna et al. (2014) and Rania et al. (2015), Using of laser radiation increased the nitrogen content that caused increase in protein content which is necessary for increasing plant organs such as branches and number of umbels (Osman et al. 2009). According to Mahmoud and Brahem (2000), they indicated that the cell number increased by laser radiation that increased nucleic acids and phospholipids membranes as well as increased phosphorus and potassium contents that led to cell elongation irradiated with laser radiation.

The current research concluded that the laser irradiations can affect plant morphology, flowering, chemical constituents, and gene mutagenesis. The highest survival % of acclimatized plants (95%) and highest values of number of branches, branches length (cm), were obtained from treated plantlets by 20 min green laser, while most of the highest floral parameters, anthocyanin pigment contents in flower, and anatomical structural parameters recorded increased by using 20 min blue laser and 20, 25 min of green and red laser respectively. Contrary, the lowest values of photosynthetic pigments and carotenoids were obtained from 20 min green exposure time.