Research Article

Efficient in Vitro Plant Regeneration Through Somatic Embryogenesis from Callus Induction Method for Brassica carinata

Rizwan Ullah Shah* and Iqbal Munir

Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar, Khyber Pakhtunkhwa, Pakistan.

Received | May 22, 2018; Accepted | January 22, 2019; Published | March 03, 2019

*Correspondence | Rizwan Ullah Shah, Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar, Khyber Pakhtunkhwa, Pakistan; Email: rizwanullahs@yahoo.com

Citation | Shah, R.U. and I. Munir. 2019. Efficient in vitro plant regeneration through somatic embryogenesis from callus induction method for Brassica carinata. Sarhad Journal of Agriculture, 35(1): 314-319.

DOI | http://dx.doi.org/10.17582/journal.sja/2019/35.1.314.319

Keywords | Somatic embryogenesis, Brassica carinata, NAA, BAP, IBA, Phytohormone, Hypocotyl, Cotyledon

Introduction

Ethiopian mustard (Brassica carinata) is an amphi-diploid species with its origin rising of interspecific hybridization between Brassica oleracea and Brassica nigra. B. carinata is famous for possessing many desirable traits like high rusticity and adaptability, strong resistance to disease and water scarcity, low pod dehiscence or delayed pod shattering, most studied plant physiology, extended crop residue and simple insertion in cereal rotation, drought and salt tolerance, and large seed size (Lazzeri and Avino, 2009; Rakow, 2004). Other advantages of the crops include cultivation on boundaries of agriculture fields as cover crops by suppressing diseases, nematodes and insects (Al-khatib and Boydston, 1999).

Studies have proven several factors such as genotype and various growth conditions (biotic and abiotic) that influence in vitro culture (Vincente and Dias, 1996). Brassica carinata seeds were known for containing high content of toxic compounds, erucic acid, but through various tissue culture techniques this toxic content has deceased within the present cultivars of B. carinata. Through tissue culture, there have been developed inbred lines with complete homozygosity, which have facilitated other biotechnological approaches like genetic engineering for production of new varieties for selection of stable resistant lines against biotic and abiotic stresses (Galli et al., 1998; Velasco et al., 2004). Recent report declared B. carinata seed oil can be easily diverted for production of biodiesel and the toxic compounds derivatives can be used as chemical additives in tannery, cosmetic and plastic industries (Bozzini et al., 2007).

To overcome these hurdles in cultivating the crop by modern biotechnologies techniques such as somatic embryogenesis, a suitable protocol is necessary. Thus, for these reason, the present study was conducted for assessment of plant regeneration of Brassica carinata by optimizing basal medium for callus formation and regeneration of shoot and root.

Materials and Methods

Plant material and germination procedure

Brassica carinata seeds were obtained from the Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar. Experimental work was accomplished in laminar flow for prevention of contamination. First step included sterilization of seeds (Bano et al., 2010). In laminar flow, the media was poured into plates and left for solidification. Sterilized seeds when properly dried were transferred to the media and incubated under standard conditions (Figure 1).

Callus induction medium

Germinated plants hypocotyls and cotyledons of about one week were exercised under sterile condition (Liu et al., 2015). These explants of 0.5 to 1 cm were transferred to MS medium supplemented with various concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzyl amino purine (BAP).

Shoot induction

The callus of various phytohormones formation was subcultured further on shoot induction media supplemented with similar concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzyl amino purine (BAP) with addition of silver nitrate (AgNO3) (Khan et al., 2010).

Elongated shoots were transferred to root induction medium containing different level of indole butyric acid (IBA) (Ravanfar et al., 2009).

Results and Discussion

Percent of callus formation

Results of percent callus formation from cotyledon and hypocotyl of B. carinata by different level of NAA and BAP showed that maximum percent was observed with NAA 0.05 level and BAP 0.7 mg/l in both cotyledon (61.11%) and hypocotyl (88.89%). These results were followed by significant different percent of callus formation within NAA0.05 along with BAP 1.5mg/l in cotyledon (50.00%) whereas BAP 1mg/l in hypocotyl (55.56%). As the NAA level increase the percent of callus formation of cotyledon and hypocotyl dropped highly with first and last level of BAP, whereas the middle level of BAP showed fair percent of callus formation. The percent of callus formation of both cotyledon and hypocotyl showed significant variation with least significant difference (LSD) value of 6.84 at p<0.05 of cotyledon and 6.08 at p < 0.05 of hypocotyl (Table 1).

Days to callus formation

Mean days to callus formation was observed with NAA0.05 mg/l and BAP 0.7 mg/l both in cotyledon (21.67 days) and hypocotyl (23.00 days). These results were followed by higher level of NAA (0.1 mg/l) however similar level of BAP (0.7 mg/l) with 23.67 days that of cotyledon and 24.00 days of hypocotyl. The analyzed data showed significant variation with application of hormones level (NAA and BAP) where the LSD value of 0.78 at p < 0.05 was calculated for cotyledon and 0.67 at p < 0.05 for hypocotyl.

Regeneration efficiency of shoot

Regeneration efficiency of Shoot was observed maximum with NAA 0.1 mg/l and BAP 1mg/l in both cotyledon (72.22) and hypocotyl (83.33). A drastic decrease in regeneration efficiency of shoot was observed in all the other treatments level in the present study. In cotyledon NAA 0.5mg/l with BAP 0.7 mg/l and BAP 1mg/l showed fair regeneration efficiency of shoot (38.89). On the other hand, NAA 0.5mg/l with combination of BAP 1mg/l in hypocotyl showed fair regeneration efficiency (44.44). The LSD recorded for shoot regeneration efficiency in cotyledon and hypocotyl were 6.48 and 8.31 at p <0.05 (Table 2).

Table 1: Effect of different level of phytohormones (NAA and BAP) on callus induction by cotyledon and hypocotyls of B. carinata.

The regeneration efficiency of root response to different levels of IBA from cotyledon callus formation showed the highest regeneration efficiency at IBA 0.2 mg/l (61.11) followed by IBA 0.5 mg/l (38.89). As for hypocotyl, the highest significant regeneration efficiency of root was observed at IBA 0.2 mg/l (66.67) which was followed by IBA 0.5 mg/l (44.44). Of the others combination that were IBA 0.5 and IBA 0.1 mg/l showed similar results in both cotyledon and hypocotyl (5.56 and 22.22). The results significantly varied with LSD value of 7.85 in cotyledon callus and 6.80 in hypocotyl callus at p <0.05 (Table 3).

Table 2: Effect of different level of phytohormones (NAA and BAP) on regeneration of shoot by cotyledon and hypocotyls of B. carinata.

Table 3: Effect of different level of IBA on regeneration of root by cotyledon and hypocotyls of B. carinata.

In the present study, explant (cotyledon and hypocotyl) showed highest response of callus formation with NAA 0.1mg/l and BAP 0.7mg/l, whereas hypocotyl response higher than cotyledon regard to callus formation from explant as well as days to callus formation. Similar findings were determining by Ali et al. (2007), who studied effect of 2,4 D effect on callus induction of hypocotyl and cotyledonary leaves of B. napus. According to Cheng et al. (2001), high regeneration capacity is highly dependent up on tissue type which give variant output at various levels of treatments. The present study findings were also supported by Zeynali et al. (2010) and Khan et al. (2010). Zeynali et al. (2010) determine hypcotyl explants being more suitable than cotyledon for somatic embryogenesis. Khan et al. (2010) studied various Brassica genotypes callus induction at different level of NAA and BAP, along with AgNO3. The results with NAA 0.5mg/l and BAP 1mg/l in the study were in line with the findings of Munir et al. (2008), who studied effect of different level of callus media and regeneration media supplement with B. napus.

The highest regeneration efficiency and days to shoot initiation from cotyledon and hypocotyl was observed with 0.1 mg/l NAA, 1mg/l BAP and 0.1 mg/l AgNO3, however overall results showed that cotyledonous callus had higher efficiency compare to hypocotyl callus in developing shoot. These results were in line with the finding of Bano et al. (2010), who evaluate in vitro response of three genotypes of Brassica juncea callogenesis and organogenesis at different level of phytohormones. Moreover, the study also indicated that hypocotyls needed longer callus phase for shoot production and were produced with higher concentration of plant growth regulators (PGRs) of 3 mg/l BAP and 0.5 mg/l NAA. Neha and Ashutosh (2014) observed BAP 0.5-1.0 mg/l and NAA 0.5-1.0 mg/l showed highest number of shoots produce from callus. Similar results were also observed by Kamboj et al. (2015), who determine highly efficient and reproducible plant regeneration and transformation system in Brassica juncea genotypes. According to Tang et al. (2003) addition of AgNO3 very benefits shoot regeneration but Ag2S2O3 have shown superior effect over AgNO3 when used in developing efficient regeneration protocol of Brassica species.

In the study, results showed that IBA 0.2 mg/l was efficient level in regenerating roots from callus of cotyledon and hypocotyl. These findings were in support of Ali et al. (2007), Singh et al. (2009), Ravanfar et al. (2009), Das et al. (2010) and Liu et al. (2015). Ali et al. (2007) and Ravanfar et al. (2009) showed that the highest efficiency regeneration of root was observed with IBA 0.3 mg/l, where others reported that at 0.5 mg/l IBA root regeneration from callus was efficient.

Acknowledgments

Institute of Biotechnology and Genetic Engineering (IBGE) provided all facilities and the seeds being program was kindly provided by Agriculture farm, The University of Agriculture Peshawar.

Author’s Contribution

Rizwan Ullah Shah: Principal Author who conducted study and research. Analyzed the data.

Iqbal Munir: Major Supervisor who perceived the study. Analyzed the data and wrote final draft of the manuscript.

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