High frequency plant regeneration from desiccated calli of indica rice ( Oryza Sativa l . )

An efficient and reproducible protocol is required to achieve high frequency transformation from transformed calli. We report here high frequency plant regeneration from mature seed derived embryogenic calli of two recalcitrant indica rice cultivars HKR-46 and HKR-126 after partial desiccation treatment. Embryogenic and nodular callus was initiated on MS basal medium supplemented with 2.5 mg l 2,4-D, 500 mg l proline, 500 mg l casein hydrolysate, 30 g l sucrose and 2.5 g l gelrite. Several media with different combinations of growth regulators were tried. Maximum shoot regeneration frequency (63%) was observed in partially desiccated calli for 48 h in cv. HKR 46 and 82.1 per cent in cv. HKR-126 on the MS modified medium supplemented with 2 mg l kinetin + 0.5 mg l NAA + 30 gl sucrose + 6 g l gelrite followed by in the medium supplemented with 1 mgl 2ip + 30 g l sucrose + 6 g l gelrite (61% in cv. HKR-46 and 79.2 % in cv. HKR-126). Highly significant regeneration differences were observed in partially desiccated calli (48 h) in comparison to non-dehydrated (0 h desiccation) calli. Shoot regeneration frequency increased from 1.2 to 5.6 fold after 48 h of desiccation in both the cultivars on different regeneration media. Shoot regeneration frequency declined at 72 h desiccation treatment as compared to 48 h treatment. Well-developed plantlets were hardened and transferred to the green house.


INTRODUCTION
Production of callus and its subsequent regeneration are the prime steps in crop plant to be manipulated by biotechnological means.Different tissues have been used in rice as explants (Bhaskaran and Smith, 1988).However, embryos are easily amenable to in vitro techniques due to high totipotency of calli produced from them (Maggioni et al., 1989).Regeneration from callus was achieved long back in japonica varieties (Nishi et al., 1973).The potential for callus formation and regeneration have been reported to be varietal characteristic and efficient regeneration in indica rice is still poses a major *Corresponding author.Email: saharan@bgumail.bgu.ac.il. Tel: 972-8-6461902. Fax: 972-8-6472984.Abbreviations; 2,4-D: 2,4-Dichlorophenoxy acetic acid, 2-iP: 2isopentenyl adenine, MS: Murashinge and Skoog medium, TDZ: Thidiazuran, NAA: Nepthalene acetic acid, Kinetin: 6 Furfuryl amino purine, Z: Zeatin problem for genetic manipulation through innovative approaches (Toki, 1997).Strategies to improve plant regeneration frequency in cereals, including rice, have been steadily evolving during the last decade (Kyozuka et al., 1988;Datta et al., 1992;Raman et al., 1999).
While it has been possible to obtain high plant regeneration frequencies in japonica rice varieties, the success for reproducible fertile plant regeneration has been limited in indica rice varieties, especially those belonging to group 1 (Kyozuka et al., 1988;Raman et al., 1994).As a result progress to wards the transfer of useful genes in to indica rice has been slow.Many factors have been examined to improve the frequency of plant regeneration in rice.Different reports have shown that many factors affect plant regeneration frequency in rice: genotype, development stage of callus in the explant, and hormonal composition of medium (Jain, 1997;Kyazuka et al., 1998).Partial desiccation treatments have been reported to be beneficial for embryogensis and plant regeneration in several plant species.Tsukahara and Hirosawa (1992) have reported that dehydration for 24 h of cell suspension derived calli of japonica rice increased shoot regeneration from 5 to 47 per cent.Jain et al. (1996) have reported three fold increase in shoot regeneration frequency following partial desiccation for 24 h of suspension cells in indica rice.Chand and Sahrawat (2000) carried out partial desiccation of embroygenic calli prior to transfer to regeneration medium and observed increased regeneration frequency of desiccation treatment to callus cultures of cv.safari-17 and cv.kasturi, In this paper we report high frequency plant regeneration from mature seed derived calli of two recalcitrant indica rice varieties after desiccation treatment.

MATERIALS AND METHODS
Mature seeds of indica rice cvs.HKR-46 and HKR-126 were collected from CCS Haryana Agricultural University, Rice Research Station, Kaul, Haryana (India).These are dwarf and high yielding varieties.Dehusked seeds were washed in 70 per cent (w/v) ethanol for 60 sec and then rained with sterilized water to remove traces of ethanol.Sterilization of seeds was carried out on Shaker using a solution of sodium hypochloride (with 2% active chloride) and a drop of tween-20.After 40 min the solution was removed and seeds were thoroughly washed 5-6 times with sterilized water.

Partial desiccation
Desire extent of desiccation was obtained by transferring 3 weeks old calli to sterile empty petri dishes containing two sterile whatman-1 filter papers.The petri dishes were sealed with parafilm and kept at 25±1°C in dark for 48 and 72 h to obtain the desiccation of calli.After desiccation treatment, the partially desiccated calli were transferred to various regeneration media (Table 1).The regenerated plants were taken out from the petri dishes and transferred to Magenta boxes or culture bottles for shoot elongation on the MS medium.After 3-4 weeks, the plantlets were transferred to tubes containing water for hardening and incubated at 25±1°C in the culture room for a week.The plants were finally transferred to the pots.

Callus induction
Callus formation invariably developed from the scutellar region of the seeds and was visible with in 7-10 days.The mean callus induction frequency was 60.5 per cent in cv.HKR-46, whereas in HKR-126 it was 83.5 per cent.The 3-4 old weeks old calli of two cultivars used for regeneration.Saharan et al. 257 Table 1.List of media used for plant regeneration.

Plant regeneration
Plantlet/shoots regeneration started within one week of transfer of partially dehydrated calli to MSTDZ 1 , MSTDZ 2 , MSTDZ 5 , MSIP 1 , MSIP 2 , MSIP 5 and MSKN media (Table 1).Within 4 weeks, calli were entirely covered with green shoot buds.During sub culture the shoot buds further elongated and multiplied vigorously.Shoots multiplication rates was comparatively low in 0 h desiccation (without desiccation) treatment and regenerated plantlets were comparatively not so green and healthy as compared to plantlets regenerated from partially desiccation calli.The regeneration frequency in cv.HKR-46 was 63.7 and 61.6 per cent on MSKN and MSIP 1 media, respectively.In cv.HKR-126 maximum regeneration frequency was observed when calli were desiccated for 48 h (82.0 and 79.2 per cent on MSKN and MSIP 1 media, respectively).For both the cultivars maximum average number, 8-10 plantlets per callus were observed in partially desiccation calli.In 0 h desiccation 1-6 plantlets per callus were recorded (data not shown).In both cultivars the regeneration frequency and the average number of plantlets decreased in 72 h desiccation treatment.An increase of 1.2 to 5.6 fold in shoot regeneration frequency was obtained in both the cultivars with 48 h desiccation treatment, as compared to 0 h desiccation (Figures 1 and 2).The plants were hardened and transferred to the pot for further growth.These plants were found fully fertile as compared to normal plants.Induction of embryogenic calli in rice is considered most critical step.Several different media including MS medium have been used for rice tissue culture.Mostly 2, 4-D has been used as the only growth regulator in callus induction media (Katiyar et al., 1999;Zhenyu et al., 1999).Use of casein hydrolysate was found to be beneficial for generation of embryogenic calli in japonica (Hiei et al., 1994;Toki 1997) as well as in Indica rice varieties (Zhang et al., 1996).The use of proline in the medium has been reported to be effective for the initiation and maintenance of embryogenic calli (Datta et al., 1992;Kishor et al., 1999) in present investigation, MS basal medium containing 2, 4-D (2.5 mg l -1 ), casein hydrolysate (300 mg l -1 ) proline (500 mg l -1 ) and sucrose (30 g l -1 ) have been successfully used for induction of embryogenic calli from mature seed acutella of two rice cultures.Partial desiccation has been found promotive to plant regeneration (Diah and Bhalla, 2000;Chand and Sahrawat, 2001).Partial desiccation treatment (48 h) gave maximum shoot regeneration, 82 per cent in cv.HKR-126 and 63.1 per cent in cv.HKR-46.
The results of this study showed that in both indica rice cultivars maximum average number of plantlets (8 to 10 per callus) were observed in 48 h desiccation.Shoot regeneration frequency was also higher by 1.2 to 5.6 fold in both cultivars in 48 h desiccation whereas in 72 h desiccation treatment regeneration frequency declined.These findings were in conformation with the result reported by Diah and Bhalla (2000) and Chand and Sahrawat (2001).

Figure 1 .Figure 2 .
Figure 1.Effect of partial desiccation (0, 48, 72 h) on shoot regeneration from matured seeds derived calli of indica rice cultivars HKR-126 cultured on different regeneration media.Values are the mean of 6 ± SE.