This research was conducted in ICAR-Indian Institute of Horticultural Research, Bengaluru. Six month old putative mutant seedlings of polyembryonic mango variety Nekkare used in this study were generated by treating seed kernels of Nekkare with five different doses of gamma irradiation viz., 15Gy, 20Gy, 25Gy, 30Gy and 35Gy (280 kernels per treatment) (unpublished data). From the putative mutant population thus generated, a total of 100 putative mutant seedlings (20 from each treatment) along with control (untreated nucellar seedlings) of short, medium and tall stature (based on plant height) were selected for taking further observations.

Morphological observations on plant height (cm), stem girth (mm), number of internodes, inter-nodal length (cm), number of leaves, leaf blade length and leaf blade width for 100 selected putative mutant progenies of Nekkare along with control were recorded based on mango descriptor (IPGRI 2006).

Volatile profiling was used to ascertain the nucellar origin of control seedlings (unpublished data).

Again, for these 100 putative mutants and control seedlings, stomatal size and number was determined using the procedure as described by Hamil et al. (1992). For measuring number of stomata, fully matured and expanded leaves were selected and plucked between 12-01 PM. Freshly plucked leaves were kept in butter paper, labelled properly and brought to laboratory for further analysis. For determining the number of stomata, abaxial surface of fully matured and expanded leaves plucked between 12 noon to 1 PM coated with transparent nail polish and left for 30 minutes to dry. Glass slides were prepared by peeling off the imprinted layer from the abaxial surface of the leaf sample. The slides were then examined under the microscope (OLYMPUS; Light microscope digital camera; DP-22/DP-27) at a magnification of 10x and stomata numbers were recorded and expressed as number of stomata per mm2. For measuring stomatal size, slides were examined at a magnification of 40x and length and width of stomata was recorded and expressed in µm.

Further, morphologically distinct dwarf and tall statured putative mutant seedlings along with control plants were selected for phytohormone profiling using Liquid chromatography-mass spectrometry (LC-MS).

Phytohormones were extracted using the method described by Pan et al. (2008). For extraction, 3g leaf sample was completely homogenized using 1- propanol/H2O/concentrated HCl (2:1:0.002, v/v/v) followed by sonication for 30 minutes at 4°C keep and kept overnight. Next day dichloromethane was added to the homogenate and sonicated for 30 min followed by centrifugation at 12,000 rpm for 10 minutes. After centrifugation, the bottom layer was transferred to a conical flask containing sodium sulphate to remove any traces of water and then evaporated using flash evaporator. Once the sample was completely dried, it was dissolved in 80% methanol and loaded through C18 solid-phase extraction (SPE) cartridges.

The SPE process included: (a) Pre-conditioning: It included washing the column with 5 mL methanol and 5 mL 80% methanol. (b) Adsorption: After the extract was loaded to the column, all matrices were adsorbed by the cartridge and (c) Elution: In this step, the residual plant hormones were eluted with another 5 mL 80% methanol.

The obtained elute (5 mL) was then evaporated using flash evaporator at 35°C and finally the dried sample was dissolved in 500 μL methanol–0.05% formic acid (1:1, v/v). The solution was filtered using a nylon filter paper and injected into LCMS for further analysis.

The initial gradient was composed of 85% solvent A and 15% of solvent B with a hold time of 1 min. At 12 minutes, the gradient was changed to 15% of solvent A and 85% of solvent B, with a hold time for 1 minute and at 14 minutes, linear gradient was followed by 85% of solvent A and 15% of solvent B with a hold time of 0.5 minute. The system was then returned to the initial conditions at 15 mins and equilibrated for 1 minute before the next injection. The flow rate was 0.2 mL/minute. The analytical column was 2.1 x 50 mm UPLC BEH-C18 column (Waters, USA) with 1.7μm particles, protected by a vanguard 2.1 X 5 mm BEH C-18 with 1.7μm. Guard column (Waters, USA) was used with column temperature maintained at 25°C. Identification and quantification of the elute hormones was done using a TQDMS/ MS (Waters, USA) system, optimized for the hormone analysis.

(i) Solvent - A: water/Acetonitrile/acetic acid (95/5/ 0.05, v/v/v) and (ii) Solvent - B: Acetonitrile/water/ acetic acid (95/5/0.05, v/v/v)

Descriptive statistics was calculated for morphological and stomatal parameters using standard procedure.

Gamma rays are the most widely used physical mutagen employed in mutation breeding of crop plants and are well known for bringing about morphogenetic and endomorphic changes in plants (Ali et al., 2016; Yasmeen et al., 2020). In the present study considerable variation was created for different morphological traits in the putative mutant population of polyembryonic mango genotype Nekkare generated by gamma irradiation (Table 1a and 1b; Fig 1a-f).

Plant height was found to be the most affected trait by irradiation (Fig. 2 and 3). A reduction in plant height (in comparison to control) with increasing dosage of irradiation was recorded with highest mean plant height in control being 44.55 cm while among the treatments, mean plant height ranged from 28 cm (15 Gy) to 21.35 (25 Gy). Further, although the mean plant height of population generated through 35 Gy treatment was 23.12 cm, the progenies it produced were as short as 11.50 cm and the maximum plant height recorded in this treatment was 33 cm in contrast to control where the plant height ranged from 33.50 to 56 cm. The use of gamma irradiation for developing dwarf statured mango varieties to be used for high density planting has been attempted for a very long time (Sharma and Majumder, 1985, 88; Sharma, 1987).

A decrease in stem girth with increasing doses of irradiation was also observed and the highest mean stem girth was recorded in control (9.07 cm) which declined to 6.53 cm in 25 Gy treatment. However, the progeny with largest stem girth (11.32 cm) was produced by 35 Gy treatment. Mutation affects the process of cell division and cell elongation along with disruption of protein synthesis, hormonal and enzymatic balance and water and gaseous exchange ability of plants thus leading to reduction in plant height as well as overall growth of plants (Ali et al., 2016; Asare et al., 2017). The results of the present study are in confirmation with earlier findings reporting the reduction in plant height with increasing doses of irradiation in mango (Rime et al. 2019), guava (Zamir et al., 2003), grapes (Surakshitha et al., 2017) and apple (Atay et al., 19).

Further, the variation for number of nodes was highest in control along with highest mean number of nodes being 7.2 while the lowest mean number of nodes was recorded in 15 Gy being 5.76. Mean inter-nodal length also declined with increasing doses of irradiation. The number of leaves in all the treated populations was less than control where the mean number of leaves was 24.60 ranging from 17 to 35. Among the treated populations, the lowermost treatment (15 Gy) produced highest mean number of leaves ranging from 9 to 35 while the lowest mean number of leaves (15.93). Reduction in number of leaves with increasing doses of gamma irradiation has been reported in Arumanis mango of Indonesia (Karsinah et al., 2012) and EMS derived population of mango hybrid Arka Puneet (Rime et al., 2019).

Gamma irradiation resulted in the production of smaller and narrower leaves exhibiting a reduction in leaf blade length with increasing dosage of irradiation (Fig. 4). The highest mean blade length was recorded in control (18.15 cm) which decreased to 11.62 cm in 35 Gy treatment while the lowest leaf blade length (11.21 cm) with highest CV (17.13) was observed in 25 Gy treatment. Following the same trend, the mean leaf width was found to be highest in control being 5.56 cm which decreased in the entire gamma irradiated population exhibiting highly profound reduction in the higher dosage of irradiation and the lowest leaf blade width was recorded for 35 Gy treatment being 3.65 cm along with highest CV (14.23). In contrary to our results, Rime et al., (2019) reported production of longer and wider leaves in the EMS derived putative mutant population of mango hybrid Arka Puneet while it is in congruence with the findings of Surakshitha et al. (2017) where they observed a gradual reduction in leaf length and leaf width with increased dosage of gamma rays in grape cultivars Red Globe and Muscat.

Fig. 1a :
Nekkare Control

Fig. 1b :
Treatment 1 (15 Gy)

Fig. 1c :
Treatment 2 (20 Gy)

Fig. 1d :
Treatment 3 (25 Gy)

Fig. 1e :
Treatment 4 (30 Gy)

Fig. 1f :
Treatment 5 (35 Gy)
Fig. : Seedlings of Nekkere plants raised from gamma irradiated seeds

Fig. 2 :
Dwarf putative mutant of Nekkare

Fig. 3 :
Dwarf putative mutant of Nekkare

Fig. 4 :
Narrow leaf shape in Nekkare mutant

Table 1a :
Morphological variability induced in polyembryonic mango variety Nekkare through different doses of gamma irradiation.

Table 1b :
Morphological variability induced in polyembryonic mango variety Nekkare through different doses of gamma irradiation

In our study we observed high variability for different stomatal parameters like stomatal density, length and width of stomata (Table 2; Fig. 5a-d). Highest mean stomatal length (65.07 µm) was recorded in control (64.55 to 65.43 µm) with a CV of 0.65. Among the putative mutant populations, the highest (63.39 µm) and lowest (61.9 µm) mean stomatal length was recorded in 15 Gy and 35 Gy irradiation treatments respectively (Table 2).

Variations in stomatal width was also recorded in the putative mutant populations and the mean stomatal width of all the treatments except the putative mutant population generated through 25 Gy irradiation was found to be higher than control plants which showed less variation for this trait having a CV of 0.99 and mean stomatal width of 60.83 µm varying between 60.15 to 61.57 µm. Among the putative mutant populations, the mean stomatal width increased at lower doses (15 and 20 Gy) while it declined at higher doses (30 and 35 Gy) while remaining higher than control. The highest (63.12 µm) and lowest (60.92 µm) mean stomatal width was recorded in population generated through 20 Gy and 25 Gy respectively. An increase in stomatal length and width was recorded in sugarcane at lower doses of gamma irradiation (10 and 20 Gy) which declined further with increased doses (30 and 40 Gy) (Yasmeen et al. 2020). Similarly, a significant reduction in stomata length and number at higher doses of gamma irradiation was observed in Kinnow (Mallick et al., 2016), grapes (Kok, 2011) and mango (Rime et al., 2019).

Stomatal density was found to decrease with increasing dosage of irradiation and a profound variation was observed among the populations developed by different treatments as revealed by the values of coefficient of variability. The mean stomatal density for control plants was recorded to be 14.33 (per 20000 µm2) with a very low coefficient of variability being 4.94. Among the treatments, mean stomatal density decreased in lower doses viz., 15 Gy and 20 Gy of being 12.64 (per 20000 µm2) and 12.43 (per 20000 µm2) respectively while it increased at 25 Gy (13.3 per 20000 µm2) and 30 Gy (13.85 per 20000 µm2). Our results are in contrary to the findings of Yasmeen et al. (2020) who reported an increase stomatal density at lower doses of irradiation. However, the highest dose of irradiation (35 Gy) resulted in marked reduction in the stomatal density being 11.82 (per 20000 µm2). A lower stomatal density is indicative reduced plant vigour and stature (Majumder et al., 1981) and this result is also confirmed by the production of highly reduced plant height at 35 Gy treatment in this study. Dwarfing pear variety, ‘601D’ is reported to have shorter intermodal length, thick and broad leaves, lower stomatal density and larger stomatal size as compared to it vigorous mutant ‘601T’ (Liu et al., 2022). The decline in stomatal number at higher doses of irradiation has also been reported in citrus (Mallick et al., 2016), mango (Rime et al., 2019) and sugarcane (Yasmeen et al., 2020).

Correlation analysis of physical mutagenesis with the stomatal and plant morphological parameters gave interesting insights into the data (Table 3). In general, the overall correlation between doses of gamma irradiation and stomatal (except stomatal width) and plant morphological parameters seemed to be negative and traits like plant height, stem girth, leaf blade length, leaf blade width and stomatal length showed highly strong negative correlation with irradiation doses (-0.899, -0.815, -0.887, -0.914 and -0.919 respectively). Contrarily, a strong positive correlation was observed between stomatal length and morphological traits viz., plant height, stem girth, number of leaves, leaf blade length and leaf blade width (0.919, 0.902, 0.843, 0.907 and 0.875 respectively). Further, a positive correlation between stomatal length and stomatal density was seen (0.611) while stomatal width was negatively correlated with stomatal density (- 0.600). Different morphological traits like plant height, stem girth, number of leaves, leaf blade length, leaf blade width divulged positive association among each other and significant positive correlations were observed between plant height and stem girth (0.961), number of leaves (0.957), leaf blade length (0.994), leaf blade width (0.964). A positive correlation between stomatal density and tree height has been reported previously (Camargo and Marenco, 2011, Yasmeen et al., 2020) and increased stomatal density has been found associated with reduced tree height (Barrientos-Pérez and Sánchez-Colín, 1982).

Table 2 :
Variability in stomatal density, stomatal length and width induced in polyembryonic mango variety Nekkare through different doses of gamma irradiation

Fig. 5a :
Nekkare Mother Plant

Fig. 5b :
Nekkare Control plant

Fig. 5c :
Putative mutant seedling N 63

Fig. 5d :
Putative mutant seedling N 91
Fig. : Stomata character of Nekkare plants subjected to gamma irradiation.

Table 3 :
Correlation analysis of doses of gamma radiation, stomatal parameters, morphological traits

Values in bold are different from 0 with a significance level alpha=0.05PH-Plant height; SG-Stem girth; NN-Number of nodes; NL-Number of leaves; LBL-Leaf blade length; LBW-Leaf blade width; SD-Stomatal density; SL-Stomatal length;SW-Stomatal width

Cell elongation and proliferation are the key processes determining height of a plant and plant hormones regulate these processes in a species dependent manner (Yue et al., 2016). Among the putative mutant population created by different dosage of gamma radiation 10 morphologically distinct dwarf and tall seedlings were selected to determine the levels of two growth promoting (IAA and GA7) and one growth inhibiting (ABA) phytohormones. IAA has been found to be associated with cell division, expansion and differentiation thus regulating the plant height (Liu et al., 2022). GA is another important phytohormone that regulates plant height and most of the dwarf phenotypes are results of disruption in GA biosynthesis or signal transduction (Magome et al., 2010). Nekkare is a vigorous genotype and the concentration of IAA was recorded to be highest (4.16 ng/gm) in control samples and tall (putative mutant) progenies (1.56 ng/gm) while dwarf (putative mutant) progenies were found to contain the lowest (0.78 ng/gm) amount of IAA. Similar to IAA, the highest (0.26 ng/gm) concentration of GA7 was recorded in control followed by tall (putative mutant) progenies while the lowest amount of GA7 occurred in dwarf (putative mutant) progenies (Table 4). The interaction between auxin and gibberellin has been known to have implications on plant growth where auxins act as a messenger compound that links the apical bud with biosynthesis of active GAs in the expanding inter-nodes (Ross and O’Neill, 2001). This suggests the possibility that the main growth-regulating function of endogenous IAA could be to maintain the levels of GA in the internode (Ross et al., 2001). This relationship between IAA and GA was also observed in our study where the high levels of IAA in tall (putative mutant progenies) corresponded to the increased levels of GA7 in the same. Great variation for the concentration of ABA was also recorded in the studied samples which was found to occur in concentration as high as 429.1 ng/ gm in dwarf (putative mutant) progenies to as low as 47.61 ng/gm in control plants. Dwarfing apple and citrus trees are reported to contain more bark ABA concentration than the vigorous trees and exogenous application of ABA has resulted in shortened inter- nodes and decreased growth in apple (Noda et al. 2000; Tworkoski and Fazio 2011). Dwarf stature of pear variety ‘601D’ is also considered to be a result of over accumulation of ABA (Liu et al., 2022). The highest concentration of ABA in dwarf (putative mutant) progenies in our study further endorses the role of ABA in regulating plant height.

Table 4 :
Concentration of plant hormones in control, tall and dwarf plants of polyembryonic mango variety Nekkare