Indian Journal of Biotechnology Vol 15, October 2016, pp 581-588

In vitro multiplication of pointed gourd ( dioica) through nodal explant culture, and testing the genetic fidelity of micropropagated using RAPD markers

Sanjeev Kumar1,2, Hemant Singh1, Vikramaditya Pandey3 and B D Singh1* 1School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221 005, 2Division of Crop Improvement, Indian Institute of Sugarcane Research, Rai Bareli Road, Lucknow 226 002 India 3Division of Horticulture, Indian Council of Agricultural Research, Krishi Anusandhan Bhawan II, Pusa Campus, New Delhi 110 012 India Received 10 April 2015; revised 28 August 2015; accepted 3 September 2015

An efficient in vitro propagation system was developed for large-scale multiplication of a high yielding line (IIVRPG-102) of pointed gourd (Trichosanthes dioica Roxb.) utilizing nodal segments. A procedure for surface sterilization, involving a combination of treatments with cetrimide (1.0%) for 30 min, trimethoprim (0.05%) and dimethycarbendazim (0.2%) for 2 h, and finally with 0.1% HgCl2 for 3 min, was standardized yielding 87% contamination-free cultures. growth regulator (PGR)-free MS medium was adequate for inducing bud break in up to 81.5% of the explants. Addition of PGRs like NAA, and thiadizuron had a small but significant effect on the frequency of responding explants. The highest number of shoots per explant (7.6) was obtained on MS medium supplemented with 1.0 mg L-1 BA. Root induction was achieved with NAA or IBA treatment. The best rooting responses were obtained on a medium containing 0.1 mg L-1 (89.0%) or 0.2 mg L-1 IBA (87.0% rooted shoots). The micropropagated shoots with well-developed roots were acclimatized and thereafter successfully transplanted to a glasshouse with 90.6% survival rate. PCR analysis with 25 random decamer primers yielded monomorphic products in the micropropagated plants identical to those of the mother clone, confirming the genetic uniformity of the regenerated plants. This protocol could be applied to produce a large number of propagules of high-yielding selections to fulfill commercial demand.

Keywords: Genetic fidelity, micropropagation, nodal culture, pointed gourd, Trichosanthes dioica

Introduction dioecious nature, and vegetative means of Pointed gourd (Trichosanthes dioica Roxb.), propagation. It is typically multiplied through stem6 commonly known as Parwal or Patal, is one of the and root cuttings2. Propagation through seeds is not most nutritive cucurbit vegetables. In tropical and sub- practicable due to poor seed germination and late tropical regions of the world, particularly in India and flowering of seed-derived plants, and presence of high Bangladesh, it holds a coveted position in the market frequency of non-fruiting male plants in the during the summer and rainy seasons1. Pointed gourd is population7,8, while only 10% male plants are required a perennial crop, and is highly accepted due to its for a good fruit set9. The use of stem and root cuttings availability for about 8 months (February-September) is labour intensive because ~7000 vine cuttings of per year2. Fruits of pointed gourd are consumed in a ~90 cm each are required for planting one hectare variety of ways including as vegetable curry, pickle area6,10; this restricts the rate of vegetative and various confections3; its young leaves and shoot- multiplication1. There are only few elite selections of tips are also consumed as vegetables4. Fruits are rich this difficult-to-breed crop and there is inadequate in proteins and , and some reports indicate supply of the planting material so that farmers its role in lowering blood sugar and serum generally use local selections for commercial triglycerides5. cultivation11. Therefore, in vitro multiplication of elite T. dioica is morphologically distinct from the other clones would be a more attractive approach for cucurbitaceous species due to its perennial and producing quality propagules at commercial scale. In vitro regeneration in T. dioica using shoot-tip and ______nodal segments has been reported10,12-16 but, so far, the *Author for correspondence: Tel: +91-542-2368331; Fax: +91-542-2368694 genetic uniformity of the micropropagated plants was [email protected] not investigated. Screening micropropagated plants for 582 INDIAN J BIOTECHNOL, OCTOBER 2016

somaclonal variants at an early stage is essential. The Netherlands), dispensed ~20 mL in culture tubes detection of “off-types” among micropropagated (25×150 mm2, Borosil, Mumbai, India) and sterilized plants, especially perennial crops, using by autoclaving at 121°C for 20 min. The cultures morphological traits or physiological behaviour is were incubated at 25±2°C, under 16 h light period of labour-intensive and time-consuming task. On the white light from fluorescent tubes at unit of irradiance other hand, DNA markers are more informative and 50 E m-2 s-1 and 8 h dark period. can detect DNA variations that may be present in plants derived from tissue culture. Among the Culture Establishment, Shoot Multiplication and Root Induction different techniques available to detect DNA For multiple shoot induction, single node segments polymorphisms, randomly amplified polymorphic excised from established cultures were transferred on DNA (RAPD) markers have been widely used to to MS medium supplemented with different concentrations (0.1 to 5.0 mg L-1) of 6-benzyladenine study clonal integrity and to detect genetic and -1 somaclonal variations in Momordica dioica17 and (BA) alone or in combination with 0.1 or 0.2 mg L 18 naphthalene acetic acid (NAA), or with different Spondias mangifera . In view of the above, the -1 present investigation was carried out to develop a concentrations (0.1 to 0.5 mg L ) of thidiazuron (TDZ). For root induction, in vitro-raised shoots from protocol for in vitro multiplication of an elite -1 selection (IIVRPG-102) of pointed gourd. The genetic MS medium containing 1.0 mg L BA were initially integrity of the in vitro regenerated plants was cultured on to PGR-free MS basal medium for 1 wk checked using RAPD analysis. and, thereafter, shoot pieces of ~2.5 cm containing at least 2 nodes were cultured on MS medium Materials and Methods supplemented with different concentrations of indole 3-butyric acid (IBA; 0.1 to 2.0 mg L-1) or NAA Explant Source and Surface Sterilization -1 The plant material consisted of a high yielding elite (0.1 to 1.0 mg L ). Observations were recorded in selection (acc. no. IIVRPG-102) obtained from Indian terms of (i) days to bud break, (ii) number of explants Institute of Vegetable Research (IIVR), Varanasi, India. showing bud break or shoot/root development, Young vine cuttings (30-40 cm) were collected from (iii) number of shoots per responding explant, field-grown plants. Stem segments of 6.0-8.0 cm (iv) mean length of elongated shoots, (v) mean containing 3-4 nodes were excised, washed thoroughly internode length, (vi) number of roots per shoot, in running tap water, and then treated with 1.0% (w/v) (vii) mean root length, and (viii) number of plantlets cetrimide (Merck, Mumbai, India) solution for 30 min. survived in the field, as applicable to different experiments. After thorough rinsing in running tap water, the explants were treated with a solution containing an antibiotic Experimental Design (Trimethoprim; 0.05%, Merck, Mumbai, India) and a The experiments were laid out according to a fungicide (Dimethyl-carbendazim, 0.2%, BASF, randomized complete block design with 3 replications; Mumbai, India) for 2 h. Finally, the explants were each replication had 18 explants per treatment. The surface sterilized with 0.1% mercuric chloride cultures were observed periodically and the data as (HgCl2) for 3 min inside a laminar air-flow cabinet stated above was recorded after 21 d. Analysis of followed by 4-5 rinses with sterile double distilled variance (ANOVA) appropriate for the design was water. In the end, the stem pieces were cut to generate carried out to detect the significance of differences single-node explants of ~1.5 cm, which were among the treatment means, and treatment means inoculated onto plant growth regulator (PGR)-free were compared using Duncan’s Multiple Range Test full- and half-strength Murashige and Skoog19 (MS) (DMRT). Both the analyses were carried out using the medium with 3% sucrose (Himedia, Mumbai, India) software Statistical Package for Agricultural for culture establishment. Research; SPAR20.

Nutrient Medium and Culture Conditions Hardening of Plantlets and Transfer to Soil MS medium was used in all the experiments and it For hardening and establishment, healthy plantlets was supplemented with different concentrations of obtained from shoots rooted on MS medium with PGRs as per the requirement. The medium pH was 0.1 mg L-1 IBA were removed from culture tubes, adjusted to 5.8 using 1 N NaOH or HCl, solidified washed under running tap water to remove agar from using 0.8% agar (Plant Agar; Duchefa, The their roots, transplanted in to plastic cups filled with KUMAR et al: MICROPROPAGATION OF POINTED GOURD 583

sterilized Soilrite mix (KelPerlite, Bangalore, India) high (60-90%). It has been reported that the routine and covered with polyethylene bags. The pots were sterilization procedures practiced during in vitro then kept in a culture room at 25±2°C, and 16 h light culture operations seldom control systemic and 8 h dark period. The pots were irrigated every microorganisms present in plants22. Therefore, alternate day with distilled water. After 7 d, the explants were pre-treated for 2 h with a solution polyethylene bags were gradually removed over a containing trimethoprim (0.05% w/v; HiMedia, period of 15 d. The plantlets were kept in the culture Mumbai, India) and dimethyl carbendazim (0.2% w/v; room for 10 d, and then transferred to room BASF, Chennai, India); such a pre-treatment with temperature for another 10 d before they were final surface sterilization with 0.1% HgCl2 for 3 min transferred to a glasshouse. yielded the highest (86.7%) frequency of contamination-free cultures as judged by visual Genomic DNA Extraction and PCR Amplification screening. Although no contamination was recorded The genomic DNA was isolated from leaf tissue of when explants were treated with HgCl2 for 5 min, 54 in vitro-raised plants grown in a glasshouse, and these explants died due to bleaching and necrosis. It from the parental clone using DNeasy Plant Mini Kit has been advocated to use a combination of an (Qiagen, Germany) according to the manufacturer’s antibiotic and a fungicide to target more than one type instructions. The quality and quantity of genomic of endophytic microorganisms22. DNA were assessed by spectrophotometric Culture medium salt strength had a significant effect measurement and agarose gel electrophoresis. A total on bud break and shoot development. Full-strength of 25 random decamer primers (Series OPA01 to PGR-free MS medium was more suitable for in vitro OPA20, and OPD01 to OPD05; Eurofins MWG shoot initiation since it supported bud break in 77.8% of Operon, Alabama, USA) were employed to amplify explants compared to half-strength PGR-free MS the genomic DNA. Polymerase chain reaction (PCR) medium (40.7%). Shoot bud development was visible amplification was performed according to the 21 within 8 d inoculation of explants (Fig. 1a). Nodal protocol of Kumar et al . The amplification reaction segments were used as explants for micropropagation was carried out in 25 µL volume containing 50 ng since they yield true-to-type plants, and such explants genomic DNA, 2.5 µL PCR buffer (Fermentas, were reported to be more suitable for in vitro Madison, USA), 200 µM dNTPs (Bangalore Genei, Bangalore, India), 1.5 U Taq DNA polymerase (Fermentas, Madison, USA), and 0.4 µM primer using a thermal cycler (PTC 1000, MJ Research). The first amplification cycle consisted of the following steps: at 94°C for 4 min, at Tm-5°C for 1 min and at 72°C for 1 min. This was followed by 39 cycles for 1 min at 94°C, 1 min at Tm-5°C, and 1 min at 72°C; the final extension was allowed for 10 min at 72°C. The amplified DNA fragments were resolved through electrophoresis in 1.5% agarose gel using TAE buffer and visualized in a gel documentation system (Alfa Imager 2200, Alfa Innotech Corporation, California, USA). The 100 bp to 3 kb DNA Ruler Plus (Merck, Mumbai, India) was used as molecular size marker.

Results and Discussion

Culture Establishment Fig. 1 (a-f)—Axillary shoot initiation from in vitro established A high degree of contamination (46.7-100%) was nodal explants after 12 d of culture on PGR-free MS medium observed in explants treated with either 5 or 10% (a); multiple shoot formation after 21 d of culture on MS -1 sodium hypochlorite (NaOCl) for 5 or 10 min. medium+1.0 mg L BA (b); root induction from tissue culture- raised shoots on MS medium+0.1 mg L-1 IBA (c); malformed Treatment with 0.1% HgCl2 for 2-5 min also was thick roots on MS medium+1.0 mg L-1 NAA (d), & tissue culture- ineffective as the contamination frequency was very raised plantlets transferred to plastic cups for acclimatization (e, f). 584 INDIAN J BIOTECHNOL, OCTOBER 2016

Table 1—Effect of different concentrations of BA, NAA and TDZ on shoot development from nodal segments of T. dioica

PGR concentration Days to bud Frequency of responding Number of shoots/ Mean shoot length* Mean internode (mg L-1) break* explants* responding explant* (cm) length* (cm) (%) BA 0.0 7.3a 81.5ab 1.0e 7.8a 1.7a 0.1 6.5ab 63.0cd 2.4d 1.2f 0.7d 0.2 5.4bc 68.5cd 2.8cd 1.7e 1.0c 0.5 6.8ab 74.0bc 5.3b 2.5c 1.1b 1.0 5.3bc 81.5ab 7.6a 4.8b 0.5e 2.0 4.8c 85.2a 5.1b 2.2d 0.3f 5.0 6.3abc 57.4e 3.2c 0.8g 0.1g LSD (0.05) 1.55 7.63 0.68 0.27 0.10 BA NAA 0.0 0.0 7.5b 81.5c 1.0e 7.7a 1.6a 0.0 0.1 8.0a 83.3bc 1.1e 6.6b 1.6a 1.0 0.0 5.0f 85.2bc 7.1a 4.2c 0.5cd 1.0 0.1 5.0f 92.6a 1.1e 1.0f 0.3d 2.0 0.1 5.4e 90.7ab 1.4d 0.9f 0.1d 1.0 0.2 6.0d 88.9abc 2.2cd 2.0d 0.8c 2.0 0.2 6.6c 81.5c 2.6b 1.6e 1.2b LSD (0.05) 0.49 8.18 0.16 0.33 0.14 TDZ 0.0 7.5a 70.4b 1.0c 7.9a 1.6a 1.0** 5.2d 80.2a 7.0a 4.5b 0.6b 0.1 6.8c 40.7e 3.0c 0.9c 0.5b 0.2 6.6cd 48.1d 3.3c 1.0c 0.5bc 0.3 6.4d 51.6cd 3.3c 0.7cd 0.5b 0.4 7.1b 53.8c 4.2b 0.6d 0.4bc 0.5 7.6a 38.9e 4.0b 0.5d 0.3c LSD (0.05) 0.27 5.18 0.25 0.39 0.17

Data recorded after 21 d of inoculation; each treatment had three replications, each comprising of 18 explants *Means having different letters in their superscript are significantly different from each other (P=0.05); comparison by DMRT within columns only **1.0 mg L-1BA only multiplication of T. dioica15,16. Axillary bud proliferation when cultured on MS medium containing 1.0 mg L-1 ensures clonal fidelity of the regenerants and, therefore, BA23. Similarly, a high frequency (93.86 & 100%) of can avoid the limitations of callus cultures. shoot regeneration from nodal explants cultured on MS medium containing 2.0 mg L-1 BA and 0.3 mg L-1 Shoot Multiplication -1 -1 -1 NAA, and with 2.0 mg L BA and 0.1 mg L NAA, Different concentrations (0.0 to 5.0 mg L ) of BA was reported by Malek and co-workers15 and Debnath had significant effects on shoot development. Days to and associates13, respectively. shoot initiation on different media ranged from 4.8 d in the case of MS medium supplemented with Shoot development from axillary buds of nodal 2.0 mg L-1 BA, or 5.3 d on medium containing explants was observed on all the media tested, but 1.0 mg L-1 BA to 7.3 d on PGR-free MS medium multiple shoots developed only when BA was added (Table 1). Similarly, the frequency of cultures to the medium (Table 1). The number of shoots showing shoot development was the highest (85.2%) increased with increasing BA concentration; the in cultures maintained on MS medium supplemented highest (7.6) number of shoots per explant was - with 2.0 mg L-1 BA (Table 1), closely followed by obtained on the medium supplemented with 1.0 mg L those cultured on the medium containing 1.0 mg L-1 1 BA (Fig. 1b), followed by 5.3 shoots per explant on BA or PGR-free MS medium (81.5%); while only medium supplemented with 0.5 mg L-1 BA. A further 57% of the explants responded when cultured on MS increase in BA concentration beyond 1.0 mg L-1 had a medium having 5.0 mg L-1 BA. These results are in suppressive effect on the number of shoots per agreement with the reports in pointed gourd where explant. A similar trend was reported by Malek and 92% of the shoot-tips showed shoot development co-workers15 for nodal explants, by Yadava and Dhir KUMAR et al: MICROPROPAGATION OF POINTED GOURD 585

for shoot-tip explants12, and by Hoque and supportive for shoot proliferation. Such effects could associates23 for cotyledon explants from be attributed to the use of a genotype from a different physiologically mature and immature seeds of T. geographical region or origin; in the present case it is dioica. However, Mythili and Thomas reported that, from Bangladesh. in the case of T. dioica shoot-tip cultures, 0.20 mg L-1 The different TDZ concentrations had a significant -1 BA and 0.35 mg L GA3 in MS medium resulted in effect on bud break and axillary shoot development. shoots with poor elongation coupled with high Days to bud break on different media ranged from 6.4 d -1 vitrification10. In case of some other cucurbits like on MS medium containing 0.2 mg L TDZ to 7.6 d on -1 spp., multiple shoot induction has been the medium containing 0.5 mg L TDZ. The frequency reported from shoot-tips on MS medium containing of explants showing shoot development declined 1.0 mg L-1 BA24. However, direct organogenesis in significantly with increase in TDZ concentration, and Coccinia indica was achieved on MS medium only 38.9% of the explants on the medium having -1 containing a much higher concentration of cytokinins 0.5 mg L TDZ showed shoot development (Table 1). -1 -1 -1 These findings are in agreement with the results of (2.5 mg L BA+0.5 mg L Kinetin) and 0.1 mg L 12 15 IBA25. The requirements of variable exogenous levels Yadava and Dhir , and Malek and co-workers who of individual growth regulators may reflect the reported that TDZ inhibited the number of responding differences in in vivo levels and/or degradation/ shoot-tips of T. dioica. Media containing TDZ supported utilization rates of these substances in the tissues of development of significantly more shoots per different species and genotypes26. responding explant than PGR-free MS medium, and the number of shoots/explant tended to increase with TDZ Single node explants were cultured on media concentration. However, TDZ containing media were supplemented with combinations of two different -1 significantly less supportive of shoot proliferation and concentrations of BA (1.0 & 2.0 mg L ) and NAA -1 -1 elongation than the medium having 1.0 mg L BA. (0.1 & 0.2 mg L ). These explants showed initiation of axillary shoot development between 5 d (1.0 mg L-1 Root Induction BA) and 8 d (2.0 mg L-1 BA+0.1 mg L-1 NAA) after Presence of NAA or IBA in the medium culture (Table 1). Addition of 0.1 mg L-1 NAA alone significantly enhanced both rooting frequency and to the MS medium did not have any significant root number per shoot (Table 2). The frequency of positive influence on the frequency of explants rooted shoots was the highest (>87%) on MS medium showing shoot proliferation, but it significantly supplemented with 0.1 or 0.2 mg L-1 IBA (Table 2); delayed bud break and suppressed shoot elongation as the average number of roots/shoot was just over 5 and compared to the PGR-free MS medium (Table 1). the mean root length was more than 2.0 cm (Fig. 1c). Addition of 0.1 or 0.2 mg L-1 NAA to the BA In general, number of responding cultures decreased supplemented media suppressed the number of shoots with increased NAA or IBA concentration, while the per explant as well as shoot length, resulting in close- number of roots per responding shoot increased up to set nodes. In the presence of NAA, the highest shoot a level of NAA and IBA, beyond which uncountable number per explant was only 2.6, and average shoot number of thick abnormal roots were produced length was 1.6 cm on the medium having 2.0 mg L-1 (Table 2, Fig. 1d). Root length was significantly BA+0.2 mg L-1 NAA. In contrast, 1.0 mg L-1 BA reduced with increases in NAA or IAA alone produced 7.1 shoots per explant, having average concentrations. In contrast, shoot length increased length of 4.2 cm. In fact, the combinations of BA and with NAA or IBA concentrations, except in the NAA were only marginally superior to PGR-free MS presence of 9.8 µM IBA where a significant reduction medium or MS medium having 0.1 mg L-1 NAA alone was noted (Table 2). Thus a lower concentration of in terms of number of shoots per explant, while they NAA (0.1 mg L-1) or IBA (0.1 or 0.2 mg L-1) was all yielded shoots drastically reduced in length. considered the most suitable for root induction. These Clearly, the combinations of BA and NAA were findings disagree with those of Yadava and Dhir12, inferior to BA alone, and they were not suitable for Debnath and associates13, and Malek and co-workers15, multiple shoot proliferation from nodal explants. who reported root induction in in vitro-raised shoots These findings are in contrast to the observations of of T. dioica on MS medium having a much higher Debnath and co-workers13 who found MS medium (0.5 or 1.0 mg L-1 NAA, or 0.5 mg L-1 IBA) auxin containing 2.0 mg L-1 BA+0.1 NAA to be the most concentrations; while Mythili and Thomas reported 586 INDIAN J BIOTECHNOL, OCTOBER 2016

Table 2—Effects of different concentrations of NAA or IBA on root induction in shoots derived from nodal segments of T. dioica

PGR conc. Frequency of Mean shoot No. of roots Mean root (mg L-1) rooted shoots* length* per responding length* (%) (cm) shoot* (cm) 0** 55.6cd 7.96de 7.2d 3.6a NAA 0.1 55.6cd 7.76e 10.0c 2.7c 0.2 66.7b 8.24d 20.8a† 2.9b 0.5 51.9 de 8.90c UC - 1.0 42.6e 9.30bc UC - IBA a f f d 0.1 88.9 6.65 5.1 2.5 0.2 87.0a 6.75f 5.7e 2.3e Fig. 2—Agarose gel image showing the amplification profile 0.5 64.8bc 5.11g 16.6b† 1.9f generated by primers OPA01 from genomic DNA of 54 in vitro- 1.0 50.0de 9.47b UC - cultured plants of T. dioica. [Lane M is 100 bp to 3 kb DNA Ruler 1.5 44.4e 10.52a UC - Plus, mother-stock plant (lane S), & in vitro-cultured plants grown f h 2.0 31.5 2.87 UC - in a glasshouse (lanes 1-54)]. LSD (0.05) 10.67 0.55 0.44 0.20 reported 80% survival of plantlets obtained from Data recorded after 21 dof culture 12 The experiment had three replications, each comprised of 18 explants shoot-tip explants . However, Mythili and Thomas *Means having different letters in their superscript are significantly had to resort to ex vitro establishment of rooted different from each other (P=0.05); comparison by DMRT within stumps containing 2-3 nodes, since they could not get columns only 10 **Growth regulator free MS medium good rooting of the in vitro-raised shoots . Similar UC: Uncountable profuse and thick abnormal roots; †Thick abnormal high rates of survival of micropropagated plants have roots been reported in other cucurbitaceous plants, viz., 28 32 poor rooting from in vitro-raised shoots on MS media M. charantia (90% survival) and M. dioica supplemented with 0.2 mg L-1 IAA or NAA10. (85% survival). In other members of family , root Genetic Fidelity of Micropropagated Plants induction often occurs on PGR-free MS medium, but All the 25 random primers used in this study a small auxin concentration promotes the frequency of produced reproducible and scorable bands. The root induction. For example, in the case of Cucumis number of amplified bands produced by each primer sativus, root induction was achieved on PGR-free 27 ranged from 3 (primer OPA10) to 8 (OPD04). The medium but, in bitter gourd (M. charantia), MS primers produced the amplification in following medium with 0.5 mg L-1 NAA28, or 0.1 mg L-1 IBA or 29 manner; OPA01 (6 amplification product), OPA02 NAA was reported to be the best for root initiation. (5), OPA03 (4), OPA04 (3), OPA05 (5), OPA06 (5), Chee reported rooting in C. pepo shoots on MS -1 OPA07 (3), OPA08 (4), OPA09 (3), OPA10 (3), medium containing 0.05 mg L NAA, while in case OPA11 (4), OPA12 (4), OPA13 (), OPA14 (), OPA15 of C. melo the same was achieved on MS medium (4), OPA16 (9), OPA18 (4), OPA19 (5), OPA20 (4), having 0.01 mg L-1 NAA30,31. OPD01 (4), OPD02 (5), OPD03 (3), OPD04 (8), and Acclimatization and Plantlet Transfer to Field OPD05 (1 product). All the 25 primers produced A total of 160 plantlets were acclimatized, of monomorphic bands confirming the genetic which 150 (93.8%) plants survived at the end of uniformity of the in vitro-raised plants. Amplified acclimatization and hardening (Figs 1e & f). These genomic DNA profile of in vitro-raised plants and 150 plantlets were transferred to a glasshouse, where that of the mother clone were identical with respect to 136 (90.6%) plants survived and developed into all the amplification products (Fig. 2). The Fig. 2 normal plants that flowered in 245-335 d from the day depicts profiles generated from the primer OPA01 with of culture establishment and bear normal flowers and 54 micropropagated plants, which were acclimatized fruits. Thus the overall survival rate of the in vitro- and grown in a glasshouse. The amplification profile of raised shoots was 85%. These findings are similar to mother plant compared to 54 in vitro multiplied plants the reports where 70-90% survival of cotyledon- did not detect any polymorphism. These results derived plantlets of T. dioica was achieved upon revealed that variations have not been induced during transfer to field23. Similarly, Yadava and Dhir the four cycles of in vitro multiplication of T. dioica. KUMAR et al: MICROPROPAGATION OF POINTED GOURD 587

Among the different type of molecular markers, RAPD 9 Maurya K R, Barooah S, Bhattacharya R K & Goswami R K, marker is quite useful for the detection of variation that Standardization of male plant population in pointed gourd, Ann Agric Sci, 30 (1985) 1405-1411. is induced due to in vitro culture process mainly due to 10 Mythili J B & Thomas P, Micropropagation of Trichosanthes their simplicity, speed and relatively low-cost. dioica Roxb., Sci Hortic, 79 (1999) 87-90. 17 18 Momordica dioica and S. mangifera are some of the 11 Singh H P, Rai M, Pandey S & Kumar S, Vegetable varieties of examples where RAPD markers have been successfully India (Studium Press India Pvt. Ltd., New Delhi, India) 2010. employed for assessing genetic fidelity of 12 Yadava U L & Dhir S K, In vitro regeneration of Trichosanthes dioica from shoot tips, Hortscience, 30 (1995) 871. micropropagated plants. 13 Debnath R K, Roy SK, Ahmed G & Hossain M, The present study describes a simple yet efficient Micropropagation of patal (Trichosanthes dioica Roxb.) protocol for in vitro propagation of T. dioica for from nodal segment and shoot tip, Plant Tissue Cult, 10 producing plant material suitable for commercial (2000) 125-130. 14 Kumar S, Singh M, Singh A K, Srivastava K & Banerjee M K, cultivation. In this investigation, ~8 shoots per nodal In vitro propagation of pointed gourd (Trichosanthes dioica explant were obtained, which is better than the earlier Roxb.), Cucurbit Genet Coop Rep, 26 (2003) 74-75. reports in terms of various parameters of in vitro 15 Malek M A, Miah M A, Amin M A, Khanam D & Khatun M, multiplication. The significant aspect of this work is In vitro regeneration in pointed gourd, Bangladesh J Agric that as compared to 6-8 plants in 10 months by Res, 32 (2007) 461-471. 16 Huq A, Akter S, Islam S & Khan S, In vitro propagation of conventional vine cutting, which is often tedious and pointed gourd (Trichosanthes dioica Roxb.) from shoot tip impractical on a large-scale, it gives a much higher and nodal segments, Bangladesh J Sci Ind Res, 47 (2012) multiplication rate (7.6 shoots per node in 21 d) with a 217-222. surety of true-to-the type plants. In this way, using the 17 Rai G K, Singh M, Rai N P, Bhardwaj D R & Kumar S, In vitro propagation of spine gourd (Momordica dioica standardized protocol, a large number of quality Roxb.) and assessment of genetic fidelity of micropropagated propagules of high yielding/hybrid selections, or plants using RAPD analysis, Physiol Mol Biol Plants, 18 parthenocarpic lines could be multiplied to fulfill the (2012) 273-280. propagule demand. 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