1. SYNERGISTIC EFFECT OF GIBBERELLIN AND CYTOKININ FOR A RAPID NODAL MULTIPLICATION SYSTEM OF AVOCADO
J. C. A. Hiti Bandaralage, A. Hayward, W. Nak , N. Mitter |
Results
Bud breaking and observations
No bud breaking was observed at 8 day with hormone –free media.
T2, T3 & T6 showed 100% bud breaking.
Introduction
Avocado (Persea americana Mill.) is grown as a grafted crop to combine beneficial rootstock and shoot/scion characters for maximum tree performance.
Clonal rootstocks produced by vegetative propagation of elite selections are genetically uniform and offer superior on-farm productivity and higher returns for farmers.
However, clonal rootstock propagation employs an arduous double grafting technique (Ernst 1999) that is laborious, lengthy and expensive.
This is a major bottleneck for the avocado industry
Tissue culture offers an alternative for efficient and effective mass propagation of avocado.
At present however, avocado tissue culture suffers from problems such as poor regeneration, poor shoot elongation, stunted shoots, defoliation, necrosis, tip die back and poor rooting ability.
These effects can be associated with suboptimal supplementation of commonly used hormones including the cytokinin benzyl adenine (BA).
The current study tested for the first time the use of Meta- topolin (MT), a natural aromatic cytokinin, to improve avocado in vitro regeneration capacity while eliminating problems caused by commonly used cytokinins.
Specific Objectives
Identify positive effects of MT, and synergistic effects with gibberellic acid (GA3), for shoot regeneration of avocado.
Optimise a hormone regime to support shoot multiplication from physiologically mature nodal explants of an industry important cultivar (cv. Velvick).
Methods and Materials
Leaf Expansion
Shoots in T7 had significantly more open leaves (P<0.001) than all other treatments at day 15, but at day 45 the highest mean value was recorded on T6 (P< 0.000).
15 D
45 D
30 D
Healthier and larger green shoots were produced in all media containing hormones.
Conclusions
MT, together with GA3 offer a promising hormone combination for avocado shoot multiplication in vitro.
Results of this study agree with the beneficial effects of MT reported in other woody plants such as pine (De Diego, Montalbán, and Moncaleán 2010) , apple (Magyar-Tábori et al ), pistachio( Benmahioul et al. 2012), and sweet orange (Niedz and Evens 2011).
Recommendations
1. The optimal hormone regime for shoot regeneration was 0.1 mg/L MT with 0.1 mg/L GA3.
2. MT can effectively eliminate leaf necrosis, defoliation, and senescence. MT also acts synergistically with GA3 to improve shoot heath in regeneration of avocado.
3. Subculturing for nodal multiplication should be completed at 45 days in optimum hormone concentration.
Acknowledgements
Neena Mitter group, ARC linkage grant, Industry partners (Delroy orchards, Jasper farms & Anderson’s horticulture).
“This project has been jointly funded by the Department of Agriculture and Fisheries and the University of Queensland”
References
Ernst, A. A “Micro Cloning: A Multiple Cloning Technique for Avocados Using Micro Containers.” Revista Chapingo Serie Horticultura 5: 217–20.
Platt, R. G “Current Techniques of Avocado Propagation.” Proceedings of the First International Tropical Fruit Short Course: The Avocado. Gainesville (FL): Florida Cooperative Extension Service and Institute of Food and Agricultural Services, University of Florida. P, 92–95.
De Diego, N., I.A. Montalbán, and P. Moncaleán “In Vitro Regeneration of Adult Pinus Sylvestris L. Trees.” South African Journal of Botany 76 (1): 158–62.
Magyar-Tábori, Katalin, Judit Dobránszki, Jaime A. Teixeira da Silva, Sean M. Bulley, and Ildikó Hudák “The Role of Cytokinins in Shoot Organogenesis in Apple.” Plant Cell, Tissue and Organ Culture (PCTOC) 101 (3): 251–67.
Niedz, Randall P., and Terence J. Evens “The Effects of Benzyladenine and Meta-Topolin on In Vitro Shoot Regeneration of Sweet Orange.” ARPN J Agric Biol Sci 6: 64–73.
Benmahioul, Benamar, Noëlle Dorion, Meriem Kaid-Harche, and Florence Daguin “Micropropagation and Ex Vitro Rooting of Pistachio (Pistacia Vera L.).” Plant Cell, Tissue and Organ Culture (PCTOC) 108 (2): 353–58.
Shoot quality
At day 45, T6, T7 & T8 supported highest quality shoots based on overall visual observation and statistical significance (P<0.001). T1 T2 T5 T4 T3
T10 T9 T8 T7 T6
Hormone-free
15 D
45 D
30 D
Material collection
Sterilization
Initiation
Shoot regeneration
50-60 cm twigs (budwood) of mature cv. Velvick plants.
Wash under running tap water for 45 min.
Immerse in 70% ethanol for 3 min.
Wash with sterile distilled water.
Beach with 3% commercial bleach + 2 drops of Tween 20 for 3 min.
Rinse with sterile distilled water.
½ strength Lloyd & McCown woody plant medium (WPM), incubate for 7 days.
Transfer to full strength WPM with treatment hormones (Table 1.)
20 sections per treatment.
Culture incubation – 25 0C , 16 h photoperiod.
Shoot height
Height was greatest in T6 at all three time points and was significantly greater at day 45. (P<0.000)
Table 2. Mean height of regenerated shoots at the three time points measured
Treatment
Mean Height
(15 D)
(30 D)
(45D) 1
2.1a
6.2a
9.25a 2
4.1a
5.6a
7.15a 3
3.8a
8.25b
9.45a 4
3.75a 7b
8.45a 5
4.05a
8.4b
9.6a 6
5.05a
10.8b
15.6b 7
4.15a
9.2b
10.1a 8
4.2a
7.5b
9.35a 9 5a
7.9b
8.1a 10
3.7a
8.3b
9.1a
Table 1. Shoot regeneration treatment media
Treatment
MT (mgL-1)
GA3 (mgL-1)
T 1 -
T 2
0.1
T 3
1.0
T 4
T 5
T 6
T 7
0.2
T 8
0.3 T9 1
T10 2
Measurements and Data analysis
Observations for verification, callus production, shoot tip die back, defoliation and necrosis were recorded.
Percentage bud breaking at 8 days, shoot height, number of open leaves, shoot quality (0-5 scale) were recorded at day 15, 30 and 45.
ANOVA and Turkey’s HSD (0.5 significant level) was performed using IBM SPSS 23
15 D
45 D
30 D