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Thiol Compounds, Pre-Conditioning and Orientation of Explants – Important Factors Affecting Regeneration from Cotyledons of Legume Crop Sesbania Aculeata

Thiol Compounds, Pre-Conditioning and Orientation of Explants – Important Factors Affecting... Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 Original paper DOI: 10.2478/agri-2021-0009 THIOL COMPOUNDS, PRE-CONDITIONING AND ORIENTATION OF EXPLANTS – IMPORTANT FACTORS AFFECTING REGENERATION FROM COTYLEDONS OF LEGUME CROP SESBANIA ACULEATA NIKHIL MEHTA, PRIYANKA RAO AND RAMAN SAINI* Kurukshetra University, Kurukshetra, India Mehta, N., Rao, P. and Saini, R. (2021). Thiol compounds, pre-conditioning and orientation of explants ‒ important factors affecting regeneration from cotyledons of legume crop Sesbania aculeata. Agriculture (Poľnohospodárstvo), 67(3), 95 – 102. Sesbania aculeata is a multipurpose legume crop grown primarily for green manuring in the rice-based cropping system. Besides this, it is an industrial crop and is also used as food in many parts of the world. The present work reports for optimization of various parameters (growth medium, plant growth regulators, pre-conditioning, orientation of explant, and presence of thiol compounds) affecting in vitro regeneration using mature cotyledon explants. The 5-day-old mature cotyledon explants excised from seedlings grown on Murashige and Skoog (MS) salts and Gamborg (B5) vitamins medium containing 15 µM 6-benzylaminopurine were cultured with its adaxial side facing on medium containing 2.5 µM 6-benzylaminopurine and 50 mg/L thiourea and produced multiple shoots (7 ‒ 8) in 100% cultures within 28 days. Healthy shoots were rooted on half-strength Murashige and Skoog (MS) salts and full-strength vitamins medium augmented with 2.5 µM indole-3-butyric acid. Key words: cotyledon, explant’s orientation, organogenesis, pre-conditioning, regeneration, shoot multiplication Sesbania aculeata (Pers.) is a leguminous crop some vitamins, and minerals (Hossain et al. 2002). native to India, Pakistan, and other South Asian The fat content is very low (6%), with a relatively countries but found throughout the world (Bunma high proportion of healthy, unsaturated fatty acids. & Balslev 2019). It is a multipurpose crop species But, the use of seeds as food is restricted due to the that is grown for fibre, gums (galactomannan), fuel- presence of anti-nutritional factors like phenols, tan- wood, fodder, paper, dye industry, flowers as food, nins, phytic acid, saponins, and trypsin inhibitors in ornamental, and other agroforestry purposes (Pol- a considerable amount. The problem could not be lard et al. 2011; Swami et al. 2012; Singh & Rani overcome by using traditional breeding approaches 2013; Bunma & Balslev 2019). The plant possesses due to the limited gene pool present. So, to over- various medicinal properties such as alexeteric, come such problems there is a requirement for bio- antihelmintic, diuretic, antibacterial, and anticancer technological approaches. properties and is used in the treatment of skin dis- There is one report for regeneration in S. acule- eases, snake bites, and ophthalmic disorders (Mehta ata (Bansal & Pandey 1993) using hypocotyls and et al. 2018, 2019; Bunma & Balslev 2019). Being leaf explants, but the regeneration protocol is very a legume crop, its seeds are a good source of easily time-consuming. In the current study, we have used digestible protein (33%) along with good fibre con- mature cotyledon explants for regeneration which tent (11%), carbohydrates, essential fatty acids, gave rise to multiple shoots in less time. Mature Nikhil Mehta, Priyanka Rao, Raman Saini (*Corresponding author), Department of Biotechnology, Kurukshetra University, Kurukshetra – 136119, India. E-mail: ramanksaini@rediffmail.com © 2021 Authors. This is an open access article licensed under the Creative Commons Attribution-NonComercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/). 95 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 cotyledon explants have been used previously in rent concentrations of BAP (0.0 – 15.0 µM). Other cytokinins (kinetin, TDZ ‒ Thidiazuron) effect was different Sesbania species for in vitro regeneration also assessed by using equivalent concentration to (Kapoor & Gupta 1986; Shanker & Ram 1990; Yan- BAP and the best concentration was further com- Xiu et al. 1993; Detrez et al. 1994; Mouhamad et al. bined with different concentrations of auxins (IAA 2014). In this perspective, the present study reports ‒ Indole-3-acetic acid, NAA ‒ α-Naphthaleneacetic the first development of an efficient regeneration acid). system using mature cotyledons explants of S. acu- The effect of the orientation of the explants on leata. regeneration was assessed by culturing 5-day old explants horizontally with either the adaxial side or abaxial side facing the medium or by dipping the MATERIAL AND METHODS 2 ‒ 3 mm proximal cut end in the medium. The effect of BAP preconditioning of seedlings Plant material and explant preparation on regeneration was assessed by germinating seeds Sesbania aculeata seeds were procured from the on MSB V medium supplemented with different government seed distribution centre, Kurukshetra, concentrations of BAP (0.0, 5.0, 10.0, 15.0, and 20.0 India. Healthy seeds were segregated and sterilized µM) before excision of explants and then cultured using 70% (v/v) ethanol for 2 minutes followed by the 5-d-old mature cotyledons on optimized shoot using 0.2% (w/v) mercuric chloride solution for up regeneration medium (Figure 1a). Mature cotyl- to 5 minutes. Sterilized seeds were washed with au- edons excised from seedlings germinated on toclaved distilled water several times to remove any MSB V basal medium were used as control. mercuric chloride left. Seeds were then blotted dry Effect of different thiol-containing compounds on sterilized filter paper (GE Healthcare (Whatman), (dithiothreitol, thiourea, L-cysteine, sodium thiosul- Mumbai, India) and transferred to sterilized MSB V phate) on shoot regeneration of S. aculeata was also medium (pH-5.8) composed of MS salts (Murashi- assessed by culturing the 5-day old explants on op- ge & Skoog 1962) and B vitamins (Gamborg et timized shoot regeneration medium supplemented al. 1968), sucrose (30 g/L) and agar-agar (7.5 g/L). with different concentrations of thiol-containing Seeds were germinated at 26 ± 2ºC temperature compounds (0.0 ‒ 125 mg/L). and 16-h illumination of light intensity 80 µE/m /s given by cool white fluorescent lamps (Philips, Rooting and transplantation Delhi, India). Healthy regenerated shoots 3 ‒ 4 cm long were Mature cotyledons were excised by cutting from excised from the base and transferred to the rooting the point of attachment of cotyledons with the em- medium composed of half-strength MS salts and bryonic axis from different age-old seedlings (4-d, full-strength MS vitamins with 30 g/L sucrose and 5-d, 6-d, 7-d, and 8-d). All of the chemicals used in 3.75 g/L Phytagel supplemented with different con- the study were obtained from HiMedia , Mumbai, centrations of indole-3-butyric acid (IBA; 0.0 – 2.5 India unless otherwise indicated. μM). Shoots with well-developed roots were select- ed after 2 weeks and transferred to pots containing Optimization of multiple shoot regeneration Soilrite™ (Keltech Energies Ltd., Bengaluru, In- Optimum media for efficient shoot regeneration dia). A high level of humidity was maintained ini- was assessed by culturing excised explants in Plan- tially which was gradually decreased and ultimately ton containers (Tarsons products ) having different plants were transferred to soil and grown to matu- media viz. MS media (Murashige & Skoog 1962), rity. B media (Gamborg et al. 1968), and MSB V me- 5 5 dia containing Murashige and Skoog (MS) salts Data analysis All the in vitro regeneration experiments were and Gamborg (B ) vitamins, supplemented with repeated thrice, using 20 replicates each time. Quan- 2.5 µM BAP (6-Benzylaminopurine). To determine tification of each treatment was based on the percen- the effective concentration of BAP, explants were tage of regenerated explants, percentage of rooted cultured on MSB V medium augmented with diffe- 96 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 shoots and the average number and length of shoots plants. This combined media had not been tested regenerated per explant. Newman-Keuls multiple before for S. aculeata but had been found effective range test (Brunning & Kintz 1977) was used for the regeneration medium on other legumes (Saini et al. variance and significant differences analysis of the 2005; Mekala et al. 2016). The addition of cytoki- data related to the number, and the length of shoots nin resulted in multiple shoot regeneration within per explant. 7 ‒ 8 days of culture from the proximal end of the 5-day old explant. The best multiple shoot regenera- tion response was obtained at 2.5 µM BAP concen- RESULTS AND DISCUSSION tration (Table 1). Moving towards the higher BAP concentration resulted in the decreased regeneration In vitro multiplication of shoots potential. Substitution of BAP with equimolar con- Mature cotyledon explant cultured on basal me- centration of other cytokinins viz kinetin and TDZ dium without any growth regulator did not give rise (Thidiazuron) resulted in decreased regeneration to any shoot rather it remained green and no growth potential but the decrease with kinetin addition was was observed. The addition of cytokinin in the me- non-significant as compared to significant reduction dium resulted in multiple shoot formation. The most by addition of TDZ in the medium. There is no ear- effective medium for shoot induction was found to be lier report of regeneration from mature cotyledon in the MSB V (Murashige and Skoog’s salts and Gam- S. aculeata but the explant has been used in other borg’s vitamins) medium (5.1 shoots per explant) Sesbania species. The results are in accordance with followed by MS (Murashige and Skoog’s) medium earlier reports where BAP alone had been reported (4.6 shoots per explant) and B (Gamborg) medium efficient for regeneration using cotyledon explants (4.2 shoots per explant) supplemented with 2.5 µM in S. bispinosa (Kapoor & Gupta 1986), Leucaena BAP (6-Benzylaminopurine), using 5-day old ex- leucocephala (Saafi & Borthakur 2002), and Dal- T a b l e 1 Effect of different growth regulators on multiple shoot regeneration from 5-d-old mature cotyledon explants of Sesbania aculeata Regeneration Mean average number Mean average length of Growth regulators [μM] [%] of shoots/explants* shoots [cm] a a 0.0 100 0.0 ± 0.00 0.0 ± 0.00 cd d 1.0 100 3.5 ± 0.35 4.4 ± 0.27 e d 2.5 100 5.1 ± 0.39 3.8 ± 0.41 BAP cde c 5.0 95 4.2 ± 0.30 2.4 ± 0.36 cd bc 10.0 90 3.7 ± 0.29 1.9 ± 0.30 b b 15.0 70 1.3 ± 0.12 1.1 ± 0.30 cd d KIN 2.5 95 4.4 ± 0.34 4.2 ± 0.46 cd c TDZ 2.5 90 3.5 ± 0.29 2.3 ± 0.19 c c BAP + IAA 2.5 + 0.5 70 3.3 ± 0.27 3.3 ± 0.39 c c BAP + IAA 2.5 + 1.0 60 3.1 ± 0.34 3.2 ± 0.24 c c BAP + NAA 2.5 + 0.5 65 3.2 ± 0.21 3.6 ± 0.12 c c BAP + NAA 2.5 + 1.0 60 2.9 ± 0.53 3.5 ± 0.46 Data scored after 4 weeks of cultures on MSB V medium supplemented with or without growth regulators. *Values are the mean ± SD (standard deviation), followed by same letter within columns are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: BAP ‒ 6-Benzylaminopurine; KIN ‒ kinetin; MSB V ‒ Murashige and Skoog salts and Gamborg vitamins; TDZ ‒ thidiazuron. 97 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95−102 bergia sissoo (Singh et al. 2002) but required high old explants were found most responsive towards concentration of BAP. Combining auxin (IAA ‒ In- multiple shoot regeneration. With the increasing age dole-3-acetic acid and NAA ‒ α-Naphthaleneacetic of the explants, a decline in regeneration potential acid) at different concentrations (0.5 and 1.0 µM) was observed on MSB V medium supplemented with the BAP (2.5 µM) in the medium resulted in with 2.5 µM BAP (Table 2). Similar results were ob- a decrease in the regeneration potential significantly. served in S. grandiflora (Detrez et al. 1994) where These results are in converse to the earlier reports in 3-day old explants resulted in efficient shoot multi - other Sesbania species where the addition of aux- plication using cotyledon explants. However, con- ins had resulted in increased regeneration potential verse with other reports in Sesbania species where in cotyledons explants (Yan-Xiu et al. 1993; Detrez older explants resulted in better shoot multiplication et al. 1994). However, the results are in accordance than the younger ones (Kapoor & Gupta 1986; Yan- with the earlier reports in Vigna mungo (Sainger et Xiu et al. 1993; Shanker & Ram 1990). al. 2015) and Annona squamosa (Nagori & Purohit Orientation of the 5-day old explants in the cul- 2004) where the addition of auxins didn’t increase ture significantly affects the regeneration potential. the regeneration potential. Explants cultured horizontally with their adaxial Among the different age tested (4-d, 5-d, 6-d, side facing the medium gave the best response of 7-d, and 8-d) for mature cotyledon explants, 5-day T a b l e 2 Effect of age of mature cotyledon explants of Sesbania aculeata on in vitro regeneration Regeneration Mean average number of Mean average lenth of shoots Age of explant [%] shoots/explants* [cm] c b 4-d 100 4.7 ± 0.42 4.2 ± 0.27 c b 5-d 100 5.1 ± 0.39 3.8 ± 0.41 bc b 6-d 90 4.2 ± 0.55 3.7 ± 0.28 ab ab 7-d 80 3.6 ± 0.75 3.3 ± 0.71 a a 8-d 70 2.7 ± 0.53 2.4 ± 0.43 Data scored after 4 weeks of cultures on MSB V medium supplemented with 2.5 μM BAP. *Values are the mean ± SD (standard deviation), mean values within columns followed by same letter are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: BAP ‒ 6-Benzylaminopurine; MSB V ‒ Mu- rashige and Skoog salts and Gamborg vitamins. T a b l e 3 Effect of BAP preconditioning for 5 days on in vitro regeneration of mature cotyledon explants of Sesbania aculeata Concentration Regeneration Mean average number of Mean average length of shoots [μM] [%] shoots/explants* [cm] a a 0.0 100 5.1 ± 0.39 3.8 ± 0.41 ab a 5.0 100 5.4 ± 0.53 3.8 ± 0.27 ab a 10.0 100 5.7 ± 0.18 3.9 ± 0.15 b a 15.0 100 6.3 ± 0.44 3.9 ± 0.48 a a 20.0 95 5.2 ± 0.27 3.7 ± 0.62 Data scored after 4 weeks of cultures on MSB V medium supplemented with 2.5 μM BAP. *Values are the mean ± SD (standard deviation), mean values within columns followed by same letter are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: BAP ‒ 6-Benzylaminopurine; MSB V ‒ Mu- rashige and Skoog salts and Gamborg vitamins. 98 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 5.1 shoots per explant in 100% of cultures. While of Sesamum indicum (Debnath et al. 2018) when the explants with a dipped proximal end, initial- placed vertically in the medium, were reported to ly formed callus followed by the formation of 3.6 have better regeneration potential. shoots per explant in 70% of cultures whereas hor- The effect of pre-conditioning for 5-days with izontal explants with their abaxial side facing me- a high dose of BAP was assessed and it was found dium produced 2.4 shoots per explant preceded by to increase the regeneration assessed by the num- callus in 45% of cultures. The results are in accor- ber of shoots formed per explant. The increase in dance with the previous report with cotyledon ex- BAP concentration in the germination medium in- plants in Vigna subterranea (Koné et al. 2013) and creased the number of shoots formed per explant up Helianthus annuus (Sujatha et al. 2012) where hori- to the concentration of 15 µM BAP that was found zontal explants with adaxial side facing medium most responsive (Table 3). Above this concentration gave the best results. However, cotyledon explants (20 µM BAP), there was a decrease in regeneration T a b l e 4 Effect of different concentration of thiol containing compounds on in vitro regeneration of mature cotyledon explants of 1,2 Sesbania aculeata Regeneration Mean average number Mean average length of Thiol compounds [mg/L] [%] of shoots/explants* shoots* [cm] d a None (Control) 0 100 6.3 ± 0.44 3.9 ± 0.48 a a 25 100 6.4 ± 0.22 4.0 ± 0.16 de a 50 100 6.5 ± 0.42 4.0 ± 0.32 de a L-cysteine 75 100 6.6 ± 0.50 4.1 ± 0.31 de a 100 100 6.7 ± 0.65 4.0 ± 0.45 b a 125 70 4.4 ± 0.22 3.6 ± 0.54 de a 25 100 6.8 ± 0.32 4.0 ± 0.17 e a 50 100 7.3 ± 0.22 4.0 ± 0.39 bc a Thiourea 75 80 5.2 ± 0.42 3.8 ± 0.24 b a 100 65 4.0 ± 0.18 3.6 ± 0.29 a a 125 50 2.8 ± 0.56 3.5 ± 0.64 de a 25 100 6.4 ± 0.18 4.0 ± 0.27 de a 50 100 6.5 ± 0.50 3.9 ± 0.44 bc a DTT 75 80 5.0 ± 0.35 4.1 ± 0.51 b a 100 70 4.2 ± 0.44 3.8 ± 0.32 a a 125 55 3.0 ± 0.25 3.6 ± 0.14 d a 25 100 6.4 ± 0.28 4.1 ± 0.29 de a 50 100 6.5 ± 0.59 4.0 ± 0.54 STS de a 75 100 6.7 ± 0.38 4.1 ± 0.18 bc a 100 75 5.4 ± 0.24 3.9 ± 0.22 Exokabts excised from 5 day old seedlings germinated on MSB5V medium supplemented with 15μM BAP. Data scored after 4 weeks of cultures on MSB V medium supplemented with 2.5 μM BAP and various concentration of thiol compounds. *Values are the mean ± SD (standard deviation), followed by same letter within columns are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: DTT ‒ dithiothreitol; STS ‒ sodium thiosulphate; BAP ‒ 6-Benzylaminopurine; MSB V ‒ Murashige and Skoog salts and Gamborg vitamins. 99 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 −102 potential but the difference was non-significantly present naturally in the cotyledons are required for higher than the non-pre-conditioned explants. This the solubilization of the stored nutrients. Thus, to enhancing effect of pre-conditioning on shoot re- counter the effect of auxins, pre-conditioning using generation has been reported earlier in different ex- a high amount of BAP was applied that helps to plants of several legumes like Cajanus cajan (Shiva move the auxin-cytokinin ratio towards the cytoki- Prakash et al. 1994), Phaseolus spp. (Santalla et al. nin side, which is required for multiple shoot proli- 1998), Vigna radiata (Avenido et al. 2001), Vigna feration. mungo (Saini & Jaiwal 2005), Vigna unguiculata The addition of different thiol-containing com- (Chaudhury et al. 2007). In our study, pre-conditio- pounds has been found beneficial for shoot organo- ning was found to increase the size and thickness of genesis. Thiol-containing compounds are natural the cotyledons. This might be due to the increase in anti-oxidants that have been reported to protect the stored nutrients in the cotyledon by mobiliza- plants against stress by removing the free radicals tion from the growth medium in presence of BAP. from the tissues (Włodek 2002). The addition of the According to Nanda and Anand (1970), high auxins thiol compounds reduces the effect of phenolics re- Figure 1. Plant regeneration from mature cotyledon explants of Sesbania aculeata: (a) Mature cotyledon explant excised from pre-conditioned (15 µM BAP) 5-day old seedling; (b) Multiple shoot regeneration from mature cotyledon explants after 21 days of culture on MSB5V medium (Murashige and Skoog salts and Gamborg vitamins) supplemented with 2.5 µM BAP (6-Benzylami- nopurine) and 50 mg/L thiourea; (c) Induction of roots from shoot on rooting medium containing half strength MS (Murashige and Skoog medium) salts and full MS vitamins supplemented with 2.5 µM IBA (Indole-3-butyric acid); (d) Fertile plant growing in pot. 100 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 −102 leased in the medium by tissue thus preventing the tion with BAP, age of explant, different media (MS necrosis of the explants. The best multiple shoot- ‒ Murashige and Skoog’s medium /B ‒ Gamborg ing response was found with 5-day old explants on medium /MSB V ‒ Murashige and Skoog’s medium pre-optimized shooting medium (MSB V + 2.5 µM salts and Gamborg medium vitamins), the orienta- BAP), supplemented with 50 mg/L thiourea (Figure tion of explant on culture medium, BAP pre-condi- 1b; Table 4). The effect of other thiol compounds tioning, and addition of thiol compounds that affect was non-significantly less than the thiourea giving the regeneration potential has been assessed and their best response at 50 mg/L DTT (dithiothreitol), optimized. Mature cotyledon explants excised from 75 mg/L STS (sodium thiosulphate), and 100 mg/L 5-day old seedlings germinated on MSB V medium L-cysteine. The further increase in concentration containing 15 µM BAP when cultured on MSB V leads to a decrease in regeneration potential. The medium containing 2.5 µM BAP and 50 mg/L results are in accordance with the previous report, thiourea gave rise to multiple shoots (average 7.3 where low concentrations of thiol-containing com- shoots per explant), in 28 days. This is a direct meth- pounds resulted in increased regeneration potential, od of shoot regeneration which avoids any chances whereas at high concentration, tissue necrosis oc- of somaclonal variations. The developed system is curred (Sivanandhan et al. 2015). This decrease in simple and fast, giving rise to plantlets in just 6 ‒ 7 the regeneration potential at higher concentrations weeks. In the future, the developed efficient and re- must be related to the formation of thiyl radicals producible regeneration protocol could be used for that reacts with metals such as iron in the media and transformation experiments to manipulate the crop form transition metal ions that are toxic to the plant for desirable agronomically important traits. tissues (Włodek 2002). Acknowledgement: Nikhil Mehta and Priyanka Rooting and transplantation Rao are thankful to the University Grant Commis- Healthy, thick, and branched roots were induced sion, New Delhi for the award of Senior Research in 100% of healthy shoots transferred to the rooting Fellowship. medium containing 2.5 µM IBA (Indole-3-butyric acid) (Figure 1c). Hormone-free rooting medium Authors’ contributions: NM- performed experi- also resulted in shoot formation in 70% of culture, ments and drafted the original manuscript; PR and but the roots so formed were thin and less branched. NM ‒ collected and analysed the data; RS-designed The addition of 0.5 µM IBA concentration increased the research, made available the lab facilities, edit rooting to 80% of culture but the quality of the roots the manuscript. was not changed significantly. However, 1 µM IBA Declaration of conflicts of interest/competing concentration resulted in increased branching and interest: There are no conflicts of interest in this rooting in 90% of cultures, though roots remained work. thin. The results are in accordance with the earlier reports in different Sesbania species (Jha et al. 2004; Mouhamad et al. 2014) where the addition of auxin REFERENCES in the medium significantly increased the formation of the roots. Seventy percent of the rooted shoots Avenido, R., Motoda, J. and Hattori, K. (2001). 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Received: April 6, 2021 Nanda, K.K. and Anand, V.K. (1970). Seasonal changes in auxin Accepted: June 16, 2021 effects on rooting of stem cuttings of Populus nigra and its relationship with mobilization of starch. Physiologia Plan- tarum, 23(1), 99 ‒ 107. DOI:10.1111/j.1399-3054.1970. tb06396.x. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agriculture de Gruyter

Thiol Compounds, Pre-Conditioning and Orientation of Explants – Important Factors Affecting Regeneration from Cotyledons of Legume Crop Sesbania Aculeata

Agriculture , Volume 67 (3): 8 – Oct 1, 2021

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Abstract

Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 Original paper DOI: 10.2478/agri-2021-0009 THIOL COMPOUNDS, PRE-CONDITIONING AND ORIENTATION OF EXPLANTS – IMPORTANT FACTORS AFFECTING REGENERATION FROM COTYLEDONS OF LEGUME CROP SESBANIA ACULEATA NIKHIL MEHTA, PRIYANKA RAO AND RAMAN SAINI* Kurukshetra University, Kurukshetra, India Mehta, N., Rao, P. and Saini, R. (2021). Thiol compounds, pre-conditioning and orientation of explants ‒ important factors affecting regeneration from cotyledons of legume crop Sesbania aculeata. Agriculture (Poľnohospodárstvo), 67(3), 95 – 102. Sesbania aculeata is a multipurpose legume crop grown primarily for green manuring in the rice-based cropping system. Besides this, it is an industrial crop and is also used as food in many parts of the world. The present work reports for optimization of various parameters (growth medium, plant growth regulators, pre-conditioning, orientation of explant, and presence of thiol compounds) affecting in vitro regeneration using mature cotyledon explants. The 5-day-old mature cotyledon explants excised from seedlings grown on Murashige and Skoog (MS) salts and Gamborg (B5) vitamins medium containing 15 µM 6-benzylaminopurine were cultured with its adaxial side facing on medium containing 2.5 µM 6-benzylaminopurine and 50 mg/L thiourea and produced multiple shoots (7 ‒ 8) in 100% cultures within 28 days. Healthy shoots were rooted on half-strength Murashige and Skoog (MS) salts and full-strength vitamins medium augmented with 2.5 µM indole-3-butyric acid. Key words: cotyledon, explant’s orientation, organogenesis, pre-conditioning, regeneration, shoot multiplication Sesbania aculeata (Pers.) is a leguminous crop some vitamins, and minerals (Hossain et al. 2002). native to India, Pakistan, and other South Asian The fat content is very low (6%), with a relatively countries but found throughout the world (Bunma high proportion of healthy, unsaturated fatty acids. & Balslev 2019). It is a multipurpose crop species But, the use of seeds as food is restricted due to the that is grown for fibre, gums (galactomannan), fuel- presence of anti-nutritional factors like phenols, tan- wood, fodder, paper, dye industry, flowers as food, nins, phytic acid, saponins, and trypsin inhibitors in ornamental, and other agroforestry purposes (Pol- a considerable amount. The problem could not be lard et al. 2011; Swami et al. 2012; Singh & Rani overcome by using traditional breeding approaches 2013; Bunma & Balslev 2019). The plant possesses due to the limited gene pool present. So, to over- various medicinal properties such as alexeteric, come such problems there is a requirement for bio- antihelmintic, diuretic, antibacterial, and anticancer technological approaches. properties and is used in the treatment of skin dis- There is one report for regeneration in S. acule- eases, snake bites, and ophthalmic disorders (Mehta ata (Bansal & Pandey 1993) using hypocotyls and et al. 2018, 2019; Bunma & Balslev 2019). Being leaf explants, but the regeneration protocol is very a legume crop, its seeds are a good source of easily time-consuming. In the current study, we have used digestible protein (33%) along with good fibre con- mature cotyledon explants for regeneration which tent (11%), carbohydrates, essential fatty acids, gave rise to multiple shoots in less time. Mature Nikhil Mehta, Priyanka Rao, Raman Saini (*Corresponding author), Department of Biotechnology, Kurukshetra University, Kurukshetra – 136119, India. E-mail: ramanksaini@rediffmail.com © 2021 Authors. This is an open access article licensed under the Creative Commons Attribution-NonComercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/). 95 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 cotyledon explants have been used previously in rent concentrations of BAP (0.0 – 15.0 µM). Other cytokinins (kinetin, TDZ ‒ Thidiazuron) effect was different Sesbania species for in vitro regeneration also assessed by using equivalent concentration to (Kapoor & Gupta 1986; Shanker & Ram 1990; Yan- BAP and the best concentration was further com- Xiu et al. 1993; Detrez et al. 1994; Mouhamad et al. bined with different concentrations of auxins (IAA 2014). In this perspective, the present study reports ‒ Indole-3-acetic acid, NAA ‒ α-Naphthaleneacetic the first development of an efficient regeneration acid). system using mature cotyledons explants of S. acu- The effect of the orientation of the explants on leata. regeneration was assessed by culturing 5-day old explants horizontally with either the adaxial side or abaxial side facing the medium or by dipping the MATERIAL AND METHODS 2 ‒ 3 mm proximal cut end in the medium. The effect of BAP preconditioning of seedlings Plant material and explant preparation on regeneration was assessed by germinating seeds Sesbania aculeata seeds were procured from the on MSB V medium supplemented with different government seed distribution centre, Kurukshetra, concentrations of BAP (0.0, 5.0, 10.0, 15.0, and 20.0 India. Healthy seeds were segregated and sterilized µM) before excision of explants and then cultured using 70% (v/v) ethanol for 2 minutes followed by the 5-d-old mature cotyledons on optimized shoot using 0.2% (w/v) mercuric chloride solution for up regeneration medium (Figure 1a). Mature cotyl- to 5 minutes. Sterilized seeds were washed with au- edons excised from seedlings germinated on toclaved distilled water several times to remove any MSB V basal medium were used as control. mercuric chloride left. Seeds were then blotted dry Effect of different thiol-containing compounds on sterilized filter paper (GE Healthcare (Whatman), (dithiothreitol, thiourea, L-cysteine, sodium thiosul- Mumbai, India) and transferred to sterilized MSB V phate) on shoot regeneration of S. aculeata was also medium (pH-5.8) composed of MS salts (Murashi- assessed by culturing the 5-day old explants on op- ge & Skoog 1962) and B vitamins (Gamborg et timized shoot regeneration medium supplemented al. 1968), sucrose (30 g/L) and agar-agar (7.5 g/L). with different concentrations of thiol-containing Seeds were germinated at 26 ± 2ºC temperature compounds (0.0 ‒ 125 mg/L). and 16-h illumination of light intensity 80 µE/m /s given by cool white fluorescent lamps (Philips, Rooting and transplantation Delhi, India). Healthy regenerated shoots 3 ‒ 4 cm long were Mature cotyledons were excised by cutting from excised from the base and transferred to the rooting the point of attachment of cotyledons with the em- medium composed of half-strength MS salts and bryonic axis from different age-old seedlings (4-d, full-strength MS vitamins with 30 g/L sucrose and 5-d, 6-d, 7-d, and 8-d). All of the chemicals used in 3.75 g/L Phytagel supplemented with different con- the study were obtained from HiMedia , Mumbai, centrations of indole-3-butyric acid (IBA; 0.0 – 2.5 India unless otherwise indicated. μM). Shoots with well-developed roots were select- ed after 2 weeks and transferred to pots containing Optimization of multiple shoot regeneration Soilrite™ (Keltech Energies Ltd., Bengaluru, In- Optimum media for efficient shoot regeneration dia). A high level of humidity was maintained ini- was assessed by culturing excised explants in Plan- tially which was gradually decreased and ultimately ton containers (Tarsons products ) having different plants were transferred to soil and grown to matu- media viz. MS media (Murashige & Skoog 1962), rity. B media (Gamborg et al. 1968), and MSB V me- 5 5 dia containing Murashige and Skoog (MS) salts Data analysis All the in vitro regeneration experiments were and Gamborg (B ) vitamins, supplemented with repeated thrice, using 20 replicates each time. Quan- 2.5 µM BAP (6-Benzylaminopurine). To determine tification of each treatment was based on the percen- the effective concentration of BAP, explants were tage of regenerated explants, percentage of rooted cultured on MSB V medium augmented with diffe- 96 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 shoots and the average number and length of shoots plants. This combined media had not been tested regenerated per explant. Newman-Keuls multiple before for S. aculeata but had been found effective range test (Brunning & Kintz 1977) was used for the regeneration medium on other legumes (Saini et al. variance and significant differences analysis of the 2005; Mekala et al. 2016). The addition of cytoki- data related to the number, and the length of shoots nin resulted in multiple shoot regeneration within per explant. 7 ‒ 8 days of culture from the proximal end of the 5-day old explant. The best multiple shoot regenera- tion response was obtained at 2.5 µM BAP concen- RESULTS AND DISCUSSION tration (Table 1). Moving towards the higher BAP concentration resulted in the decreased regeneration In vitro multiplication of shoots potential. Substitution of BAP with equimolar con- Mature cotyledon explant cultured on basal me- centration of other cytokinins viz kinetin and TDZ dium without any growth regulator did not give rise (Thidiazuron) resulted in decreased regeneration to any shoot rather it remained green and no growth potential but the decrease with kinetin addition was was observed. The addition of cytokinin in the me- non-significant as compared to significant reduction dium resulted in multiple shoot formation. The most by addition of TDZ in the medium. There is no ear- effective medium for shoot induction was found to be lier report of regeneration from mature cotyledon in the MSB V (Murashige and Skoog’s salts and Gam- S. aculeata but the explant has been used in other borg’s vitamins) medium (5.1 shoots per explant) Sesbania species. The results are in accordance with followed by MS (Murashige and Skoog’s) medium earlier reports where BAP alone had been reported (4.6 shoots per explant) and B (Gamborg) medium efficient for regeneration using cotyledon explants (4.2 shoots per explant) supplemented with 2.5 µM in S. bispinosa (Kapoor & Gupta 1986), Leucaena BAP (6-Benzylaminopurine), using 5-day old ex- leucocephala (Saafi & Borthakur 2002), and Dal- T a b l e 1 Effect of different growth regulators on multiple shoot regeneration from 5-d-old mature cotyledon explants of Sesbania aculeata Regeneration Mean average number Mean average length of Growth regulators [μM] [%] of shoots/explants* shoots [cm] a a 0.0 100 0.0 ± 0.00 0.0 ± 0.00 cd d 1.0 100 3.5 ± 0.35 4.4 ± 0.27 e d 2.5 100 5.1 ± 0.39 3.8 ± 0.41 BAP cde c 5.0 95 4.2 ± 0.30 2.4 ± 0.36 cd bc 10.0 90 3.7 ± 0.29 1.9 ± 0.30 b b 15.0 70 1.3 ± 0.12 1.1 ± 0.30 cd d KIN 2.5 95 4.4 ± 0.34 4.2 ± 0.46 cd c TDZ 2.5 90 3.5 ± 0.29 2.3 ± 0.19 c c BAP + IAA 2.5 + 0.5 70 3.3 ± 0.27 3.3 ± 0.39 c c BAP + IAA 2.5 + 1.0 60 3.1 ± 0.34 3.2 ± 0.24 c c BAP + NAA 2.5 + 0.5 65 3.2 ± 0.21 3.6 ± 0.12 c c BAP + NAA 2.5 + 1.0 60 2.9 ± 0.53 3.5 ± 0.46 Data scored after 4 weeks of cultures on MSB V medium supplemented with or without growth regulators. *Values are the mean ± SD (standard deviation), followed by same letter within columns are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: BAP ‒ 6-Benzylaminopurine; KIN ‒ kinetin; MSB V ‒ Murashige and Skoog salts and Gamborg vitamins; TDZ ‒ thidiazuron. 97 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95−102 bergia sissoo (Singh et al. 2002) but required high old explants were found most responsive towards concentration of BAP. Combining auxin (IAA ‒ In- multiple shoot regeneration. With the increasing age dole-3-acetic acid and NAA ‒ α-Naphthaleneacetic of the explants, a decline in regeneration potential acid) at different concentrations (0.5 and 1.0 µM) was observed on MSB V medium supplemented with the BAP (2.5 µM) in the medium resulted in with 2.5 µM BAP (Table 2). Similar results were ob- a decrease in the regeneration potential significantly. served in S. grandiflora (Detrez et al. 1994) where These results are in converse to the earlier reports in 3-day old explants resulted in efficient shoot multi - other Sesbania species where the addition of aux- plication using cotyledon explants. However, con- ins had resulted in increased regeneration potential verse with other reports in Sesbania species where in cotyledons explants (Yan-Xiu et al. 1993; Detrez older explants resulted in better shoot multiplication et al. 1994). However, the results are in accordance than the younger ones (Kapoor & Gupta 1986; Yan- with the earlier reports in Vigna mungo (Sainger et Xiu et al. 1993; Shanker & Ram 1990). al. 2015) and Annona squamosa (Nagori & Purohit Orientation of the 5-day old explants in the cul- 2004) where the addition of auxins didn’t increase ture significantly affects the regeneration potential. the regeneration potential. Explants cultured horizontally with their adaxial Among the different age tested (4-d, 5-d, 6-d, side facing the medium gave the best response of 7-d, and 8-d) for mature cotyledon explants, 5-day T a b l e 2 Effect of age of mature cotyledon explants of Sesbania aculeata on in vitro regeneration Regeneration Mean average number of Mean average lenth of shoots Age of explant [%] shoots/explants* [cm] c b 4-d 100 4.7 ± 0.42 4.2 ± 0.27 c b 5-d 100 5.1 ± 0.39 3.8 ± 0.41 bc b 6-d 90 4.2 ± 0.55 3.7 ± 0.28 ab ab 7-d 80 3.6 ± 0.75 3.3 ± 0.71 a a 8-d 70 2.7 ± 0.53 2.4 ± 0.43 Data scored after 4 weeks of cultures on MSB V medium supplemented with 2.5 μM BAP. *Values are the mean ± SD (standard deviation), mean values within columns followed by same letter are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: BAP ‒ 6-Benzylaminopurine; MSB V ‒ Mu- rashige and Skoog salts and Gamborg vitamins. T a b l e 3 Effect of BAP preconditioning for 5 days on in vitro regeneration of mature cotyledon explants of Sesbania aculeata Concentration Regeneration Mean average number of Mean average length of shoots [μM] [%] shoots/explants* [cm] a a 0.0 100 5.1 ± 0.39 3.8 ± 0.41 ab a 5.0 100 5.4 ± 0.53 3.8 ± 0.27 ab a 10.0 100 5.7 ± 0.18 3.9 ± 0.15 b a 15.0 100 6.3 ± 0.44 3.9 ± 0.48 a a 20.0 95 5.2 ± 0.27 3.7 ± 0.62 Data scored after 4 weeks of cultures on MSB V medium supplemented with 2.5 μM BAP. *Values are the mean ± SD (standard deviation), mean values within columns followed by same letter are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: BAP ‒ 6-Benzylaminopurine; MSB V ‒ Mu- rashige and Skoog salts and Gamborg vitamins. 98 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 − 102 5.1 shoots per explant in 100% of cultures. While of Sesamum indicum (Debnath et al. 2018) when the explants with a dipped proximal end, initial- placed vertically in the medium, were reported to ly formed callus followed by the formation of 3.6 have better regeneration potential. shoots per explant in 70% of cultures whereas hor- The effect of pre-conditioning for 5-days with izontal explants with their abaxial side facing me- a high dose of BAP was assessed and it was found dium produced 2.4 shoots per explant preceded by to increase the regeneration assessed by the num- callus in 45% of cultures. The results are in accor- ber of shoots formed per explant. The increase in dance with the previous report with cotyledon ex- BAP concentration in the germination medium in- plants in Vigna subterranea (Koné et al. 2013) and creased the number of shoots formed per explant up Helianthus annuus (Sujatha et al. 2012) where hori- to the concentration of 15 µM BAP that was found zontal explants with adaxial side facing medium most responsive (Table 3). Above this concentration gave the best results. However, cotyledon explants (20 µM BAP), there was a decrease in regeneration T a b l e 4 Effect of different concentration of thiol containing compounds on in vitro regeneration of mature cotyledon explants of 1,2 Sesbania aculeata Regeneration Mean average number Mean average length of Thiol compounds [mg/L] [%] of shoots/explants* shoots* [cm] d a None (Control) 0 100 6.3 ± 0.44 3.9 ± 0.48 a a 25 100 6.4 ± 0.22 4.0 ± 0.16 de a 50 100 6.5 ± 0.42 4.0 ± 0.32 de a L-cysteine 75 100 6.6 ± 0.50 4.1 ± 0.31 de a 100 100 6.7 ± 0.65 4.0 ± 0.45 b a 125 70 4.4 ± 0.22 3.6 ± 0.54 de a 25 100 6.8 ± 0.32 4.0 ± 0.17 e a 50 100 7.3 ± 0.22 4.0 ± 0.39 bc a Thiourea 75 80 5.2 ± 0.42 3.8 ± 0.24 b a 100 65 4.0 ± 0.18 3.6 ± 0.29 a a 125 50 2.8 ± 0.56 3.5 ± 0.64 de a 25 100 6.4 ± 0.18 4.0 ± 0.27 de a 50 100 6.5 ± 0.50 3.9 ± 0.44 bc a DTT 75 80 5.0 ± 0.35 4.1 ± 0.51 b a 100 70 4.2 ± 0.44 3.8 ± 0.32 a a 125 55 3.0 ± 0.25 3.6 ± 0.14 d a 25 100 6.4 ± 0.28 4.1 ± 0.29 de a 50 100 6.5 ± 0.59 4.0 ± 0.54 STS de a 75 100 6.7 ± 0.38 4.1 ± 0.18 bc a 100 75 5.4 ± 0.24 3.9 ± 0.22 Exokabts excised from 5 day old seedlings germinated on MSB5V medium supplemented with 15μM BAP. Data scored after 4 weeks of cultures on MSB V medium supplemented with 2.5 μM BAP and various concentration of thiol compounds. *Values are the mean ± SD (standard deviation), followed by same letter within columns are not significantly different according to Newman-Keul´s multiple range test (P = 0.05). Note: DTT ‒ dithiothreitol; STS ‒ sodium thiosulphate; BAP ‒ 6-Benzylaminopurine; MSB V ‒ Murashige and Skoog salts and Gamborg vitamins. 99 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 −102 potential but the difference was non-significantly present naturally in the cotyledons are required for higher than the non-pre-conditioned explants. This the solubilization of the stored nutrients. Thus, to enhancing effect of pre-conditioning on shoot re- counter the effect of auxins, pre-conditioning using generation has been reported earlier in different ex- a high amount of BAP was applied that helps to plants of several legumes like Cajanus cajan (Shiva move the auxin-cytokinin ratio towards the cytoki- Prakash et al. 1994), Phaseolus spp. (Santalla et al. nin side, which is required for multiple shoot proli- 1998), Vigna radiata (Avenido et al. 2001), Vigna feration. mungo (Saini & Jaiwal 2005), Vigna unguiculata The addition of different thiol-containing com- (Chaudhury et al. 2007). In our study, pre-conditio- pounds has been found beneficial for shoot organo- ning was found to increase the size and thickness of genesis. Thiol-containing compounds are natural the cotyledons. This might be due to the increase in anti-oxidants that have been reported to protect the stored nutrients in the cotyledon by mobiliza- plants against stress by removing the free radicals tion from the growth medium in presence of BAP. from the tissues (Włodek 2002). The addition of the According to Nanda and Anand (1970), high auxins thiol compounds reduces the effect of phenolics re- Figure 1. Plant regeneration from mature cotyledon explants of Sesbania aculeata: (a) Mature cotyledon explant excised from pre-conditioned (15 µM BAP) 5-day old seedling; (b) Multiple shoot regeneration from mature cotyledon explants after 21 days of culture on MSB5V medium (Murashige and Skoog salts and Gamborg vitamins) supplemented with 2.5 µM BAP (6-Benzylami- nopurine) and 50 mg/L thiourea; (c) Induction of roots from shoot on rooting medium containing half strength MS (Murashige and Skoog medium) salts and full MS vitamins supplemented with 2.5 µM IBA (Indole-3-butyric acid); (d) Fertile plant growing in pot. 100 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 95 −102 leased in the medium by tissue thus preventing the tion with BAP, age of explant, different media (MS necrosis of the explants. The best multiple shoot- ‒ Murashige and Skoog’s medium /B ‒ Gamborg ing response was found with 5-day old explants on medium /MSB V ‒ Murashige and Skoog’s medium pre-optimized shooting medium (MSB V + 2.5 µM salts and Gamborg medium vitamins), the orienta- BAP), supplemented with 50 mg/L thiourea (Figure tion of explant on culture medium, BAP pre-condi- 1b; Table 4). The effect of other thiol compounds tioning, and addition of thiol compounds that affect was non-significantly less than the thiourea giving the regeneration potential has been assessed and their best response at 50 mg/L DTT (dithiothreitol), optimized. Mature cotyledon explants excised from 75 mg/L STS (sodium thiosulphate), and 100 mg/L 5-day old seedlings germinated on MSB V medium L-cysteine. The further increase in concentration containing 15 µM BAP when cultured on MSB V leads to a decrease in regeneration potential. The medium containing 2.5 µM BAP and 50 mg/L results are in accordance with the previous report, thiourea gave rise to multiple shoots (average 7.3 where low concentrations of thiol-containing com- shoots per explant), in 28 days. This is a direct meth- pounds resulted in increased regeneration potential, od of shoot regeneration which avoids any chances whereas at high concentration, tissue necrosis oc- of somaclonal variations. The developed system is curred (Sivanandhan et al. 2015). This decrease in simple and fast, giving rise to plantlets in just 6 ‒ 7 the regeneration potential at higher concentrations weeks. In the future, the developed efficient and re- must be related to the formation of thiyl radicals producible regeneration protocol could be used for that reacts with metals such as iron in the media and transformation experiments to manipulate the crop form transition metal ions that are toxic to the plant for desirable agronomically important traits. tissues (Włodek 2002). Acknowledgement: Nikhil Mehta and Priyanka Rooting and transplantation Rao are thankful to the University Grant Commis- Healthy, thick, and branched roots were induced sion, New Delhi for the award of Senior Research in 100% of healthy shoots transferred to the rooting Fellowship. medium containing 2.5 µM IBA (Indole-3-butyric acid) (Figure 1c). Hormone-free rooting medium Authors’ contributions: NM- performed experi- also resulted in shoot formation in 70% of culture, ments and drafted the original manuscript; PR and but the roots so formed were thin and less branched. NM ‒ collected and analysed the data; RS-designed The addition of 0.5 µM IBA concentration increased the research, made available the lab facilities, edit rooting to 80% of culture but the quality of the roots the manuscript. was not changed significantly. However, 1 µM IBA Declaration of conflicts of interest/competing concentration resulted in increased branching and interest: There are no conflicts of interest in this rooting in 90% of cultures, though roots remained work. thin. The results are in accordance with the earlier reports in different Sesbania species (Jha et al. 2004; Mouhamad et al. 2014) where the addition of auxin REFERENCES in the medium significantly increased the formation of the roots. Seventy percent of the rooted shoots Avenido, R., Motoda, J. and Hattori, K. (2001). 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Journal

Agriculturede Gruyter

Published: Oct 1, 2021

Keywords: cotyledon; explant’s orientation; organogenesis; pre-conditioning; regeneration; shoot multiplication

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