Maturation, conversion and metabolic profile of somatic embryos derived from Klussia odoratissimaMozaff as affected by light spectra ​

doi:10.21203/rs.3.rs-4938119/v1

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Maturation, conversion and metabolic profile of somatic embryos derived from Klussia odoratissimaMozaff as affected by light spectra

https://doi.org/10.21203/rs.3.rs-4938119/v1

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published 04 Nov, 2024

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The present study investigated potential application of somatic embryogenesis (SE) for conservation of Klussia odoratissima Mozaff. As the efficiency of SE greatly varies depending on the culture conditions including light, effects of five light spectrums including a fluorescent light and four types of light - emitting diode (LED) (red, blue, 3red-1blue, and3blue:1red) for an exposure time of a month on the maturation of K.odoratissima were also studied. To better understand the effect of light treatment on conversion of K.odoratissima cultures, two months after treatment, fresh and dry weight and metabolite contents were evaluated. Samples of the same age in situ plant and air-dry plant were also considered for relative comparison. Study of developmental stage of K.odoratissima under in vitro conditions revealed that a combination of 3red:1blue LED provided the highest number of cotyledonary (mature) embryos per callus. Hence, this light treatment was identified as the best treatment for the conversion of somatic embryos in K.odoratissima. Consequently, the highest fresh and dried weights were recorded in the cultures grown under 3blue:1red and blue LED treatment. Phthalide content of in situ plant was significantly greater than other treatments. Interestingly, the phthalide content was significantly higher in tissue culture samples compared to air-dry plants, which suggests tissue culture as an alternative technique for the production of bioactive compound in K.odoratissima. Cultures were incubated in 3blue:1red LED followed by blue LED possessed higher contents of phthalides. Application of LEDs is promising approach in micopropagation of K.odoratissima.

Iranian wild celery, known as mountain celery (MC) or “Kelus” (Klussia odoratissima Mozaff), is a perennial plant belonging to the family of Apiaceae. The plant has enormous economic and industrial applications, albeit being considered an endangered species. K. odoratissima grows in central Zagros Mountain ranges of Iran. It possesses a wide range of medicinal properties including sedative, antimicrobial, hypotensive and antioxidative activities (Ahmadipour et al. 2015 and references therein). The health benefits of K. odoratissima are mainly conferred by phthalides. Phthalides, including Butylidene phthalides and Ligustilides, are a group of secondary metabolites, which are recognized as the major flavoring component in K. odoratissima. Unlike most endemic plants that are vulnerable to herbivores’ browsing (Cubas et al. 2019), K. odoratissima is threatened by excessive and illegal harvest at early growth stage (Seifipour Naghneh et al. 2022). The production of secondary metabolites in plants is naturally influenced by some regional and environmental factors, which limit the commercialization of K. odoratissima production. The extraction of secondary metabolites from K. odoratissima in nature poses a risk of extinction. Plant tissue culture techniques have been recently highlighted as the means of in vitro production of secondary metabolites (Hashim et al. 2021). Somatic embryogenesis (SE) is a tissue culture technique in which somatic cells gain the capacity to produce embryos that finally develop into a whole plant (Aguilar-Hernández and Loyola-Vargas 2018; Garcia et al. 2019). This technique plays a vital role in plant regeneration and rapid reproduction (Tomiczak et al. 2019). The SE process includes several distinct stages: induction, embryo proliferation, maturation, and germination/conversion of embryos into plantlets (Ramírez-Mosqueda, 2022). In dicotyledonous plants, somatic embryos follow the same successive morphological developmental steps as described for zygotic embryogenesis: globular, heart-shaped, torpedo and cotyledonary (Ramírez-Mosqueda, 2022). According to recent studies, somatic embryos in suspension culture are useful tools for the generation of secondary metabolites (Murthy et al. 2023). This method has been successfully used to produce biomass and various secondary metabolites in several medicinal plant species such as Salvia officinalis (Kintzios et al.1999), Gentiana straminea (Cai et al. 2009), Rauwolfia serpentine (Harisaranraj et al. 2009), and Eurycoma longifolia Jack (Iriawati et al. 2014). These studies have demonstrated that the production of plant bioactive metabolites is affected by different biotic and abiotic elicitors. Application of elicitors appears to be a viable method for increasing secondary metabolites in medicinal plants. In this respect, light has emerged as one of the unique and efficient elicitors for enhancing the in vitro production of pharmacologically important secondary metabolites (Hashim et al. 2021).

Various types of light (UV, fluorescent, and LEDs) have been reported as stimulators for the production of plant secondary metabolites (Hashim et al. 2021). In recent years, the application of light emitting diodes (LEDs) has attracted a significant attention as an alternative artificial light source for controlled environmental agriculture as well as plant tissue culture experiments (Almeida et al. 2019). LEDs have several advantages relative to traditional light sources including: small size, durability, long operating lifetime, and relatively cool emitting surfaces, which make them an ideal light source for indoor environments. An outstanding influence of LED lighting on plant regeneration and secondary metabolite production has been reported by numerous studies (Fan et al. 2013; Cio´c, et al. 2018; Almeida et al. 2019; Hashim et al. 2021). However, the response of different plants to LEDs has been inconsistent and there was no specific pattern among various species (Bula et al. 1999; Gupta and Jatothu 2013).

In 1995, Munoz estimated that between18000-25000 wild plant species were at risk of extinction, and in vitro culture techniques were proposed as the sole solution for satisfactory conservation of the majority of endangered species. As for K. odoratissima, attempts to conserve the plant had little progress due to poor seed setting and germination as well as long-time seed dormancy (Askari-Khorasgani et al. 2013). Hence, tissue culture seems to be an effective method for conservation of K. odoratissima. Few reports have studies in vitro culture in K. odoratissima. Most researches have focused on traditional methods such as adjusting the basic medium and growth regulators, and its artificial light source was the traditional fluorescent lamp. Therefore, the objective of the present study was to investigate in vitro tissue culture in tandem with different LED spectra on maturation and development of somatic embryogenesis in K. odoratissima. Since areal parts of K. odoratissima are rich sources of phthalides, we have also compared the concentration of phthalide across samples grown under different light conditions. This study provides an effective regeneration method for conservation of K. odoratissima and could pave the way for the proliferation of this endangered plant in bioreactor systems.

Plant Material and Seed Germination

Seeds of K.odoratissima were collected from its natural habitat in Iran's “Chaharmahal and Bakhtiari” province. Seeds were first treated with 70% ethanol for 1 min, and then washed with sterile distilled water. Subsequently, seeds were dipped in freshly prepared hypochlorite sodium solution (1% w/v with a few droplets of tween-20) for 15 min. Seeds were then rinsed at least three times with sterile distilled water and cultured in a hormone-free (HF) MS basal medium (Murashige and Skoog) (pH 5.8) containing 3.0% (w/v) sucrose before incubating in a dark condition for 2 months at 4°C.

Induction and Proliferation of Embryogenic Callus

For the induction of embryogenic callus, we used three different explants including; cotyledonary leaves, hypocotyls (both derived from in vitro germinated seeds), and zygotic embryos. The explants were cultured in MS medium supplemented with 1 mg/L 2,4-dichlorophenoxyaceticacid (2,4-D) and 0.25 mg/L Kinetin as described by Ebrahimi et al.( 2018). The induction medium was supplemented with 30 g/L of sucrose and 7 g/L of agar and dispensed into petri dishes. All treatments had three replicates, each consisted of 10 explants. The cotyledonary leaves were cut in half and cultured with the adaxial side in the medium (Ebrahimi et al. 2018). The cultures were incubated at 25 ± 2°C in a dark culture room. After a month, the SE induction rate (% of explants containing SE/total explants) and the mean number of somatic embryos per explant were recorded. The embryo-forming capacity (EFC) index was also calculated using the following equations according to Raomai et al. (2014):

EFC = SE induction rate × mean number of SEs per explant/100

Besides, embryogenic calli were sub-cultured in three cycles on MS basal medium containing 1mg/L of 2,4-D for embryogenic callus proliferation. To avoid genetic variation, we used single-embryogenic callus for the proliferation and the provision of samples for the light treatment experiments. The cultures were kept in the same condition as mentioned above.

Light treatments

In order to investigate the effects of different light spectra on maturation of somatic embryos derived from K. odoratissima, five light treatments were applied including a fluorescent lamp, red LED (660-675nm), blue LED (460-475nm), 3red:1blue LED and 3blue:1red LED (Fig. 1). Somatic embryos were incubated for a month on MS medium (pH 5.8) enriched with 30g/L sucrose and 6.2 g/L agar under diurnal temperature of 23 ± 1°C, the relative humidity of 60%, a 16L:8D photoperiod, and a photosynthetic photon flux density of 50 µmol m^− 2s^− 1. The experiment was conducted in a completely randomized design (CRD) with three replications containing 200mg somatic embryogenesis mass (approximately 80–110 embryos). Within each LED environment, petri dishes were rotationally switched their locations on a daily basis to assure of the homogeneity in the light intensity for all samples. After a month, the number of somatic embryos at each developmental stage (globular, heart, torpedo, and cotyledonary) was recorded. In addition, the number of regenerated embryos at each LED treatment was recorded. The samples were transferred on fresh MS medium and regenerated embryos were grown under the same LED conditions for another additional two months. Following this period, different characteristics including fresh weight (FW), dry weight (DW), leaf area index (mm²) and chlorophyll content (mg/g FW) of samples were measured. Leaf area index was calculated using ImageJ software. The total chlorophyll content (Chl) and the content of carotenoids (Car) were quantified spectrophotometrically using the Lichtenthaler equations and represented as mg/g FW (Lichtenthaler 1978).

Secondary metabolites

To understand the effect of LEDs on metabolite profile of K. odoratissima, 2.5g fresh leaves from in vitro cultured plantlets as well as in situ plants were harvested for the GC-MS analysis. These plants had the same age (two months). Likewise, a sample of air-dry of K. odoratissima was also used to measure the concentration and composition of bioactive compounds. It is noteworthy that dry powder of K. odoratissima is available in the market year around. The headspace (HS) sampling technique was implemented to ensure preserving the volatile constituents and avoiding any loss. The procedures for GC-MS analysis of K. odoratissima, were previously described by Seifipour Naghneh et al. (2022). The relative percentage of metabolic constituents was evaluated from the total peak area (TIC) by apparatus software. Metabolic compounds were identified by comparing their mass spectra with those stored in Wiley NBS75K.L and NIST/EPA/NIH (2002 version, National Institute of Standards and Technology, Gaithersburg, MD, USA) mass spectral libraries using various search engines (PBM, Nist02). Given the area under the peak is a function of the compound's concentration in the sample, the GC-MS data were presented as the area under the peak (Trivedi et al. 2017; Khazaeie et al., 2023).

Statistical analysis

Data analysis was performed using the SAS software (v.9.3), and the averages with the standard errors were compared by one-way ANOVA. The Duncan’s multiple range test was used to separate the treatment means (P < 0.05).

Induction and Proliferation of Embryogenic Callus

Somatic embryogenesis (SE) is a pivotal process in plant biotechnology that enables the regeneration of plants from vegetative or non-reproductive tissues. The efficiency of SE greatly varies depending on the plant species, type of explant, the culture conditions, and the growth regulators involved.

In our study, the callus induction was started 12–14 days following the culture of explants and the percentage of SE after a month was evaluated. Our finding revealed a significant difference (P ≤ 0.05) in the induction of somatic embryos on explants derived from germinated seeds and zygotic embryos under laboratory conditions (Fig. 2a). Although callus formation occurred more rapidly in hypocotyl explants compared to others, none of resulting calli exhibited an embryogenic potential. Therefore, this explant was excluded from further analysis. Similarly, Ebrahimi et al. (2018) reported that K. odoratissima explants were unable to induce somatic embryos irrespective of callus formation on the hypocotyls.

Based on our results, the percentage of explants containing embryos exceeded 60 and 40% in zygotic embryos and cotyledonary leaves, respectively. Moreover, the average number of embryos per cotyledonary leaf reached 16, whereas this number was eight for zygotic embryos (Fig. 2b). Although the EFC index was higher in cotyledonary leaves compared to zygotic embryos but the change was insignificant (Fig. 2c).

Following the formation of embryogenic calli, somatic embryos were formed on lateral edges and adaxial surface of cotyledonary leaves in the fourth week. In contrast, the formation of embryo on zygotic embryos was induced after three weeks albeit without callus formation (Fig. 3).

In the study reported by Hu et al. (2016), zygotic embryos produced callus in indirect SE, but they had a lower frequency of callus formation compared to other explants such as leaves or immature embryos. Alternatively, the callus formed from zygotic embryos may have a lower capacity to develop into somatic embryos compared to callus from other explants. While there is no strong evidence to support this hypothesis, this may be due to the lack of dedifferentiation ability of zygotic embryos (von Arnold et al. 2002). In particular, mature and well-developed embryos are less inclined to deviate towards callus formation. The ability of an explant to undergo direct or indirect embryogenesis was historically thought to be determined by the age of the explant. The further the explant is from the zygotic embryo stage, the more reprogramming (callus formation) needed to convert the explant into a somatic embryo (Merckle et al. 1995). On the other hand, young tissues of cotyledonary leaves contain higher levels of totipotency, which efficiently respond to laboratory culture conditions. Therefore, such tissues become less differentiated compared to zygotic embryos, making them more favorable for SE (Bhojwani and Razdan 1996; Thorpe 2007).

Investigation into in vitro propagation of Quassia amara L. (Simaroubaceae) using the vegetative embryogenesis revealed that leaf and internode explants from older plants were ineffective due to severe phenolic exudation (Martin and Madassery 2005). On the other hand, cotyledon explants were superior for the induction of vegetative embryos. Embryos were developed from both axial side and cutting point of the embryonic axis, with more embryos forming at the proximal end compared to the middle and distal regions of the cotyledons.

The effect of LED on developmental stages and conversion of somatic embryos

In tissue culturing, environmental factors play a central role in plant morphogenesis (Gupta and Jatothu 2013). Developmental stages of somatic embryos have been shown to be influenced by light spectrum (Almeida et al. 2019). In the present research, the influence of light wavelength on development of somatic embryos of K. odoratissima formed per callus (200 mg of initial FW) was evaluated after a month (Fig. 4). Our morphological studies indicated that light treatment significantly affected the development and regeneration of somatic embryos in the K. odoratissima (Table 1). In this way, the number of cotyledonary (mature) embryos per callus under 3red:1blue treatment was significantly (P ≤ 0.05) greater than that under other light treatments (Table 2). Additionally, the somatic embryos subjected to 3red:1blue treatment, were able to undergo conversion phase (Table 2).

Table 1

Analysis of variance for effect of different light treatments on embryogenic cell maturation and regeneration of *K. odoratissima*
Source of variation	df	Mean of square
		Globular	Heart	Torpedo	Cotyledonary	Regenerated
Light treatment	4	126.67^ns	17.17^**	13.43^**	417.57^**	12.4^*
Error	10	313.3	0.47	0.67	17.07	2.53
ns, , * means non-significant, significant at p < 0.05 and significant in p < 0.01, respectively.

Table 2

Mean comparisons for effect of different light treatments on development and regeneration of somatic embryos in *K. odoratissima*
Light treatment	Mean number of somatic embryos
Light treatment	Globular	Heart	Torpedo	Cotyledonary	Regenerated
Flourescent	60^a ± 10.01	3.67^b ± 0.33	3.67 ^cd ±0.67	22.3^b ± 2.96	9.33^ab ± 0.67
Red LED	70 ^a ±10.4	3.33^b ± 0.34	5.33 ^b ±0.33	20.4^b ± 0.66	6.67^bc ± 1.67
Blue LED	60 ^a ±10.05	1.67^c ± 0.67	4.67 ^b ±0.66	12.7^c ± 2.67	5.67^bc ± 0.66
3Red:1Blue LED	66.7 ^a ±12.02	7.64^a ± 0.31	8^a ± 0.001	41.7^a ± 3.35	10.7^a ± 1.61
3Blue:1Red LED	53.3 ^a ±8.83	2^c ± 0.001	2.33^d ± 0.33	13 ^c±1.01	7.33^c ± 0.33
Data are expressed as mean ± SE; Different letters indicate significant differences using the Duncan’s Multiple Range Test (p < 0.05).

The minimum number of cotyledonary embryos was observed under blue LED and 3blue:1red LED (Table 2). In Fritillaria cirrhosa, the maximum number of SEs with cotyledonary leaves was observed in white light followed by a combination of 8red:1blue LED (Chen et al. 2020). On the other hand, in sugarcane, the highest number of somatic embryos reaching to maturation phase was reported under LED lights with the medium blue wavelength (Heringer et al. 2017). Therefore, depending on plant species, different types of growth chambers equipped with LED lights can be considered to achieve the best performance.

LED lamps have become a common approach for optimizing plant growth conditions, and this approach can successfully be used for SE of several species such as Dimocarpus longan (Li et al. 2018), Saccharum spp. (Heringer et al. 2017) and Vitis vinifera (Tittmann et al. 2015). A review of literature showed that light is selectively perceived by plant photoreceptors including cryptochromes and phototropins. Although cryptochromes react with blue and UV-A lights, phytochromes are reactive to red and far-red light (Huang et al. 2014). The plant photoreceptors perceive different wavelengths and trigger complex signal transduction to modulate gene expression profile and subsequently induce developmental responses such as induction, maturation and conversion in somatic embryos (Pedmale et al. 2016; Li et al. 2018; Gupta and Jatothu 2013). The present findings showed that 3red:1blue LED played an important role in maturation of SE in K. odoratissima so that application of this light combination in SE could coordinate photoreceptors with proteins, ions, hormones and other factors to regulate gene expression patterns with accompanying changes in SE phases.

Interestingly, we noticed that light treatment could affect synchronization of somatic embryos. In this regard, the embryos grown under red LED and 3red:1blue LED had more uniformity compared to other light conditions (Fig. 4). Synchronization of somatic embryo cultures could greatly increase the efficiency of SE for in vitro propagation, which makes the technique ideal to produce artificial seeds (Nadel et al. 1990; Alwael et al. 2017). Thus, if all resultant cultures could be synchronized, this would have shortened the duration of culture with concomitant increase in propagation efficiency and uniformity (Nadel et al. 1990). A review of literature indicated that researchers prefer to use hormonal treatments to increase synchronization in SE cultures. To the best of our knowledge, this report is the first to note that light treatment could also influence the uniformity of SE in plant species. Therefore, we suggest to consider light as an underlying factor in synchronization of SE in plant tissue culture technique.

To better understand the effect of light treatment on conversion of K. odoratissima cultures, fresh and dry weight, leaf area index with chlorophyll and carotenoid contents were evaluated (Fig. 5). Our findings showed a significant variation in the fresh and dry weights for light treatments following additional two months of the experiment (Table 3).

Table 3

Analysis of variance for effect of different light treatments on conversion of somatic embryos derived from *K. odoratissima*
Source of variation	df	Mean of square
		FW (mg)	DW (mg)	LAI (mm²)	Chl (mg/g FW)	Car(mg/g FW)
Light treatment	4	9473.33^**	93.45^**	83.85^*	0.017^*	0.000037^**
Error	10	1044.33	8.83	20.51	0.004	0.000004
The symbols , * means significant at p < 0.05 and significant in p < 0.01, respectively.

The highest fresh weight was recorded in the cultures grown under combination of 3blue:1red and blue LEDs followed by Fluorescent lamp. In contrast, the lowest fresh weight was obtained under the red LED (Table 4). The dry weights were significantly enhanced in the cultures treated under 3blue:1red LED and blue LED (p ≤ 0.05) whereas no significant difference was observed in the dry weight of the samples incubated under the red LED and 3red:1blue LED treatments (Table 4). Likewise, the same trend was recorded for leaf area index in the samples grown under the combination of 3blue:1red and blue LED lights (Table 4).

Table 4

Mean comparisons for effect of different light treatments on growth performance of converted cultures in *K. odoratissima*
Light treatment	Mean number of somatic embryos
Light treatment	FW (mg)	DW (mg)	LAI (mm²)	Chl (mg/g FW)	Car (mg/g FW)
Flourescent	587.01^a ± 19.43	52.51^b ± 1.61	22.16^ab ± 1.75	0.84 ^ab±0.051	0.02^c ± 0.0011
Red LED	503.33^b ± 11.47	45.77^c ± 1.33	16.67^b ± 0.88	0.73^b ± 0.023	0.015^c ± 0.0006
Blue LED	587.04^a ± 33.12	54.75^a ± 2.89	25.88^a ± 4.27	0.85^a ± 0.029	0.021^ab ± 0.0011
3Red:1Blue LED	513.67 ^b±7.84	46.63^c ± 0.88	17.02^b ± 0.58	0.75^b ± 0.023	0.018^bc ± 0.0003
3Blue:1Red LED	627.01^a ± 8.54	59.67^a ± 2.78	28.56^a ± 3.42	0.92^a ± 0.052	0.023^a ± 0.002
Data are expressed as mean ± SE; Different letters indicate significant differences using the Duncan’s Multiple Range Test (p < 0.05).

The chlorophyll content is a measure of well-being of a plant under different conditions. Our finding showed that light quality significantly affected total chlorophyll and carotenoid contents in the leaves of K. odoratissima plantlets (Table 3). The chlorophyll content was correlated with the culture growth in the present study. Consequently, cultures exposed to the 3blue:1red LED and blue LED exhibited the highest content of chlorophyll and carotenoids and the cultures grown under red LED showed the lowest content of the pigments (Table 4). Our results are consistent with those reporting plants grown under blue light had a higher chlorophyll content in comparison to plants grown under red light (Fan et al. 2013; Hung et al. 2016). Blue light seems to play a crucial role in the activity of photosystem I and II as well as the photosynthetic electron transport capacity (Miao et al. 2016). In plant species such as Gerbera jamesonii and Myrtus communis, the accumulation of photosynthetic pigments was reduced when plants were subjected to red LED (Pawłowska et al. 2018; Cio´c et al. 2018). In line with our findings, the accumulation of carotenoids in Sinningia speciosa (Simlat et al. 2016) and Stevia rebaudiana (Zheng and van Labeke 2017) cultures was stimulated by blue light spectrum. Findings from the present study, corroborate earlier literature that reported light quantity, quality and duration were involved in controlling morphogenesis, growth and differentiation of plant cells, tissues and organ cultures (Moshe and Dalia 2007). Different sources of LED provide wavelengths that are matched with plant photoreceptors, which leads to optimal production and metabolome (Bourget 2008; Massa et al. 2008; Morrow 2008). Plants apparently sense changes in light quality through photoreceptors, which in turn regulate growth and development through modulating signaling pathways (Rechenmacher et al. 2010).

Light treatments and secondary metabolites

In plants, environmental factors have a significant role in the biosynthesis of secondary metabolites (Khajali and Rafiei 2024). Several studies have identified the influence of light related factors such as light intensity, light quality, and light period on metabolite accumulation of different medicinal plants (Khazaeie et al. 2023 and the references therein). Despite a great deal of information on the application of LEDs on plant secondary metabolites, limited information is available on optimizing LEDs to improve secondary metabolites in endangered plant species. We demonstrated the effect of different light treatments on the metabolic compounds in in vitro grown aerial parts of the K. oddoratisma. Chemical composition of K. oddoratisma among different conditions including in situ, dried powder sample from in situ plant, florescent, red, blue, 3red:1blue and 3blue:1red indicated 14, 6, 9, 5, 8, 8 and 9 bioactive constituents, respectively (Table 5, 6). A more diverse variation of bioactive compounds was recorded for two-old-month fresh plant grows in situ (Table 6). The composition of plant secondary metabolites is affected by different factors including environmental condition, interaction between genotype and environment, method of distillation, storage condition, physiological stage, time of harvest, and season (Khajali and Rafiei 2024).

Table 5

The composition and concentration (AUP/FW) of bioactive compounds in *K. odoratissima* cultures grown under different light treatment and two-month-old dried sample
RT	Compound’s name	KI _Cal	Flourescent	Red	Blue	3Red:Blue	3Blue:Red	Dried sample
14.209	3,9-Epoxy-p-mentha-1,8(10)-diene	1199						247181 ± 8421
19.895	Neophytadiene	1817						280265 ± 10023
18.8	(Z-Butylidenephthalide)	1675	3880705 ± 32421	1238428 ± 17400	3617628 ± 40221	2839095 ± 18664	4374798 ± 18728	1192851 ± 13235
19.447	((E)-Ligustilide)	1830	6294434 ± 29671	1694471 ± 23551	6819459 ± 33265	4956396 ± 50210	6364303 ± 37425	1020396 ± 29840
16.955	isoledene	1373		-		-	442749 ± 2655
14.978	(α-Copaene)	1377	320392 ± 9885	-	986654 ± 7677	481636 ± 8965	325730 ± 10245	336192 ± 5922
16.94	(β-Isocomene)	1412		-	-	-		170230 ± 8545
15.679	(β-Copaene)	1430	2621460 ± 4744	357727 ± 11250	7446177 ± 12565	4300660 ± 6700	3090324 ± 12024
15.553	.beta.-ylangene (β-Ylangene)	1423	1225541 ± 5860	289332 ± 8600	4901094 ± 12433	2773455 ± 14254	2599152 ± 7855
15.158	(Germacrene D)	1484	374215 ± 13442		894846 ± 14000	431077 ± 1977	444565 ± 2166
16.58	6.alpha.-Cadina-4,9-diene, (-)-		-	-	2122920 ± 12922	1239421 ± 8500	1572100 ± 23412
16.585	.gamma.-Muurolene (γ-Muurolene)	1485	1197285 ± 11971	-	-	-	-
15.927	trans-.beta.-Farnesene ((E)-β-Farnesene)	1473	6052262 ± 26329		20644815 ± 21459	12431334 ± 54200	9108300 ± 32950
15.942	cis-.beta.-Farnesene ((Z)-β-Farnesene)	1476	-	1341066 ± 8710	-	-	-
16.955	(-)-.alpha.-Panasinsen (α-Panasinsanene)	1527	945045 ± 10200			-	-
	Others		3468376 ± 75980	3238994 ± 47684	3238994 ± 67234	2456886 ± 33865	693234 ± 20124	2594982 ± 21000
Data are expressed as mean ± SE

Table 6

The composition and concentration (AUP/FW) of bioactive compounds in two-month-old *in situ K. odoratissima*
RT	Compound’s name	KI _Cal	Two-month-old
13.371	D-Limonene	1046	65608 ± 446
23.239	β-Cubebene	1390	74028 ± 4235
24.437	Elixene	1453	1988650 ± 24004
24.812	(E)-β-Farnesene	1473	1311422 ± 14221
25.377	Germacrene D	1484	440701 ± 6412
25.274	γ-Cadinene	1497	49992 ± 2810
25.820	δ-Cadinene	1526	28151 ± 108
28.322	Z-Butylidene phthalide	1675	1234885 ± 28760
29.398	(E)-3-Butylidene phthalide	1744	194856 ± 4650
29.715	Z-Ligustilide	1765	24982212 ± 78700
30.625	E-Ligustilide	1830	702727 ± 9695
30.907	Caffeine	1582	8972991 ± 38439
6.876	(E)-2-Hexenal	866	1669101 ± 19010
15.951	Nonanal	1105	786261 ± 8218
Data are expressed as mean ± SE

The natural habitat of K. oddoratisma is recognized by an altitude of > 2500m and the temperature of barely above 20°C during the vegetative stage (Seifipour Naghneh et al. 2022), which are totally different from a controlled condition. It is presumed that such unique growth environment could differently affect emission and composition of secondary metabolite in K. oddoratisma grown in situ compare to those obtained from tissue culture technique.

In the present study, the composition and the relative concentration of bioactive compounds were significantly changed across different light conditions (Table 5). Phthalides constituted a large amount of secondary metabolites in K. odoratissima. These compounds are known to have a wide range of health benefits including antibacterial, antifungal, insecticidal, cytotoxic, anti-inflammatory properties (Seifipour Naghneh et al. 2022 and the references therein). In Drosera rotundifolia, the amount of secondary metabolites was significantly higher, in the field grown samples compared to in vitro plantlets (Tienaho et al. 2021). Wilken et al. (2005) used different tissue culture techniques in three different plant species including Hypericum perforatum, Cymbopogon citratus and Fabiana imbricate. They reported that the concentration of secondary metabolites produced in tissue culture technique was significantly lower when compared to the field grown plants.

Interestingly, phthalide content was significantly (p ≤ 0.05) higher in tissue culture samples compared to dried sample derived from in situ plant (Table 5).

In our study, under in vitro condition, 3blue:1red LED followed by blue LED and fluorescent light were the best treatments for production of phthalides. However, the concentration of phthalides was minimal in cultures grown under red LED condition (Table 5).

Secondary metabolites are compounds that play an important role in the interaction of plants with their environment (Oksman-Caldentey and Inzé 2005; Khajali and Rafiei 2024). Therefore, it is perceived that their biosynthesis in plant tissues is modified by changing environmental conditions including light (Tomaszewiczet al., 2022). In C. delgadii plants, blue light was the most efficient for the accumulation of flavonoid glycosides, such as astragalin and rutin. A stimulatory effect of this light quality on the accumulation of secondary metabolites was also noticed for other species, e.g., Rhodiola imbricate (Kapoor et al. 2018) and Capsicum annuum (Yap et al. 2021).

It has been reported that blue light stimulates the biosynthesis of secondary metabolites in plants and in the meantime, enhances the synthesis of compounds such as antioxidants. Such increase is attributed to the specific wavelength of blue light that affects plant metabolic pathways (Larsen et al. 2022).

Under in vitro condition, Manivannan et al. (2015) demonstrated that blue LED treatment significantly increased the total phenol and flavonoid contents in the aerial parts of Rehmannia glutinosa. Moreover, Lian et al. (2019) reported that blue LED was associated with increased antioxidant activity and enhanced contents of total flavonoid and phenolic compounds in Gynura procumbens, with a notable increase in cyanidin-monoglucosides under blue light.

A recent study indicated that high-intensity blue light supplementation for 4 hours during the night could induce the expression of MYBs, CRY2/3, SPAs, and HY5, thereby enhancing anthocyanin and catechin accumulation in tea leaves (Zheng et al. 2019). Furthermore, Wang et al. (2020) reported that blue light extensively regulates multiple physiological processes and secondary metabolism in young tea shoots, significantly affecting gene transcription primarily in the pathways of photosynthesis, lipid metabolism, and flavonoid synthesis under high-intensity blue light. Recent studies shows blue light has a significant impact on the biosynthesis of terpenes and cannabionoids in plants by enhancing the activity of key biosynthetic pathways and enzymes (Morello 2022; Desaulniers Brousseau 2021).

Although application of LEDs in plant regeneration is promising, but any given plant species needs ascertaining wavelengths in order to maximize the yield and quality (Khazaeie et al. 2023). Based on analysis of GC–MS data, we found that 3blue:1red LED followed by blue LED and fluorescent light were the best environments for the production of phthalide, the most outstanding secondary compound in K. odoratissima. It seems that these light treatments can favorably affect light-responsive genes and such changes corresponded to biosynthesis of phthalides in K. odoratissima.

In general, it was noticed that the cotyledonary leaves were the most competent explant for somatic embryogenesis in K. odoratissima under the conditions of this study. The developmental stage of K. odoratissima under in vitro conditions requires a specific lighting treatment. In this regard, a combination of 3red:1blue LED provided the highest number of cotyledonary (mature) embryos per callus. This light treatment was the best treatments for conversion of somatic embryos in K. odoratissima. Consequently, the highest fresh and dried weights were recorded in the cultures grown under 3blue:1red and blue LED treatment. Interestingly, phthalide content was significantly (p ≤ 0.05) greater in tissue culture samples compared to dried powder of two-month-old in situ plants, which suggests tissue culture as an alternative technique for the production of bioactive compound in K. odoratissima. Cultures were incubated in 3blue:1red LED followed by blue LED and florescent light possessed higher contents of phthalides. Based on this research, it was shown that LEDs can be an effective alternative to florescent lamps in micropropagation of K. odoratissima.

Acknowledgment

Authors acknowledge Shahrekord University and Agricultural Biotechnology Research Institute of Iran-Central Branch (ABRII-CB) for spiritual and financial supporting. This research was part of a project in ABRII-CB entitled “Optimization of shoot culture and production of Kelus (Kelussia odoratissima Mozaff.) in bioreactor- ID: 4-05-05-010-010551”.

Funding

This research was part of a project in ABRII-CB entitled “Optimization of shoot culture and production of Kelus (Kelussia odoratissima Mozaff.) in bioreactor- ID: 4-05-05-010-010551”. Morteza Ebrahimi has received research support from “Agricultural Biotechnology Research Institute of Iran-Central Branch (ABRII-CB) “

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Morteza Ebrahimi, Fariba Rafiei, Farnoosh Khosravi and Mohammad Rabiei. The first draft of the manuscript was written by Fariba Rafiei and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Maturation, conversion and metabolic profile of somatic embryos derived from Klussia odoratissimaMozaff as affected by light spectra

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Introduction

Material and Methods

Plant Material and Seed Germination

Induction and Proliferation of Embryogenic Callus

Light treatments

Secondary metabolites

Statistical analysis

Results and Discussion

Induction and Proliferation of Embryogenic Callus

The effect of LED on developmental stages and conversion of somatic embryos

Light treatments and secondary metabolites

Conclusion

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References

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Maturation, conversion and metabolic profile of somatic embryos derived from Klussia odoratissimaMozaff as affected by light spectra ​

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Material and Methods

Plant Material and Seed Germination

Induction and Proliferation of Embryogenic Callus

Light treatments

Secondary metabolites

Statistical analysis

Results and Discussion

Induction and Proliferation of Embryogenic Callus

The effect of LED on developmental stages and conversion of somatic embryos

Light treatments and secondary metabolites

Conclusion

Declarations

References

Supplementary Files

Status:

Journal Publication

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Maturation, conversion and metabolic profile of somatic embryos derived from Klussia odoratissimaMozaff as affected by light spectra