Abstract

Keywords

Introduction

Canola, Brassica napus L. is one of important crops in Australia. The application of genetic engineering to B. napus varieties will lead to the generation of plant varieties possessing more agriculturally and economically viable genetic traits. Genetic engineering relies on the development of efficient methods for the regeneration of viable shoots from cultured tissues, and the successful application of transformation techniques.

A variety of plant tissues have been used for regeneration of B. napus shoots including hypocotyl (Dietert et al., 1982; Radke et al., 1988) and cotyledons ( Narasimhulu and Chopra, 1988) root segments (Sharma and Thorpe, 1989 ), stem and leaf segments of one-month old seedling (Khehra and Mathias, 1992; Ovesna et al., 1993). For Agrobacterium-mediated genetic transformation of Brassica, hypocotyl, cotyledon and flowering stalks are commonly used explants. In the case of canola, hypocotyl sections derived from young seedlings have been used for transformation (De Block et al. 1989). Recently, transformation of other Brassica crop varieties such as cabbage and kale was achieved using cotyledonary petioles (Metz et al. 1995, Bhalla and Smith, 1998), and transformation protocols of Arabidopsis thaliana, also a member of the Brassicaceae family, rountinely uses root explants (Valvekens et. al. 1988). However, no study has been conducted to compare the regeneration ability of hypocotyl, cotyledonary and root explants of commercial canola cultivars in order to optimise the recovery of viable transformed shoots from genetic transformation experiments.

The aim of this study was to develop a procedure for the regeneration of viable shoots from hypocotyl, cotyledonary and root explants of commercial Australian canola varieties. Seven commercial cultivars of Canola Brassica napus L. were successfully regenerated. Differences in the regeneration capacity among different cultivars were observed.

Materials And Methods

Seven commercial genotypes (Dunkeld, Grouse, RK7, RI25, Oscar, Rainbow, and Monty) of oilseed Brassica napus L. were used. Seeds of these varieties were stored in 4⁰C untill used.

Seeds were surface-sterilised in 12.5% (w / v ) sodium hypochlorite by vigorous shaking for 20 minutes. The seeds were placed onto germination medium after rinsing five times in sterile distilled water. Tissue culture media including germination medium used in the study were essentially the same as described by Bhalla and Smith, (1998).

Cotyledon, hypocotyl and root segments from 4 days old seedlings were used as explants. These explants were carefully excised from the seedling without including any of the meristematic axillary bud. Hypocotyl (2-3 mm long section ), root segments (5-7 mm long each) and cotyledons with 1-3 mm long petioles were used. Fourty seedlings per cultivar were used. The explants were placed on the callus induction medium for one week and then transferred onto shoot induction medium. Well developed shoots were transferred onto shoot outgrowth medium for 2 weeks. The numbers of shoots per explant were counted after a total period of 13 weeks in culture. Sixty shoots from each genotype (20 shoots for each explant type) were transferred onto rooting induction medium. Most of the shoots formed roots on root induction medium and then were transferred in five inch plastic pots using a commercially available potting mix. These plantlets were placed in an intermittent mist chamber for two weeks before being transferred to glasshouse conditions.

Results And Discussion

Shoots were regenerated from cotyledons, hypocotyl and root segments of seedling explants of seven commercial Australian cultivars of oilseed Brassica napus seedlings. Response of explants to culture was observed within one week on callus induce medium. Shoots started to appear on shoot induction medium within 3-4 weeks in culture. In general, a high percentage (85-100%, Table 1) of explants formed callus. No significant difference in callus induction between different explant of a cultivar and among the cultivars was observed. In contrast, the percentage of explants that formed shoots varied greatly between the cultivars (Table 1). In addition, different explants of a cultivar also showed a varied response to shoot regeneration (Table 1).

Table 1: Shoot regeneration from seedling explants of Australian cultivars of canola, Brassica napus .

B. napus cultivar	Explant type no.	formed callus no. %	formed shoots no. %	number of shoots range mean ± S.E.
Dunkeld	Cot. 80 Hyp. 120 Root 160	78 97.5 119 99.2 152 95.0	36 46.2 49 41.2 22 14.5	1-3 1.50 ± 0.80 1-4 1.45 ± 0.82 1-5 1.42 ± 0.67
Grouse	Cot. 80 Hyp. 120 Root 160	68 85.0 120 100.0 142 88.8	30 44.1 75 60.0 58 40.8	1-4 1.57 ± 0.85 1-2 1.16 ± 0.58 1-4 1.40 ± 0.52
RK7	Cot 80 Hyp. 120 Root 160	71 88.8 118 98.3 154 96.3	13 18.3 32 27.1 23 14.9	1-5 2.50 ± 1.38 1-4 1.27 ± 0.80 1-3 1.40 ± 0.63
RI25	Cot 80 Hyp. 120 Root 160	80 100.0 120 100.0 151 94.3	29 36.3 41 34.2 20 13.2	1-6 1.83 ± 0.72 1-4 1.25 ± 0.46 1-2 1.29 ± 0.49
Oscar	Cot. 80 Hyp. 120 Root 160	79 98.8 118 98.3 157 98.1	35 44.3 50 42.4 56 35.7	1-5 2.00 ± 1.17 1-4 1.40 ± 0.52 1-5 1.96 ± 0.98
Rainbow	Cot. 80 Hyp. 120 Root 160	77 96.3 119 99.2 146 91.3	42 54.5 61 51.3 71 48.6	1-6 2.47 ± 1.64 1-5 2.10 ± 0.99 1-12 2.05 ± 1.16
Monty	Cot. 80 Hyp. 120 Root 160	73 91.3 109 90.8 144 90.0	25 34.2 74 67.9 66 45.8	1-5 1.73 ± 1.01 1-3 1.33 ± 0.49 1-9 1.60 ± 0.82

The average number of shoots from each explant type varied from 1.13 to 2.50. Total number of shoots regenerated varied significantly among the seven varieties tested. During this study a maximum number of shoots (>300) were produced from Rainbow cultivar of canola. However, mean separation by Turkey Pairwise Comparison showed that varieties Rainbow, Oscar and Monty were not statistically different in their shoot regeneration capacity. Cultivar RK7 showed a poor response to shoot regeneration; total number of shoot regenerated (<100) from this cultivar was less than one third of Rainbow cultivar. Variations of shoot regeneration among different explant types (cotyledon, hypocotyledon and root) and their sub-types (dorminate and recessive cotyledon; upper, middle and lower position segments of hypocotyl; root segment 1, 2, 3 and 4, from root tip to the base) were also investigated. No significant differences in shoot regeneration of these explants was observed when the results were analysised using oneway anova at α=0.05.

In the present study, a combination of BAP (cytokinin), NAA (auxin) and GA3 (gibberellin) was used. This combination was used after a number of preliminary experiments with these hormones. Growth hormones such as cytokinins, auxins and gibberellic acid are commonly used in conjunction to stimulate in vitro shoot regeneration. Murata and Orton (1987) used NAA (auxin) and kinetin (cytokinin) to regenerate shoots from hypocotyl and cotyledon explants of 7 Brassica species with a shoot regeneration capacity of 1.2 to 20 %.

Cotyledons were first to initiate callus and shoot production while root explants were slow in culture, indicating that cotyledons may be more suitable for transformation experiments. This difference in the response of canola genotypes may be due to different requirements of the explants for optimal callus and shoot regeneration. Khehra and Mathias (1992) studied the importance of explant type, genotype and growth hormone regime on shoot regeneration of a number of B. napus genotypes. Their study found that the most important factors for shoot regeneration were explant type and genotype and the influence of the hormone regime was negligible.

Sixty rooted shoots were transferred to soil under glasshouse condition. All shoots survived this transfer. No difference between the varieties was observed during root initiation and acclimatisation to glasshouse conditions. The plants produced flowers and set seeds. FCR test was used to check pollen viability. Seed germination test showed that ninty percent of the seeds produced were viable.

In conclusion, we have developed an efficient method for viable shoot regeneration from seedling explants of commercial Australian cultivars of canola. Based on our study, the method was more successful when applied to cotyledon and hypocotyl explants than root explants indicating that these could be the preferred explants for genetic transformation experiments.

References

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