Stomatal Studies on Colchicine Treated Bajra Napier Hybrids (Pennisetum glaucum x P. purpureum)

Poaceae family contains Pennisetum which is considered as one of its key genera. Pearl millet [Pennisetum glaucum (L.) R. Br.] and Napier grass (Pennisetum purpureum (K.) Schum) are the two economically significant cultivars. Napier grass is an allotetraploid species with 2n = 4x = 28 and Pearl millet is a diploid with 2n = 2x = 14 (genomes AA). Because of their close kinship and strong genetic compatibility, these two species can produce interspecific hybrids with 2n= 3x = 21 (genomes A’AB). This kind of hybridization was an attempt to combine the traits of pearl millet and Napier grass, such as disease resistance, high vigour, fodder quality and perennial nature, with the traits of Napier grass, such as high biomass, aggressiveness and high dry matter production. Cattle have preferred the interspecific hybrids that were produced over Napier grass. However, their infertility as a result of meiotic abnormalities has been viewed as a significant barrier for breeding programmes. The term “polyploidy” describes the presence of multiple chromosomal sets. Autopolyploids are produced by a species’ genome doubling, whereas allopolyploids are produced via interspecific hybridization and genome doubling. Genome duplication is one of the most crucial evolutionary events leading to the huge expansion of genetic diversity; thus, allowing species to adapt to varying environmental habitats. The evolutionary success of polyploids has been greatly influenced by various adaptive modifications, including increased tolerance to extreme environmental fluctuations, effective vegetative reproduction methods, apomixes, disease resistance and changes in morphology, flowering period and biomass. As a result, they are an excellent choice for many agricultural and breeding practises (Ramsey and Schemske, 2002).

A powerful approach for creating unique germplasm appropriate for selective breeding is inducing polyploidy. Autopolyploid plants have a number of potential benefits over allopolyploid plants, which are more common, since they have more genetic variability and higher gene doses (Soltis and Soltis, 2009). Additionally, polyploid plants typically have thicker and bigger leaves, stalks, flowers, fruits and seeds, which results in a higher yield (Verma et al., 2017). Recent research has revealed that polyploidy genomes are very dynamic and exhibit quick structural and functional changes (Soltis and Soltis, 2009); Dar et al., 2013).

The most widely used antimitotic drug, colchicine, is derived from the angiosperm Colchicum autumnale plant and binds selectively to tubulins to block microtubule polymerization and hence cause polyploidy in cells (Murali et al., 2013). Chromosome doubling has been used to restore fertility in sterile hybrids between napier grass (Pennisetum purpureum) and pearl millet (Pennisetum glaucum) (Falerio et al., 2015). Stomata count and size measurements are a reliable screening method for determining the ploidy level effectively. Stomatal size has been used successfully as an indicator of altered ploidy by Murali et al., (2013) in sorghum, Quesenberry et al., (2010) in bahia grass,Compos et al. (2009) in pearl millet napier hybrids, Ramesh et al., (2014) in mulberry and Falerio et al., (2015) in pearl millet napier hybrids. This was kept in mind when the current work was carried out to induce polyploidy in two kinds of bajra napier hybrids using two distinct techniques of colchicine application and to analyse the putative polyploids using the stomatal measurements.  

During the year 2021-2022, this study was carried out in the forage department of CPBG, TNAU, Coimbatore. Two alternative techniques were used to administer the colchicine treatment to one nodded setts of pearl millet napier hybrids. One nodded setts were given various treatments utilising cotton swabs and the complete immersion procedure. The planting material (setts) were prepared and planted individually in polybags filled with soil. The buds were cleaned with distilled water before the application of colchicine. Buds were covered with dry swabs of cotton and colchicine solution was applied (concentrations mentioned in materials and methods) from 6.00 A.M to 6.00 P.M for two continuous days at three hour intervals while the buds of control were given distilled water. The concentrations used were 0.05%, 0.10%, 0.15%, 0.20% and 0.25%. The growth parameters and stomatal dimensions (30 days after transplanting) were recorded and subjected for analysis. Several one nodded setts were also used in the full immersion technique and kept for 3 and 6 hours in five different colchicine concentrations: 0.05%, 0.10%, 0.15%, 0.20% and 0.25%. In this way, a total of 10 treatments were done. Growth metrics and stomatal investigations were carried out in the same manner as in the cotton swab approach.
 
Stomatal studies
 
Approximately one cm² strips from the middle of the leaf’s epidermis were coated with a thin layer of quick fix or natural nail polish. The stomata impressions were carefully removed with forceps or nails when dried and put on the glass slide. Longitudinal and transverse dimensions were recorded for adaxial epidermal stomata in the leaves of all survived and established plants at 30 days after transplanting in the field. Scope Image programme was used to measure five stomata from each leaf and determine the mean value

Putative colchiploids were identified by preliminary screening based on stomatal size so that they could be chosen for further field experiments. When the adaxial stomatal length and width of each plant were examined, a significant degree of variance was found among these colchiploids. In case of the whole immersion method, the control plant of the genotype CO 6 recorded an average stomatal length and width of 52.927±0.579 µm and 22.046±0.290 µm respectively (Table 1).



The highest mean value of 55.470±1.128 µm for length of stomata was observed at 0.20% for 6 hours treatment with a range of 46.008-61.334 µm, followed by 0.25% for 3 hours treatment with a mean value of 52.9740.510 µm having the range between 43.010-59.201 µm. The highest stomatal length of 61.334 µm was observed in plants that were treated with 0.20% colchicine for 6 hours (Fig 1a) indicating that this concentration was capable of producing variants in this genotype.



In case of width of stomata, the highest value of 23.768±0.643 µm was observed at 0.25% concentration for 3 hours treatment where the stomatal width ranged from 17.326-27.77 µm. As the concentration and period of the treatment increased, the range of stomatal length and breadth also expanded. The highest range for stomatal length (36.005-54.116) (Fig  1c) and width (17.628-28.135) were observed for 0.25% concentration for 6 hours duration. It indicates that as the concentration increased, the stomatal size started to fluctuate. Hence, it can be seen as a reliable factor for determining variants produced during colchicine application.

In case of genotype TNCN 1534 (Table 1), the control plant had the mean value of 46.2480.834 µm and 24.2860.376 µm for stomatal length and width respectively. As the concentration increased, the range of variation for stomatal length and width increased. The highest range of stomatal length and width of 31.003-50.353 µm and 17.334-31.201 µm was observed at 0.25% concentration for 6 hours duration. The second highest range of stomatal length (33.334-46.702 µm) was recorded at 0.25% concentration for 3 hours duration (Fig 1d). The mean of stomatal length and width decreased with increase in concentration and duration, the lowest being seen in plants treated with 0.20% concentration for 6 hours duration (40.3360.732 µm). The increase in range of variants decreased the mean of stomatal length and width of plants which were treated at higher concentration.

The swab method also generated some variations in genotypes CO 6 and TNCN 1534. In case of genotype CO 6, all of the concentrations had similar range of stomatal length when compared to the control (530.759 µm) (Table 2).



The highest range of stomatal length was observed for 0.25% concentration (45.188-58.034 µm) (Fig 1b) followed by 0.20% concentration where the plants had the values between 46.021-56.100 µm. The highest mean value for stomatal width of 23.124±0.648 µm was recorded for 0.15% concentration. In the case of genotype CO 6, the range of stomatal length and width gradually increased with increase in colchicine concentration, hence, proving the fact that stomatal measurements can be used as an indirect tool to analyse induced polyploids in plants.

In case of genotype, TNCN 1534 (Table 2), the highest mean value for stomatal length of 49.8680.919 µm was recorded at 0.20% concentration followed by 0.25% concentration of 49.186±1.083 µm. The highest stomatal range between 42.187-56.021 µm was observed at 0.25% concentration. The highest mean value for stomatal width was also observed at 0.25% concentration (23.080±0.399 µm). However, the highest stomatal range between 18.320-28.116 µm was observed at 0.20% concentration. This indicated increment in variations as concentrations increased.

The scatter plot diagram indicated that the genotype CO6 produced a whole range of variation when the setts were treated with whole immersion method (Fig 2a). The genotype TNCN 1534 had plants which showed a less scattered pattern (Fig 2b) and also didn’t had much range as compared to genotype CO 6. Majority of the genotypes in CO 6 were concentrated at a particular range of stomatal length and width but some genotypes were scattered more than TNCN 1534.



The genotypes (CO6 and TNCN 1534) when treated with cotton swab method showed a range between 48 µm to 52 µm. The plants in both the genotypes were much scattered over ranges but didn’t had a wide range as compared to the plants treated with whole immersion method (Fig 3a and b). This indicates that even the method used for colchicine treatment can cause variations in the plants. The longer the chemical comes in contact with the plant material, more will be the variation induced.



Stomatal size has been used to distinguish between diploid and polyploid regenerants of several plants, including orchids, Stevia rebaudiana and many varieties of Lilium (Nouraddin et al., 2019). According to Raghunath et al., (2014), the stomatal dimension (length and width) of the colchicine-treated plants in African marigold seemed to be higher than the untreated control. According to Dario and Paul (2009), guard cells in Vaccininium darrowii treated with colchicine grew longer and were bigger, making them a useful tool for checking colchiploid alterations. It can be summarized that colchicine was efficient in generating variants at higher concentrations which was evident from the above results. In whole immersion method, 0.20% and 0.25% concentration for 3 and 6 hours generated considerable variants having largest width and length of stomata and also covered the largest range for mean values. In cotton swab treatment, highest range for stomatal measurements were seen at higher concentrations of 0.20% and 0.25%. However, genotype CO 6 recorded the highest stomatal width at 0.15% concentration. Although stomatal dimensions can be utilized as a primary selection criteria for colchiploids, further verification is necessary as there might be a chance of occurrence of mixoploids as was indicated by (Zhang et al., 2010) in crape myrtle (Lagerstroemia indica). Similar results were revealed by Campos et al., (2009) in bajra napier hybrids as well. It can be stated that colchicine can be used as a potential antimitotic agent to produce variability in triploids and can be seen as a key tool in overcoming the sterility of these triploid hybrids to produce fertile hybrids and also generate variants that are having superior forage characteristics. 

Colchicine application usually results in increased stomatal length and width. This revealed that variation in the stomatal traits due to colchicine treatments can act as an indicator for the variation induced through colchiploidy. Different genotypes react differently to colchicine treatments as from the results, it could be interpreted that genotype CO 6 showed wide variation for stomatal measurements as compared to TNCN 1534 genotype. This indicates how some genotypes are better adapted to chemical treatments that can induce changes in their cellular functions and structure. The method applied to induce polyploidy in these genotypes also generated a wide difference in the stomatal measurements. Whole immersion method was more effective in generating variants than the cotton swab method. Similarly, the concentration also plays a vital role in inducing polyploidy. It is established from previous studies as well as from the present study that more the concentration of the chemical used, more are the chances for inducing polyploidy in the plants. This indicates that when appropriate methods and concentrations are selected for inducing polyploidy, it can generate useful and true variants.

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