Development of an Embryogenic Suspension Culture of Bitter Melon (Momordica Charantia L.) M

Scientia Horticulturae 109 (2006) 123–129 www.elsevier.com/locate/scihorti

Development of an embryogenic suspension culture of bitter melon (Momordica charantia L.) M. Thiruvengadam a,*, S. Varisai Mohamed b, C.H. Yang a, N. Jayabalan c a Graduate Institute of Biotechnology, National Chung-Hsing University, 250, Kuo-Kuang Road, Taichung 402, Taiwan, ROC b Laboratory of Molecular Genetics, Agronomy Division, Agriculture Research Institute, Wufeng, Taichung 413, Taiwan, ROC c Department of Plant Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli 24, Tamilnadu, India Received 31 October 2004; received in revised form 23 March 2006; accepted 23 March 2006

Abstract We have optimized a system for the somatic embryogenesis via embryogenic suspension cultures in bitter melon (Momordica charantia L.). Friable calli could be induced in 30-day-old leaves on semi-solid MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.] medium supplemented with 1.0 mg/l 2,4-D. Large number of globular embryos (24.6%) were noticed when the calli was subcultured in liquid medium containing 1.5 mg/l 2,4-D. The complete removal of 2,4-D in the later stages of culture, stimulated their further development to heart and torpedo stages. Microscopic examination revealed the ontogeny of somatic cell development via the formation of cell clusters, which then enlarged to pro-embryos, and gave rise to heart and torpedo stages within a period of 2 weeks. Somatic embryos successfully germinated on agariﬁed MS medium with no additional growth regulators. An effect of media, other components and stimulating factors such as carbohydrates, amino acids has also been evaluated for somatic embryogenesis. The full strength MS medium containing 50 mg/l PVP and 40 mg/l glutamine was effective to achieve a high frequency of somatic embryo induction, maturation and further development. An average of 6.2% young plants was achieved from friable callus and was phenotypically normal. To our knowledge, there is no published report on somatic embryogenesis of bitter melon (M. charantia L.) via embryogenic callus or cell suspension cultures. These results are likely to facilitate genetic transformation of bitter melon. # 2006 Elsevier B.V. All rights reserved.

Keywords: Embryogenic callus; Cell suspension culture; Somatic embryos; Growth regulators; Momordica charantia L

1. Introduction antiseptic (Ambasta, 1986). Recently, few reports have also shown that this vegetable has significant anti-HIV (Lee-Huang Bitter melon (Momordica charantia L.) is one of the most et al., 1990, 1995), and anti-tumor growth (Okabe et al., 1980). important species belonging to family Cucurbitaceae. It is However, bitter melon is extremely susceptible to damage by believed to be originated in Asia and in the tropical areas of many pathogens such as fungus, virus and insects, which severely Africa, the Caribbean and South America and is commonly limit the yield (Telang et al., 2003). Furthermore, improvement consumed as a vegetable. It’s fruits, leaves and roots containing via genetic transformation pre-requisites the establishment of an two alkaloids (momordicine and cucurbitacins) which have efficient, fast and reproducible plant regeneration system. There shown to be highly effective components in preventing are reports of limited in vitro studies in Momordica tissue abortifacious, anti-diabetic, anti-rhematic, carminative, febri- cultures i.e. shoot bud differentiation in long term callus cultures fuge medicine for jaundice, leprosy and applied for bleeding of Momordica and Cucumis (Halder and Gadgil, 1982), in vitro piles, urinary complaints, snake bite, scorpion sting and as an flowering of M. charantia (Wang et al., 2001) and regeneration from shoot tips of M. dioica (Thiruvengadam and Jayabalan, 2001). Attempts at optimizing somatic embryogenesis of the Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, (Æ)-cis, bitter melon (M. charantia) have apparently been unsuccessful. trans-abscisic acid; CH, casein hydrolysate; PVP, polyvinylpyrrolidone; This paper identifies factors important for the successful ASCA, ascorbic acid * Corresponding author. Tel.: +886 4 22840328x757. standardization of somatic embrogenesis in M. charantia via E-mail address: [email protected] (M. Thiruvengadam). cell suspension cultures.

2. Materials and methods 1965) and SH medium (Schenk and Hildebrandt, 1972) were tested for their efficacy in the induction of embrogenic calli. 2.1. Collection of seeds and germination The effect of different carbohydrates (sucrose, fructose, and maltose at concentration ranging from 0% to 5%) was tested for Seeds of bitter melon (M. charantia cv. Coimbatore-1) were somatic embryogenesis. The full strength MS media with 3% obtained from Arignar Anna farm, Kudimianmalai, Pudukkot- sucrose was treated with various amino acids (alanine, proline, tai, Tamilnadu, India. Seeds were potted in a soil less mix 1:2:1 glutamine at concentrations ranging from 0 to 50 mg/l) and (peat:vermiculite:soil) and maintained in growth chambers at additives (0–100 mg/l of casein hydrolysate, polyvinylpyrro- 27 8C day/22 8C light under a 16 h photoperiod. The plants lidone, ascorbic acid and 0–1.0 mg/l of abscisic acid) to were fertilized and watered at weekly intervals. Leaves were determine their efficiency on induction and maturation of excised from highly proliferating (30-day-old) plants in growth somatic embryogenesis. chamber and rinsed thoroughly in running tap water for 2 h. The leaves were then surface sterilized by agitation in 5% 2.5. Transplantation laboline and 0.1% HgCl2 for 10 min and rinsed five to seven times with sterile distilled water. After the plantlets were grown to the top of the culture tube, they were removed from the medium and transferred to a small 2.2. Explant preparation and callus induction paper pot (6.5 cm Â 6.5 cm) containing mixture of sterilized vermiculite, sand and soil (2:1:1). The plants were covered with The leaves were cut into 3–5 mm2 which included the midrib polythene bags and nourished with 1/4 MS nutrient solution. portion. They were inoculated with their adaxial surface in The hardened plants were established in a plant growth contact with the semi-solid callus induction medium in culture chamber under a 16 h photoperiod at 25 8C. tubes (25 mm Â 150 mm, Borosil, Mumbai) plugged with non- absorbent cotton plugs. The callus induction medium com- 2.6. Statistical analysis prised of MS salts, MS vitamins plus 3% sucrose, 0.8% Bacto agar (Hi media Co, Mumbai, India) and 2,4-D (0.5–3.0 mg/l) Each treatment consisted of at least 50 explants and the with adjusted pH 5.8 prior to autoclaving. The cultures were experiment was repeated thrice. All experiments were analyzed maintained at 25 Æ 2 8C under 16 h light 8 h dark photoperiod by variance analysis (ANOVA), followed by Dunnett Multiple with a light intensity of 150 mmol mÀ2 sÀ1. Comparison and Tukey tests at P = 5% level by SPSS software version (SPSS 14 for windows, SPSS, Chicago, IL, USA) were 2.3. Development of somatic embryos from liquid culture used to compare each treatment group with control.

Suspension cultures were initiated by transferring 500 mg 3. Results (approximately) of friable calli in a 125 ml Erlenmeyer flask (Pyrex) containing 25 ml MS liquid medium supplemented with 3.1. Initiation of callus thiamine HCl (0.1 mg/l), pyridoxine HCl (0.5 mg/l), nicotinic acid (0.5 mg/l), myo-inositol (100 mg/l), sucrose (30 g/l) and After 21 days on MS medium containing 1.0 mg/l 2,4-D, different concentrations (0.0–2.0 mg/l) of 2,4-D. The cultures approximately 90% of leaf segments gave rise to a well- wereplacedonagyratoryshaker(100–110 rpm)atalightintensity organized friable calli. They were found to be comprised of of 25 mmol mÀ2 sÀ1, 16:8 h photoperiod and a temperature of dense cytoplasm, small vacuoles and large nuclei with deeply 25 Æ 2 8C. Sub-culturing was done once in a week by the stained nucleoli (Fig. 1a) when observed under a compound replacement of 10 ml of spent medium with fresh medium of the microscope. Occasionally, a portion of callus cells was non- samecomposition.Cellsfromthesuspensionwereobservedunder embryogenic, showing highly vacuolated, non-nucleated cells a compound microscope during the culture period. After 2nd and was deemed not suitable for further liquid cultures. These week of subculture, the suspension culture were filtered through a sectors were physically removed from the friable calli and were sterile 200 mM sterile stainless steel sieve and the filtered subcultured for establishing suspension cultures. Callus suspension was allowed to settle under the force of gravity in a proliferation was found to vary with different 2,4-D concentra- graduate tube. Filtered cells of 50 mg (Packed cell volume) were tion. On varying concentrations tested, 2,4-D 1.0 mg/l was subcultured into fresh liquid medium comprising of MS salts, MS found to be best for callus induction. An increase in the 2,4-D vitamins supplemented with 2,4-D (1.5 mg/l) in 125 ml Erlen- concentration beyond 1.0 mg/l resulted in visible lower growth meyer flasks containing 25 ml medium. For tracing the ontogeny rates and proportion of embryogenic tissue (data not shown). of somatic embryos, several samples from suspension cultures were observed microscopically at varying periods of growth. 3.2. Suspension culture and ontogeny of somatic embryos

2.4. Optimization of media conditions Friable calli were sub-cultured on MS liquid medium containing different concentrations of 2,4-D (0.0–2.0 mg/l). Four different basal medium MS salts, MS + B5 vitamin Following transfer to liquid medium, the cells of the calli (Gamborg et al., 1968), LS medium (Linsmaier and Skoog, dispersed to give rise to a ﬁne suspension and displayed rapid M. Thiruvengadam et al. / Scientia Horticulturae 109 (2006) 123–129 125

Fig. 1. Different stages in somatic embryogenesis of bitter melon (Momordica charantia): a: friable callus originating from leaf explants; b: pro-embryogenic mass; c: globular staged embryo; d and e: heart shaped embryos; f and g: torpedo-staged embryos; h: germination of embryos; i: hardened plant; j: young plantlets from somatic embryos. Bar represents 0.5 mm (a–g); 0.4 mm (h–j). 126 M. Thiruvengadam et al. / Scientia Horticulturae 109 (2006) 123–129 cell division. The best frequency of cell division and growth characteristics were recorded in 1.5 mg/l 2,4-D. Microscopic observation of suspension cultures, after a week, revealed that the suspension comprised of morphologically two distinct cell types, namely spherical cells with visible nucleus, dense cytoplasm and highly vacuolated elongated cells with no apparent nucleus and thin cytoplasm. The spherical cells, proliferated in the form of tubular outgrowth, followed by transverse division, resulting in two and four cell stages. These later underwent divisions in various planes to produce a pro- embryogenic mass (Fig. 1b). The pro-embryogenic masses subsequently gave rise to circular globular-staged embryos (Fig. 1c) over a period of 10 days. Globular stages further differentiated into heart shaped embryos within 7 days of transfer to 2,4-D free liquid MS medium (Fig. 1d and e). Torpedo-shaped embryos were recovered with an additional 2 Fig. 3. Influence of 2,4-D concentrations on induction of somatic embryogenesis weeks of sub-culture onto the media of the same composition fromleafderivedcallus ofMomordicacharantiainMSliquidmedium.Eachvalue (Fig. 1f and g). Mature torpedo shaped embryos successfully represents the treatment mean of 10 independent replicates. The frequency of different stages of somatic embryos were determined by taking the samples from germinated into tiny plantlets on hormone-free semi-solid MS 10 replicate flasks per 2,4-D treatment. Embryo percentage denotes the number of medium (Fig. 1h and j). About 6.2% of germinating embryos embryos to the total number of embryos formed per flask. Bars represent the could be raised into young plantlets on hormone-free MS mean Æ S.E.M. Significant treatment by ANOVAwith comparison to the control vs. 2,4-D treatment by Dunnett’s multiple comparison test [*significantly greater medium solidified with gel-rite and hardened to plantlets onto ** soil mixture in the greenhouse (Fig. 1i). than the control (P =0.5), significantly less than the control (P =0.5)].

3.3. Effect of media, amino acids, and other components somatic embryo development decreased significantly with the use of glucose, fructose and maltose (Data not shown). Of the The four different media (MS, MS + B5 (vit.), LS and SH) additives (PVP, CH, ASCA, and ABA) tested for their possible were used to assess their efficiency to induce somatic stimulatory effects on somatic embryogenesis, only PVP at embryogenesis from leaf segments. On comparison with different media, MS media exhibited highest frequency in Table 1 the formation of somatic embryogenesis (Fig. 2). The highest Effect of various media additives on somatic embryogenesis in MS liquid frequency of globular stage was achieved in MS liquid medium medium with 1.5 mg/l 2,4-D with increasing concentration of 2,4-D until 1.5 mg/l, whereas Additives Concentrations Different stages of somatic embryos (%) on increasing concentration of 2,4-D (0.25–2.0 mg/l) exhibited (mg/l) gradual decrease in the frequency of developmental stages of Globular Heart Torpedo heart and torpedo shaped embryos (Fig. 3). Among the four stage stage stage different carbohydrates tested, sucrose at 3% was most PVP 0 24.6 aa 14.4 aa 12.6 aa favorable for the induction of somatic embryogenesis while 25 24.2 a 13.8 a 11.2 a 50 27.4 a 19.5 a 18.2 a 75 12.2 b 7.8 b 4.4 b 100 5.2 c 3.2 c 1.2 c CH 0 24.6 a 14.4 a 12.6 a 25 23.2 a 10.4 a 7.6 b 50 14.2 b 7.2 b 3.8 c 75 3.2 c 1.8 c 1.2 cd 100 – – – ASCA 0 24.6 a 14.4 a 12.6 a 25 13.2 b 7.6 b 3.8 b 50 5.2 c 2.8 c 1.2 c 75 3.2 d 1.0 cd – 100 – – – ABA 0 24.6 a 14.4 a 12.6 a 0.25 6.8 b 3.2 b 1.6 b 0.50 3.2 bc 1.8 bc 1.2 bc 0.75 1.6 c 1.0 c – 1.00 – – – Fig. 2. Effect of different basal media with or without 1.5 mg/l 2,4-D on a Means within an additive followed by the same letters are not significantly somatic embryogenesis. Means within a somatic embryo stage followed by the different as indicated by Tukey’s test (a = 5%). (À) Not determined due to nil same letter are not significantly different as indicated by Tukey’s test (a = 5%). response. M. Thiruvengadam et al. / Scientia Horticulturae 109 (2006) 123–129 127

Table 2 Effect of amino acids on somatic embryogenesis in MS liquid medium with 1.5 mg/l 2,4–D Amino acid types Concentrations (mg/l) Different stages of somatic embryos (%) Globular stage Heart stage Torpedo stage Alanine 0 24.6 a 14.4 a 12.6 a 10 2.6 bc 1.2 b 1.0 b 20 3.4 b 1.2 b – 30 1.2 c – – 40 – – – 50 – – – Glutamine 0 24.6 aa 14.4 aa 12.6 aa 10 24.2 a 16.2 a 12.2 a 20 26.2 a 18.4 a 13.4 a 30 32.6 a 22.2 a 18.2 a 40 34.2 a 24.6 a 21.6 a 50 25.2 a 17.6 a 13.4 a Proline 0 24.6 a 14.4 a 12.6 a 10 21.2 a 14.4 a 11.2 b 20 14.6 b 8.8 b 6.4 c 30 4.2 c 1.8 c 1.2 d 40 – – – 50 – – – a Means within an additive followed by the same letters are not signiﬁcantly different as indicated by Tukey’s test (a = 5%). (À) Not determined due to nil response.

50 mg/l was noticeably beneficial (Table 1). Three different effective in the induction of somatic embryogenesis from amino acids, viz alanine, glutamine and proline were used to different explants of C. pepo. We found that 2,4-D (1.0 mg/l) assess their effects on the development of somatic embryos. was found to be most effective for induction of friable callus Fortification with proline and alanine proved to be marginally after 21 days of culture, while lower concentration of 2,4-D effective while glutamine at 40 mg/l significantly enhanced the (0.5 mg/l) failed to induce embryogenic calli from cultured frequency of induction and maturation of somatic embryos explants. We observed that the concentration of 2,4-D is the key (Table 2). factor governing the induction of a friable embryogenic callus. Further growth of the callus and subsequent embryogenesis 4. Discussion were inhibited by an increase in 2,4-D concentration (>1.5 mg/ l). Supplementation of 2,4-D with low concentrations of Somatic embryogenesis has been reported from diverse cytokinins interfered with induction and proliferation of explants such as cotyledon and shoot tip explants of Cucumis somatic embryos in bitter melon (data not shown). This is, melo on MS medium fortified with BAP 2.0 mg/l and NAA however, in contrast to the results obtained by Punja et al. 0.1 mg/l (Halder and Gadgil, 1982; Young et al., 1983). (1990) in C. sativus were somatic embryogenesis was achieved However, no reports of successful somatic embryogenesis exist on MS medium supplemented with 2,4-D or NAA supple- for M. charantia. In the present investigation, somatic mented with BAP. embryogenesis has been achieved for the first time in bitter Wetherell and Halperin (1963) reported that key element for melon. Our preliminary experiments revealed that only the the induction of somatic embryogenesis is the presence of high explants excised from 1-month-old ex vitro plants were highly levels of nitrogen in the form of ammonium nitrate that enhance competent for induction of friable, embryogenic callus. Leaf embryo initiation and maturation. Of the various media (MS, segments cultured on MS medium swelled within 10 days, and MS + B5 vit, LS and SH) compared, MS medium was found to in 21 days over 80% of them proliferated into friable callus. be best for the friable callus production and embryo Moreover, 80% of the friable calli inoculated in liquid medium differentiation. SH medium failed to produce torpedo embryos differentiated into somatic embryos. The growth regulator most from embryogenic cell cultures of 2,4-D. Our results suggest commonly used to induce somatic embryogenesis is 2,4-D. For that the high nitrate content of the MS medium is contributory instance, MS medium supplemented with 2,4-D (0.4 mg/l) was towards enhanced somatic embryogenesis obtained on this effective for the induction of somatic embryogenesis from leaf medium. The source of carbohydrates often plays an important and stem explants of C. sataivus (Malepszy et al., 1982; role in somatic embryogenesis. Guis et al. (1997) reported that Malepszy and Nadolska-Orczyk, 1983) and some pulses like glucose was the most important sugar for somatic embryogen- Cajanus cajan (Anbazhagan and Ganapathi, 1999), Vigna esis in melon. However, our experiments confirmed sucrose to unguiculata (Premanand et al., 2000; Ramakrishnan et al., be the carbohydrate of choice for somatic embryogenesis in 2005). On the other hand, Jelaska (1972, 1974) noted that MS bitter melon. Our results support similar observations made in medium fortified with 2,4-D (1.0 mg/l) and IBA (1.0 mg/l) was cucumber tissue culture (Lou and Kako, 1995; Lou et al., 1996) 128 M. Thiruvengadam et al. / Scientia Horticulturae 109 (2006) 123–129 in which sucrose was essential for somatic embryogenesis, under green house conditions. The entire procedure from the while mannitol was not found to be efficient. More recently, induction of somatic embryos till the formation of plantlet Nakagawa et al. (2001) reported the interaction between sugars recovery could be completed within 3 months, thereby resulting and abscisic acid on somatic embryogenesis from mature in a rapid yet robust protocol. We believe that this protocol cotyledons in C. melo. However, in bitter melon, the would hasten the development of genetic transformation supplementation of sugars with abscisic acid on medium studies in bitter melon. resulted in decreased frequency in somatic embryo induction and further development. Diverse amino acids are capable of modulating the Acknowledgements efficiency of somatic embryogenesis (Bela and Shetty, 1999). It has also been observed that somatic embryogenesis The authors wish to thank the Arignar Anna farm, is associated with an increase in the endogenous levels of Kudimianmalai, Pudukkottai, Tamilnadu, India, for supplying proline (Thorpe, 1993). Medium supplemented with proline the seed materials. We gratefully acknowledge Dr. Prasanna was found to be beneficial for somatic embryo production in Bhomkar, University of Lethbridge, Canada and Mrs. T. Rekha, Medicago sativa (Shetty and McKersie, 1993) and Vigna Research Assistant, Institute of Biotechnology, National Chung radiata (Girija et al., 2000). In contrast to this report, amino Hsing University, Taichung, Taiwan for their valuable acid supplementation was found to have neutral effects in suggestions in completing the manuscript. Pisum sativum (Chengalrayan et al., 1994). In this study glutamine was most effective in enhancing the frequency of References somatic embryogenesis in bitter melon while proline and alanine had marginal promotery effects. Similar results have Ambasta, S.P., 1986. The Useful Plants of India. Publication and Information been reported by Varisai Mohamed et al. (2004), in which Directorate, CSIR, Hillside Road, New Delhi. glutamine (50 mg/l) enhanced the efficiency of somatic Anbazhagan, V.R., Ganapathi, A., 1999. Somatic embryogenesis in cell sus- embryogenesis i.e. Callus induction, embryo differentiation pension culture of Pigeonpea (Cajanus cajan L.). Plant Cell Tissue Org. Cult. 56, 179–184. and embryo maturation of horse gram. These results suggest Bela, J., Shetty, K., 1999. 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