Media Genotype Died Callused Type of callus produced Andersons Tayberry 20 0 Sunberry 20 0 None Loch Ness 20 0 WPM Tayberry 4 16 Moderate Sunberry 2 18 production Loch Ness 6 14
RM Tayberry 14 6 Small amount of
Sunberry 12 8 unhealthy callus Loch Ness 15 5 produced
A Tayberry 3 17 Large amount of
Sunberry 0 20 healthy callus
Loch Ness 5 15
Medium A was the most successful for producing callus (see results). This medium was further simplified to produce medium NA and the two were compared in
experiment 3.4. Experiment 3.4. Genotypes: Tayberry Sunberry Loch Ness 8242E6
Number of discs: 20 discs / 4 genotypes / 2 treatments at 10 discs per plate.
Choice of media: Media A and NA were compared
Choice of hormones: Hormone combination 0.2 mg/1 BAP and 0.2 mg/1 2,4-D.
Experimental conditions: As exp.l
Comment: Medium A and a simpler version NA were examined here.
Results Experiment 3.4.
Callus production was achieved on both media, however the simpler one, NA, was the most successful giving more profuse and healthy callus than on medium A (there were 20 discs per 4 genotypes and 2 treatments).
Table 3.4. Comparison of the amount and description of callus produced by leaf discs on media A and NA.
Media Genotype Died Callused
A Tayberry. 4 16 Sunberry 2 18 Loch Ness 5 15 8242E6 8 12 NA Tayberry 2 18 Sunberry 0 20 Loch Ness 3 17 8242E6 5 15 77
The results obtained from this experiment suggest that medium NA was superior at sustaining cell division, and
it was subsequently used in all further experiments.
Experiment 3.5.
Genotype: Sunberry (this had shown the greatest potential for cell division of leaf tissue ).
Number of discs: 40 discs / 1 genotype / 16 treatments at 10 discs per plate.
Choice of Media: Media NA
Choice of hormones: 0.05 IBA mg/1 0.1 0.2 2.0 0.2 0.2 0.2 0.2 BAP 2.0 2.0 2.0 2.0 mg/1 2.5 2.5 2.5 2.5 4.0 4.0 4.0 4.0
Experimental conditions: As exp.3.1
Comment: Medium NA with a range of IBA and BAP
concentrations were examined for potential to induce plantlet regeneration. IBA was substituted for 2,4-D which appeared very effective at callus production but not shoot regeneration.
Results Experiment 3,5.
In this experiment, for the first time plantlet regeneration occurred from the genotype Sunberry. This only occurred, however, on media NA containing 4 of the 16 hormome combinations used, but was achieved
successfully at only one of the 16 combinations, this being 0.1 mg/1 IBA and 2 mg/1 BAP.
Table 3.5. Comparison of callus production and
regeneration potential of the 4 hormone combinations which did induce regeneration.
Hormone combination Recreneration $ Callus only
A) 0.1 mg/1 IBA and 18/40 45 16 2 mg/1 BAP B) 0.05 mg/1 IBA 1/40 2. 5 22 2 .5 mg/1 BAP c) 0.05 mg/1 IBA and 1/40 2. 5 29 2 mg/1 BAP D) 0.1 mg/1 IBA and 1/40 2. 5 28 2 .5 mg/1 BAP
Regeneration in this experiment was observed to be occurring mainly along the mid-vein, with further regeneration occurring to a lesser extent, and at a later stage along the cut outer surface of the leaf discs.
The figures on the following page, show cell division occurring along the mid-vein (3.3) of leaf tissue of the hybridberry Sunberry, leading to plantlet formation
(3.4). Callus production can be observed along the cut outer surface of the leaf.
Fia. 3.3
Distortion along mid-vein of a leaf disc of the hybrid
berry Sunberry due to cell division. Fig. 3.4
Callus production and plantlet formation on leaf discs of the hybrid berry Sunberry on media NA with the
Experiment 3.6.
Genotype: Sunberry
Number of discs: 140 discs / 1 genotype / 2 treatments at 10 discs per plate.
Choice of Media: NA
Choice of hormones: 0.1 mg/1 IBA and 2 mg/1 BAP
Experimental conditions: As exp.3.1
Comment: This experiment was carried out to examine the importance of incorporating the mid-vein in the discs.
Results experiment 3.6.
The importance of regeneration occurring along the mid-vein was examined and found to greatly contribute to the regeneration potential of the discs (140 discs, 1 genotype, 2 treatments).
Table 3.6. Comparison of callus production and
regeneration potential of leaf discs with and without the mid-vein. Mid-vein No raid-vein No. discs 140 140 Callus 76 57 No. regn. 62 4 % regn. 44 3 81
The percentage regeneration from leaf discs
containing the mid-vein in exps. 3.5 and 3.6 were found to be almost identical. Experiment 3.7. Genotype: Sunberry Tayberry Loch Ness Autumn Bliss 8242E6.
Number of discs: 80 discs / 5 genotype / 1 treatment at 10 discs per plate.
Choice of media: NA
Choice of hormones: 0.1 mg/1 IBA and 2 mg/1 BAP
Experimental conditions: As exp.3.1
Comment: Regeneration potential of a range of plant genotypes was examined here.
Results Experiment 3.7.
This experiment was a repeat of experiment 3.6, using a wider range of plant genotypes including again
Sunberry, to ensure that regeneration was not genotype- specific.
Regeneration was extended to all plant genotypes tested, with varying levels of success. The specificity of conditions required by each cultivar, was
demonstrated by variation in the level of plantlet regeneration.
(80 discs per 5 genotypes per 1 treatment)
Table 3.7. Comparison of regeneration from 5 different genotypes of Rubus spp.
Plant material No. discs Callus Recreneration %
Sunberry 80 65 37 46
Loch Ness 80 50 23 29
Tayberry 80 62 35 44
8242E6 80 57 23 29
Autumn Bliss 80 27 7 9
Using chi-squared, results from this experiment suggested that there were significant differences
(P<0.01) between the genotypes in their ability to
regenerate. Sunberry is the best at regenerating, Autumn Bliss is the poorest.
In this and all the previous leaf disc experiments, leaf discs had been placed abaxial surface uppermost. The next experiment examines the effect of leaf disc orientation on plantlet regeneration.
Experiment 3,8
Genotype: Sunberry
Number of discs: 60 discs/ 1 genotype / 2 treatments / 10 discs per plate.
Choice of media: NA
Choice of hormones: 0.1 mg/1 IBA and 2 mg/1 BAP
Experimental conditions: As exp.3.1
Comment: The importance of leaf disc orientation was examined, 60 discs being placed on the media with their abaxial surface uppermost and 60 discs adaxial surface uppermost.
Results Experiment 3.8.
This study of the effect of leaf disc orientation on the regeneration potential of 120 discs in all showed a significant difference (P<0.001) between those with the abaxial surface uppermost and those with the adaxial surface uppermost.
Table 3.8. Effect of leaf disc orientation on the regeneration potential of Sunberry.
Surface Number of discs Regeneration Uppermost % regeneration Abaxial 60 31 62 Adaxial 60 7 12 Experiment 3.9. Genotype: Sunberry
Number of discs: 100 discs / 1 genotype / 2 treatments at 10 discs per plate.
Choice of media: Activated charcoal (routinely used in Rubus micropropagation media) was incorporated into medium NA.
Choice of hormones: 0.1 mg/1 IBA and 2 mg/1 BAP
Experimental conditions: As exp.3.1
Comment: In an attempt to improve regeneration, activated charcoal was added to the medium to
absorb phenolic compounds, produced by the explants in response to tissue damage and which may have been inhibitory to cell division.
Results Experiment 3.9,
Table 3.9. The effects of activated charcoal on the regeneration potential of leaf discs.
Charcoal No charcoal No. discs 100 100 No. died 63 20 Callused 14 70 Regenerated 0 51 % Regn. 0 51
As illustrated in table 3.9, plantlet regeneration was totally inhibited in the presence of activated charcoal, though a small amount of callus was produced on 14 of the 100 leaf discs on the charcoal medium (100 discs per 1 genotype per 2 treatments).
Experiment 3.10.