Effect of gamma ray irradiation on in vitro shoot regeneration from leaf explants

Shoot regeneration from the leaf explants irradiated by gamma rays was achieved in an 8-wk period. Significant interaction of gamma ray dosage × cultivar in shoot regeneration was observed (Table 1). In the first 4 weeks, the cultures were placed in the dark and no shoots were observed from any explants; however, calluses were developed from the explants that were irradiated with 0–50-Gy gamma rays. In the 100-Gy treatment, no callus was developed from ‘Amethyst’ and ‘Polana,’ whereas calluses were seen in the leaf explants of ‘Joan J’ (data not shown). After 4 weeks of dark treatment, all cultures were subcultured to the fresh shoot regeneration medium and placed in the tissue culture room with a 16/8 hr (day/night) photoperiod. In vitro shoots were gradually developed. Shoot regeneration was significantly affected by gamma ray dosage and cultivars (Fig. 1). The gamma ray irradiation dosage significantly inhibited shoot regeneration. An average of 27.8, 12.2, and 2.2% of leaf explants that were irradiated with 25-, 50-, and 100-Gy gamma rays developed shoots, while 51.1% non-irradiated leaf explants formed shoots. Three cultivars showed different responses to gamma ray irradiation. The regeneration rate of ‘Amethyst’ dropped from 83.3 to 46.7%, 23.3%, and 3.3% after the leaf explants were irradiated with 25-, 50-, and 100-Gy gamma rays. The regeneration rate of ‘Joan J’ was also dropped from 46.7 to 33.3%, 13.3%, and 3.3% when irradiated with 25-, 50-, and 100-Gy gamma rays, respectively. There is no significant difference in decreasing regeneration rates between ‘Amethyst’ and ‘Joan J.’ However, a significant decreasing regeneration rate of ‘Polana’ at 25 and 50 Gy irradiation was observed compared to ‘Amethyst’ and ‘Joan J’ (Fig. 1).

Table 1. Analysis of variance (ANOVA) on shoot regeneration of gamma ray–irradiated three raspberry cultivars ‘Amethyst’ (Rubus occidentalis × R. idaeus ‘Amethyst’), ‘Joan J’ (R. idaeus ‘Joan J’), and ‘Polana’ (R. idaeus ‘Polana’)Figure 1.

Effect of gamma ray dosage on shoot regeneration from leaf tissues of ‘Amethyst’ (Rubus occidentalis × R. idaeus ‘Amethyst’), ‘Joan J’ (R. idaeus ‘Joan J’), and ‘Polana’ (R. idaeus ‘Polana’). Vertical bars represent standard errors.

Effect of gamma ray irradiation on in vitro shoot regeneration from petiole explants

In vitro shoots were also regenerated from petioles that were irradiated by gamma rays. SAS analysis showed that there was no significant interaction of gamma ray dosage × cultivar in shoot regeneration. The shoot regeneration from petiole explants was solely significantly affected by gamma ray dosage (Table 1). ‘Polana’ was most sensitive to gamma ray irradiation and no shoots were regenerated from the gamma ray–irradiated petioles (Fig. 2). An average of 26.7% of non-irradiated petiole explants produced shoots, whereas shoot regeneration only occurred in 11.1%, 8.9%, and 2.2% of petiole explants that were irradiated with 25-, 50-, and 100-Gy gamma rays, respectively (Fig. 2).

Figure 2.

Effect of gamma ray dosage on shoot regeneration from petiole tissues of ‘Amethyst’ (Rubus occidentalis × R. idaeus ‘Amethyst’), ‘Joan J’ (R. idaeus ‘Joan J’), and ‘Polana’ (R. idaeus ‘Polana’). Vertical bars represent standard errors.

Morphological Variations in ‘Amethyst’ Plants

The regenerated shoots were rooted in the rooting medium, grown in the greenhouse, and finally transplanted in the field. The majority of ‘Joan J’ and ‘Polana’ plants were lost to the harsh winters; therefore, variations were screened only for ‘Amethyst,’ which survived in the field.

Leaf Variations

The original leaves of ‘Amethyst’ are opposite, odd-pinnate compound consisting of three leaflets, ovate with closely serrate or serrulate margins. Leaf arrangement is alternate on canes. Under the field conditions, the mature leaf was about 12 to 15 cm long and 12 to 15 cm wide with three opposite and odd-pinnate compound leaflets separating from each other, dark green above and glabrous silver beneath (Fig. 3A). Leaf variations included additional leaflets (Fig. 3B), wide and/or overlapped leaflets (Fig. 3B, C), and small and overlapped leaflets (Fig. 3E). These variations were generally observed on the different plants.

Figure 3.

Variations in Rubus occidentalis × R. idaeus ‘Amethyst’ leaves. (A) Original leaf, (B) additional leaflets, (C) and (D) wide and overlapped leaflets, and (E) small and overlapped leaflets.

Cane Variations and Plant Stature

The original cane of ‘Amethyst’ is round with small spines. Cane variations include less nodes per cane and shortened internodes (Figs. 4 and 5). Changes in the length of internodes and leaflet number resulted in the change of the overall structure of the mutant plants. Some plants had a small and compact stature (Fig. 5B, C), while others were long and drooping, similar to the original ‘Amethyst’ plant in the field (Fig. 5A).

Figure 4.

Variations in Rubus occidentalis × R. idaeus ‘Amethyst’ canes. (A) Original primocane of Amethyst, (B) and (C) less node and/or shortened internodes resulted in short and/or erect canes, and (D) short cane with small leaves.

Figure 5.

Variations in Rubus occidentalis × R. idaeus ‘Amethyst’ plant stature. (A) Original Amethyst plant with long and drooping canes (5 to 7 feet long) and (B) and (C) a small plant stature with short and erect canes (2 to 4 feet high).

Fruit Variations

Fruit variations included changes in size of drupelets, deformed, and crumbly fruits (Fig. 6). In the raspberry field, the average weight of an ‘Amethyst’ fruit is about 2.0 g, while the fruit weight in mutant plants ranged from 0.6 to 2.8 g per fruit and the content of total soluble solid ranged from 7.9 to 13.7 Brix (data not shown). Variations were also detected in the drupelet number of the fruit. The number of drupelets per berry in mutant ‘Amethyst’ plants varied from 4 to 50, compared to an average of 35 to 50 drupelets in a normal ‘Amethyst’ fruit. It has been noticed that the drupelet size rather than the number plays a major role in fruit size. In this study, deformed and crumbly fruits were also observed (Fig. 6C). The fruit color varied from dark red to purple, and some fruits never turned to purple even after fully ripened.

Figure 6.

Variations in the Rubus occidentalis × R. idaeus ‘Amethyst’ fruit. (A) Original fruits of Amethyst, (B) large fruits with big drupelets, and (C) small fruits with big and loose drupelets.