African Crop Science Journal, Vol. 9, No. 1, March 2001, pp. 1-8
Endale Gebre and B. N. Sathyanarayana1
Code Number: CS01028
The efficiency of in vitro shoot regeneration and microtuber production of potato (Solanum tuberosum L.) from nodal explants was studied using agar and other new and cheaper gelling agents- tapioca and sago in Murashige & Skoog (MS) salt medium. For shoot regeneration, agar was maintained at 8 mg l -1, tapioca and sago were varied between 9-18 and 10-14 % (w/v), respectively supplemented with 3% sucrose, 0.03 mg l-1 NAA, 0.25 mg l-1 GA3 and 2.5 mg l-1 Ca-panthothnate. The microtuberisation study was done using agar, tapioca and sago at 0.8, 14 and 10% concentration (w/v), respectively, in the presence of benzylaminopurine and paclobutrazol. Type of gelling agent significantly affected in vitro plant regeneration. After 32 days, shoot height and number of nodes respectively were 8.86 cm and 10.5 in agar, and 8.9 -12.1 in 11-15% tapioca, respectively. Tapioca at 14-15% stimulated growth giving significantly (P<0.01) higher root fresh weight gain (36.3%) over agar. Microtuber fresh weight was influenced more by the type of gelling agent than microtuber number. The highest (53.3%) biomass conversion was found with sago in the medium containing paclobutrazol followed by agar (43.3%) in BAP medium. A deferential increase in weight occurred in media solidified with tapioca and sago. Within 11-14% of tapioca, the existence of a favourable osmotic environment and better carbohydrate and/ or ionic supplement to the culture medium than agar is suspected causing improved cell growth and morphogenesis. The result showed the possibility of a successful use of tapioca, a cheaper gelling substance, for in vitro culture of potato.
Key Words: Microtubers, sago, shoot regeneration, Solanum tuberosum, tapioca, tissue culture
L'efficacité de la régéneration des pousses in vitro et de la production des microtubercules de la pomme de terre (Solanum tuberosum L. ) à partir d'explants nodaux a été étudiée en utilisant l'agar et d'autres nouveaux et moins chers agents colloïdaux; tapioca et sago dans le milieu salé de Murashiga &Skoog (MS). Pour la régéneration des pousses, l'agar a été maintenu à 8 mg l-1 , tapioca et sago ont varié entre 9-18 et 10-14 (w/v) respectivement supplémentés avec 3% de sucrose, 0.03mg l-1NAA, 0.25mg l-1 GA et 2.5ml l-1 de pantothenate de Ca. L' étude de la microtuberization a été conduite en utilisant l'agar, tapioca et sago à une concentration de 0.8, 14 et 10(w/v) respectivement, en présence de la benzylaminopurine et de la paclobutrazole. Le type d'agent colloidal a affecté significativement (P<0.01) le plus haut gain du poids frais des raciness (36.3%) plus que l'agar. Le poids frais des microtubercules a été influencé par le type d'agent colloïdal plus que le nombre de microtubercules. La conversion la plus élevée de la biomasse (53.3%) a été obtenue avec le sago dans les tubercules moyens contenant du paclobutrazole suivi de l'agar (43.3%) dans le milieu BAP. Une augmentation artificielle en poids a apparu dans un milieu soldifié avec tapioca et sago. Entre 11-14% de tapioca, il est prévu l'existence d'un environnement osmotique favourable et de meilleurs hydrates de carbone et/ou un supplément ionique au milieu de culture plus que l' agar, favorisant une amélioration de la croissance des cellules et de la morphogénèse. Les résultats ont montré aussi la possibilité d'utilisation avec success du tapioca, une substance colloïdale moins chère, pour la culture in vitro de la pomme de terre.
Mots Clés: Microtubercules, sago, régéneration
des pousses, Solanum tuberosum, topioca, culture de tissu
Potato (Solanum tubersum L.) is grown in the highly populated, intensively cultivated high and mid altitude in Ethiopia. As a crop of high biological value for its protein and a substantial amount of vitamins, minerals and trace elements, it is undoubtedly a very important crop in the country and also in many parts of Sub-Saharan Africa. In all potato growing regions the availability of high quality clean seed tuber has been the most limiting owing to the conventional clonal propagation that favours disease build-up that drastically reduces yield. However, the recent advancement in tissue culture and the flexibility of organ development in potato allows for alternative methods of propagation through in vitro techniques. Potato seed production programmes in many countries have been boosted by using these techniques. In recent years the first multiplication steps in seed production programmes are speeded up by using in vitro plantlets, microtubers (Bizarri and Ranalli, 1995) or minitubers (Hussey and Stacey 1981).
In the rapid multiplication of clean material in vitro, the use of single nodal cuttings is the most preferred method of propagation since it ensures higher propagation rates with maximum genetic uniformity in potato (Chandra and Naik, 1993). The major factors limiting the rates of multiplication in nodal culture are the short height of the plantlets and the low number of nodes on the plantlets obtained. Improvement has been made possible by addition of growth regulators to the medium. GAs stimulated development of nodal cutting on MS but at high concentration it produced narrow and elongated shoots (Novak et al., 1980) depending on genotypes. Longest main shoot and highest node numbers are reported to be obtained in medium containing NAA and BAP (Yousef et al., 1997). Among these methods, the direct use of microtubers has gained a considerable interest owing to their ease of handling, storage and transport of germplasm and reduced period to produce seed tubers (Jones, 1994). Experiments have shown that while tuberisation is under the tight control of hormonal stimulation, environ-mental factors such as photoperiod, temperature, irradiance and C02 affect in vitro tuberisation. Media conditions such as N concentration, sucrose or osmolarity of the medium have either a direct or indirect effect on the induction or developmental processes of in vitro produced microtubers (Garner and Blake, 1989; Khuri and Moorby, 1995).
However, there are limitations both in shoot regeneration and microtuber production. The limitations in many ways are ascribed to the components of the culture environment and to the low photosynthetic ability of the explants or plantlet. Most current systems of microtubers production have problems of obtaining sufficient number and size of microtubers produced per cycle. Thus, both shoot and microtuber production systems are still less competitive and economical compared with in vitro rapid multiplication.
Some studies have been made in order to reduce tissue culture media costs such as for agar. Batacharaya et al. (1994) used a cheaper gelling agent sago for chrysanthemum tip culture while Nene et al. (1997) used tapioca for tobacco and chickpea. The later in a pioneering work on the use of tapioca, reported a higher (66.7%) rooting for chickpea as compared with 40.0% on agar. Percent plant establishment in pot culture was also higher (83.3) in tapioca than agar (77.8).
The aim of this study was to compare the efficacy of alternative cheaper gelling agent(s) namely tapioca and sago to costly item agar (in the presence or absence of growth retardant) for in vitro shoot regeneration and microtuber production using nodal explants of potato. In this paper we report on two alternative gelling agents; tapioca obtained from tubers of cassava (Manihot esulenta Crantz.) (Nene and Sheila, 1997) and sago from the stem pith of Meroxylon sagu Rottb (Bhattachary et al., 1994) for in vitro shoot and MT regeneration.
MATERIAL AND METHODS
Shoot proliferation. The shoot proliferation study was done using a potato
genotype MF-2, obtained as one year old sprouted microtubsers stored at 5 °C.
The sprout cuttings of about 5-10 mm with a leaf axle were aseptically prepared
under sterile condition and serially sub-cultured every four weeks using 10
mm long nodal plants having one bud and a leaf axle.
The medium was prepared using MS (full strength ) salt dissolved in double distilled water and consisted of 3% sucrose. The pH was adjusted to 5.8 before boiling the medium. Agar was maintained at the standard concentration (8 mg l-1) and tapioca was varied from 9 - 18 (9, 10, 11, 12, 14, 15, 17, 18) and sago from 10-14 (10,12,14) percent (w/v). The rates were fixed based on prior information and pilot investigations. Two nodal cuttings of cultivar MF-2 taken from four weeks old plantlets were cultured in 30 ml MS medium supplemented with 0.25 mg l-1 GAs, 0.03 mg l-1 NAA and 2.0 mg l-1 (Ca) panthothenate. The hot medium was immediately dispensed into culture vessels (baby jars of 300 ml capacity) and covered with polypropylene cap. In case of tapioca and sago, the required concentration (w/v) were calculated per volume of media to be dispensed in each culture vessel following the protocol developed by Nene and Sheila (1997). All jars were then autoclaved at 1.06 kg cm-2 and a temperature of 121 °C for 20 minutes. The experiment was laid out as CRD and replicated eight times. The cultures were incubated for 35-40 days at 26° 3° C and 16 h photoperiod.
In vitro tuberisation. The three gelling agents, agar at 0.8%, tapioca at 14% and sago at 10 % were prepared. Nodal sections of about 10 mm having a leaf axle were used as explants from a virus free platelets of cultivar Kufri Ashoka after removing the apical and basal parts. The nodes were transferred onto MS (full strength) basal tuberisation medium which consisted of 8% sucrose, and BAP at 2.0 mg 1-1. The pH of the medium was adjusted to 5.8 prior to boiling with agar or dispensing it into jars containing tapioca or sago each with or without the presence of paclobutrazol (0.001 mg 1-1). A liquid culture included as a treatment was maintained in static condition supported by filter paper (1 FP: Whattman No. 1) bridge to keep explants above the liquid medium. A treatment was replicated 20 times. The cultures were incubated at 26±3°C, and 16 h photoperiod and illumination of 60 Mmol m-2 s-1 for 32 days. Subsequently, they were moved into a growth chamber with temperature and photoperiod adjusted at 15/15°C (day/night) and 8 h., respectively, under the same light intensity where they were grown for 6-8 weeks.
Data for shoot proliferation were analysed by one way ANOVA after Barlett's test for homo-geneity and transformation. Shoot, root and micro-tuebr fresh weight data in the tuberisation study were log transformed and microtubers numbers were square root transformed and analysed also using one way ANOVA. Mean comparison was made per six nodal cuttings (original shoot).
RESULTS AND DISCUSSION
Shoot proliferation. In vitro plantlet growth from nodal culture was significantly influenced by the type of gelling agent. After 32 days of incubation, shoots in agar reached 8.9 cm with 10.5 nodes. In tapioca at 11-15 % strength, these values ranged between 8.9 - 9.8 cm and 11.3 - 12.1, respectively, for shoot length and number of nodes and were not statistically different from agar based medium (Table 1). These results are comparable or even better than the most rapid node production (x 8 to x 10 per month) reported earlier using agar (Hussey and Stacey, 1981). The increase in plantlet growth in tapioca based medium represents a substantial increase in propagation rates. Sago based medium was generally unfavourable for in vitro shoot proliferation. Tapioca 9 - 14 % strength supported higher root growth than agar. At 18 % , however, growth was reduced and almost no roots (0.5 cm) were formed. A notable low root mass occurred in sago 12 and 14 % (Table 1). The roots produced in tapioca and sago were thicker than those in agar medium after 40 days of incubation.
Tapioca at 12 and 14 % had 58.9 and 31.2% biomass increase over agar, respectively. A strong stimulation of shoot and root growth occurred in tapioca based medium up to 14% strength (Table 2). Biomass in sago was significantly lower than that in tapioca or agar.
The differences in growth between tapioca and sago (Table 2) was possibly due to the differential osmotic effects varying at different concentrations (gelling matrices) in addition to their varying nutritional supplement. The significant fresh weight (shoot and root) gain in tapioca medium at 12 and 14 % concentrations was 36.3% more over agar. Nene and Sheila (1997) explained that tapioca, unlike agar, can be used as a gelling agent and a carbohydrate component in the nutrient media.
Such better growth of tissue in tapioca at 12 -15% strength than the rest of the concentrations, is also associated with the improved gelling consistency providing a favourable osmotic and matric potentials. In 9 or 10 % of tapioca gelling was poor and in 17-18% of tapioca and 12-14% of sago, solidity of gelling was a problem. Such changes to either lower or higher concentrations lead to changes in the plant tissue such as hyperhydric condition (observed at 9 - 10 tapioca) on short internodes and highly reduced leaf expansion (at higher concentrations) as a result of greater osmotic stress and possibly ionic contamination, which impaire optimum growth. Bhattacharya et al. (1994) reported a significantly higher ionic contamination of sago than Difco agar.
Growth was retarded at concentrations > 15% of tapioca possibly because of elevated sucrose from the starch granules. Increased sucrose beyond 2% in the plant tissue of Raphanus sativus was shown to be a reason for lack of rooting (Lovell et al., 1972) and similar results were reported by Rahman and Blake (1988). Perata et al. (1997) showed that sugar negatively interact with signal transduction pathway of GA. It is possible that the poor growth at high concentration of carbohydrate rich gelling substances in our study is a result of repression of growth hormones such as GA in addition to its direct osmotic interference in the medium.
In vitro tuberisation. Microtuberisation study using new and cheaper alternative gelling agents such at tapioca and sago has not been reported hitherto. Microtuber initiation occurred within 10-20 days in 50% of the cultures in agar based medium and 10 and 20% of the cultures in tapioca and sago based media, respectively. However, not all of these early initiated tiny swelling tubers succeeded in developing into a microtuber. Most of them were resorbed. The re-utilisation of these tuber initials could be due to the slightly higher (26± 3°C) temperature with 16 hr photoperiod that occurred before transferring the culture into a tuber inducing condition (8 hr photoperiod and 15°C). Such environment provokes shoot growth (Hussey and Stacey, 1981) while high temperature condition revert developing tuber to stolen growth under light condition (Ewing, 1995).
Gelling agents significantly influenced shoot and root fresh weight (Table 2). A highly significant (P<0.01) plantelt biomass (shoot + root) (533.8 mg per shoot) was obtained in tapioca compared with 83.7 mg in agar in the presence of only BAP (2.0 mg-1) in the medium (Plate 1). Even in the presence of a potent inhibitor, paclobutrazol, shoot and root growth was not hindered in tapioca as in agar or sago based medium. The inhibitory role of paclobutrazol was previously reported by Simok (1993) who found no stem or root formation in cultures treated with 0.1 mg 1-1 paclobutrazol but no such effects were found at 0.001 g 1-1. However, the reason for the buffering effect of tapioca to the inhibitory action of paclobutrazol was not clear. Tapioca based tuberisation medium showed relatively higher shoot proliferation. The suppressing effect of paclobulrazol was more pronounced in agar and sago media with almost no roots formed on the plantlets (Table 3).
Microtuber number was not different between the gelling agents. In the presence of paclobutrazol, however, tapioca medium gave significantly higher number (13.4) of micro tubers although about 40% of these tubers weighed below 50 mg (Table 4) while the liquid medium gave the least number (3.6) of tubers per original shoot. Average microtuber weight was significantly (P<0.01) higher in tapioca (97.0 mg) than sago (62.66 mg) or the liquid medium (44.4 mg) (Table 3) without paclobutrazol in the medium but its presence increased microtuber weight significantly in sago medium. Moreover, biomass conversion (biomass recovered in the form of microtuber) was significantly improved by the presence of paclobutrazol in all the media types, the highest (82.6) being in sago than agar (78.0) or tapioca (56.3). The increase in sago was due to a relatively low plantlet biomass (shoot + root) than increase in microtuber weight in the medium. The increase in % biomass conversion in paclobutrazol treated medium over the medium containing only BAP was 176.4, 114.8 and 46.7%, respectively, for tapioca, agar and sago based medium. Such a role of paclobutrazol to promote tuberisation per se and tuber fresh weight of microtubers comes from its inhibitory effect on oxidation of ent-kaurene sequence reactions in GA biosynthesis. This lowers GA levels, which is a prerequisite of tuber formation (Graebe, 1987), and its ability to divert assimilates to the tuberous organs (Deng and Prange, 1988). Biomass conversion ranged between 14.4-82.6%.
The frequency distribution of microtubers size showed significant variation among gelling substances. Most (72%) tubers formed in agar were < 50 mg . In tapioca and sago based medium a higher proportion (87 and 56%, respectively) of tubers formed were >50 mg all in paclobutrazol free medium(Table 5). The frequency of tubers of size >200 mg, was low except in tapioca containing paclobutrazol. The differential increase in size of microtubers in the medium solidified with tapioca is possibly because of a better contribution of inorganic and organic components compared with agar while maintaining a favourable osmotic concentration.
Tapioca is expected to have a greater ionic supplement to ingredients of the culture medium than agar. Such conditions are expected to influence the rate of cell division (success of morphogenesis) of the tissues they support. Increasing sucrose supply, within a limit, can be a means of stimulating rooting and overall plantlet growth in vitro. Sago seemed to lack the virtues of a good gelling agent for tissue culture work.
In general tapioca at 11-15% gave comparable and/or significantly higher results of in vitro shoot proliferation than the agar and sago. The best concentration (14%) was found to be higher than the report of Nene and Sheila (1997) for Tobacco and Chickpea culture. Tapioca based medium was also better than agar based medium for microtuber development. The results showed the possibility of using tapioca as an alternative cheaper gelling substance (40x cheaper than agar at equal concentration) in micropropagation of potato through production of plantlets or microtubers. This may have a significant commercial implication.
We are very grateful to CIP Regional Office in Nairobi, Kenya, for financial support to carry out this study. We also thank Dr. Hailemichael Kidanemariam, Dr. Berga Lemaga and Mr. Robert Nüssli for their encouragement and co-operation.
TABLE 1. Effects of agar and various tapioca and sago concentrations (w/v) on shoot and root proliferation after 32 and 40 days of culture
Figures in parenthesis are transformed values
TABLE 2. Efficacy of gelling agents and paclobutrazol on microtuber number and fresh weight, and shoot and root fresh weight and percent biomass conversion
aBAP at 2.0 mg1-1, bPaclobutrazol at concentration 0.001 mg 1-1, cTapioca at 14 percent concentration, dSago at 10 percent concentration.
Figures in parenthesis are transformed values
TABLE 3. Effects of agar, tapioca and sago gelling agents at various concentrations (w/v) on shoot and root fresh weight and root: shoot ratio after 40 days of culture
** Significant P< 0.01; NS: Non-significant;
Figures in parenthesis are transformed values
TABLE 4. Effect of media type and paclobutrazol on frequency distribution of microtubers in various size categories
aTreatment concentrations are the same as indicated in Table 1
Figures in parenthesis are transformed values
TABLE 5. Effect of media type and paclobutrazol on frequency distribution (%) of microtubers in various size categories
aTreatment concentrations are the same as indicated in Table 1
Plate 1. Higher shoot and root growth observed in tapioca than agar tuberisation media.
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