Kenya Agricultural Research Institute (KARI). National Potato Research Centre, Tigoni, Kenya
Author
Correspondence author
International Journal of Horticulture, 2014, Vol. 4, No. 15 doi: 10.5376/ijh.2014.04.0015
Received: 01 Aug., 2014 Accepted: 17 Sep., 2014 Published: 30 Oct., 2014
This is an open access article published under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Muthoni et al., 2014, Multiplication rate of selected potato cultivars in vitro through single node culture, International Journal of Horticulture, 2014, Vol.4, No.15 1-4 (doi: 10.5376/ijh.2014.04.0015)
A study was carried out in the tissue culture laboratory at the National Potato Research Centre (NPRC), Tigoni, Kenya to determine the multiplication rate of selected potato cultivars when micropropagated through single node culture. Seven potato cultivars were subcultured on solid media for three consecutive cycles (each cycle took three weeks). This was repeated twice. Data collected was the number of single-node cuttings that were generated from each node after subculturing. Generally, there were significant differences (P≤0.05) on the number of single node cuttings generated among the potato cultivars, among the subculture cycles and in the interaction between genotypes and subculture cycle.
The main applications of in vitro plant tissue culture in crop production are in plant sanitation and micropropagation. For plant sanitation, meristem tip culture is the most commonly used for virus elimination in crop plants (Ng et al., 1992; Naik and Karihaloo, 2007; Badoni and Chauhan, 2010). Usually, meristem tips, about 0.5-1 mm long and consisting of the meristematic dome and two leaf primordial, are excised from surface-disinfected apical or axillary buds and allowed to grow into plantlets on artificial nutrient media under controlled conditions. Generally, the percentage of virus-free plants obtained is inversely proportional to the size of the tips cultured. This technique is used for elimination of viruses in the planting materials as many viruses are unable to infect the apical/axillary meristems of a growing plant and a virus-free plant can therefore be produced if a small piece of meristematic tissue is propagated (Wang and Hu, 1980; Kassanis, 1957). Elimination of viruses through tissue culture is possible because the vascular system through which viruses are spread is not well developed in the meristematic region. The high chromosome multiplication (due to high cell division) and high auxin content in the meristematic tissue possibly inhibit virus multiplication through interference with viral nucleic acid metabolism. Also, there exists virus inactivating system with greater activity in the apical region than elsewhere (Kassanis, 1957; Wang and Hu, 1980).
Micropropagation through tissue culture has played a major role in mass production of propagating material (Murashige, 1974). Micropropagation can be divided into three stages: 1) establishment of an aseptic in vitro culture and development of an explant by cell division, 2) a series of subcultures to achieve rapid multiplication of the propagules and, 3) rooting of established plantlets and their hardening to impart some tolerance to moisture stress and pathogens (Waithaka, 1992). Multiplication of propagules may be through somatic embryogenesis, adventitious organogenesis or axillary shoot development. While adventitious organogenesis may result in faster multiplication of the propagules, the occurrence of genetically aberrant plant is common. Axillary shoot multiplication may be slow but genetically aberrant propagules are rare (Waithaka, 1988). Therefore, production of plants from axillary shoots is the most applicable and reliable method of in vitro propagation (Ng, et al., 1992). With axillary shoots, the two methods which are usually used are shoot tip culture and single node culture. The shoot tip is usually taken from the tender tip of the growing shoot (about 2 cm long), while the single node cuttings are from either terminal or axillary buds with the stem segment attached. These two types of explants are preferred over meristem-tip culture in micropropagation when virus elimination is not part of the objective. Micropropagation allows large scale asexual multiplication of pathogen-free potato cultivars. At an interval of 21 days, at least 3 single node cuttings can be obtained from a single microplant. Therefore, theoretically 315 (14 million) micro plants can be obtained from a single virus free mericlone in a year. Single node culture is important in potato production because rapid multiplication of disease-free seed potatoes is the main bottleneck in production of certified seeds in Kenya. Although single node culture is the main approach employed in micropropagation of potatoes in Kenya, little is known about the multiplication rate of different potato genotypes when they are multiplied through single-node culture. This study was therefore undertaken to determine the multiplication rate of selected potato genotypes when they are micropropagated through single node culture.
Materials and methods
Seven potato cultivars (Table 1) commonly grown by farmers in Kenya and currently conserved in in vitro form at the National Potato Research Centre, Tigoni were involved in the trial.
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Table 1 Potato cultivars used in the trial
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The study was carried out in the plant tissue culture laboratory at the National Potato Research Centre, Tigoni between July and December 2013 (season 1) and between January and May 2014 (season 2).
Preparation of growth media
The solid media consisted of the Murashige and Skoog (1962) basal salts supplemented with glycine (0.2 g/l), nicotinic acid (0.05 g/l), pyridoxine (0.05 g/l), inositol (10 g/l), thiamine (0.01 g/l), giberellic acid (0.001 g/l), and sucrose (30 g/l). The pH of the media was adjusted to 5.8 using 1N NaOH or 0.1 m HCl. Two and a half grams of phytagel was added to one litre of the media as a solidifying agent. The culture media was dispensed into 1 litre pyrex jars, the jars were covered using aluminium foil and the auctoclaved at 121℃ for 15 minutes. Thereafter, 20 ml of the media was poured into each 500 ml plastic jars.
Preparation of the experimental materials
Seven tubers of each of the seven popular Kenyan potato cultivars (Table 1) were potted. Four weeks after potting, the plants were transferred to thermotherapy chamber for heat treatment for one month for virus elimination. The thermotherapy room was maintained at a constant temperature of 38℃ ± 2 and photoperiod of 168 during the entire period; the high temperature increased the rate of production of virus-free materials. Thereafter, 4-5 nodes from the apical end of the sprouted plants were excised and placed in 5% hypochlorite solution for 10 minutes and then rinsed 3-4 times with distilled water. The meristems were then dissected from each node under a microscope and the meristem tips were carefully transferred into plastic jars containing standard culture media (Murashige and F. Skoog, 1962). Thirty meristem tips of each variety were cultured (each jar had 10 meristem tips), they were then incubated in a growth chamber under 16 hrs of light and 8 hrs of darkness at 22 ± 2 ℃. After 10 weeks the meristems developed into plantlets; these formed the experimental materials.
Culturing of the single node cuttings
For each of the seven potato cultivars, the three plastic jars containing the ten-week old in vitro plantlets were used. Each jar, containing 10 plantlets was treated as a replicate. From each jar, the 10 plantlets were cut into single node cuttings and from each plantlet; one single node cutting was selected and cultured for three weeks in another jar containing solid media. There were 10 such single node cuttings in each jar. This was also done for the two remaining jars to make three replicates per potato cultivar. Each single node cutting measured 5 mm and had one axial bud and the subtending leaf.The single node cuttings were dissected using sterile blades and forceps under a sterilized laminar hood chamber. After culturing, the jars (containing the single node cuttings) were placed in the growth chamber; the chamber was maintained at 22 ± 2℃, 16 hours photoperiod and light intensity of 3000 lux.
After three weeks, the ten plantlets having grown from the 10 single node cuttings were cut again and the number of single nodes cuttings from each plantlet was counted and recorded (subculture interval 1). From each of the 10 plantlets, one single node cutting was selected and cultured for three weeks in another jar containing solid media. There were 10 such single node cuttings in each jar. This was also done for the two remaining jars to make three replicates. After three weeks, the ten plantlets having grown from the 10 single node cuttings were cut again and the number of single nodes cuttings from each plantlet was counted and recorded (subculture interval 2). The cycle was repeated again for another three weeks to subculture 3.
Data analysis
The experiment was analysed as a split-plot design where the potato cultivar was the main plot and the subculturing interval (three of them) was treated as the subplot. Data was analysed using Genstat statistical package, 14th edition (Payne et al., 2011) and means separated using Fisher’s Protected LSD Test (Steel and Torrie, 1980).
Results and Discussion
There were significant differences (P≤0.001) in the number of single node cuttings obtained among potato cultivars, subculturing cycles and in the interaction between potato cultivars and the subculturing cycle in season 1 (Table 2).
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Table 2 Skeletal ANOVA: Number of single node cuttings obtained from seven potato cultivars in three consecutive subculture intervals in season 1
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There was no significant difference in the number of single node cuttings among potato cultivars between subculture cycle 1 and 2 in the first season except for Desiree (Figure 1). Generally, the third subculture cycle gave higher number of single node cuttings than the first two subculture cycles. In addition, Purple Gold gave the least number of single node cuttings (2.367) while Dutch Robyjn gave the highest (6.644).
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Figure 1 Number of single node cuttings obtained from seven potato cultivars over three consecutive subculture intervals in season 1
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In the second season, there were significant differences (P≤0.05) in the number of single node cuttings obtained among potato cultivars, subculturing cycles and in the interaction between potato cultivars and the subculturing cycle in season 2 (Table 3).
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Table 3 Skeletal ANOVA: Number of single node cuttings obtained from seven potato cultivars in three consecutive subculture intervals in season 2
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There was no significant difference in the number of single node cuttings among potato cultivars between subculture cycle 1 and 2 in the second season except for Desiree (Figure 2). Generally, the third subculture cycle gave higher number of single node cuttings than the first two subculture cycles. In addition, Purple Gold gave the least number of single node cuttings (2.300) while Dutch Robyjn gave the highest (7.144).
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Figure 2 Number of single node cuttings obtained from seven potato cultivars over three consecutive subculture intervals in season 2
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All the potato cultivars followed a similar trend in production of single node cuttings (Figure 3). In all cases, the third subculture interval gave more cuttings than the first two.
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Figure 3 Average number of single node cuttings obtained from seven potato cultivars over three consecutive subculture intervals across the two seasons
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Differences in the number of single node cuttings generated from potato cultivars could be due to genetic differences among the potatoes; the harder and woody the potato stem, the slower the generative rate and the fewer the number of single nodes generated. Differences in the number of single node cuttings generated in different subculturing cycles could not be explained.Badoni, A. and J. S. Chauhan. 2010. Conventional vis -a- vis biotechnological methods of propagation in potato: A review. Stem Cell 1: 1-6
Naik, P. S. and J. L. Karihaloo. 2007. Micropropagation for the production of quality potato seed in Asia-Pacific. Asia-Pacific Consortium on Agricultural Biotechnology. New Delhi, India
Ng, S. Y. C., G. Thottapily, and H. W. Rossel. 1992. Tisue culture in disease elimination and micropropagation. p. 171-182. In G. Thottapilly, L.M. Monti, D.R. Mohan Raj and A.W. Moore(ed) Biotechnology: Enhancing Rsearch on Tropical Crops in Africa. CTA/IITA co-publication. International Institute of Tropical Agriculture, Ibadan, Nigeria
Payne, R. W., D. A. Murray, S. A. Harding, D. B. Baird, and D. M. Soutar. 2011. GenStat for Windows 14th ed. Introduction. VSN International, Hemel Hempstead
Steel, R. G. D. and J. H. Torrie. 1980. Principles and procedures of statistics: A biometrical approach. 2nd ed. McGraw-Hill, New York
Waithaka, K. 1988. Application of plant tissue and cell culture in horticultural production. Acta Horticulturae 218: 131-139
Waithaka, K. 1992. Micropropagation techniques and the production of pathogen-free plants. p. 183-187. In G. Thottapilly, L.M. Monti, D.R. Mohan Raj and A.W. Moore (ed) Biotechnology: Enhancing Rsearch on Tropical Crops in Africa. CTA/IITA co-publication. International Institute of Tropical Agriculture, Ibadan, Nigeria
Wang, P. J. and C. Y. Hu. 1980. Regeneration of virus-free plants through in vitro culture.p. 61-99. In A. Flechter (ed.) Advances in biochemical engineering. Springer-Verlag, Berlin
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