IN VITRO PLANT LET REGENERATION OF TAMATO (SOLANUM LYCOPERISCAN MILL. CV. PUSA RUBY) FROM SHOOT TIP EXPLANTS USING FIVE CYTOKININS
*Ugandhar T1, Venkateshwarlu. M2, Odelu G3 Rajendra Prasad B4and Anitha Devi5
1Department of Botany, Govt. Degree College Mahabubabad -506101
2Department of Botany University College Kakatiya University Warangal-505006
3Department of Botany, Govt. Degree College Parkal-506164
4Department of Botany, UCS, Saifabad, Osmania University, Hyderabad-500004
5Department of Botany Govt. Degree College Peddapally-505172
*Email: [email protected]
Direct In Vitro Plant propagation was achieved in Pusa ruby cultivar of Tamato (Solanum lycoperiscan Mill). Using shoot tip explants (0.5cm) has been developed on Murashige and Skoog medium. Among various concentrations of five cytokinins tested Adenine (Ad), N6-Benzyladenine (BA), Kinetin (Kn), Zeatin (Ze) and Thidiazuron (TDZ) individually. Highest shoots/explants (18.0± 034) were obtained on MS medium supplemented with (2.0mg/L) TDZ. Multiple shoot elongation occurred upon transfer to TDZ (2.5 mg/L each). The elongated shoots were transferred to Indole Butyric Acid (IBA) (0.5-1.0 mg/L) and Indole Acetic Acid (IAA) (0.5-1.0 mg/L) for root induction. Rooting was observed within two weeks of culture. MS medium supplemented with (1.0 mg/L) IBA proved better with seventy percent rooting after 25 days of implantation. Plantlets obtained from TDZ containing media were normal diploid (2n = 24) Most of the roots were long and healthy. Rooted plantlets were successfully hardened under culture conditions and subsequently established in the field conditions. The recorded survival rate of the plants was 86.3%. Plants looked healthy with no visually detectable phenotypic variations.
Keywords: Cytokinins, Multiple shoots, Tamoto, Plant regeneration and Rooting
Tomato (Lycopersicon esculentum L.) is an economically important vegetable crop and widely consumed vegetable in many countries around the world, including India. At present, tomato is grown in an area of around 4.4 million ha. Worldwide with an annual production of 115 million tons. United States of America, China, Turkey and Italy are the major producers of tomato in the world, with India ranking 5th in the world production. In India, it is cultivated in Orissa, Andhra Pradesh, West Bengal, Bihar and Karnataka. The estimated area and production of tomato in India during 2006-07 were about 593.6 thousand ha and 9878.3 thousand tons, respectively. In Karnataka, it is cultivated in an area of 45.8 thousand ha. with a production of 1223.7 thousand tons (Anon., 2008b).
The regeneration capacity of tomato was significantly influenced by explant type (Gubis et al., 2005; Hanur et al., 2007). The type of explants used not only determines the proportion of explants, which show organogenesis, but also the number of shoots produced per explant. Although almost all explants are amenable for regeneration, cotyledons (Gunay and Rao, 1980; Dai et al., 1988; Garcia and Luque, 1988; Hamza and Chupeau, 1993; Fari et al., 2000; Park et al., 2003) and hypocotyls (Gunay and Rao, 1980; Zelcer et al., 1984; Garcia and Luque, 1988; Newman et al., 1996) are the most widely used explants due to their high organogenic ability. Other explants such as leaf tissue (Dwivedi et al., 1990; Chandra et al., 1995; Chandel and Katiyar, 2000), shoot tips (Garcia and Luque, 1988; Mirghis et al., 1995), root and epicotyl have been used occasionally for regeneration. The purpose of this work was to study the effect of different five types of growth regulators on direct plantlet regeneration of tomato from shoot tip explants.
Materials and methods:
Seeds of Tamato (Solanum lycoperiscan Mill) cv Pusa ruby obtained from Regional Agricultural Research Station, Lam, Guntur, India, were imbibed in distilled water for 24 h, then surface sterilized with 0.1% HgCl2 for 3-5 min, rinsed in several changes in sterile distilled water and germinated aseptically on Murashige and Skoog (1962) basal medium. About one cm shoot tips were excised from 3-week-old aseptic seedlings. Dissecting out shoot meristems (~ 0.5 cm long) from the above shoot tips was done using a low power stereomicroscope in a laminar flow cabinet. Freshly excised shoot meristems were placed on a moist paper in a Petri dish (sterile) until planting them on medium.
Culture media and conditions:
MS basal medium was supplemented with various plant growth regulators and 3.0% sucrose. The pH of the media was adjusted to 5.8, solidified with 0.8% Difco-bacto agar and autoclaved at 103.4 kPa or 121oC for 15-20 min. A single explant was placed in each culture tube, and incubated at 25 + 1oC with a 16 h photoperiod under fluorescent light (40-50 ? Mol m2 S-1).
Shoot proliferation media:
MS medium supplemented with (0.05-5.0 mg/L) of Adenine (Ad), N6-benzyladenine (BA), Kinetin (Kn), Zeatin (Zn), and Thidiazuron (TDZ) individually was used for shoot regeneration.
Explants with multiple shoots proliferated on TDZ-containing media were transferred to MS medium containing different concentrations of Ad/BA/Kn/Ze/TDZ (2.0 mg/L) were used for shoot elongation.
Rooting and acclimatization:
Shoots obtained only from TDZ-containing media were used in rooting experiments; and were transferred to MS medium supplemented with (0.5- 2.0mg/L) (Table-2) IAA/IBA. MS medium supplemented with (2.0 mg/L) IBA (Fig- I c).proved better with seventy percent rooting after 25 days of implantation. After 3 weeks of culture, plantlets recovered were washed with running tap water and agar sticking to the roots removed. Plantlets (a height of 6 – 10 cm) with fully expanded leaves and well-developed roots were transferred to soil and vermiculite (50: 50 v/v) mixture and covered with plastic bags, they were kept in the culture room for 2 weeks before being transferred to soil.
All data were statistically analyzed by ANOVA followed by Duncan’s multiple range test for mean comparison. Data pertaining to shoot regeneration was obtained from 10 explants in each of two replicates for each treatment and the experiment was repeated twice.
In the present study five kinds of cytokinins (Ad, BA, Kn, Ze and TDZ mg/L) were used alone only to investigate in vitro microprpoagation in S. lycoperiscan.
Effect of Ad:
The meristem containing explants shoot tip were excised from the surface sterilized, In vitro grown, 8-day old seedlings and cultured on MS medium augmented with Ad (0.05-5.00 mg/L) for multiple shoot induction of all the different concentrations of Ad tested, (2.0 mg/L) Ad was found to be more effective in inducing (14.0 ± 0.32 shoots/explants) with 70 percent responded. (Fig- I a). But at high concentration of Ad (5.0 mg/L) considerably the number of shoot induction was found to be reduced. As the concentration of Ad was increased up to (0.5 mg/L) the multiple shoots number was increased but as the concentration of Ad (3.0 mg/L) to (5.0 mg/L) Ad resulted the number of shoots was reduced.
Effect of BAP:
Shoot tip explants were capable of directly developing multiple shoots on MS basal medium containing different concentrations of BAP (0.5 – 5.0 mg/L). Multiple shoot initiation from shoot tip explants was observed within 20 – 25 days after inoculation. Highest number of shoots inducing (16.0 ± 0.42 shoots/explants) was observed with 78 percent response. In the medium concentration of BAP was increased up to (2.0 mg/L) the multiple number of shoots was also decreased (Table 1) (Fig- I b).
Effect of Kn:
Kn showed shoot bud induction from shoot tip explants. Medium containing Kn induced formation of shoot buds predominantly at the cut surface of the cultured explants within 1-2 weeks of culture. Shoot buds were proliferated and developed into shoot primordia. Among the tested concentrations of Kn (0.05 – 5.0 mg/L), lower (0.05 mg/L) and higher (5.0 mg/L) concentrations showed least regenerative response with fewer shoots produced per explant compared to (0.5 mg/L) and (1.0 mg/L) of Kn (Table-1) Kn at (2.0 mg/L) proved to be the optimal concentration producing a maximum number of shoots from shoot tip (16.4 ± 0.32) explants with 80 percent responded. Kn At (5.0 mg/L) numbers of shoots produced from shoot tip producing (3.2 ± 0.32 shoots/explants) with average percentage of response 45 respectively (Table-1).
Effect of Zeatin;
The results on shoot tip explant culture of S. lycoperiscan on MS medium + Ze (0.05-5.0mg/L) alone are presented in (Table-1). The medium containing (2.0 mg/L) Ze induced maximum number of shoots induced (17.0± 0.42 shoots/explants) and also showed high percentage (85%) of responding cultures. As the concentration of Ze was increased up to (2.0 mg/L) gradually the shoot bud proliferation was found to be decreased and when Ze concentration was increased above (2.0 mg/L) the rate of shoot multiplication was reduced.
Effect of TDZ:
The meristem containing explants shoot tip were excised from the surface sterilized, In vitro grown, 8-day old seedlings were cultured on MS media supplemented with different concentration of TDZ (0.05-5.0mg/L) and result were presented (Table -1) Maximum percentage of response 86 was observed at (2.0mg/L) with (18.0±0.34 shoots/explants) shoots/explants TDZ followed by (1.50mg/L) more number of multiple shoots was found at the (2.0 mg/L) TDZ low percentage of response with low number of shoots per explants was recorded at lower and higher concentration of TDZ used whereas callus with multiple shoot induction was recorded at higher concentration of TDZ.( Table-1). (Fig- I a).
Rooting ability of regenerated shoots from shoot tip explants culture of Solanum lycoperiscan cultured on MS medium supplemented with IAA / IBA.
Growth Hormones (mg/L) Percentage of response Average no of roots (S.E)*
70 1.0 ± 0.12
2.3 ± 0.37
4.2 ± 0.38
2.6 ± 0.38
4.3 ± 0.36
8.3 ± 0.87
6.3 ± 0.36
* Mean ± Standard Error
In the present study also, best results were obtained by using TDZ. About 86% shoot tip exhibited direct plantlet regeneration. (Costa et al., 2000) used cotyledonary-derived explants of two processing tomato cultivars (L. esculentum cvs. IPA-5 and IPA-6). Thidiazuron (TDZ) a substituted phenylurea (N-phenyl-N ?-(1, 2, 3-thidiazol-5- yl) urea) was developed originally by Schering AG for utilization as a defoliant for Gossypium hirsutum L. (cotton) for the mechanical harvest of cotton bolls (Arndt et al., 1976). Later it has proven to be a highly effective regulator of plant morphogenesis in the tissue culture of many plant species. The endogenous changes in response to TDZ application include levels of phytohormones such as cytokinins and auxins, cell signaling molecules, nutrients and markers of physiological stress (Murthy et al., 1998). This compound has both cytokinin-like and auxin like effects (Mok et al., 1982; Visser et al., 1992), and facilitates efficient micropropagation of many recalcitrant species. Hence, it was used as an effective growth regulator to stimulate shoot organogenesis in a large number of species as well as diverse experimental systems (Van Nieuwkerk et al., 1986; Thomas and Katterman, 1986; Chalupa, 1988; Yusnita et al., 1990; Murthy et al., 1998; Mithila et al., 2001; Ricci et al., 2001). It is used more frequently to stimulate shoot proliferation through direct organogenesis than for caulogenesis or indirect organogenesis. The high cytokinin activity and possible response of several species to TDZ have established it as among the most active cytokinins for in vitro manipulation of many species. However, the dose of TDZ is known to be critical in shoot organogenesis (Chalupa, 1988; Van Nieuwkerk et al., 1986; Yusnita et al., 1990). At lower concentrations it can stimulate the formation of callus, adventitious shoots or somatic embryos.
These results are also in agreement with those on Tectona grandis (Gupta et. al., 1980) Abizzia lebbeck (Gharyl and Maheshwari 1982) multiple shoot induction was also observed in Ziziphus manritiana (Sudharshan et. al., 2000) and Vanilla plantifolia (Geetha et. al., 2000) shoot tips cultured on MS + cytokinin alone as it was observed in the present studies. Zamen et.al., (1996) have studied the effect of different cytokinins viz, BAP, Kn, 2-ip and Zeatin on multiple shoot induction from shoot tip culture in mulberry. According to their observation, BAP and Kn were superior to 2-ip and Zeatin these superiority of BAP over other cytokinins for multiple shoot formation has been reported as it was observed in the present investigations in Mulberry by Hossian et. al., (1991).
Das and Mitra (1990) and Roy et. al., (1993) have also reported the requirement of both auxin and cytokinin for induction of multiple shoos in Eucalyptus tereticorvis and Jack fruit respectively. Thus, a combination of both auxin and cytokinin improved the efficiency of multiple shoots development although it depended on the Khanan et. al., (1998) have recorded the maximum number of shoots / culture (22.5) on MS medium containing 5.0 mg/L BAP in strawberry shoot tip culture. Gupta et. al., (1997) reported the multiple shoot bud induction from shoot apex cultured on MS medium containing BAP in cotton. Nasir et. al., (1997) have studied the shoot meristem culture in 16 cultures of cotton using several media formation. They observed the best shoot developmentation MS media containing Kn alone compared to other media with NAA / IAA in combination with Kn. These results are to the present observation in S. lycoperiscan which contain with cytokinins showed the increased number of shoots/ explant. Sharma and Dhiman (1998) have also observed the similar results when they have cultured the shoot tips of F1 hybrids of Paulownia.
However, at higher concentrations inhibits shoot elongation and induce fasciated shoots. Thidiazuron-induced regeneration has been reported in various species. TDZ showed specific responses that were dependent on the genotype and the type of tissue (Passey et al., 2003). TDZ is able to induce diverse morphogenic responses, ranging from tissue proliferation to adventitious shoot and somatic embryo formation. In tomato var. Alice, Ricci et al. (2001) found that TDZ at 5 ?M and 10 ?M concentrations induced shoot regeneration in 82.9 and 92.1 per cent of the explants cultured, respectively. Thidiazuron induces shoot differentiation in woody species (Huetteman and Preece, 1993), and it has been shown to be effective on shoot regeneration in apple (Van Nieuwkerk et al., 1986), silver maple (Preece et al., 1991), pear (Singh and Bhatia, 1988) and peach (Zimmerman and Scorza, 1992).
Direct Plantlet was observed in shoot tip explants. Shoot tip explants showed to be better explants for direct regeneration. Maximum plantlets were noted on MS medium supplemented with Sd/BAP/Kn/Ze/TDZ (2.0 mg/L). Maximum regeneration was observed on MS medium containing TDZ (2.0 mg/L). This study is a baseline to carry further research on this tomato variety for improvement by using gene transfer technology.
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