Effect of macronutrients and micronutrients on somaclonal variation and biochemical properties in  Paulownia shantung

doi:10.21203/rs.3.rs-1217342/v2

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Effect of macronutrients and micronutrients on somaclonal variation and biochemical properties in Paulownia shantung

https://doi.org/10.21203/rs.3.rs-1217342/v2

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Paulownia (Paulownia sp) is a fast-growing, industrial and ornamental tree, which has medicinal usages. The effect of nutrients in in vitro regenerated plantlets and somaclonal variation was investigated. Biochemical traits (soluble leaf protein, anthocyanin, flavonoid, proline, carotenoid contents and photosynthetic pigment amounts) in induced embryos and regenerated plantlets were measured. The media of A/2 MS, ½ MS, ½&_A/4 MS supplemented with a combination of TDZ, 2,4-D, Kin, BAP and GA₃ hormones, totally eight media, were used. The shortest duration for direct and indirect somatic embryogenesis was observed in media number eight (½&_A/4 MS + 0.3 mg/l 2,4-D + 2 mg/l Kin + 4 mg/l BAP + 0.3 mg/l GA₃). Moreover, this medium showed the highest frequency in calli induction, somatic embryos and regenerations. We showed the privilege of full-length medium in an in vitro propagation of paulownia with no-somaclonal and genomic alterations. For the media with reduced nutrient strength to one-fourth and one-second, somaclonal variation and genomic changes were recorded. Media with decreased macronutrients to half and a quarter amount showed a higher ratio of somatic embryos induction, regeneration, and antioxidant activity. Meanwhile, proline and protein contents were decreased. An increase in Chlorophyll content was observed in media with reduced macronutrients amount. Overall, elements may be linked to the frequency of somaclonal variation in an in vitro culture.

Anthocyanins

Flavonoids

Phyllotexy

Regeneration

Somatic embryo

By reducing macronutrients, especially nitrate and ammonium in in vitro Paulownia plant, somatic embryo induction, regeneration and antioxidant activities increase while free radicals decrease.

As a major species and commercial plant in the world, Paulownia belongs to the Paulowniaceae family (Scrophulariaceae) and is endemic to China. Paulownia wood is used in the production of chipboard, matchstick production, paper and cartoon production, layering, furniture production, music instruments production, medicine boxes, and manufacturing decorative appliances, to name a few. Paulownia tree has been used to develop landscape, decorative accessories, and co-cultivate with a variety of crops in many countries (Yadav et al. 2013). Paulownia trees with 8 to10 years old produce about 100 kg of green compound (leaves) and about 2.8-3% nitrogen and 4% potassium (Woods 2008). A larger demand for wood and the shrinking of wood resources has caused a rise in forestry with fast-growing species. The paulownia tree has the potential for forestry and growth in poor soils (Swamy et al. 2006). Paulownia will be the main source of wood in the world, so European farmers are gradually expanding the cultivation of fast-growing plants by following mixed-farming policies and developing cultivated areas of fast-growing trees (Zuazo et al. 2013). Paulownia species are rich in secondary metabolites including phenolic compounds and flavonoids with high antioxidant activity and other pharmacological activities which have been distributed in the various tissues of the tree (Smejkal et al. 2007; Si et al. 2013). Paulownia blossom tea and syrup can cure liver and spleen maladies and alleviate bronchitis. Furthermore, in traditional Chinese medicine, Paulownia’s bark is used for infectious skin diseases (Smejkal et al. 2007). This species has a proven value for the production of green fertilizer. Also, the honey produced from the flowers of this plant has medicinal applications. Paulownia leaves are soil boosters due to their high percentage of the dry weight of nitrogen and are fed to animals because of high nutritional value (Yadav et al. 2013). The average fat content of the leaves collected from bioenergy and timber plot is 2.87 percent and 2.80 percent, respectively. On average, the fat content recorded in paulownia leaves was reported to be 2.83 percent. In addition, the leaf, fruit, and wood extracts of paulownia are effective for bronchitis in particular by soothing coughs and reducing sputum and blood pressure. Also, its leaves contain Ursolic acid and Matteucinol (He et al. 2016). The Kyoto Convention has recommended planting the paulownia tree in polluted and industrial areas due to high oxygen production (each tree produces 6 kg of oxygen annually) and high absorption of carbon dioxide (each hectare absorbs 1,250 tons of carbon dioxide annually) (Icka et al. 2016; Huseinovic et al. 2017). As a main tool of asexual proliferation, plant tissue culture may not have the challenges of proliferation by cutting. Antioxidants with low molecular weights, such as anthocyanins, polyphenols, flavonoids, and carotenoids could eliminate the harmful effect of free radicals that trigger lipid stability in turn (Basu et al. 2010). Somatic embryogenesis is a powerful tool for the regeneration of plants in the in vitro medium with agricultural techniques, by forming somatic embryos, lowering the percentage of dispersion in the regenerated plants, and minimizing somaclonal diversity (Heringer et al. 2018). Population growth, income, wood consumption products, and the use of wood for energy and cellulose biofuels are determining factors in this regard. The basis of the propagation of many species of trees is the seed. However, traditional proliferation by seeds has low efficiency per area unit. Also, the seed germination and the cutting method have many hardships that drive modern farmers to opt for cell tissue culture. Moreover, sensitivity to pests and disease, poor germination, and slow seed growth are the main problems regarding proliferation by seed. For these reasons, the emphasis is on developing reliable methods for the production of somatic embryos (Jain and Gupta 2018). This study aimed to determine the best medium to induce callus formation, embryogenesis, and to measure and analyze the content of chlorophyll, protein, proline, and antioxidant of plantlet that regenerated from the embryogenic callus in various media. The results of the present study are the first report of somatic embryogenesis of Paulownia shantung. Besides, the effect of different types of MS medium especially different concentrations of ammonium and growth regulators on somatic embryogenesis and biochemical characteristics were evaluated.

For callus induction, embryogenesis and regeneration of Paulownia shantung species, the present study was carried out in the tissue culture laboratory of the college of Aburaihan, University of Tehran, Iran. Petiole explants were obtained from the petiole attachment to the leaf and the petiole attachment to the seedling hypocotyl (axil). The leaf and petiole explants were obtained from young and healthy six-month-old seedlings. Explants were selected from the top leaf of the mother plant grown in the greenhouse. In order to disinfect the explants, they were soaked in a solution of two-grams-per-liter captain fungicide containing four drops of Tween 60%. Then explants were washed in 70% alcohol for one minute and then washed with distilled water. The explants were then placed in a container of a solution of sodium hypochlorite 2% for 10 minutes and they were transferred to a laminar flow hood, afterwards. Then, the explants were placed on sterile filter paper. The Murashige-Skoog (MS) medium was used as a basal medium. Hormone compounds were prepared according to table 1 and the pH was set to 5.8. The media was sterilized in an autoclave at 121 °C and an atmospheric pressure of 1.5 bar. Vitamins and hormones were added to the media before autoclaving. A factorial experiment was conducted in a completely randomized design with two factors, explants (leaf and petiole) and eight media containing different hormone compounds in four replications. Cultivated explants were kept in a growth chamber with a temperature of 24-25 °C and a light intensity of 2720 lux. The explants and plantlets were subcultured every 4 weeks. After the formation of embryos on the culture media, young embryos and regenerated seedlings were selected for extraction of chlorophyll, carotenoids, soluble protein, anthocyanins, proline, and flavonoids. This experiment was performed in a completely randomized design with three replications.

Different stages of measuring embryogenic cell/callus induction formation and regeneration

In the first week, callus induction was observed in the media. This was more evident for culture media numbers four and eight. Nine days after placing explants (dap) in the culture medium, traits such as callus formation and callus percentage were measured. Notably, a stereomicroscope, 15 dap was used to detect stages of embryonic cells, especially the globular stage. Regeneration and subculture took place in the same culture media. During the heart-shaped stage, when embryonic cells were transferred to the hormone-free culture medium, there were no more stages and regeneration. This caused chlorosis and necrosis of the induced embryos. In addition, heart-shaped and torpedo-shaped stages, 20 dap were observed. On day 26, cotyledon and seedling stages were seen. Callus induction is difficult in paulownia. Moreover, seedling regeneration from calluses is a time-consuming process (six months) and due to the high production of phenol, most of the regenerated seedlings are destroyed. In addition, the application of PRs during organogenesis may cause somatic variations. To overcome the shortcomings, of proliferation (producing a large number of seedlings per explant) and the high rate of regeneration and growth of seedlings, somatic embryos were considered (Dumani et al., 2022).

Measurement of photosynthetic pigments content and anthocyanin content

The chlorophyll content and soluble carotenoids were determined by the method proposed by Lichtenthaler (1987). For this purpose, 0.2 gr of the plantlet regenerated from the embryogenic callus was excised and ground in 80% acetone solution for extraction (chlorophyll and carotenoids). Also, for the determination of anthocyanin concentration, 0.2 gr of the plantlet that was regenerated from the embryogenic callus was taken and extracted in 0.3% HCI in methanol at 25 °C and stored in the dark for one night. The amount of anthocyanin was calculated by extinction coefficient (ε = 33000 cm² mol^-1) (Wagner, 1979). All spectrophotometric measurements were carried out through OPTIZEN 3220UV spectrophotometer.

The concentration of chlorophylls a and b is obtained from the following formulas (Lichtenthaler, 1987):

= 12.7A₆₆₃ - 2.69 A₆₄₅ Chlorophyll a

= 22.9A₆₄₅ - 4.68A₆₆₃ b Chlorophyll

Total chlorophyll= 20.2 A₆₄₅ - 8.02A₆₆₃ l

Determination of Flavonoids content

Flavonoids of the plantlet regenerated from the embryogenic callus were estimated according to Krizek et al.’s method (1993). Tissues were homogenized in a mortar and pestle with 3 ml one percent acetic acid-ethanol solvent (1:99, v:v). The homogenate was centrifuged at 18000 rates per minute for 30 min, and then the supernatant was incubated in a water bath for 10 min at 80 °C and then allowed to cool to room temperature. The amount of flavonoids was determined from the absorbance at 270, 300 and 330 nm. Flavonoid content was expressed as µmol/g FW and the concentration of flavonoids was calculated using an extinction coefficient of flavonoids _ε550 = 33000 cm² mol

Measurement of protein content

Soluble protein content in leaf samples was determined through bovine serum albumin (BSA) as a standard by Bradford’s (1976) method. BSA standard curve was used for measuring the soluble protein concentration, was calculated and the values were expressed as mg/g^-1 FW.

Two buffers were used to determine the concentration of proteins by Bradford method as follows.

Buffer No. 1: 0.01 gr of Coomassie Brilliant blue (R-250) was dissolved in 15 cm³ of 96% ethanol. Then, 10 cm³ of phosphoric acid (85%) was added to it. Following that, the resulting solution or distilled water reached a volume of 100 cc. The filter paper separated the Coomassie brilliant blue particles from the prepared solution.

Buffer No. 2: 1.77 gr of Na₂HPO₂ reached a volume of 400 cm³. Also, 1.95 gr of NaH₂PO₄ reached a volume of 400 cm³. The buffer of these two substances was prepared in an Erlenmeyer flask in a volume of 800. Then, the pH was adjusted to 6.8. After the buffers were prepared, 0.5 gr of the plantlet regenerated from the embryogenic callus was pounded with seven mg/L of buffer number two in a mortar. The samples were centrifuged at 12000 rpm for 15 min at 5 °C. After centrifugation, the supernatants were removed and placed in the refrigerator. Moreover, 100 cm³ of each sample was taken and one cc of buffer number one was added to them.Using a spectrophotometer (UPLAB model), absorbance was measured at 595 nm. The supernatants were analyzed in the same way as bovine serum albumin (BSN) samples (Dumani et al., 2022).

Measurement of proline content

The method introduced by Bates et al. (1973) was applied for the measurement of the amount of proline. For this reason, sulfosalicylic acid (3 % w/v) was used for extracting 0.2 gr of the plantlet regenerated from the embryogenic callus, and then the obtained solution was centrifuged at 12000 rates per minute for 15 min. Afterwards, 2 ml of acid ninhydrin solution and 2 ml of glacial acetic were added to the extract. The extract was transferred to a tube and heated in boiled water at 100 ˚C for 1 hour. When the tube cooled at room temperature, 4 cm³ toluene was added to the tube and mixed well. After the formation of two phases, the top phase was collected carefully by a sampler and the absorbance was measured at 520 nm by the spectrophotometer. The standard curve was consulted for calculating the proline content and was expressed as mg g^-1 FW.

Cluster analysis and K-mean of culture media were performed to identify the highest and lowest similarity in the media with respect to all chlorophyll and antioxidant traits (Figure 9). After controlling data adequacy and ensuring the normality of the dates, statistical calculations of the data were performed by R software. The diagrams were plotted by R and Orgin.pro software. The mean of treatments was compared by Dunnett test at 1% Statistical significance level.

Callus induction, somatic embryo and regeneration

Somatic embryos were obtained directly and indirectly from both leaf and petiole explants. Somatic embryogenesis is used for vegetative proliferation of uniform clones of commercial species in a short time. Regeneration of trees is a major challenge that can be addressed through somatic embryogenesis in plant tissue culture (Jain and Gupta 2018). The present study shows that by reducing the concentration of MS medium to half and reduction of the macronutrients to half and a quarter, the percentage of callus induction, the induction of somatic embryo, and the percentage of the regeneration increased (Fig. 1). However, in the media in which MS medium was not changed and macronutrients were not reduced, the percentage of callus induction, somatic embryo induction, and regeneration decreased (Fig. 1). This may be due to a larger or smaller sources of nitrogen (ammonium) in the medium. In media six, seven, and eight, with the reduction of macronutrients and MS medium to half, an increase was observed in callus induction, somatic embryogenesis, and regeneration (Fig. 1). According to the study of Bao et al. (2012) on Rosa hybrida, it was found that the highest regeneration and formation of somatic embryos was observed in culture medium ½ MS with the association of the hormonal compounds of TDZ and BA. In the present study, complete MS medium reduced the percentage of regeneration and induction of somatic embryos. Also, most of the somatic embryos were formed in ½ MS medium in which the amount of macronutrients was reduced to half and one quarter, and almost no infirmity was observed in these media. Globular, heart, and torpedo stages were visible in these media too. Embryonic cells were observed directly and indirectly in these culture media in the shortest time, which was more evident in culture medium number eight. However, in complete MS media, callus induction was difficult to observe and somatic embryo induction was indirect. Also, most embryos in these culture media were destroyed or stopped in the heart-shaped stage. The transfer of calli from the complete MS medium to the ½&A/4 MS medium (three-quarter reduction in macronutrients and half in the MS medium) causes the calli to change to the embryogenic state and also makes somatic embryos grow in a shorter time in comparison to other media. Tissues of an organ show different concentrations of hormones and elements. Paulownia leaves have been shown to contain 3% nitrogen (Wood, 2008). Improved somatic embryo induction and regeneration and smaller nitrogen source (reduction of MS medium concentration) can be explained by the high concentration of endogenous nitrogen of the explant and by the toxicity caused by an excess nitrogen in the full MS medium. It can be stated that the concentration of macronutrients (ammonium) had a significant effect on callus induction percentage, somatic embryogenesis percentage, and regeneration. Chen et al. (2010) reported that culture media ½ MS and ¼ MS did not significantly affect the induction of somatic embryogenesis, but had a significant effect on seedling regeneration. Somatic embryo induction and regeneration of Olive plants were performed in ¼ MS medium (Cerezo et al. 2011). With the emergence of seedlings in culture medium number two, three and four at the junction of seedlings to the explant, a snow-white mass inside the plantlets began to become active. This white mass tore the outer shell of the seedlings and caused their destruction. Some calli or embryos were regenerating in which the white mass inside them began to become active and the regeneration of seedlings was inhibited. This phenomenon occurred mostly on both petiole and leaf explants (Fig. 11). This also happens in some (not all) of the replications of medium number four. This problem was probably due to the lack of formation of phloem tissues which itself arose from cytokinin deficiency. It should be noted that tissues contain different concentrations of cytokinin (Bishopp et al. 2011). In the absence or deficiency of the cytokinin hormone, phloem tissues do not differentiate. In other words, protoxylem tissue cells will not develop into metaxylem cells and will not mature (Fig. 11). In media six, seven, and eight, despite richer cytokinin content and reduction of macronutrients, especially ammonium, this phenomenon was absent. Other studies have shown that the conversion of protoxylem to metaxylem in the root zone and phloem formation is due to the cytokinin hormone. The cytokinin hormone makes phloem tissue mature (Bishopp et al. 2011). Phloem formation and movement of the raw sap in the phloem occurs by cytokinin signaling (Bürkle et al. 2003; Kudo et al. 2010). Cytokinins cause cell division in the stomata and phloem tissues and also affect the differentiation of woody cells (Wybouw and Rybel 2019). Regenerated seedlings in media where macronutrients were reduced to half and a quarter usually had broad leaves and extensive pigmentation. The leaves were also young and fresh, and this was more evident in medium eight (Fig. 12). Regenerated seedlings in media five and three contained almost oval leaves and the leaves had few chlorophylls. A possible explanation for this could be the signaling of the cytokinin hormone that affected the phyllotexy of leaves. The shape of the leaves and stems changed in culture media five and three probably because of the change in cytokinin homeostasis which was due possibly to more ammonium in the media. In general, it can be stated that in the MS medium, mineral salts, such as nitrate, are converted to ammonium, causing the production of ethylene in the medium and increasing the activity of free radicals, which trigger the activity of polyphenol oxidase enzyme. The phenolic content is located in the vacuole of the cells and because the explants were prepared in the shape of round discs with the area of one square centimeter, phenolic content penetrated more into the culture medium at the cutting sites. In addition to destroying pre-embryonic cells and causing high chlorosis and necrosis of explants and calli, this disrupts cytokinin homeostasis. Studies by other researchers have shown that manipulating the level of the cytokinin hormone leads to the tubularity of the leaves. It also causes leaf morphogenesis in the tomato plant (Shani et al. 2010). Genetic analysis has shown that smaller cytokinin levels inhibit the activity of Knotted1 proteins such as Homeobox (KNOXI) that are involved in leaf morphogenetic, and the activity of cytokinin can replace the activity of KNOXI protein in leaf brink (Hake et. al. 2004; Shi et al. 2013). Leaf phyllotexy depends on transcription factors and hormones. It is reported that the clau gene is a myb gene transcription factor that plays an important role in the development of compound leaf types (Bar et al. 2016; Skalák et al. 2019). The clau gene reduces cytokinin signaling and increases the sensitivity of cytokinin-responsive plants. Studies have shown that with the addition of ammonium in the medium, polyphenol oxidase enzyme becomes more active that leads to more free radicals. This phenomenon can disrupt hormonal and nutrient uptake and can have negative and destructive effects on cultured explants as well as regenerated plants (Chakroun et al. 2007). In this study, we found that a half or three-quarter reduction in the nitrogen level induced somatic embryogenesis in the cultured explants.

The results showed that the percentage of somatic embryo induction in medium one was zero. Somatic embryos were induced in both direct and indirect forms in medium eight faster than the other media, and calli and embryos induced in medium eight had more elongated cells. Somatic embryo induction time was also reduced in the media in which mineral elements were reduced by half and three quarters compared to full media MS (Fig. 2). These differences between the culture media can be explained by pH balance maintenance in media where macronutrients were depleted and by pH imbalance in full MS media. One of the key elements affecting the balance of the medium is the concentration of ammonium in the medium. The presence of ammonium in the medium causes an imbalance in the pH of the medium (Chen et al. 1997). In addition to light and temperature, several factors, such as nutrition, can affect the biosynthetic stages of the hormone auxin. For example, if nitrogen is absorbed, it can inhibit the biosynthetic stages. The amino acid tryptophan is a precursor to the hormone auxin. The findings of the present study agree with those of earlier research (Mashiguchi et al. 2011; Wang et al. 2016). It can be said that in media where macronutrients are reduced to half and a quarter due to ammonium non-toxicity and maintaining the pH of the medium, nitrate uptake activates the amino acid tryptophan and cell growth in the shortest time. This is more evident in the case of medium number eight. In the study of (Robert and Friml 2009; Carranza et al. 2016; Romani 2017), it is stated that auxin on the membrane surface binds to the ABP1 receptor. By activating ABP1, protons move from the cytosolic surface to the cell wall, causing the cell wall to become more acidic. As the cell walls become acidic, potassium channels become active, creating an osmotic potential. Water and nutrients move into the cell, and on the other hand, the loosened cell walls eventually grow. Cell elongation is also due to auxin uptake and loosening of the cell wall. It is noteworthy that in the eight types of the studied media, the leaf explants that accompanied the petiole had high activity in comparison to other types of explants. This high activity may have been due to more endogenous hormones auxin and cytokinin. These explants produced direct and healthy embryos. It was reported that auxin is homogeneously distributed throughout the leaves but found more in younger leaves and the highest amount is in the young leaves at the junction of petiole to leaf (Edlund et al. 1995). With this interpretation, the leaf explant along with the petiole probably had high cell growth activity due to more internal auxin.

Flavonoids, including anthocyanins

The present study showed that the samples grown in the culture medium eight, among the studied media, had the highest levels of flavonoids, including anthocyanins even more than their parents (Figs. 3 and 4). Furthermore, regenerated seedlings in these media had richer pigments; this is more evident in medium eight (Figs. 3 and 4). In media where mineral compounds, especially ammonium, have been reduced (media two, four, six, seven, and eight), an elevation can be seen in antioxidants (anthocyanins and flavonoids) such that even in some treatments, their levels were higher than the parent plant (Figs. 3 and 4). The increase in antioxidants in these media can be due to the ammonium non-toxicity in the culture medium. The amount of antioxidants increase with smaller nitrogen source and higher carbon uptake (sucrose). In other words, with an increasing ratio of carbon to nitrogen, there will be more antioxidants. While in complete MS media, the amount of antioxidants was less than that in the parent plant and this difference was significant (Figs. 3 and 4). Wan et al. (2015) showed that the flavonoids, including anthocyanins compounds and the expression of their genes in the leaves and calli of Malus sp cultivars increased when there was larger carbon to nitrogen ratio and this difference was significantly different from the control plant. They also stated that the amount of flavonoids increased in treatments with a higher carbon to nitrogen ratio. The reason of this phenomenon was reported to be the higher expression of chalcone synthase (chs) gene; uridine diphosphate-glucose: flavonoid 3-O-glycosyltransferase (ufgt) gene and flavonol synthase (fls) gene. Among these genes, the anthocyanidin synthase (ans) gene had the largest effect on anthocyanin synthesis. It can be said that in the present study, the synthesis of ufgt, chs, fls and fls genes has probably increased in media where there was higher carbon/nitrogen ratio. In the present study, media with higher levels of antioxidants contained calli, milky yellow and white embryos, and healthy regeneration. In these media, calli and embryos continued to grow without burning or browning. High levels of antioxidants prevent accumulation of free radicals and inhibit cell growth by reducing the activity of polyphenol oxidase. The results of the study by Domznguez et al. (2008) showed that with the increase of ammonium, antioxidants are reduced and on the other hand, the increase of free radicals triggers more ammonium ions. In a study on the cell suspension of Vitis vinifera, it was found that ammonium ions produce free radicals (ROS) and hence oxidize antioxidants (Skopelitis et al. 2006). The use of the two types of polysaccharides in the tissue culture of Phellinus pini showed that these two polysaccharides lower free radicals and produce more antioxidants (Jiang et al. 2017). Flavonoids, including anthocyanins, are secondary metabolites that play an important role in plant physiology. Antioxidants coordinated action with a low molecular weight such as anthocyanins, polyphenols, flavonoids, and carotenoids can effectively neutralize harmful free radical compounds and thereby cause lipid stability (Basu et al. 2010). Flavonoids, including anthocyanins, as major antioxidant compounds, not only eradicate free radicals but also prevent their further production in the plant (Nasibi and Kalantari 2005). Increased flavonoids, including anthocyanins, appear to promote the growth of early embryonic cells and prevent them from being destroyed or inhibited at one embryonic stage by free radicals and the enzyme polyphenol oxidase. In complete MS medium, most calli became brown and disappeared. Also, the embryos stopped in the heart-shaped stage and changed from yellow or milky white to snow-white. Also, most of the calli induced in these media were non-embryonic calli. This can be due to the reduction of antioxidants and the high activity of free radicals, as well as the activity of the enzyme polyphenol oxidase.

The amount of proline

The present study showed that the amount of proline in media six, seven and eight reached its lowest level and had a significant difference with the parent plant (Fig. 4). In these media, due to the lowest amount of nitrate and high concentrations of cytokinin (2 mg/l Kin and 4 mg/l BAP) and gibberellic acid (0.30 mg/l), nitrate stimulates cytokinin and causes its high absorption. Accumulation of cytokinin in cells and plastids reduces plant resistance to stress conditions (Fig. 12). In a study on Medicago truncatula, it was found that at low nitrogen concentrations, the uptake and stimulation of the hormone cytokinin rose, but high concentrations of nitrogen prevented the stimulation of the hormone cytokinin (Ariel et al. 2012). According to a study by Ruffel et al. (2011), high nitrate concentrations inhibit cytokinin stimulation. The researchers stated that lower levels or lack of cytokinin could help plants tolerate stress, but the presence of cytokinin would reduce plant tolerance to stress (Rivero et al. 2007; Ha et al. 2012). As the hormone gibberellic acid is absorbed by the explants in the culture medium, it inhibits the function of Della protein, which reduces ethylene. Eventually, the plant becomes more sensitive to stress. The present study was in harmony with the research conducted by (Magome et al. 2004; Achard et al. 2006; Cao et al. 2007). In media four (half MS medium), three and five (full MS medium), the amount of proline is equal to the parent plant (Fig. 5). This may be due to the larger amount of nitrate that has prevented further absorption of cytokinin. Culture medium two (containing half- strength of macronutrients) also showed the richest proline content, which was higher than that of the parent plant. This result is consistent with the study of Ariel et al. (2012) and Ruffel et al. (2011). It can be said that medium number 2 lacked the hormone cytokinin and the plant's resistance to stress increased, i.e., medium two reduced the sensitivity of seedlings and induced embryos. On the other hand, reducing ammonium toxicity has a positive effect on high auxin uptake. With high auxin uptake, the hormone ethylene increases and ultimately reduces the plant's sensitivity to stress. The relationship between auxin and ethylene is synergistic and with more auxin, ethylene increases too (Liu et al. 2017). The high percentage of somatic embryogenesis and regeneration is likely to be highest in the media where proline levels are minimal. In other words, reducing the percentage of somatic embryo induction and regeneration in the media where the amount of proline has reached its maximum is that proline acts as an inhibitor in the in vitro culture medium. Besides, proline changes in embryo maturity may vary in different developmental stages. Proline not only acts as an osmotic pressure agent but also affects a wide range of plant growth processes (Kishor and Sreenivasulu 2014).

Amount of protein

The present study showed that among the studied media, medium three causes the highest amount of protein accumulation and at the same time does not differ from the amount of protein of the parent plant (Fig. 6). Also, media five, two and three showed richer protein content. Protein content in media eight, seven, sex and four had its lowest level. As vital growth agents, proteins play important physiological roles such as structural and enzymatic roles. The cell protein content is considered a major cell growth factor (Hasanuzzaman et al. 2013). Any factor that inhibits protein synthesis stops the cell growth (Adamczyk et al. 2010). Growth in medium one may be stopped due to the inhibition of proteins by the enzyme polyphenol oxidase and the high toxicity of ammonium in this medium (Fig. 6). It should be noted that the increase in somatic embryogenesis through proteins and amino acids uptake is due to the fact that these compounds provide an available source of nitrogen for cell growth (Pawar et al. 2015). In the media where macronutrients are reduced by half or three-quarters, ammonium toxicity is prevented and high nitrogen is made available to the embryo. For this reason, these media had the highest percentage of somatic embryos. This is especially evident in the case of medium eight. Researchers have observed that carrot cell wall protein is secreted in the growth medium and regulates somatic embryonic growth (Hall and Cannon 2002). The protein content of cells in pre-embryonic calli is richer than that of embryonic calli. As callus enters the somatic embryonic stage, its protein level decreases, indicating protein consumption for somatic embryogenesis (Jariteh et al. 2015). Therefore, it can be concluded that in media two, three, and five, where most of the formed embryos either perished or stopped in the heart-shaped stage, they could not use the protein well. However, in media six, four, seven, and 8 in particular where the amount of protein is minimized (Fig. 6), the reason is the high consumption of protein by induced embryos. Studies have shown that cells use their protein to initiate embryogenesis, so it can be said that cellular protein, as important macromolecules, trigger somatic embryogenesis. The inception and progression of somatic embryogenesis also encourage protein intake (Adamczyk et al. 2010; Jariteh et al. 2015).

Content of chlorophyll and carotenoids

The content of total chlorophyll, chlorophyll a, chlorophyll b, carotenoids, chlorophyll a/b and total carotenoid/chlorophyll ratio increased in the media containing half- or quarter-strength of macronutrients (Figs. 7 and 8). In these media, many pigments were observed on regenerated leaves and seedlings. Among the studied media, medium eight had the highest amount of carotenoids (Fig. 8). The larger number of pigments is due possibly to the absorption of more sucrose than nitrogen, which has caused more activity of antioxidant genes and antioxidants that prevented the degradation of chlorophyll (Figs. 7 and 8). Growth analysis at different N/C ratios in Arabidopsis showed that it plays an important role in regulating seedling growth, translocation of stored lipids, and photosynthetic gene expression (Marti et al. 2002). Media three and five had lower chlorophyll a and b levels because of fewer pigments (Fig. 7). Therefore, antioxidant activity in these media was low and on the other hand, chlorophyll degradation occurred. Chlorophyll concentration in living plants is a main factor in maintaining photosynthetic capacity (Jiang and Huang 2001). A principal reason for the reduction of chlorophyll is degradation by means of reactive oxygen types (Navari-Izoo et al. 1990). Deterioration of chlorophyll in media three and five is due to free radical activity. Another class of antioxidant compounds in plant cells is carotenoids. These compounds reduce oxidative damage to the plant through non-enzymatic pathways.

Cluster and K-mean analysis of culture media showed that parental treatment and medium eight had the highest intra-group similarity and the lowest inter-group similarity amongst the experimental groups. Media three, six and five bore the closest resemblance. However, media four, seven and two were the most similar and were placed in one cluster (Fig. 9).

The effect of nitrate reduction on somatic embryo induction and regeneration to improve cell growth in plants, especially in trees, is a nascent science and requires more extensive research. In the present study, by reducing nitrate in culture media, fundamental physical and chemical changes were made in differentiated cells. As a result, our study introduced a functional guideline for the induction of somatic embryos and regeneration of somatic embryo calli in the shortest time using the reduction of macronutrients (especially nitrate source) in the culture media. Also, in the media where macronutrients were reduced to one-fourth and one-second, an increase in the percentage of somatic embryo induction, somatic embryo regeneration, and high antioxidant activity was observed. Nevertheless, hile the amount of proline and protein in these media decreased. In addition, in complete MS medium, proline and protein levels increased compared to lower-concentration MS media. Also, in complete MS media, the percentage of somatic embryo induction and regeneration decreased compared to lower-concentration MS media.

A Absorbance

BAP 6-Benzylaminopurine

GA₃ Gibberellic acid

MS Murashige and Skoog

A/2 MS Reduction to half of macronutrients in the MS medium

½ MS Reduction to half the entire MS medium content

½&_A/4 MS Reduction to half than twice the entire contents of the medium and reduction to a quarter of the macronutrients in the MS medium

2,4-D 2,4-Dichlorophenoxyacetic acid

Kin Kinetin.

V Volume

Acknowledgements

We thank and appreciate the University of Tehran science & technology park for financial support.

Compliance with Ethical Standards and Data Availability

The authors declare that they have no competing interests. The authors declare that the data supporting the findings of this study are available within the article.

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Table 1 containing various hormonal compounds used in the study of Paulownia shantung tissue culture
Media	2.4-D (mg/l)	TDZ (mg/l)	KIN (mg/l)	BAP (mg/l)	GA₃(mg/l)
media	0.3	2	-	-	-
_A/2 media	0.3	2	-	-	-
media	0.5	-	0.5	-	-
½ media	0.5	-	0.5	-	-
media	0.3	-	2	4	0.30
_A/2 media	0.3	-	2	4	0.30
_1/2 media	0.3	-	2	4	0.30
½&_A/4 media	0.3	-	2	4	0.30

Figure 12 is not available with this version.

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Effect of macronutrients and micronutrients on somaclonal variation and biochemical properties in Paulownia shantung

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Abstract

Figures

Key Message

Introduction

Materials And Methods

Results And Discussion

Callus induction, somatic embryo and regeneration

Flavonoids, including anthocyanins

The amount of proline

Amount of protein

Content of chlorophyll and carotenoids

Conclusion

Abbreviations

Declarations

References

Tables

Figure 12

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