Development of Somaclones through Callus Culture in Fenugreek (Trigonella foenum-graecum) Variety Gujarat Methi-2

Fenugreek (Trigonella foenum-graecum) a widely cultivated herb, belongs to the family Fabaceae (Balodi and Rao, 1991.) For centuries, it has grown wild in India, the Mediterranean and North Africa where it is mainly cultivated. In India it is known as “methi,” in Moroccan “helba” and in Ethiopia “abesh.”It was used by the ancient Egyptians to combat fever and grown in classical times as cattle fodder. In India, it is used medicinally and as a yellow dyestuff. Fenugreek shoots and leaves used into salads and its powder, add flavor to cuisine and chutneys.
 
The seeds and leaves of fenugreek have potential to reduce blood sugar and blood cholesterol levels in both animals and human (Acharya et al., 2007). The medicinal properties of crop are due to the presence of various phytochemicals, including trigonelline, diosgenin, fenugreekine and 4-Hydroxyisoleucine (Losso et al., 2009a; Losso et al., 2009b and Zandi et al., 2015). Thus, the demand of fenugreek in pharmaceutical and nutraceutical industries is quite high (Zandi et al., 2017). The production of fenugreek is limited by various aetiological agents among them, powdery mildew caused by Erysiphe polygoni DC is a potential threat (Petropoulos, 2002). It causes damage to all above ground plant parts. It usually appears during later stage of the crop growth and becomes serious during flowering and pod formation stage (Prakash and Saharan, 2002). In this disease, white powdery patches appear on the lower and upper surface of leaves and other parts of the plant. Powdery mildew affects both biomass and seed yield and can result in seed yield loss up to 50 per cent (Rathi et al., 2002). Disease resistance offers a sustainable solution to control plant disease due to economic and environmental factors (Komarek et al., 2010). However, limited data on genetic diversity of Trigonella species (Marzaugui et al., 2009), non-availability of resistance source for powdery mildew (Raje et al., 2002) and lengthy conventional breeding procedures hinder the production of powdery mildew resistance varieties in fenugreek. Somaclonal variations can be used to generate new genetic diversity in existing genepool and to overcome the constraints of conventional resistance breeding approach. 
 
Plethora of research has been conducted on in vitro regeneration, callus induction and production of secondary metabolites in fenugreek (El-Bahr, 1989, Qaderi et al., 2016 and Valizadeh, 2018). Genetic variability have also been introduced through in vitro culture techniques to isolate fenugreek variants with superior yield and seed quality (Basu et al., 2008), yet critical studies to screen somaclones resistant to powdery mildew have not been done in the past. However, attempts were made by several workers to screen and isolate powdery mildew resistance fenugreek lines among the existing natural genetic diversity in the germplasm (Raje et al., 2003; Kumar, 2009 and Mulat, 2017). Since fenugreek has extensive adoption in industrial sectors and powdery mildew being an important disease on fenugreek, an extensive study was conducted to induce and isolate somaclones resistant to powdery mildew in popular variety Gujarat Methi-2.
 

The experiment was conducted at Plant Biotechnology Laboratory, Department of Genetics and Plant Breeding, College of Agriculture, Navsari Agricultural University, Campus Bharuch in the year 2019. Healthy, bold and fresh crop seeds of fenugreek var. Gujarat Methi-2 (GM-2) released from Sardarkrushinagar Dantiwada Agricultural University (SDAU), S. K. Nagar, Gujarat in 2006 were used for regeneration of tender and disease-free leaves and shoots in vitro. The grains are bold, uniform in size, attractive and have more test weight. On an average GM-2 yielded 1920 kg/ha and showed its superiority by producing 10.03 and 20.32 per cent higher than the varieties GM-1 and Hisar Sonali, respectively
 
The seeds were treated by dipping in 1% teepol detergent solution for 5 minutes for removing dirt and dust particles adhered on the seed surface and thoroughly washed with double distilled water (DDW). Surface sterilization fenugreek seedswere done with 70% Alcohol for 30 seconds and thoroughly washed with DDW. Finally, the material was taken into Laminar Air Flow Cabinet for sterilization, rinsed with 0.1% HgCl₂ solution for 10 minutes to avoid all the contaminants from the seeds. Seven seeds were inoculated in each glass bottle with 5 repetitions in Agar Agar semi solid MS medium (Murashige and Skoog, 1962) with BAP 2.0 mg/l in addition to 1.0 mg/l Adenine Sulphate under aseptic condition for regeneration. The cultures were incubated at 25±1^oC temperature with 2000 lux light intensity. The regenerated hypocotyl and cotyledonary segments were placed in callus media (semi solid MS medium with different concentration of 2,4-D and NAA i.e. 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l) for callus induction. The induced calluses were further transferred in to the regenerated semi solid MS medium with various levels of BAP i.e. 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l with same concentration of Adenine sulphate i.e. 1.0 mg/l for development of calliclones. For root induction, shoots were transferred in White’s rooting media with different concentrations of IAA such as 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l).
 
Regenerated tissue culture seedlings were planted for hardening process using low polythene tunnels. After hardening, seedlings were transplanted in the earthen pots in the screen house, all the planted material was labelled and necessary management practices were adopted. Potted plants were artificially inoculated by dusting conidia of Erysiphe polygoni DC.  Powdery mildew severity was visually scored, as suggested by Kumar, 2009 on 0-5 scale and lines were categorized based on disease reaction, experimental material was further categorized based on percent disease intensity (Kumar, 2009).
 
Statistical analysis
 
Significance was determined by analysis of variance (ANOVA) and the differences between the means were compared by Duncan’s Multiple Range Test using SPSS for Windows computer program. Data given in percentages were subjected to arcsine (Vx) transformation (Snedecor and Cochran, 1967) before statistical analysis.
 

Callus induction
 
The effects of plant growth regulators (2,4-D and NAA)in MS medium on explants (hypocotyl and  cotyledonary nodal segments) and their comparison on initiation of callus, % callusing, callus weight (g), callus colour, callus texture and morphogenetic response showed in Table 1, Fig 1-4 and Chart 1A-1C.
















 
The treatment MS medium containing 2.0 mg/l 2,4-D was significantly superior for callus initiation in hypocotyl (12.80) and  cotyledon (13.40) segments had taken less number of days, while in the treatment MS medium containing 1.5 mg/l NAA taken less number of days for callus initiation in hypocotyl (13.20) and cotyledon (14.60) segments. The comparison of both the auxins, 2,4-D had given early response as compare to NAA for callus induction. The results obtained through experimentation were in accordance to EL Nour et al., (2013).
 
Callus weight is important character for regeneration of calliclones and development of more numbers of somaclones. The maximum callus formation in hypocotyl segment was obtained 2.0 mg/l 2,4-D (1.456 g) and it was significantly superior over all the treatments, whereas in cotyledon (1.322 g) in the same 2,4-D level. Similarly, callus weight was highest in 1.5 mg/l NAA in hypocotyl segments (1.268 g) as compare to cotyledon (1.144g). The callus formation was increased from 0.5-2.0 mg/l 2,4-D and it was fall down after 2.5 mg/l 2,4-D. Similar trend was examined in NAA after 2.0 mg/l. Minimum callusing were recorded in hypocotyl and  cotyledonary nodal segments by the concentration 0.5 mg/l 2,4-D i.e. 0.374g and 0.292g respectively and in NAA i.e. 0.312g and 0.276g respectively (Bahram et al.,2005). The various kinds of callus colour and callus textures and morphogenetic responses were observed during the experimentation mentioned in the Table 1, the yellowish green colour and friable texture callus reported good callus induction and gave high callus weight in hypocotyl and cotyledonary nodal segments. Our results revealed that 2,4-D gave high frequency of callus induction, these results agreed with finding of Rezaeian (2011).

Shoot regeneration
 
The study presents the effect of cytokinin on frequency shoot regeneration (%), shoots/gm callus and shoot length (cm) in fenugreek using cotyledonary nodes and hypocotyl explants were cultured on MS medium containing BAP i.e. 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l with same concentration of Adenine sulphate 1.0 mg/l depicted in Table 2, Fig 5 and Chart 2.







Result showed that hypocotyl segment responded better than cotyledon for shoot regeneration. The shoot regeneration frequency of hypocotyl and cotyledon segments ranged from 39.80% to 73.20% and 33.80% to 71.40%, respectively. In hypocotyl, maximum shoot regeneration frequency (73.20%) was found significantly superior over all the treatments in MS medium supplemented with 2.0 mg/l BAP + 1.0 mg/l Adenine sulphate, where as it was noticed highest (71.40%) in 2.0 mg/l BAP + 1.0 mg/l Adenine sulphate in cotyledon segment. The minimum shoot regeneration frequency, shoots/gm callus and shoot length (cm) was observed in 0.5 mg/l BAP + 1.0 mg/l Adenine sulphate in both cotyledonary nodes and hypocotyl segments.

In hypocotyl segments, maximum 9.40 shoots was found in MS medium supplemented with 1.5 mg/l BAP + 1.0 mg/l Adenine sulphate and this treatment was highly significant over remaining treatments, where as it was noticed that highest 8.20 shoots in 1.5 mg/l BAP + 1.0 mg/l Adenine sulphate in cotyledon segment. The less number of shoots (2.80) were found in the treatment 0.5 mg/l BAP+ 1.0 mg/l Adenine sulphate in hypocotyl explant. Thus, the shoot regeneration frequency reported more in 2.0 mg/l BAP, while numbers of shoots were achieved higher in 1.5 mg/l BAP. The number of shoots increasing was restricted after the BAP concentration 1.5 mg/l. The result was in accordance with the research finding of Aasim et. al., (2010).
 
The shoot length of the shoots obtained from hypocotyl segment, ranged from 1.41 to 1.89 cm and from cotyledon, it was ranged from 1.37 to 1.68 cm. Shoot length increased with increase in BAP up to the level of 1.5 mg/l in both types of the explants but it was decreased after the increasing the levels from 2.0 mg/l BAP and above. Similarly, as above, the shoot regeneration frequency reported more in 2.0 mg/l BAP, while the shoot length was achieved higher in 1.5 mg/l BAP for both explants.

Root formation
 
The results of root induction obtained in regenerated plantlets showed in Table 3, Fig 6 and Chart 3. Morphogenetic root response ranged from 9.80 to 74.00% between the IAA concentration of 0.1 to 0.4 mg/l. After IAA concentration of 0.4 mg/l, morphogenetic response of root was not observed even up to the 0.6 mg/l. Maximum morphogenetic root response was observed on White’s medium supplemented with 0.2 mg/l IAA (74.00%) with thick and long roots, whereas minimum was noticed in 0.4 mg/l IAA (9.80%) with thin and short roots. These results were confirmed with Burdak et al., 2017. It is true that addition of auxins in the culture medium produced roots in crop species like carrot (Pant and Manandhar, 2007) and brinjal (Narayanaswami, 1994) .







Number of roots per plantlet was obtained highest in the treatment White’s medium with 0.2 mg/l IAA (2.6) and it was significantly superior over all the treatments whereas lowest was reported in 0.4 mg/l. IAA (1.0). The concentration above 0.4 mg/l IAA, could not showed root induction up to the 0.6 mg/l IAA. Same kind of response was observed by Burdak et al., (2017).
 
Downy mildew resistance
 
The somaclones regenerated were evaluated in comparison with parent variety GM-2, under screen house condition for powdery mildew resistance. After inoculation, 20 somaclones of 160 (11.12%) produced less severe symptoms than parental material, while eight somaclones (5%), showed the highest levels of resistance, under controlled conditions (Table 4). Though complete resistance has not been observed, yet five somaclones showed resistance reaction and least disease intensity, can be further evaluated to incorporate in the powdery mildew resistance breeding program in fenugreek or to improve the resistance level of parental line which showed moderate susceptible reaction in our experimental design. The effectiveness of somaclones in disease resistance have also been established by various researchers in different crops, including chickpea (Parkash et al., 1994), barley (Li et al., 2001), peanut (Yusnita et al., 2005), garden pea (Sharma et al., 2010) and sugarcane (Kona et al., 2019).


 

It can be concluded that in vitro induction of somaclonal variation can be a viable and sustainable alternative in developing powdery mildew resistant fenugreek. Among all the treatments, 2.0 mg/l 2,4-D resulted in regeneration  of maximum number of somaclones in GM-2 variety. The resistant somclones compared to parental line can be screened under field conditions for various agronomic feautures to develop powdery mildew resistant varieties with competitive yield in fenugreek.

All authors declare that they have no conflicts of interest.