regulators on 
direct shoot regeneration are shown in Table 3. Direct shoot 
regeneration was observed within 14 days of culture. Shoot 
buds emerged directly from the cut end of the region near to 
petiole on cotyledon explants. Multiple shoot was formed 
directly within 21 day of culture initiation (Fig. 1). According 
to percentage of direct shoot regeneration , different plant growth regulators combinations showed different responses. 
Thus, the best result was obtained in M1 and M7 medium 
with 0.54 µM NAA, 0.44 µM BA (72% direct shoot 
regeneration) and 0.1 µM NAA, 0.44 µM BA (65% direct 
shoot regeneration), respectively. Higher concentrations of 
NAA and BA did not increase the number of direct shoot 
regeneration. These results are similar to those obtained by 
Hunter and Burritt (2002), who produced the greatest number 
of shoots when 0.54 µM of NAA used in combination with 
0.44 µM of BA. Similarly, Kanamoto et al. (2006) obtained 
high shoot regeneration from leafy explants cultivated on 
medium supplemented with 0.1 mgl-1 NAA and 0.1 mgl-1
BA. Webb et al. (1984) showed that cotyledon age can 
influence the regeneration response; with older cotyledons has less ability for direct shoot regeneration than younger 
ones. According to Hunter and Burritt (2002), cotyledon age 
influences the shoot-forming ability of cotyledon explants but 
in a genotype manner. But in the present study, explants age 
did not have a significant effect on direct shoot regeneration 
in two lettuce landraces. The present study also showed that 
lettuce genotypes did not have a significant effect on direct 
shoot regeneration. The relative influence of the genotypes on 
tissue response has been a matter of controversy. Vasil and 
Vasil (1986) suggested that the differential response of the 
genotypes may be due to differential expression, which in 
turn, depends upon the spatial and temporal distribution of 
their physiological and developmental stages. To identify the 
relationship between callus induction and direct shoot regeneration, Pearson’s correlation coefficient was computed. 
Pearson’s correlation coefficient identified that callus 
induction was correlated with direct shoot regeneration. 
These variables were positively correlated (0.408), and the 
correlation was statistically significant at the 0.05 level. This 
result is not similar to that of Ampomah-Dwamena et al. 
(1997), who reported no correlation between callus 
production and shoot regeneration. Three regions (middle, 
near to petiole and petiole) on cotyledon explants were also 
tested to identify the best region for direct shoot regeneration. 
According to obtained results to identify the best region for 
direct shoot regeneration, petiole of cotyledons did not show 
significant response to growth regulators. On the other hand, 
middle parts of cotyledon showed significant response to 
growth regulators and produced callus. But the best region 
for direct shoot regeneration in lettuce was the region near to 
petiole on cotyledon explants which showed high significant 
response to growth regulators (Fig. 1). In conclusion, our 
study revealed that the effect of growth regulators 
combinations on callus induction was genotype and explant 
age-dependent. The highest number of callus was obtained 
using 2.7 µM NAA and 4.4 µM BA. It also showed that 
direct shoot regeneration significantly decreased with 
increasing BA concentration. However, the use of BA less 
than 1 µM increased direct shoot regeneration in two 
genotypes. Positively significant correlation was observed 
between callus induction and direct shoot regeneration. It was 
also shown in our study that the best region for direct shoot 
regeneration on cotyledon explants of lettuce is near to 
petiole. This adventitious origin of shoots directly from the 
region near to petiole of cotyledons may aid the use of 
genetic engineering to improve horticultural characteristics. If 
propagation through tissue culture can be done efficiently on 
a large scale in plant breeding programs, it may be used to 
produce F1 hybrids to use the hybrid vigour. This 
regeneration protocol will be useful not only for further 
research studies such as Agrobacterium mediated genetic 
transformation or protoplast fusion studies, but also for 
commercial nurseries to produce virus-free plants and 
agricultural practices that could reduce pesticides application 
and increase production.
 
regulators on direct shoot regeneration are shown in Table 3. Direct shoot regeneration was observed within 14 days of culture. Shoot buds emerged directly from the cut end of the region near to petiole on cotyledon explants. Multiple shoot was formed directly within 21 day of culture initiation (Fig. 1). According to percentage of direct shoot regeneration , different plant growth regulators combinations showed different responses. Thus, the best result was obtained in M1 and M7 medium with 0.54 µM NAA, 0.44 µM BA (72% direct shoot regeneration) and 0.1 µM NAA, 0.44 µM BA (65% direct shoot regeneration), respectively. Higher concentrations of NAA and BA did not increase the number of direct shoot regeneration. These results are similar to those obtained by Hunter and Burritt (2002), who produced the greatest number of shoots when 0.54 µM of NAA used in combination with 0.44 µM of BA. Similarly, Kanamoto et al. (2006) obtained high shoot regeneration from leafy explants cultivated on medium supplemented with 0.1 mgl-1 NAA and 0.1 mgl-1BA. Webb et al. (1984) showed that cotyledon age can influence the regeneration response; with older cotyledons has less ability for direct shoot regeneration than younger ones. According to Hunter and Burritt (2002), cotyledon age influences the shoot-forming ability of cotyledon explants but in a genotype manner. But in the present study, explants age did not have a significant effect on direct shoot regeneration in two lettuce landraces. The present study also showed that lettuce genotypes did not have a significant effect on direct shoot regeneration. The relative influence of the genotypes on tissue response has been a matter of controversy. Vasil and Vasil (1986) suggested that the differential response of the genotypes may be due to differential expression, which in turn, depends upon the spatial and temporal distribution of their physiological and developmental stages. To identify the relationship between callus induction and direct shoot regeneration, Pearson’s correlation coefficient was computed. Pearson’s correlation coefficient identified that callus induction was correlated with direct shoot regeneration. These variables were positively correlated (0.408), and the correlation was statistically significant at the 0.05 level. This result is not similar to that of Ampomah-Dwamena et al. (1997), who reported no correlation between callus production and shoot regeneration. Three regions (middle, near to petiole and petiole) on cotyledon explants were also tested to identify the best region for direct shoot regeneration. According to obtained results to identify the best region for direct shoot regeneration, petiole of cotyledons did not show significant response to growth regulators. On the other hand, middle parts of cotyledon showed significant response to growth regulators and produced callus. But the best region for direct shoot regeneration in lettuce was the region near to petiole on cotyledon explants which showed high significant response to growth regulators (Fig. 1). In conclusion, our study revealed that the effect of growth regulators combinations on callus induction was genotype and explant age-dependent. The highest number of callus was obtained using 2.7 µM NAA and 4.4 µM BA. It also showed that direct shoot regeneration significantly decreased with increasing BA concentration. However, the use of BA less than 1 µM increased direct shoot regeneration in two genotypes. Positively significant correlation was observed between callus induction and direct shoot regeneration. It was also shown in our study that the best region for direct shoot regeneration on cotyledon explants of lettuce is near to petiole. This adventitious origin of shoots directly from the region near to petiole of cotyledons may aid the use of genetic engineering to improve horticultural characteristics. If propagation through tissue culture can be done efficiently on a large scale in plant breeding programs, it may be used to produce F1 hybrids to use the hybrid vigour. This regeneration protocol will be useful not only for further research studies such as Agrobacterium mediated genetic transformation or protoplast fusion studies, but also for commercial nurseries to produce virus-free plants and agricultural practices that could reduce pesticides application and increase production.
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