ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2022, 27(1): 90–98 DOI: 10.53541/jams.vol27iss1pp90-98 Received 15 Jan 2021 Accepted 06 June 2021 Relationship between Induction of Novel Somaclonal Variants and Types of Organogenesis in Muskmelon (Cucumis melo L.) A.K.M. Mohiuddin1, Zaliha Christine Abdullah2, M.K.U. Chowdhury3, K. Harikrishna4, and Suhaimi Napis5* Suhaimi Napis5*( ) suhaimi@upm.my and akmmohiu@yahoo.com, Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor D.E., Malaysia. Introduction Muskmelon (Cucumis melo L.) species is a val-ued agricultural crop and widely cultivated in Asia, America and European countries (Al Mawaali et al., 2017). However, it is highly prone to a wide range of diseases, which can result reduced yield and quality (Jelaska, 1986). Therefore, availability of useful alternative agronomically important traits will be more useful for speedy development of desired variety as compared to conventional breeding for muskmelon improvement (Pijnacker and Ramulu, 1990, Mohiuddin et. al., 1998). The in vitro-generated variation and induc- tion of somaclonal variation can improve the genetic diversity and may also amplify the pre-existing genetic heterogeneity in somatic cells, thus facilitating the over- all breeding and improvement of the economically im- العالقة بني استقراء املتغريات اجلسدية اجلديدة و أنواع األعضاء يف الشمام (Cucumis melo L.) أ.ك.م. حمي الدين1، زليحة كريستني عبد هللا2،. م.ك. تشودري3 ، ك. هاريكريشنا4، و سحيمي انبيس5* Abstract. A comparative study on induction of somaclonal variation in muskmelon (Cucumis melo L.) cv. Birdie re- generants obtained through direct and indirect organogenesis was carried out. Two types of non-meristematic explants (e.g. cotyledon and petiole) were used for this study. A significantly lower (p<0.05) frequency of variation was observed in muskmelon somaclones regenerated via direct organogenesis (MS medium with BAP) compared to indirect (MS me- dium with BAP and 2,4-D). Morphological study revealed that the somaclones regenerated from proximal cotyledon, petiole and distal cotyledon explants through direct organogenesis did not show any variation in elongation medium at the concentrations of BAP 0.1, 0.3 and 0.5 mg-l, respectively. In contrast, higher number of morphologically somaclonal variants was obtained from these explants at the same concentration of BAP obtained through indirect organogenesis. Other concentrations of BAP, on the other hand, added to the elongation medium showed higher percentage of soma- clones with different types of novel variations, e.g. early flowering including higher number of flowers, slow growth of shoots with variant shape of leaves having long and thick petioles, and stubby shoot apices including flattened stem. These variations could be the prime genetic materials to develop new varieties of muskmelon, e.g. high yielding vari- ety, early, late variety, dwarf variety, and variety with desirable body configurations. The results suggest that specific concentrations of BAP or combinations of BAP and 2,4-D have a significant (p<0.05) influence on the induction of novel somaclonal variations in muskmelon regenerants. Future cytogenetic and molecular studies reveal that the novel genetic variations at the chromosome level in somaclonal variants can exist. Keywords: Muskmelon, novel somaclones, direct and indirect organogenesis, variety development. امللخص:دراســة مقارنــة حــول حتريــض التبايــن اجلســدي يف الشــمام ).Cucumis melo L(. مت إجــراء جتديــدات الطيــور الــي مت احلصــول عليهــا مــن خــال تكويــن األعضــاء املباشــرة وغــر املباشــرة. مت اســتخدام نوعــني مــن النبــااتت املســتأصلة غــر اإلنشــائية )مثــل الفلقــة والســويقات( هلــذه الدراســة. لوحــظ تواتــر أقــل بشــكل ملحــوظ )p <0.05( يف التغــر somaclones الشــمام املتجــدد عــن طريــق تكويــن األعضــاء املباشــر )وســط MS مــع BAP( مقارنــة ابلغــر مباشــر )وســط MS مــع BAP و D-2،4(. كشــفت الدراســة املورفولوجيــة أن somaclones الــي مت جتديدهــا مــن نبــااتت الفلقــات القريبــة ، والنبــااتت الســويقية ، والنبــااتت البعيــدة مــن خــال تكويــن األعضــاء املباشــر مل تظهــر أي اختــاف يف وســط االســتطالة بركيــزات BAP 0.1 و 0.3 و 0.5 ملغــم / لــر علــى التــوايل. يف املقابــل ، مت احلصــول علــى عــدد أكــر مــن املتغــرات اجلســدية النســيلة شــكلًيا مــن هــذه النبــااتت املســتأصلة بنفــس تركيــز BAP الــذي مت احلصــول عليــه مــن خــال تكويــن األعضــاء غــر املباشــر. مــن انحيــة أخــرى، أظهــرت تركيــزات أخــرى مــن BAP املضافــة إىل وســط االســتطالة نســبة مئويــة أعلــى مــن somaclones مــع أنــواع خمتلفــة مــن االختافــات اجلديــدة، علــى ســبيل املثــال اإلزهــار املبكــر يشــمل عــدًدا أكــر مــن األزهــار ، منــو بطــيء للراعــم ذات أشــكال خمتلفــة مــن األوراق ذات أعنــاق طويلــة ومسيكــة ، ومقرمشــات قصــرة صلبــة مبــا يف ذلــك الســاق املســطحة. ميكــن أن تكــون هــذه االختافــات هــي املــواد اجلينيــة األساســية لتطويــر أنــواع جديــدة مــن الشــمام ، علــى ســبيل املثــال تنــوع عــايل الغلــة ، تنــوع مبكــر ، متأخــر ، تنــوع قــزم ، ومتنــوع مــع تكوينــات اجلســم املرغوبــة. تشــر النتائــج إىل أن تركيــزات معينــة مــن BAP أو جمموعات من BAP و D-2،4 هلا أتثر كبر )p <0.05( على حتريض االختافات اجلســدية اجلديدة يف جتديدات الشــمام. تكشــف الدراســات الوراثيــة اخللويــة واجلزيئيــة املســتقبلية أن االختافــات اجلينيــة اجلديــدة علــى مســتوى الكروموســوم يف املتغــرات اجلســدية ميكــن أن توجــد. الكلمات املفتاحية: الشمام، somaclones ، تكوين األعضاء املباشر وغر املباشر، تنمية األصناف. 91Research Paper Mohiuddin, Z. Abdullah, Chowdhury, Harikrishna, Napis portant plants (Patnaik et al., 1999). In vitro technique can also be used to induce economically useful agro- nomic characteristics into crop to increase productivity without loss of available desired genetic characters (Mo- hiuddin, 1998). The ability to produce morphologically normal plants at high frequency with little or no soma- clonal variation as well as induction of high frequency of variations in somaclones is a prerequisite to the applica- tion of tissue culture techniques in crop improvement (Mohiuddin et al., 2000, & 2003, Ren et al., 2012). Application of in vitro tissue culture techniques to crop improvement has been focused on selection for herbicide resistance, stress tolerance, amino acid over production, rice fortification having Fe and Zn, dis- ease resistance, etc. from somaclonal variants (Larkin and Scowcroft, 1981; Evenor et al., 1994; Al-Noor et al., 2019). It has been known over the five decades that cul- tured plant cell and tissue undergo various morphologi- cal and genetic changes especially in chromosome num- bers and ploidy level during in vitro culture (Murashige and Nakano, 1966, Ren et al., 2013). This change is also known as somaclonal variation displayed among in vitro tissue culture-derived plants and it has been described for several plant species (Larkin and Scowcroft, 1981; Orton 1984; Bajaj, 1990; Mofidabadi et al., 2001; Shas- three et al., 2009; Saraswat and Kumar, 2019). Considerable work in muskmelon tissue culture has been conducted (Moreno et al., 1985; Trulson and Sha- hin, 1986; Niedz et al., 1989; Oridate et al., 1992; Gray et al., 1993; Mohiuddin et al., 1998, and Ren et al., 2013) but to our best knowledge negligible report has been evalu- ated yet on relationship in production of morphological- ly normal somaclones (true-to-type) at high frequency through direct organogenesis as well as induction of nov- el somaclonal variants in muskmelon plants via indirect organogenesis. This present study was aimed to investi- gate the extent of true-to-type somaclone production (no variation) and induction of novel somaclonal variants as observed phenotypically in muskmelon regenerants and obtained through direct and indirect organogenesis. Materials and Methods Plant Materials This study focused on the Birdie cultivar of muskmelon, which is the most widely used in Malaysia as a fruit crop. Testas were removed manually from mature seeds of Birdie (Sakata Seed Corp., Japan) and sterilized by 20% Clorox (containing 5.25% sodium hypochlorite) with one drop of ‘Tween 20’ for 15 min followed by 3X wash with sterile distilled water. The seeds were then asepti- cally germinated on filter paper soaked with MS (Mu- rashige and Skoog, 1962) liquid medium in Petri dishes having 3% sucrose. After eight days cotyledons were sep- arated from germinated seedlings and cut into four piec- es by a transverse cut followed by a longitudinal cut. The proximal cotyledons (attached to hypocotyls) and distal cotyledons were used for this study. The second-top-leaf petiole was used as explant, obtained from 21-day-old muskmelon seedlings germinated in MS solid medium in Magenta boxes having 2% phytagel (Sigma, USA). Each petiole was bisected into equal pieces (4-5 mm long) and it was used in this study. Somaclone Induction and Elongation Medium Both cotyledon and petiole explants were cultured asep- tically on somaclone induction medium (SIM) consist- ing of MS nutrients, MS vitamins, 3% sucrose, 2 g-l phy- tagel, and BAP at the concentration of 1.0 and 2.0 mg-l either alone (direct organogenesis) or in combination with 2,4-D at 0.1 and 0.3 mg-l (indirect organogenesis). Ten explants of each type were cultured on SIM con- taining each concentrations of BAP alone or in combi- nations with 0.1 and 0.3 mg-l 2,4-D. This was repeated 5 (five) times. Subsequently, 15-day-old somaclones as ob- tained from direct and indirect organogenesis and these were then cultured onto somaclone elongation medium (SEM) containing MS medium supplemented with BAP at 0.07, 0.1, 0.3, 0.5 and 0.7 mg-l. Root Induction Medium and Culture Conditions The elongated somaclones derived from proximal coty- ledons, distal cotyledons and petioles were rooted in MS medium containing NAA at the concentrations of 0.01, 0.05 and 0.03 mg-l, respectively (Mohiuddin, 1998). All the media combinations were adjusted to pH 5.7 before autoclaving at 121°C for 15 minutes at 1.05 kg/cm2 pres- sure (15-20 psi). Cultures were incubated in the growth chamber at 26±1°C under 16/8 h light (39.3 µmol m-2 s-1)/dark regime. Evaluations of Somaclonal Variants The extents of variations were evaluated morpholog- ically on the somaclones after elongation in SEM for four weeks at each concentration of BAP as mentioned earlier. For the identification of morphologically true- to-type somaclones (no variation) and variants (having variation) obtained from each explant of muskmelon at each formulation of hormone(s) were closely examined. Variations in somaclones were scored of the following quantitative traits: (i) early flowering including higher number of flowers, (ii) slow growth of shoots with de- formed shape of leaves having long and thick petiole, and (iii) stubby shoot apices with flattened stem. On the other hand, normal somaclones were scored of the following quantitative traits: (i) normal organ develop- ment, (ii) rapid growth, (iii) normal flower formation and fruit development, (iv) good root induction ability and (v) ability to acclimatize to the ambient environ- mental conditions. 92 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 1 Relationship between Induction of Novel Somaclonal Variants and Types of Organogenesis in Muskmelon (Cucumis melo L.) Statistical Analysis The data were statistically analysed by analysis of vari- ance (ANOVA). Duncan’s Multiple Range Test (DMRT) was also carried out to show the significance of relation- ship among the percentage result (mean) of somaclones either morphologically normal or variant obtained through the direct or indirect organogenesis. Results Huge numbers of somaclone primordia were initiated from muskmelon explants cultured on somaclone in- duction medium (Figure 1a). Somaclones induced from proximal and distal cotyledons and petiole explants of muskmelon via direct organogenesis, elongated faster in elongation medium compared to the somaclones as obtained from identical explants via indirect organogen- esis. Moreover, proximal cotyledon-derived somaclones elongated earlier than somaclones derived from either distal cotyledon or petiole explants. The distal coty- ledon derived somaclones obtained from both direct and indirect organogenesis, however, elongated slower as compared to other two explant-derived somaclones. The proportion of morphologically normal (true- to-type) and variant somaclones induced from differ- ent explants varied at identical concentration of BAP in elongation medium (Tables 1 and 2). Similarly, the proportion of normal and variant somaclones induced through either direct or indirect organogenesis from different explants also varied to identical BAP concen- tration (Tables 1 and 2). BAP at the concentration of 0.1, 0.3 and 0.5 mg-l significantly (p<0.05) induced the highest rates (100%) of morphologically normal somaclones (Figure 1b) through direct organogenesis from proximal cotyle- don, petiole and distal cotyledon, respectively (Table 1). Other concentrations of BAP also induced normal so- maclones but at lower rates. SEM having 0.007 and 0.7 mg-l BAP induced the lowest rates of normal somaclones from all explants (Table 1). On the other hand, the rates Figure 1. Somaclone and its variation. a) Somaclone primordia initiated from proximal cotyledon, b) Shoots elongated in shoot elongation medium, c) Morphologically normal somaclones, d) Early flowering obtained in somaclones, e) Slow growth of shoots with distorted leaves having long and thick petioles, f) Stubby shoot apices and g) Successfully acclimatized somaclones. 93Research Paper Mohiuddin, Z. Abdullah, Chowdhury, Harikrishna, Napis of morphologically normal somaclones obtained via in- direct organogenesis were generally lower as compared to the direct organogenesis (Table 1). No significant difference was observed among the rates of normal so- maclones induced through indirect organogenesis from proximal cotyledon, distal cotyledon and petiole ex- plants at the different concentrations of BAP (Table 1). From the result in this study, it was found that the di- rect organogenesis at specific concentrations of BAP 0.1, 0.3 and 0.5 mg-l did not produce any somaclonal variants morphologically in proximal cotyledon (Figure 1c), peti- ole and distal cotyledon of muskmelon, respectively (Ta- ble 2). However, other concentrations of BAP induced somaclonal variants from all three explants of musk- melon. MS medium containing 0.7 mg-l BAP induced a significantly higher (p<0.05) rate of somaclonal variants from proximal cotyledon (29%) and petiole explants (20%), while MS medium having BAP 0.07 mg-l induced the highest rate of somaclonal variants from distal coty- ledon explants (27%) through direct organogenesis (Ta- ble 2). The rates of morphologically variant somaclones, on the other hand, obtained through indirect organogen- esis were higher compared to that of direct organogene- sis (Table 2). About 2 to 4 folds higher somaclonal vari- ants were found in indirect organogenesis as compared to direct organogenesis (Table 2). No significant effect was observed among the different rates of somaclonal variants induced from explants at different concentra- tions of BAP through indirect organogenesis (Table 2). The morphologically normal somaclones obtained from the three different explants via direct organogene- sis were longer height at different concentrations of BAP than those obtained through indirect organogenesis (Ta- ble 3). BAP at 0.1, 0.3 and 0.5 mg-l produced normal so- maclones from proximal cotyledon, distal cotyledon and petiole through direct organogenesis, which attained lengths of 9.2 cm, 7.9 cm and 9.1 cm, respectively. How- ever, at these BAP concentrations, normal somaclones were 6.4, 5.1 and 4.7 cm in length from respective ex- plants obtained through indirect organogenesis which is considerable lower (Table 3). Furthermore, lower height was observed in the somaclonal variants obtained from all three explants through direct and indirect organogen- esis (Table 4). On average, lowest height attained from Table 1. Variations on production of morphologically normal somaclones of muskmelon obtained through direct and indirect organogenesis BAP (mg/l) Production of precent (%) morphological normal somaclones Direct organogenesis Indirect organogenesis Proximal cotyledon Distal cotyledon Petiole Proximal cotyledon Distal cotyledon Petiole 0.07 93±2.7a 73±5.2b 85±4.7ab 74±1.6 a 80±8.1a 67±6.6a 0.1 100±0.0a 79±4.0b 91±2.3a 71±4.3 a 63±5.6a 64±1.7a 0.3 84±3.4b 86±2.1a 100±00a 62±3.7 a 75±3.9a 72±2.9a 0.5 82±3.0b 100±0.0a 86±2.8ab 68±0.8 a 76±4.3a 62±6.5a 0.7 71±3.4c 80±6.3b 80±5.9b 62±6.5 a 74±4.5a 57±4.9a Note: Percentage results are means of five replications±Standard Error. Means with the same letters (superscript) within same columns are not significantly different (p<0.05) Table 2. Variations on production of novel somaclonal variants of muskmelon attained through direct and indirect organogenesis BAP (mg/l) Production of precent (%) of somaclonal variants Direct organogenesis Indirect organogenesis Proximal cotyledon Distal cotyledon Petiole Proximal cotyledon Distal cotyledon Petiole 0.07 7±4.7 b 27±3.3 a 15±5.4 b 26±1.0 a 20±8.4 a 33±4.2 a 0.1 0.0±0.0 c 21±2.8 a 9±3.8 b 29±2.8 a 37±3.4 a 36±1.0 a 0.3 16±2.6 a 14±3.6 a 0.0±0.0 c 38±2.2 a 25±2.4 a 28±2.0 a 0.5 18±2.3 a 0.0±0.0 b 14±2.1 b 32±0.5 a 24±3.0 a 38±3.9 a 0.7 29±2.2 a 20±6.4 a 20±6.5 a 38±3.9 a 26±2.9 a 43±2.9 a Note: Percentage results are means of five replications±Standard Error. Means with the same letters (superscript) within columns are not significantly different (p<0.05). 94 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 1 Relationship between Induction of Novel Somaclonal Variants and Types of Organogenesis in Muskmelon (Cucumis melo L.) somaclonal variants regenerated from explants through indirect organogenesis in comparison to direct (Table 4). Early flowering (EF) was the most common novel variants observed in muskmelon somaclones (Figure 1d) with higher rates attained frequently from all ex- plants through direct and indirect organogenesis (Tables 5 and 6). Maximum percent of EF (e.g. 14% and 19%) was obtained at 0.7 mg-l BAP from proximal cotyle- don explants through direct and indirect organogene- sis, respectively. On the other hand, regenerated novel variants (e.g. slow growth, SG) of shoots with distort- ed leaves having long and thick petioles (Fig. 1e) and stubby shoot apices (SSA) with flatted stem (Figure 1f ) was found comparatively lower in number than EF via both direct and indirect organogenesis (Tables 5 and 6). The rate of somaclonal variants obtained from prox- imal cotyledon, distal cotyledon and petiole explants ranged from 4 to 14% of EF, 2 to 9% of SG with vari- ant shape of leaves and 2 to 6% of SSA with flattened stem via direct organogenesis (Table 5). However, these novel variants ranges increased when plants were regenerated via indirect organogenesis (Table 6). No variations were observed in the somaclones ob- tained through direct organogenesis from proximal cot- yledon, petiole and distal cotyledon explants at BAP 0.1, 0.3 and 0.5 mg-l, respectively (Table 5). However, proxi- mal cotyledon at BAP 0.1 mg-l induced 14%, 11% and 4% of EF, SG and SSA novel variants, respectively through indirect organogenesis (Table 6). Similarly, petiole ex- plants also induced above mentioned novel variants (e.g. 13%, 9% and 6%) in same order at 0.3 mg-l BAP, respec- tively via indirect organogenesis. Consequently, distal cotyledons at the concentration of 0.5 mg-l BAP induced 12% of EF, and 6% of each SG and SSA novel variants via indirect organogenesis (Table 6). The other concentra- tions of BAP, on the other hand, showed mostly a higher percentage (%) of novel somaclonal variants as obtained through indirect organogenesis (Table 6). Although different types of novel somaclonal variants were obtained via direct organogenesis from all explants but no SSA variants was attained in the medium contain- ing 0.07 mg-l of BAP from proximal cotyledon explants via direct organogenesis (Table 5), whereas same explant at same concentration of BAP produced 5% SSA novel vari- ant via indirect organogenesis (Table 6). Moreover, BAP at 0.1 and 0.5 mg-l also did not produce same (SSA) novel variant from petiole explants of muskmelon through di- rect organogenesis while the same BAP concentrations Table 3. Variations on Average Height of Normal Somaclones obtained through Direct and Indirect Organogenesis BAP (mg/l) Average height of morphological normal somaclones (in cm) Direct organogenesis Indirect organogenesis Proximal cotyledon Distal cotyledon Petiole Proximal cotyledon Distal cotyledon Petiole 0.07 8.6±0.1 7.2±0.3 8.7±0.2 5.8±0.2 4.8±0.1 4.6±0.1 0.1 9.2±0.1 6.6±0.3 8.8±0.2 6.4±0.3 4.9±0.1 4.8±0.1 0.3 8.9±0.2 7.9±0.2 7.8±0.2 6.8±0.2 5.1±0.1 4.9±0.1 0.5 8.7±0.1 7.8±0.2 9.1±0.1 6.2±0.2 5.6±0.1 4.7±0.1 0.7 6.6±0.3 7.6±0.2 7.2±0.4 6.4±0.2 5.3±0.1 4.7±0.1 Note: Average results are means of five replications±Standard Error. Table 4. Variations on average height of novel somaclonal variants obtained through direct and indirect organogenesis BAP (mg/l) Average height of somaclonal variants (in cm) Direct organogenesis Indirect organogenesis Proximal cotyledon Distal cotyledon Petiole Proximal cotyledon Distal cotyledon Petiole 0.07 7.8±0.5 0.0±0.0 6.9±0.2 7.1±0.1 5.9±0.4 4.8±0.5 4.8±0.3 6.2±0.4 0.0±0.0 6.7±0.6 6.6±0.3 7.7±0.5 0.0±0.0 6.8±0.5 4.5±0.3 5.4±0.2 4.9±0.1 4.8±0.2 5.0±0.2 5.1±0.2 4.3±0.1 4.5±0.2 4.7±0.2 4.8±0.2 4.7±0.2 4.4±0.1 4.3±0.1 4.5±0.2 4.0±0.1 4.5±0.3 0.1 0.3 0.5 0.7 Note: Average results are means of five replications±Standard Error 95Research Paper Mohiuddin, Z. Abdullah, Chowdhury, Harikrishna, Napis induced SSA novel variants in 7% and 8% of shoots obtained via indirect organogenesis (Tables 5 and 6). The mean height (in parentheses) of novel somaclonal variants obtained from different explants of muskmelon at all concentrations of BAP through direct organogene- sis was greater (Table 5) than the height (in parentheses) of novel somaclonal variants obtained through indirect organogenesis (Table 6). The ranges height of novel so- maclones initiated through direct organogenesis from proximal cotyledon was 4.9 - 9.5 cm, from distal cotyle- don was 3.8 - 8.0 cm and from petiole was 3.7 - 8.7 cm in height (Table 5). On the other hand, the height rang- es were lower when somaclonal variants regenerated through indirect organogenesis (Table 6) as compared to direct organogenesis (Table 5). Average height of nov- el somaclonal variants found in plants regenerated from proximal cotyledon through direct organogenesis is rel- atively higher as compared to distal cotyledons and pet- iole. Average lowest height was found in plants regen- erated from distal cotyledon as compared to two other explants (Table 5). A similar trend was also observed in height of novel somaclonal variants as initiated from all explants through indirect organogenesis (Table 6). The somaclones as well as novel variants induced roots vig- orously in MS medium containing NAA at the concen- trations of 0.01 and 0.03 mg-l and successfully acclima- tized to the ambient humidity level in soil (Figure 1g). Discussion There is no doubt that genetic variation especially mor- phological variations of plant cells cultured in vitro conditions is a general phenomenon (Skirvin, 1978; Constantin, 1981; Ren et al., 2013; Al-Noor et al., 2019) and has now been known for over 50 years. Larkin and Scowcroft (1981) reported that in vitro plant cell culture Table 5. Effect of direct organogenesis on production of novel somaclonal variants in muskmelon cv. Birdie BAP (mg/l) Percentage of novel somaclonal variants Proximal cotyledon Distal cotyledon Petiole ef sg ssa ef sg ssa ef sg ssa 0.07 4 (9.5±0.1) 3 (5.6±0.0) 0 13 (5.4±0.1) 9 (3.9±0.1) 5 (5.2±0.0) 8 (7.8±0.1) 4 (4.0±0.1) 3 (6.9±0.0) 0.1 0 0 0 11 (5.3±0.1) 6 (3.9±0.1) 4 (4.8±0.1) 7 (8.7±0.1) 2 (4.0±0.0) 0 0.3 9 (7.9±0.1) 5 (4.9±0.0) 2 (7.2±0.0) 7 (7.6±0.1) 5 (4.0±0.1) 2 (6.9±0.0) 0 0 0 0.5 10 (8.1±0.1) 5 (5.0±0.1) 3 (7.0±0.0) 0 0 0 9 (8.4±0.1) 5 (3.9±0.0) 0 0.7 14 (6.8±0.1) 9 (4.9±0.1) 6 (5.3±0.1) 12 (8.0±0.1) 6 (3.8±0.1) 2 (7.8±0.0) 11 (4.9±0.1) 6 (3.7±0.1) 3 (4.8±0.0) Note: ef: early flowering including higher number of flowers, sg: slow growth of shoots with variant shape of leaves having long and thick petiole, ssa: stubby shoot apices and flattened stem. The numbers in the parentheses are average heights of novel variants in cm ± Standard Error Table 6. Effect of indirect organogenesis on production of novel somaclonal variants in muskmelon cv. Birdie BAP (mg/l) Percentage of novel somaclonal variants Proximal cotyledon Distal cotyledon Petiole ef sg ssa ef sg ssa ef sg ssa 0.07 16 (5.8±0.3) 5 (4.7±0.2) 11 (4.4±0.1) 11 (3.9±0.2) 10 (4.1±0.1) 12 (4.4±0.5) 5 (4.6±0.2) 4 (5.2±0.1) 11 (5.3±0.5) 7 (4.9±0.1) 10 (5.2±0.4) 7 (4.6±0.2) 16 (5.0±0.4) 13 (5.2±0.1) 12 (5.9±0.2) 11 (5.2±0.5) 7 (3.3±0.5) 11 (3.7±0.0) 8 (3.9±0.4) 6 (3.4±0.2) 8 (3.6±0.1) 6 (5.0±0.2) 10 (4.7±0.4) 4 (4.6±0.1) 6 (5.2±0.1) 7 (5.0±0.3) 17 (5.0±0.2) 18 (4.9±0.1) 13 (5.1±0.5) 18 (4.3±0.1) 19 (5.1±0.1) 8 (3.6±0.1) 11 (3.4±0.2) 9 (3.7±0.0) 12 (3.3±0.3) 14 (3.8±0.1) 8 (4.5±0.1) 7 (4.2±0.5) 6 (4.5±0.1) 8 (4.5±0.3) 10 (4.4±0.4) 0.1 14 (5.2±0.2) 0.3 16 (5.5±0.0) 0.5 15 (5.6±0.3) 0.7 16 (5.5±0.1) Note: ef: early flowering including higher number of flowers, sg: slow growth of shoots with variant shape of leaves having long and thick petiole, ssa: stubby shoot apices and flattened stem. The numbers in the parentheses are average heights of novel variants in cm ± Standard Error. 96 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 1 Relationship between Induction of Novel Somaclonal Variants and Types of Organogenesis in Muskmelon (Cucumis melo L.) itself generated genetic variability, was so called soma- clonal variation, in the regenerants. During in vitro re- generation studies, a number of somaclones was found in R0, R1 and R2 generations in medicinally important cucurbit, Citrullus Ccolosynthis (L.) Schrad (Shasthree et al., 2009). Our experiments conducted on muskmel- on through direct and indirect organogenesis confirmed this conspicuous phenomenon. Interestingly, it was observed that greater number of morphologically normal somaclones obtained via direct organogenesis produced lower rate of plants with varia- tions compared to indirect organogenesis. This may be due to the minimal production of callus by the cytoki- nin-type-hormone, BAP, added in the direct organo- genesis protocol. High frequency multiple shoots were formed on MS nutrient medium containing cytokinins through direct organogenesis without intervening callus phase in Passiflora foetida L. (Anand et al., 2012). Plant regeneration from explants via an intermediate callus stage (indirect organogenesis) was often associated with more variations than regenerants induced via direct or- ganogenesis whereby little or no callus was produced (Harini and Sita, 1993). The addition of auxin (2,4-D) to BAP in SIM resulted in lower rates of induction of normal shoots suggesting that the presence of 2,4-D in the medium may inhibit the production of phenotypically normal somaclones, which resulted increase in the production of somaclon- al variants. On the other hand, BAP at concentrations of 0.1, 0.3 and 0.5 mg-l did not produce any somaclonal variants obtained from proximal cotyledon, petiole and distal cotyledon explants through direct organogenesis, while the reverse trend was observed in indirect organo- genesis. It can be suggested that muskmelon shoots with normal traits can be obtained at certain concentration of BAP through direct organogenesis. It should be noted that the production of morphologically normal shoots is an important factor to ensure success of genetic manip- ulation (gene transformation) experiments of cucurbits e.g. muskmelon for variety development (Mohiuddin et al., 2000). There are some more reports found available on variety developed through direct organogenesis in tomato (Ewa et al., 2000) and garlic (Taşkın et al., 2013) even production of true-to-type in vitro-propagated Aloe vera L. plants (Khatun et al., 2018) and virus free plant variety (Ahmed et al., 2019). Various types of novel variants such as early flower- ing, slow growth with long and thick petioles, and stubby shoot apices were observed among the shoots of musk- melon derived from different explants. Shasthree et al. (2009) also observed a number of variations in habit, leaf and tendrillar character, fruit number, sizes, colours, and seed coat colours. Similar variation was observed in ga- metoclonal variation in Populus euphratica (Mofidabadi et al., 2001) as well as somaclonal variation of cucumber (Mohiuddin et al., 2003). Our study confirmed that the range of morphogenic changes in somaclones was significantly higher when somaclones were obtained through indirect organogen- esis. This technique could be utilized for the production of somaclonal variants like early variety, late variety, early flowering and/ high yielding variety, dwarf variety needs less nutrients, variety with desirable body configurations of muskmelon, etc. There are several published reports indicating above mentioned achievements obtained from sugarcane early variety development (Sreenivasan and Jalaja, 1983), early flowering in both woody bam- boo (Yuan et. al., 2017) and in Swertia chirayita, an en- dangered medicinal herb (Sharma et. al., 2014). Other achievements similar to our findings were also obtained from dwarf variety generally needs less nutrients or other purpose like ornamental plant production (Leva and Petruccelli, 2011), production of high yielding va- riety observed in fruit crops (Harsimrat and Manjot, 2020), and variety with desirable body configurations to produce new genotypes for breeding purposes (Vita- mvas et. al., 2019). 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