Bioscience Journal | 2021 | vol. 37, e37019 | ISSN 1981-3163 1 Phung Thi Bich HOA1 , Nguyen Hoang TUE2 , Phan Thi Quyen TRANG2 , Le Thi HANG2 , Nguyen Quang Duc TIEN2 , Nguyen Hoang LOC2 1 Postgraduate Program in Plant Physiology, University of Sciences, Hue University, Hue, Thua Thien Hue, Vietnam. 2 Department of Biotechnology, University of Sciences, Hue University, Hue, Thua Thien Hue, Vietnam. Corresponding author: Nguyen Hoang Loc Email: nhloc@hueuni.edu.vn How to cite: HOA, P.T.B., et al. An efficient protocol for in vitro regeneration of peanut (Arachis hypogaea L.) cultivar L14. Bioscience Journal. 2021, 37, e37019. https://doi.org/10.14393/BJ-v37n0a2021-56949 Abstract The present work aims to establish an efficient protocol for in vitro regeneration of peanut (Arachis hypogaea) cultivar L14. The study showed that de-embryonated cotyledon was a suitable explant for shoot multiplication on MS medium containing 4 mg/L BAP. The highest number of shoots per explant obtained after 4 weeks of culture was up to 6.8 shoots. Shoots in vitro were able to produce a large number of approximately 11 roots on MS medium supplemented with 0.5 mg/L NAA. These results will be very useful in establishing an in vitro regeneration protocol for peanut cultivar L14 during gene transfer in the next studies to improve their disease resistance. Keywords: Arachis hypogaea. In Vitro Propagation. Rooting. Shoot Multiplication. Shoot Regeneration. 1. Introduction Arachis hypogaea (peanut, groundnut), an annual oilseed crop of the Fabaceae family, is native to South America but is currently grown in diverse environments around the world (Sharma and Bhatnagar- Mathur 2006). Peanut is also known as an important industrial and oilseed crop in many of the warmer regions of the world. Peanut seed contains about 50% oil (Cheng et al. 1992); a rich source of protein (22- 30% dry weight), many minerals (P, Ca, Mg and K), and vitamins (Savage and Keenan 1994). However, pests and diseases are two of the causes leading to poor peanut productivity (Nageshwara-Rao and Nigam 2001). Therefore, the transfer of genes for resistance to pests and diseases into peanuts is considered a promising solution to increase field productivity (Dang et al. 2019; Vasavirama and Kirti 2012). To transfer genes into plants, completing an efficient in vitro regeneration system for them is a very important first step. In literature, numerous studies on in vitro cultures of peanut were reported previously, such as micropropagation from mature cotyledons (Venkatachalam et al. 1999; Maina et al. 2010), nodals (Al-Joboury et al. 2012), de-embryonated cotyledon (Radhakrishnan et al. 2000; Pratap et al. 2018) and cotyledonary nodes (Verma et al. 2011, 2014; Limbua et al. 2019); or through organogenesis (Pestana et al. 1999), somatic embryogenesis and callus culture (Iqbal et al. 2011). However, studies have also shown that the response of peanut cultivars to the culture medium is very different. It is almost difficult to use a nutrient medium for this peanut cultivar to culture the others if a high regeneration efficiency is desired. Peanut cultivar L14 (Spanish group) has a high yield from 4-4.5 metric tons/ha and is suitable for many peanut growing areas in Vietnam. Plants have good agronomic characteristics such as vertical stem (30-50 cm high), neat canopy, good anti-falling, and dark green leaves. The growing period from 120-125 AN EFFICIENT PROTOCOL FOR IN VITRO REGENERATION OF PEANUT (Arachis hypogaea L.) CULTIVAR L14 https://pubmed.ncbi.nlm.nih.gov/?term=Sharma+KK&cauthor_id=16988358 https://pubmed.ncbi.nlm.nih.gov/?term=Bhatnagar-Mathur+P&cauthor_id=16988358 https://pubmed.ncbi.nlm.nih.gov/?term=Bhatnagar-Mathur+P&cauthor_id=16988358 https://orcid.org/0000-0002-9989-7713 https://orcid.org/0000-0003-4387-0201 https://orcid.org/0000-0001-5699-3169 https://orcid.org/0000-0002-9570-3547 https://orcid.org/0000-0002-7330-3139 https://orcid.org/0000-0002-6387-0359 Bioscience Journal | 2021 | vol. 37, e37019 | https://doi.org/10.14393/BJ-v37n0a2021-56949 2 An efficient protocol for in vitro regeneration of peanut (Arachis hypogaea L.) cultivar L14 days for a spring crop, 95-100 days for an autumn-winter crop. 100-seed weight from 58-60 g, kernel/fruit ratio from 73-75%. This peanut cultivar is highly resistant to some leaf spot diseases such as brown spots, black spots, rust, and bacterial wilt (Suu et al. 2017), but is poorly resistant to stem rot caused by white mold (Sclerotium rolfsii) (Cuong et al. 2018). Therefore, in the present study, we investigated the in vitro regeneration capacity of different types of explants from peanut cultivar L14 to serve genetic transformation in the next study to improve the white mold disease resistance of peanuts. 2. Material and Methods Studies were performed in peanut (Arachis hypogaea L.) cultivar L14 which was provided by Field Crops Research Institute, Vietnam Academy of Agricultural Sciences. Explants were used for in vitro cultures including hypocotyl, epicotyl, shoot-tip, and cotyledon node from germinated seeds, embryonated cotyledon, and de-embryonated cotyledon. Cultures of shoot regeneration and multiplication, and root induction were maintained at 25±2ºC at the cool white, the fluorescent light intensity of 27 µmol/m2/s with 12 h daylight. In vitro growth in all experiments was evaluated at week 4 of culture. Healthy peanut seeds were carefully rinsed under running water, then surface sterilized with 65% sodium hypochlorite solution for 10 minutes. Finally, the seeds are rinsed four times with sterile distilled water. Sterile seeds were germinated on basal MS (Murashige and Skoog 1962) medium containing 3% sucrose and 0.8% agar, pH of 5.8. Explants (5 days old) were cultured on MS medium containing 3% sucrose and 0.8% agar, supplemented with various concentrations of benzylaminopurine (BAP) from 1-10 mg/L and naphthaleneacetic acid (NAA) from 0.1-0.5 mg/L to induce shoot regeneration. In vitro individual shoots were then transferred to MS medium containing 3% sucrose and 0.8% agar, supplemented with BAP from 1-5 mg/L for shoot multiplication. Shoots (approx. 3 cm length) were isolated from in vitro shoot clusters and subcultured on MS medium containing 3% sucrose and 0.8% agar, supplemented with NAA or indolebutyric acid (IBA) from 0.1-0.5 mg/L for rooting. 3. Results and Discussion The present study showed that more than 81% of sterile peanuts cultivar L14 have germinated on MS medium after 3 days of incubation (Figure 1A) and by day 7 have grown into in vitro whole plant. Explants including shoot-tip, hypocotyl, epicotyl, and cotyledon nodes were isolated from seedlings to investigate shoot regeneration on different culture media (Data not shown). The observation found that MS medium containing 4 mg/L BAP and 0.1 mg/L NAA is the most suitable. The number of regenerated shoots from epicotyl (2.3) was higher than the remaining types of explants (1-1.8), however, regenerating explants were only about 50%. The highest shoot regeneration efficiency was found in the shoot-tips, shoot regenerating explants of 95% with 1.6 shoots per explant (Table 1). Table 1. Shoot regeneration from various types of explants on MS medium containing 4 mg/L BAP and 0.1 mg/L NAA. Explants Regenerating explants (%) No. of shoots per explant Shoot length (cm) Shoot-tip 95 1.6b 3.1b Epicotyl 50 2.3a 2.9b Hypocotyl 40 1.8b 2.6b Cotyledon node 100 1.0b 6.6a Different letters in a column indicate significantly different means (Duncan’s test, p<0.05). As it is well known, cytokinins and auxins such as benzyladenine (BA), BAP, 2-isopentenyladenine (2- iP), thidiazuron (TDZ), and NAA are commonly used for plant micropropagation. Cytokinins and auxins play a crucial role as promoters of cell division and act in the induction and development of meristematic centers leading to the formation of organs (Peeters et al. 1991). Shoot-tip (0.1-1 mm high) is the terminal portion of a shoot comprising the apical meristem (0.05-0.1 mm) together with primordial and developing leaves and adjacent stem tissue (Torres 1989). Hence, the majority of shoot-tip culture is essentially apical meristem Bioscience Journal | 2021 | vol. 37, e37019 | https://doi.org/10.14393/BJ-v37n0a2021-56949 3 HOA, P.T.B., et al. cultures. In our thinking, explants taken from actively growing parts of plants such as apical meristem are very suitable for shoot multiplication. And a combination of BAP and NAA in an appropriate ratio and concentration will facilitate multiple axillary shoot initiation and elongation on the main explant (Rasool et al. 2009). In vitro shoots from regenerations were subcultured on MS medium supplemented with BAP from 1- 5 mg/L. The highest shoot number obtained at 4 mg/L BAP with 3.8 shoots per explant, regenerating explants of about 40% was relatively high compared to other concentrations of BAP (Table 2 and Figure 1B). Table 2. Effect of BAP on shoot multiplication of individual shoots from regeneration cultures. BAP (mg/L) Regenerating explants (%) No. of shoots per explant Shoot length (cm) 1 18.8 2.6bc 2.4ab 2 21.9 2.9ab 2.6ab 3 31.3 3.1ab 2.7ab 4 40.6 3.8a 3.0a 5 43.8 3.0ab 2.9ab Control 12.5 1.9c 2.3b Different letters in a column indicate significantly different means (Duncan’s test, p<0.05). Control is medium without BAP. Some previous reports showed that in vitro shoot proliferation of peanut has been found at higher BAP concentrations, e.g. MS medium containing 33 µM (over 7.4 mg/L) benzyladenine (BA) in combination with 5.3 µM NAA and 23.54 µM AgNO3 recorded maximum average shoot number per regenerating explant (shoot-tip) was 6.3 (Ozudogru et al. 2005). Palanivel et al (2014) obtained the highest number of shoots (over 17 shoots) in peanut cultivar ICGV00351 (Tamil Nadu, India) on a medium containing 25 mg/L BAP and 0.5 mg/L indoleacetic acid (IAA). In another study, different explants such as zygotic embryos, plumular apices, and embryonic axes of peanut cultivar NC-7 were pretreated for 15 days on MS medium supplemented with 25 mg/L BA. Subsequent culture counted the highest shoot counts per explant ranging from 2.67 to 4.43 (zygotic embryos), 2.33 to 4.11 (plumular apices) and 1.87 to 3.33 (embryonic axes) on MS medium containing BA concentrations ranged from 0.25 to 3 mg/L after 8 weeks of observation (Özkan and Aasim 2019). To improve shoot multiplication rate, embryonated cotyledons and de-embryonated cotyledons were cultured on MS medium supplemented with different concentrations of BAP from 1-5 mg/L for investigation of shoot proliferation. Data from table 3 shows that regeneration of de-embryonated cotyledons peaked with 6.8 shoots per explant, and 1 shoot for embryonated cotyledons in medium with 4 mg/L BAP (Figure 1C). Other tested concentrations of BAP showed lower efficiency for shoot regeneration (Data not shown). Table 3. Shoot regeneration from embryonic cotyledons on MS medium containing 4 mg/L BAP. Explants Regenerating explants (%) No. of shoots per explant Shoot length (cm) De-embryonated cotyledon 23.3 6.8a 4.1b Embryonated cotyledon 86.7 1.0b 5.9a Different letters in a column indicate significantly different means (Duncan’s test, p<0.05). It has been reported that de-embryonated cotyledons of oilseed crops (e.g. peanut) are highly regenerating without callus intervention, so they were often used as a type of potential explant for shoot multiplication (Tiwari and Tuli 2008; Radhakrishnan et al. 2000). A study of Tiwari and Tuli (2008) in four peanut cultivars of India (JL-24, TMV-2, TAG-24, and Dh-3-30) indicated that adventitious shoot primordia could be induced from de-embryonated cotyledon explants were cultured in a medium containing 5 mg/L BAP and 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 4 weeks, or in medium containing only BAP at 20 mg/L for the first 2 weeks, followed by 15 mg/L BAP for the next 2 weeks. Therefore, the results in the present study have once again contributed to reinforcing the above. However, Radhakrishnan et al (2000) only obtained 57.9% of multiple shoot formation from de- embryonated cotyledons in peanut cultivar J11 in the medium containing 15 mg/L BAP. Whereas, embryo Bioscience Journal | 2021 | vol. 37, e37019 | https://doi.org/10.14393/BJ-v37n0a2021-56949 4 An efficient protocol for in vitro regeneration of peanut (Arachis hypogaea L.) cultivar L14 axes and mature whole seeds can achieve to the highest of 100% and 86% in the medium supplemented with 30 and above and 50 mg/L BAP, respectively. In a similar study subjected to multiple shoot induction in two peanut cultivars, VRI-2 and VRI-3 from Tamil Nadu (India), Palanivel and Jayabalan (2002) found that (whole) embryonated cotyledon produced more well-developed shoots in both cultivars. The (whole) de- embryonated cotyledon and sectional de-embryonated cotyledon explants showed poor response. Among the different concentrations of BAP combined with 0.5 mg/L NAA, 25 mg/L BAP was considered to be the best in terms of the highest mean number of shoots and the shortest shoot induction time. In general, shoot induction of de-embryonated cotyledons, as well as other types of explants, was very different depending on the peanut cultivar. The number of shoots produced per explant varied with the concentration of plant growth regulators in the medium. In vitro peanut shoots were transferred onto MS medium containing NAA and IBA for root induction. The data in table 4 indicate that all tested concentrations of two of these plant growth regulators stimulated rooting. However, NAA showed higher efficacy than IBA, the highest number of roots was 10.9 at a concentration of 0.5 mg/L with a regeneration rate of 100% and roots were formed earlier, only after 9-12 days of culture (Figure 1D). Whereas on the IBA-containing medium, the in vitro shoots had a smaller number of roots, and roots formed later, from day 15 to day 18 (Data not shown). Table 4. Effect of NAA and IBA on rooting of in vitro shoots. Plant growth regulator (mg/L) Shoots regenerating roots (%) No. of roots per rooted shoot Root length (cm) NAA 0.1 75 4.3b 3.8b 0.3 87.5 4.4b 4.8ab 0.5 100 10.9a 4.3ab IBA 0.1 75 3.4b 5.7a 0.3 62.5 2.6c 4.6ab 0.5 50 1.9c 5.3a Control 0.0 50 1.5c 4.9ab Different letters in a column indicate significantly different means (Duncan’s test, p<0.05). The effects of auxin group of hormones (e.g. NAA, IBA, or IAA) on rooting and plant development have been discussed in several previous studies depending on the induction of various explants or species. However, in some cases, NAA stimulated root regeneration better than IBA. For example, Limbua et al (2019) also found that NAA is the suitable growth regulator for peanut cultivars CGV 12991, CG 7, and Red Valencia, and if using a concentration of 1 mg/L can obtain the highest number of plants with roots. Some previous studies in other plant species have also shown similar results, e.g. in vitro root regeneration in sugarcane (Jagadeesh et al. 2011) or in bromeliads (Neoregelia concentrica) (Martins et al. 2013). In contrast, peanut cultivar ICGV-00351 only gave high root regeneration, up to 25 roots per explant which derived from (whole) embryonated cotyledon, when grown on medium with 5 mg/L IBA (Palanivel et al. 2014). The in vitro shoots of the E. grandis × E. urophylla hybrid had also a good response with IBA for rooting (Cid et al. 1999). Whereas the best in vitro rooting of the apple ‘Jork 9’ shoots was found in media containing IAA. Bioscience Journal | 2021 | vol. 37, e37019 | https://doi.org/10.14393/BJ-v37n0a2021-56949 5 HOA, P.T.B., et al. Figure 1. In vitro cultures of peanut. A – germination of seeds; B – shoot multiplication from individual shoots; C – shoot regeneration from de-embryonated cotyledons; D – rooting of individual shoots. 4. Conclusions In conclusion, the current study shows that the de-embryonated cotyledon of peanut cultivar L14 is a type of suitable explant and that it may give high efficiency in shoot proliferation on a medium containing 4 mg/L BAP. Rooting of individual shoots can peak at a concentration of 0.5 mg/L NAA. Authors' Contributions: HOA, P.T.B.: conception and design, acquisition of data, analysis and interpretation of data, and drafting the manuscript; TUE, N.H.: conception and design, acquisition of data, analysis and interpretation of data, and drafting the manuscript; TRANG, P.T.Q.: acquisition of data, analysis and interpretation of data, and drafting the manuscript; HANG, L.T.: acquisition of data, analysis and interpretation of data, and drafting the manuscript; TIEN, N.Q.D.: acquisition of data, and drafting the manuscript; LOC, N.H.: conception and design, analysis and interpretation of data, and drafting the manuscript. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: Not applicable. Acknowledgments: This work was supported by the National Foundation for Science and Technology Development (NAFOSTED) - Vietnam, Finance Code 106.02-2017.346. 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Peanut Science. 2014, 41(1), 25-31. https://doi.org/10.3146/PS12-13.1 Received: 20 August 2020 | Accepted: 21 October 2020 | Published: 27 April 2021 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1590/S1983-40632013000200009 https://www.researchgate.net/journal/1399-3054_Physiologia_Plantarum https://doi.org/10.1111/j.1399-3054.1962.tb08052.x https://doi.org/10.1079/IVP2004591 https://doi.org/10.21162/PAKJAS/19.8262 https://doi.org/10.9734/BBJ/2014/9237 https://doi.org/10.1104/pp.97.1.402 https://doi.org/10.1007/s11627-999-0080-3 https://doi.org/10.1111/pbr.12590 https://doi.org/10.1023/A:1026743822546 https://pubmed.ncbi.nlm.nih.gov/?term=Sharma+KK&cauthor_id=16988358 https://pubmed.ncbi.nlm.nih.gov/?term=Bhatnagar-Mathur+P&cauthor_id=16988358 https://doi.org/10.1007/s11240-007-9297-1 https://doi.org/10.1007/s10142-012-0298-8 https://doi.org/10.1590/S1677-04202011000400004 Studies were performed in peanut (Arachis hypogaea L.) cultivar L14 which was provided by Field Crops Research Institute, Vietnam Academy of Agricultural Sciences. Explants were used for in vitro cultures including hypocotyl, epicotyl, shoot-tip, and ...