Abstract: Super Basmati is a commercial cultivar and is subjected to various stresses. This study was conducted to obtain high frequency regeneration from Super Basmati which is a pre-requisite for transformation protocol. Callus induction was obtained from N6 media with 2 mg l-1 2,4-D. The frequency of callus induction was 83.3% with N6 media and 75.05% on MS media. Regeneration of calli was the best on MS media with NAA 1 mg l-1 and BAP 5 mg l-1 i.e. 81.6%.
INTRODUCTION
Super Basmati is a major commercial variety grown in rice region of Pakistan. It’s yield is affected by many diseases like others Basmati cultivars and bacterial blight is one of them. Hence there is a need to improve this commercial cultivar by biotechnological applications. Although there are many reports available on callus induction, regeneration and transgenic plant production in other Basmati cultivars like 6129, Basmati 370 and Basmati 385[1] but no report is available for Super Basmati. Super Basmati is also recalcitrant to the tissue cultures techniques. Earlier, Rashid et al.[2] reported a low regeneration frequency in Super Basmati, but still there is a need to develop an efficient regeneration system, as a pre-requisite for the transformation protocol.
The main objective of present study is to develop a reproducible regeneration system which is essential for establishing Agrobacterium mediated transformation in this cultivar.
MATERIALS AND METHODS
Mature seeds of Super Basmati were sterilized by protocol described by Rashid et al.[1]. Poisoned seeds of Super Basmati were dehusked. Seeds were surface sterilized by 70% ethanol for one minute followed by 50% Chlorox (Sodium hypochlorite) for 20 min. Seeds were continuously shaked during treatment with Chlorox and subsequently rinsed with autoclaved distilled water for 3-4 times after a regular interval of 5 min.
The sterilized seeds were aseptically inoculated on N6 media[3] with 30 g l–1 sugar, 2,4-D 2 mg l–1 and 6 g l–1 of agar which was used as solidifying agent. The pH of the media was adjusted to 5.8 before autoclaving at 121°C for 20 min. These inoculated seeds were placed in the growth room for three weeks for callus induction. The callus contained both embryogenic (white to light yellowish in color, compact and friable) and non-embryogenic (mucilaginous and smooth) part. The calli were carefully dissected and only embryogenic part was selected for further experiments and non-embryogenic part was discarded. Three weeks old scutellum derived calli were transferred to maintenance media. Maintenance media contained N6 salts and vitamins supplemented with 2 mg l–1 of 2, 4-D, 30 g l–1 sugar and agar 6 g l–1 as a solidifying agent. This process was carried out in the inoculation chamber. Callus was placed on maintenance media for about two weeks.
Media used for regeneration contained basic MS salts and vitamins[4] 3% sucrose and 0.4% gelrite. Regeneration media contained different combinations of BAP and NAA as growth regulators. Maintained calli were placed on regeneration media and were placed in the growth room (light and dark period at 23±2°C with 16/8 h photoperiod with light intensity of 16 m.mol.m.s). After shoot regeneration, plantlets were transferred to MS media without growth regulators for root initiation. In order to determine regeneration frequency, 40-60 cultures were raised for each treatment and each experiment was performed thrice. Subculture period was maintained at 2-3 weeks intervals. Observations were taken every week on the basis of %age of regeneration for the development of shoot/culture and shoot development.
RESULTS AND DISCUSSION
N6 and MS media were used for callus induction and callus growth. Sterilized seeds were placed on these media for three weeks. Both media showed callus induction and growth with 2 mg l–1 2, 4-D. Our results were similar to Azria et al.[5] that callus induction on N6 and MS media required 0.5-2.0 mg l–1 2,4-D. With MS media callus induction frequency was 70% while on N6 media callus induction and growth frequency was observed as 83.3%. Our results are contradictory to Rashid et al.[2] which reported that callus induction frequency of Super Basmati on MS media was 23.9% and on N6 47.7%. Comparing callus induction and growth frequency N6 media proved to be better (Table 1 and 2). N6 media showed better growth than MS as reported by Rashid et al.[1]. It is perhaps due to the reason that N6 media contained more nitrogen than the MS media.
Further these calli were placed on N6 media with 2 mg l–1 2,4-D for growth and proliferation. After two weeks, it was observed that 76% calli showed growth and increased 2-3 times in size (Table 3).
Maintained calli were transferred to basic MS media for regeneration. In addition to basic MS salts and vitamins different concentrations of NAA and BAP were used i.e., NAA 1 mg l–1 and BAP 1,2 and 5 mg l–1 (Table 4). Both NAA and BAP ever required for shoot initiation[5]. Pons et al.[6] reported that BA yielded more shoots than kinetin in all varieties while in case of using auxin NAA and IAA, it depends on the varieties. Callus regenerated on all the concentrations of growth hormones, but the highest frequency 81.60% was observed on NAA 1 mg l–1 and BAP 5 mg l–1. Our results are different from Rashid et al.[2] in which regeneration frequency was 45.3%.
| Table 1: | Callus induction and callus growth frequency of Oryza sativa (cv. Super Basmati) on MS media supplemented with 2,4-D 2 mg l–1, 3% sugar and 0.6% agar |
| Table 2: | Callus induction and callus growth frequency of Oryza sativa (cv. Super Basmati) on N6 media supplemented with 2,4-D 2 mg l–1, 3% sugar and 0.6% agar |
| Table 3: | Maintenance of embryogenic calli of Super Basmati on N6 media supplemented with 2 mg l–1 2,4-D |
*Number of calli shown in column I represent the repeat of the experiment |
|
There are some albinos plants also produced which are showed in the parenthesis.
In addition to growth hormones use of sorbitol as the osmoticum was also proved necessary for the growth of rice. Gelrite was used as gelling agent for the regeneration media. Water stress conditions enhance the regeneration frequency. On Media which lack casine hydrolysate and sorbitol there was proliferation and growth of calli without plant formation and media which contain sorbitol and casine hydrolysate has 81.60% regeneration frequency (Table 5). High osmolarity also stimulates the regeneration frequency[7]. For this purpose high concentration of sorbitol was used.
| Table 4: | Regeneration frequency of Basmati rice with different combination of auxin and cytokinins |
| RM1=MS + Sucrose + Casine hydrolysate + Sorbitol + NAA 1 mg l–1 + BAP 1 mg l–1 RM2=MS + Sucrose + Casine hydrolysate + Sorbitol + NAA 1 mg l–1 + BAP 2 mg l–1 RM3=MS + Sucrose + Casine hydrolysate + Sorbitol + NAA1 mg l–1+ BAP 5 mg l–1 |
|
| Table 5: | Regeneration frequency of Super Basmati on MS media with NAA 1 mg l–1 and BAP 5 mg l–1 without addition of caseine hydrolysate and sorbitol |
Regeneration from scutellum derived calli. |
| (A) | Arising of calli from scutellum |
| (B) | Callus induction and growth |
| (C) | Three week old calli on matainace media |
| (D) | Calli became green on regeneration media |
| (E) | Calli showing regeneration on MS media with NAA 1 mg L–1 and BAP 5 mg L–1 |
| (F) | Regenerated plants from scutellum derived calli |
We can conclude from the present study that now it is possible to obtained high regeneration frequency i.e. upto 80% from Super Basmati, which is shown more than other varieties. This high regeneration frequency will lead to improve the variety by genetic transformation technology.
It is concluded from the present study that a high frequency of regeneration (80%) has been achieved. This study is different from the previous ones because in this study higher frequency regeneration system is developed in super Basmati which is commercial cultivar.