Introduction

It is one of the essential unattended crops and is grown as a minor grain crop in the Indian Himalayas, particularly in the high-altitude areas (Sharma et al., 2018). Due to its frost resistance, short growth period and easy cultivation, buckwheat is common in high-altitude areas at 2000 m and in Tibet it is found at elevations of up to 4500 m (Zhang et al.,2012). It is a dicotyledonous, multipurpose, summer-growing annual plant of family Polygonaceae with knotted stem of 30-60 in height, with shallow root system. Many of the health benefits of buckwheat have been attributed to its high levels of phenolic compounds and antioxidant activity (Wijngaard and Arendt, 2006). Common buckwheat is rich in vitamins, especially those of the B group (Fabjan et al.,2003) and is significant source of microelements (Zn, Cu, Mn, Se) as well as macroelements (K, Na, Ca, Mg) (Stibilj et al.,2004). Major flavonoids in buckwheat are rutin, quercetin, orientin, homoorientin, vitexin, isovitexin, hyperin and catechins (Morishita et al.,2007). It has been reported that the ethyl acetate and ethanol extracts of the stem, seed and aerial parts of buckwheat show neuroprotective effect through acetylcholinesterase, butyrylcholinesterase and tyrosinase inhibitory and antioxidant activity (Gulpinar et al.,2012).

Plant growth regulators (PGRs) are invention of agrochemicals subsequent to pesticides and fertilizers. PGRs improve growth of plants by influencing their usual homeostasis; mainly the hormonal regulation (Ahmada et al., 2019). Number of studies is reported where PGRs enhanced shoot length (Sumathi et al., 2017; Khunte et al., 2020), root length (Amri, 2011; Galavi et al., 2013). F. esculentum has enormous, medicinal, nutritional and economic value and if promoted, could highly contribute to reduction of poverty mainly in rural areas and to the improvement of both nutritional and health status of the local populations. Till date, there are no reports regarding the effects of PGRs on common buckwheat. Keeping these facts in mind, present study has been undertaken to see the relative effect of F. esculentum to plant growth regulators.

Materials and Methods

The seeds of Fagopyrum esculentum Moench were obtained from Himachal Pradesh Agricultural University, Research Station, Sangla, Kinnaur (HP). Experiments were conducted in the laboratory and nursery area of Shoolini University of Biotechnology and Management Sciences, Solan.

2.1.  Seedling growth assays and growing conditions

Seeds of Fagopyrum esculentum selected for uniformity, damaged and insect infected seeds were discarded and the hollow ones were rejected by floating method in distilled water. Surface sterilization of seeds was done with 0.1% HgCl₂ prior to sowing, after which the seeds were rinsed three times with distilled water. Seeds were sown in the nursery beds, in the Herbal Garden of Shoolini University, Solan (Latitude 30°51’N, longitude 77°07’E and altitude 1195 m), where the average annual rainfall was 1315.6 mm. The average maximum and minimum temperatures were 32°C and 2°C, respectively. Nursery beds were watered regularly. When the first leaf appeared the seedlings were transferred to pots (20 cm diameter). The pots were filled with 3 kg uniform soil mixture containing soil: sand: farm yard manure (FYM) in 1:1:1 ratio. Three seedlings per pot in replicates of three were used for each treatment. No inorganic fertilizer and systemic pesticide were used during the experiment. The pots were arranged in a entirely randomized design and the locations of the pots were changed weekly to avoid position effect.

2.2.  Treatments with plant growth regulators

Plant growth regulators spray was done after one week of transplanting the plants to pots. Four major hormones: indole Acetic Acid (IAA), benzylaminopurine (BAP), abscisic Acid (ABA) and gibberellic Acid (GA), were used solely as well as in combinations i.e. IAA+BAP, IAA+ABA, IAA+GA, ABA+BAP, BAP+GA and ABA+GA in concentration of 25, 50 and 100 mg l^-1 through foliar spray. Shoot length (cm) and Root length (cm) were measured at different growth stages i.e., 30, 60 and 90 days.

2.3.  Statistical analysis

The data was analyzed statistically using Graph Pad Prism® 5.2. Mean values were calculated from measurements of three replicates and the standard error of means were determined. Two-way analysis of variance (ANOVA) was applied to determine the significance of results between different treatments and Bonferroni’s post tests were performed at the significance level of p<0.05.

Results and Discussion

3.1.  Shoot length

As expected progressive increase in shoot length was noted with an increase in time period. GA at 100 mg l^-1 concentration gave highest shoot length at 30 days (20.50 cm, 20.5% increase from control), at 60 days (42.30 cm, 14.32% increase from control) and at 90 days (54.60 cm, 20.26% increase from control) of plant growth. However, at 30 days ABA 25 mg l^-1 (14.30 cm, 15.9% decrease from control), at 60 days ABA 100 mg l^-1 (34.30 cm, 7.3% decrease from control) and at 90 days ABA 25 mg l^-1 (41.80 cm, 7.9% decrease from control) treated plants resulted in minimum shoot length (Figure 1). Combination of phytohormones produced, maximum shoot length at 30 days (34 cm, 100% increase from control), at 60 days (46 cm, 24.3% increase from control) and at 90 days (58 cm, 27.8% increase from control) as shown by IAA+GA 100 mg l^-1 treated plants. Minimum shoot length at 30 days was seen in IAA+ABA 100 mg l^-1 treated plants (13.50 cm, 20.6% decrease from control), at 60 days in ABA+BAP 100 mg l^-1 treated plants (30.20 cm, 18.4% decrease from control) and at 90 days in ABA+BAP 50 mg l^-1 treated plants (35.20 cm, 22.5% decrease from control) (Figure 1).

Figure 1: Shoot length in Fagopyrum esculentum on different days treated with IAA (A), BAP (B), ABA (C), GA (D), IAA+BAP (E), IAA+ABA (F), IAA+GA (G), ABA+BAP (H), BAP+GA (I) and ABA+GA (J). Values are mean±SE; n=3, analysed by Two-way ANOVA followed by Bonferroni’s multiple comparison test. Values followed by the same letter are not statistically different (p<0.05) compared with control

Figure 1: Continue...

At 30 days of growth IAA showed no variability from control (Figure 1.1 A) while ABA had reduced growth (Figure 1.1 C) and GA enhanced growth (Figure 1.1 D). Combination of growth regulators revealed synergistic effects in case of IAA+BAP and IAA+GA (Figure 1.1 G and I); while combination with ABA produced retarded effects (Figure 1 F and H). It is evident from Bonferroni post-test (Table 1) that after 90 days untreated (control) plants showed significant difference (p<0.05) with all the three concentrations (25, 50 and 100 mg l^-1) of BAP, ABA and GA. Combination of IAA+GA and ABA+BAP also showed a significant difference. IAA (25 mg l^-1, 50 mg l^-1) and GA (25 mg l^-1, 50 mg l^-1) revealed significant differences with IAA (100 mg l^-1) and GA (100 mg l^-1) treated plants, respectively. Plants given treatment of 25 mg l^-1 IAA+BAP, IAA+GA,

Table 1: Bonferroni post-test for comparison between treated and untreated plants of Fagopyrum esculentum for shoot length

Table 1: Continue...

IAA+GA, ABA+BAP also showed significant difference with 50 mg l^-1 and 100 mg l^-1 of IAA+BAP, IAA+GA, ABA+BAP treated plants. 25 mg l^-1 concentration of BAP+GA and ABA+GA showed significant difference with 100 mg l^-1 concentration of BAP+GA and ABA+GA, respectively. There was no significant difference between other treated plants.

The effects of phytohormones on morphology of F. esculentum revealed that GA and IAA+GA at higher concentration (100 mg l^-1) effectively promoted shoot length. There are many reports indicating that application of auxins and gibberellins enhance plant growth (Giannakoula et al., 2012; Ngomuo et al., 2013). Several studies have proved positive effect of GA on shoot length (Giannakoula et al., 2012; Pérez-Jiménez et al., 2015). According to Taiz and Zeiger (2010) gibberellins stimulates cell division and cell elongation which are two events that cause plant elongation. Our results are similar to Naeem et al. (2004) who observed increased shoot length with IAA+GA. The increase in plant height might be due to the stimulatory action of auxin which softens the cell wall by escalating its plasticity or due to oxidative decarboxylation of synthetic auxins which could not be catalyzed by the peroxidase enzyme. Also, the effect of IAA on cell division and cell elongation resulted in increased plant height. Many researchers have observed increased shoot length due to IAA (Mostafa and Abou Al-Hamd, 2011; Muthulakshmi and Pandiyarajan, 2015). In present investigation ABA in combination with IAA (IAA+ABA) and BAP (ABA+BAP) decreased shoot length at higher concentration (50, 100 mg l^-1). Inhibitory effects of ABA on shoot length was also reported in rice (Chen et al. 2006; Cha-um et al., 2007), wheat (Zhang and Jiang, 2002) and Cynanchum komarovii (Yang et al. 2007). Similar to our results, Bakrim et al. (2007) observed decreased shoot length in tomato after BAP treatment at higher concentration. IAA application showed decrease in shoot length of lentil (Naeem et al., 2004). 

3.2.  Root length

Root length under phytohormones treatment was observed at 30, 60 and 90 days of growth and the results revealed that there was a progressive increase in root length in plants treated with phytohormones. When plant growth regulators were used solely, IAA 50 mg l^-1 treated plants showed maximum root length at 30 days (4.70 cm, 42.4% increase from control), at 60 days (5.50 cm, 14.5% increase from control) and at 90 days (6.20 cm, 12.7% increase from control). BAP 100 mg l^-1 treated plants showed minimum root length at 30 days (2.70 cm, 18.2% decrease from control) and 60 days (4.10 cm, 12.8% decrease from control). At 90 days BAP 100 mg l^-1 and ABA 25 mg l^-1 treated plants showed lowest root length (4.40 cm, 20% decrease from control) (Figure 2).

Figure 2: Root length in Fagopyrum esculentum on different days treated with IAA (A), BAP (B), ABA (C), GA (D), IAA+BAP (E), IAA+ABA (F), IAA+GA (G), ABA+BAP (H), BAP+GA (I) and ABA+GA (J). Values are mean±SE; n=3, analysed by Two-way ANOVA followed by Bonferroni’s multiple comparison test. Values followed by the same letter are not statistically different (p<0.05) compared with control

Figure 2: Continue...

In combined treatment of plant growth regulators, IAA+GA 100 mg l^-1 treatment resulted maximum root length at all three intervals of observation. Maximum root length was 5 cm at 30 days (51.5% increase from control), 5.80 cm at 60 days (23.4% increase from control) and 6.60 cm at 90 days (20% increase over control). Minimum root length at 30 days (1.90 cm, 42.4% decrease from control), at 60 days (3.20 cm, 46.9% decrease from control) and at 90 days (4 cm, 27.3% decrease from control) was shown by ABA+BAP 100 mg l^-1 treated plants (Figure 2).

It is evident from Figure 2 that growth regulators affected root length compared to untreated plants. In general, IAA (Figure 2 A), GA (Figure 2 D) as well as their combination (Figure 2 G) and IAA+ABA (Figure 2 F) increased root length while ABA (Fig. 2 C) and BAP (Figure 2 B) decreased root length as compared to control. After 90 days Bonferroni post-test revealed (Table 2) significant difference (p<0.05) between untreated (Control) plants and growth regulators IAA, ABA, BAP, GA, IAA+GA, ABA+BAP and BAP+GA except 25 mg l^-1 IAA, 100 mg l^-1 ABA, 50 mg l^-1 GA, 25 mg l^-1 ABA+BAP treatments. All the concentration of GA (25, 50 and 100 mg l^-1) and IAA+GA (25, 50 and 100 mg l^-1) differed significantly with each other. Plants treated with 25 mg l^-1 of IAA, ABA+BAP, ABA+GA revealed significant difference with 50 mg l^-1 of IAA, ABA+BAP, ABA+GA treated plants. Plants given treatment of 25 mg l^-1 BAP, ABA, IAA+BAP, ABA+BAP, ABA+GA was also significant with 100 mg l^-1 BAP, ABA, IAA+BAP, ABA+BAP, ABA+GA. Significant difference was seen in 50 mg l^-1 of ABA and IAA+ABA with; 100 mg l^-1 of ABA and IAA+ABA, respectively. Non-significant variations were detected in rest of the treatments. Auxin increase rooting by stimulation of division of primer root cells. Auxins usually stimulate formation of root and balance morphogenesis such as shoot and root development (Rout et al., 2000). In the present study, root length was influenced positively by IAA at 50 mg l^-1 and IAA+GA at 100 mg l^-1 concentration. Similar results regarding effect of IAA on root length has been reported (Alam et al., 2012; Muthulakshmi and Pandiyarajan, 2015). Ghodrat et al. (2013) in a study also reported increased root length after application of GA on Oryza sativa. In present studies decrease in root length was seen in ABA+BAP (100 mg l^-1). Earlier reports have also shown decrease in root length due to ABA (Liao et al., 2008; Cutler et al., 2010). Our results are in agreement with Bakrim et al. (2007) who reported that BAP inhibitedroot elongation in tomato.

Table 2: Bonferroni post-test for comparison between treated and untreated plants of Fagopyrum esculentum for root length

Table 2: Continue...

Conclusion

Need for food and medicines is supposed to continue due to ever-growing world population. Exploitation of ‘underutilized’ crops can contribute effectively to promote nourishment and biological sustainability. Fagopyrum esculentum Moench is one of the essential neglected crops having high nutritive and medicinal value. From the present study it can be concluded that PGRs effectively increased shoot and root length of Fagopyrum esculentum. Combination of PGRs were more effective than solely applied PGRs. The results of the present study call for further research on mechanism of PGRs action by using molecular approaches.

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