Reviewed Proceedings of National Seminar on Internet: Applications in Research, 36-40 (2011) ISBN: 978-81-920945-1-9
Department of Zoology Maharshi Dayanand University Rohtak 124001 Haryana www.mdurohtak.com
Efficient in Vitro Regeneration System Using Cotyledon Explants in Indian Cultivar Of Sponge Gourd (Luffa Cylindrica Roem.) Compatible to Agrobacterium tumifaciens Mediated Transformation Netrapal Singh1*, Ankush Raj1 Arvind Sharma1 and Pradeep Kumar2 1
Center for Biotechnology, MDU, Rohtak-124001, Haryana, India 2 Department of Zoology, MDU, Rohtak-124001, Haryana, India Corresponding author:*
[email protected] Abstract
Publication data Received
29.3.11
Revised received
19.8.11
Accepted
19.8.11
For the first time, an efficient regeneration system for Indian cv. of Sponge gourd, compatible to Agrobacterium mediated transformation using cotyledon explants is developed. The cotyledon explants were cultured on regeneration medium consisting of MS salts, B5 vitamins supplemented with different concentrations of cytokinins. The highest rate of shoot multiplication was obtained in medium containing 10µM BAP (6-benzylaminopurine). The regenerated shootlets were rooted on half MS basal medium with different concentrations of IBA (Indole-3Butyric Acid). The maximum number of roots was produced on the medium containing 2.5µM of IBA. The plantlets, thus developed were hardened and successfully established in soil and grown to maturity. The explants were found to be competent for Agrobacterium mediated transformation showing very good transient GUS (β-Glucuronidase) activity at the site of regeneration. Keywords: Agrobacterium tumifaciens, In vitro regeneration, Sponge gourd, Luffa cylindrica, cotyledon
Introduction Among the various cultivated cucurbitaceous vegetables grown worldwide, sponge gourd (Luffa cylindrica Roem.) is very popular in the tropical and subtropical regions and commonly called loofah, vegetable sponge, bath sponge or dish cloth gourd. The number of species in the genus Luffa varies from 5 to 7. Only two species, sponge gourd (L. cylindrica Roem.) and ribbed or ridge gourd (L. acutangula (L.) Roxb) are domesticated. India is considered to be the centre of origin of Luffa. Other main commercial producers are China, Korea, Japan and Central America (Bal et al. 2004). Luffa is diploid species with 26 chromosomes (2n = 26) and an annual climbing cross-pollinated crop. It is monoecious and its fruit, a gourd, is green and has a large cylinder-like shape. The stem is green and pentagonal and grows climbing other physical solid (Lee et al. 2006). Generally, L. cylindrica can be used in virtually all areas. Factors such as high surface area per volume, strong and durable structure, low specific gravity (which makes it light) and reasonable cost are characteristics of loofa making it a suitable alternative for use as a packing medium in an attached growth system (Mazali et al. 2005). It has been suggested as an immobilization matrix for plant, algal, bacterial and yeast cells (Iqbal et al. 1993a, b; Iqbal et al. 1994). Young fruits are edible. The fibrous vascular system can be used as a bathroom sponge, as a component of shock absorbers, as a sound proof linings, as a utensils cleaning sponge, as packing materials, for making crafts, as filters in factories and as a part of soles of shoes (Bal et al. 2004). They also can be used for cleaning floors or cars without scratching. The small ones are softer and good for washing the face and larger ones for the body. They can also be recycled into mats or pillows when they finally wear down (Newton, 2006). A novel circulating loop bioreactor with cells immobilized in loofa (L.
Internet: Applications in Research
cylindrica) sponge has been used for the bioconversion of raw cassava starch to ethanol (Roble et al. 2002). Loofa sponge has been used as a medium for the culture of human hepatocyte cell line (Chen et al. 2003). It showed a very good performance as a solid substrate for the development of the biofilm aggregating microorganisms capable of metabolizing both organic and inorganic compounds adsorbed on it, particularly those responsible for nitrification (Tavares et al. 2008). It has been reported that loofa (L. cylindrica) sponge is an excellent carrier for immobilization of microorganisms and plants and animal cells (Roble et al., 2002; Chen et al., 2003; Ogbonna et al. 2001; Liu et al. 1999). Loofa sponge has been used extensively for the biosorption of heavy metals from wastewaters (Iqbal et al. 2004, 2005; Ahmadi et al. 2006a, b). Traditionally, it is used for bathing and dish washing and, recently, the fibers were used for environmental reclamation (Iqbal et al. 2004). Oil is extracted from seeds for industrial use (Bal et al. 2004). The oil extracted from L. cylindrica is finding increasing use in the production of biodiesel which is now gaining wide acceptance because of low CO2 emission and other considerations (Ajiwe et al. 2005). Loofa sponge is fast becoming an indispensable crop because of its very wide industrial applications and many medicinal properties such as emetic, diuretic, purgative and useful in asthma, skin diseases and splenic enlargement. It is used internally for rheumatism, backache, internal hemorrhage, chest pains as well as hemorrhoids. Like all other cucurbitaceous vegetables, it is highly susceptible to several abiotic (low temp, high temp, drought, salinity, acidity) and biotic stresses like aphids, gall fly plume moth, fruit fly, stem boring longicorn beetles, red spider mite, Powdery mildew, Downey mildew diseases and severely affected by several viral diseases (Bal et al. 2004). The yellow mosaic disease of sponge gourd November 2011
37 Efficient In vitro regeneration system in Indian cultivar of Sponge gourd caused by Tomato leaf curl New Delhi virus system is required which is compatible to (ToLCNDV; genus Begomovirus, family Agrobacterium tumefacience mediated method. The Geminiviridae) is a recently reported disease and objective of this study was to develop a simple and may cause yield losses up to 100% under epidemic more efficient plant regeneration system from conditions (Sohrab et al. 2003). The diseased plant cotyledon explants which could be used for genetic is characterized by yellow spots appearing on newly transformation studies. emerging leaves, followed by a mosaic appearance Materials and Methods and upward curling of the upper leaves. In cases of severe attack, the leaves of the plant are small and Plant material, Agrobacterium strain: The distorted, and misshapen fruits are produced. The commercially grown Indian cv. of sponge gourd virus is transmitted through sap as well as Whitefly (Bemisiatabaci Hemiptera-Aleyrodidae). One (Luffa cylindrica Roem.) Pusa-Chikni seeds were used for the development of regeneration system. strategy to improve resistance against diseases and The disarmed Agrobacterium tumifaciens strain insects is use of conventional breeding but it is limited due to the lack of desirable and satisfactory used for the transformation studies was EHA-105 (pCAMBIA2301), harbouring a neomycin level of genetic variability. To date, there are no phosphotransferase gene (nptII) as a bacterial virus-resistant varieties/hybrids available in sponge gourd (Islam et al. 2010). In this perspective, selection marker and an intron containing βglucuronidase (GUS) gene (uidA) as a plant genetic engineering offers an opportunity to develop selection marker – both driven by the cauliflower inherent resistance in susceptible plant species by incorporation and expression of resistance gene mosaic virus (CaMV) 35S promoter. The presence of an intron in the coding region of uidA from foreign source using Agrobacterium. Although gene ensures that the observed GUS activity occurs the transformation of other crops of cucurbitaceae family such as water melon, melons, cucumber has in the plant cell but not as a result of the presence of endogenous Agrobacterium cells. been reported but for the development of transformed Luffa plants, an efficient regeneration
Explant preparation and multiple shoot regeneration: Seeds were surface sterilized by rinsing them with 70% alcohol for 1-2 min then in 0.2% (w/v) aqueous HgCl2 for 2-3 min and thereafter they are subsequently washed with sterilized distilled water 4-5 times and the seeds were soaked in sterilized distilled water for 2 hours. After that, seeds were manually de-coated and sequentially surface-sterilized in 70% (v/v) ethyl alcohol for 2 min, 0.2% (w/v) sodium dodecyl sulphate for 10 min, 1% (v/v) NaOCl for 20 min, and 0.5%(v/v) NaOCl for 10 min (Kyung-Min Kim et al. 2010). Between each step, the seeds were rinsed three times with sterile distilled water. Most degenerated perisperms, such as a membrane, were removed from the embryos during the sterilization process. The embryos were blot dried on sterile filter paper for about 5 min, and then seeds were germinated in MS basal medium (Murashige and Skoog 1962) solidified with 0.75% agar. The cotyledons excised from different aged (4, 6, and 8 days) seedlings were used as explants and cultured on shoot regeneration medium (SRM) containing MS salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al. 1968), 3% (w/v) sucrose and 0.75% agar supplemented with different concentrations of BAP, Kinetin and TDZ in planton boxes and kept in culture room at 25±1ºC and 16/8-hrs photoperiod supplied by cool fluorescent white light having luminous intensity 80 µEm-2s-1.
For each concentration, 16 explants were used, and each experiment was repeated at least twice. Observations were made weekly and effects of different cytokinins with different concentrations were quantified on the basis of the number and length of regenerants obtained per explant, and the percentage of explants with shoots. Rooting and transplantation: After 20 days 4-6cm long shoots were excised from the point of origin and transferred to (RM) half strength MS salts, full strength MS vitamins, 3% (w/v) sucrose and 0.375% Phytagel supplemented with different concentrations of IBA (1-5µM) for rooting and after 10 days shoots with well-developed healthy roots were shifted to pots containing soilrite mix and grown to maturity. Transformation procedure and transient histochemical GUS activity: To confirm the compatibility of this regeneration system with Agrobacterium mediated transformation, a single bacterial colony of A. tumifaciens strain EHA105 (pCAMBIA 2301) was used to inoculate 20ml YEM broth (1.0 g/l yeast extract, 10 g/l mannitol, 0.1 g/l NaCl, 0.2 g/l MgSO4.7H2O, 0.5 g/l K2HPO4, 15 g/l agar) supplemented with 100µM acetosyringone (3', 5'Dimethoxy-4-hydroxyacetophenone), 50mg/l kanamycin and 10mg/l rifampicin. The culture was grown for overnight to log phase (O.D.600≈0.8) at 27ºC and 200rpm. Agrobacterium cells were then harvested by centrifugation at 4000rpm at 25ºC for 10 min and resuspended in liquid regeneration
Netrapal Singh et al. medium (SRM) having pH 5.6 and 200 µM acetosyringone. The cotyledon from 4 days old seedling were excised and then inoculated in the Agrobacterium suspension for 20 min thereafter explants were blot dried and cocultivated in cocultivation medium (SRM having pH-5.2). Histochemical staining of GUS activity (Jefferson et al. 1987) was performed. After 3days of cocultivation, the explants were washed 2-3 times with sterilized distilled water and finally with solution containig 100mg/l cefotaxime then blotted dry on sterile filter paper were immersed in freshly prepared 5-bromo-4-chloro-3-indolyl-β-Dglucuronide (X-gluc) solution and incubated for 24 hours at 37°C. The staining solution was removed after 24 hours and plant tissues were decolorized using 70% ethanol and examined under a microscope. Results and Discussion Plant regeneration through multiple shoots: The objective of present study was to develop a superior in vitro regeneration system compatible to Agrobacterium mediated transformation. Cotyledons excised from 4, 6, and 8 days seedlings were cultured on SRM basal medium with different cytokinins. We reported, the frequency and the number of shoots induced were higher in 4 days old cotyledons explants on SRM with 10µM BAP than Kinetin and TDZ. Cotyledons produced only single shoot along with profuse roots at the basal end of the shoot on SRM basal medium. The age of the donor seedlings from which cotyledons were excised showed significant differences for achieving optimization of regeneration. Albeit multiple shoots were induced in explants from different ages, conversely the regeneration frequency decreased with increase in age of the cotyledon explants. In previous studies, in vitro regeneration of Luffa sp., the age of the cotyledon explants was optimized as 6 days and lower number of shoots (1 to 2) was
38 reported (Lee et al. 2006), while in the present study, maximum number of shoots (3 to 4) was obtained from 4 days old cotyledon explants. Y.K. Lee et al. 2003 has also reported in winter squash (Cucurbita maxima Duch.) that the use of seedlings older than 4 days resulted in a dramatically decreased shoot induction in all explants. Among the various cytokinins used, BAP proved its worth for multiple shoot regeneration in various cucurbitaceous members such as Lagenaria siceraria (Han et al. 2004; Saha et al. 2007), Citrullus colocynthis (Dabauza et al. 1997), Citrullus vulgaris (Dong et al. 1991), Cucurbita maxima (Bologun et al. 2007; Haque et al. 2008), Benincasa hispida (Haque et al. 2008), and Cucumis sativus (Li et al. 2008). In the present study using cotyledon explants, TDZ and Kinetin produced only one to three shoots while BAP induced multiple shoots (Fig. 2B). Similarly, BAP at different concentrations was found more effective in inducing shoot organogenesis in Luffa cylindrica using cotyledon explants as in other plant species such as Lagenaria siceraria (Han et al. 2004; Saha et al. 2007), Citrullus colocynthis (Dabauza et al. 1997), Citrullus vulgaris (Dong et al. 1991) than other cytokinins. This indicates that not only the presence of cytokinin but type of cytokinin in the medium also influence the multiple shoot induction (Table- 1). We found that at lower concentrations of BAP the shoots were longer but less in number having abundant rooting at the base, while with the increase in concentration of BAP the shoots become smaller but more in number. The best response was observed when the explants were cultured on the SRM supplemented with BAP (10µM) which produced average 3.6 shoots per explants, with an average length of 3.2 cm, in 75% of the cultures (Fig. 2B).
Fig. 2 (A-G): In vitro regeneration of multiple shoots from Indian cv. of Sponge Gourd Pusa-Chikni. (A) Single shoot regeneration from the cotyledon explants on basal shoot regeneration medium. (B) Multiple shoot regeneration from the cotyledon explants on SRM supplemented with 10µM BAP. (C) & (D) Adventitious root induction from the elongated shoot in half strength MS medium supplemented with 2.5µM IBA. (E) Fully developed plants growing in pots. (F) Non transformed (control) cotyledon explant not showing GUS activity. (G) Transient GUS activity shown at the site of regeneration in the cotyledon explants after 3-days cocultivation with Agrobacterium.
39
Efficient In vitro regeneration system in Indian cultivar of Sponge gourd
Table -1: Effect of different cytokinins on shoot regeneration from 4 days old cotyledon Cytokinin SRM Basal + Regenerating explants Avg. no. of shoots cytokinin (µM) % per explant 00.0 100 1.0±0.00 01.0 100 1.0±0.00 02.5 100 1.0±0.00 05.0 100 1.1±0.03 BAP 08.0 87.5 2.0±0.14 10.0 75.0 3.6±0.12* 12.0 75.0 3.0±0.19 15.0 75.0 2.5±0.09 20.0 62.5 1.4±0.10
Avg. shoot length (cm) 9.8±0.52 8.3±0.43 7.4±0.52 3.9±0.26 3.1±0.26 3.2±0.34 2.7±0.30 1.0±0.20 0.7±0.17
TDZ
00.0 01.0 02.5 05.0 08.0 10.0 12.0 15.0 20.0
100 100 93.7 93.7 81.2 75.0 68.7 56.2 50.0
1.0±0.00 1.0±0.00 1.0±0.00 1.3±0.04 1.8±0.16 1.2±0.09 1.0±0.00 1.1±0.06 1.0±0.00
6.9±0.36 6.1±0.25 5.3±0.26 2.5±0.20 1.6±0.36 1.2±0.20 0.9±0.10 0.6±0.10 0.5±0.17
KINETIN
00.0 01.0 02.5 05.0 08.0 10.0 12.0 15.0 20.0
100 100 100 87.5 87.5 81.2 75.0 75.0 62.5
1.0±0.00 1.0±0.00 1.0±0.00 1.1±0.04 1.4±0.11 2.0±0.19 2.5±0.04 1.4±0.16 1.1±0.17
8.8±0.26 8.1±0.55 7.9±0.30 4.5±0.30 4.1±0.26 3.8±0.26 2.9±0.43 1.0±0.20 0.8±0.20
*Highest average number of shoots/explants. Data represented as means ± SD from 16 explants for each experiment and repeated twice.
Root Induction: For root induction at the base of the in vitro regenerated shoots, half strength MS salts, full strength MS vitamins, 3% (w/v) sucrose and IBA (1, 2.5, 5µM), were tried. In earlier reports, Haque et al. (2008) found root induction in Cucurbita maxima, Benincasa hispida on half MS with 1.5mg/l IBA. Vasudevan et al. (2007) also used 4.5µM IBA to develop roots in Cucumis sativus. Similarly, in present study, IBA was found optimal for better root development with 2.5µM concentration. Although roots were also developed on half MS without IBA but the roots were thinner than the roots developed on half MS with IBA. Here, we observed root induction after 10 days and found 100% of the inoculated shoots regenerated roots on rooting media containing 2.5µM IBA (Fig. 2C-D). Expression of the GUS gene after co-cultivation with Agrobacterium tumefaciens: Cotyledon explants resulted in transient expression of GUS gene particularly at the sites of regeneration from where the shoots developed in 100 % of the explants when these were inoculated with Agrobacterium suspension for 20 min with gentle shaking (Colocynthis citrullus Ntui et al. 2010), indicates the suitability of the explants for transformation (Fig 2G). However, Transformation systems in many cucurbitaceous members have been developed such as Colocynthis citrullus (Ntui et al. 2010), Lagenaria siceraria (Han et al. 2004), Cucumis sativus (Wako et al. 2001), muskmelon (Fang et al. 1990). The endogenous GUS activity (color) was not detected in non-transformed (control) explants (Fig. 2F). Transient GUS activity indicates the compatibility of the cotyledon explants to Agrobacterium mediated transformation assisted, which could be used as routine experiments for the manipulation of genes to enhance the production of this important cash crop. References Ahmadi, M., F. Vahabzadeh, B. Bonakdarpour and M. Mehranian: Empirical modeling of olive oil mill
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